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Isolation of human NURF: a regulator of Engrailed gene expression

Isolation of human NURF: a regulator of Engrailed gene expression The EMBO Journal Vol. 22 No. 22 pp. 6089-6100, 2003 Isolation of human NURF: a regulator of Engrailed gene expression 1,2 remodeling complexes containing the ISWI protein as the Orr Barak , Maribeth A.Lazzaro , ATPase catalytic subunit were identified initially in William S.Lane , David W.Speicher, 1 4 5 Drosophila and include NURF (nucleosome-remodeling David J.Picketts • and Ramin Shiekhattar factor), ACF (ATP-utilizing chromatin assembly and The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, remodeling factor) and CHRAC (chromatin assembly Harvard Microchemistry and Proteomics Analysis Facility, Harvard complex), and it is likely that each complex performs University, Cambridge, MA 02138, USA, The Ottawa Health unique functions (Tsukiyama et al., 1995; Ito et al., 1997; Research Institute, 501 Smyth Road, Ottawa, Ontario KlH 8L6 and 4university of Ottawa, Centre for Neuromuscular Disease and Varga-Weisz et al., 1997). Departments of Medicine, Biochemistry, Microbiology and Mammalian genomes encode two genes with close Immunology, Ottawa, Ontario, Canada homology to Drosophila ISWI, SNF2H and SNF2L Present address: Health Canada, Therapeutic Products Directorate, (Okabe et al., 1992; Aihara et al., 1998; Lazzaro and Bureau of Cardiology, Allergy and Neurological Sciences, Tunney's Picketts, 2001). In vivo, SNF2H is ubiquitously expressed, Pasteur, Ottawa, Ontario KIA 1B9, Canada with highest expression in populations of actively dividing Corresponding author cells during development and in adults (Lazzaro and e-mail: [email protected] Picketts, 2001). Biochemically, SNF2H is the ATPase O.Barak and M.A.Lazzaro contributed equally to this work catalytic core subunit of a variety of complexes including WCRF (WSTF-related chromatin-remodeling factor)/ The modification of chromatin structure is an import­ hACF (Bachar et al., 2000; LeRoy et al., 2000), RSF ant regulatory mechanism for developmental gene (remodeling and spacing factor) (LeRoy et al., 1998), expression. Differential expression of the mammalian WICH (WSTF-ISWI chromatin-remodeling complex) ISWI genes, SNF2H and SNF2L, has suggested that (Bozhenok et al., 2002), NoRC (nucleosome-remodeling they possess distinct developmental roles. Here we complex) (Strohner et al., 2001), huCHRAC (Poot et al., describe the purification and characterization of the 2000) and SNF2H--cohesin (Hakimi et al., 2002). The first human SNF2L-containing complex. The subunit SNF2H complexes all possess nucleosome spacing composition suggests that it represents the human activity that results in the formation of regular ordered ortholog of the Drosophila nucleosome-remodeling arrays. Moreover, several of these complexes associate factor (NURF) complex. Human NURF (hNURF) is with heterochromatin and facilitate its replication. Taken enriched in brain, and we demonstrate that it regu­ together, it is suggestive of a role for SNF2H complexes in lates human Engrailed, a homeodomain protein that the establishment and maintenance of heterochromatin regulates neuronal development in the mid-hindbrain. and, by extension, gene repression. Furthermore, we show that hNURF potentiates neur­ Despite a growing number of SNF2H-containing com­ ite outgrowth in cell culture. Taken together, our data plexes, relatively little is known about the closely related suggess a role for an ISWI complex in neuronal SNF2L protein. Specific antibodies to SNF2L suggest that growth. a small amount of HuCHRAC or WCRF/hACF comprises Keywords: BPTF/Engrailed/ISWI/NURF/SNF2L SNF2L rather than SNF2H, although an SNF2L-contain­ ing complex has not been purified (Bozhenok et al., 2002). The absence of a mammalian NURF complex has led to speculation that this may be an SNF2L-specific complex Introduction involved in transcriptional activation, yet this remains to Modification of histone tails or altering the placement of be determined. Furthermore, the brain-enriched expression nucleosomes within the context of a promoter can lead to pattern of SNF2L suggests a neurocentric role for this profound effects on gene expression and, ultimately, on putative complex (Lazzaro and Picketts, 2001). cell function and/or differentiation. As such, mutations in To address the physiological function of SNF2L, here genes encoding chromatin-modifying proteins cause a we report the purification of an SNF2L complex to wide range of human developmental disorders. Such a homogeneity, which corresponds to the human ortholog of wide range of defects probably reflects the specificity of Drosophila NURF (dNURF). We demonstrate that human the individual complexes that these proteins form NURF (hNURF) has nucleosome-, and to a lesser extent, (Hendrich and Bickmore, 2001). Indeed, chromatin­ DNA-stimulated ATPase activity, and that it can remodel remodeling complexes have been identified in a number a chromatin template in vitro. Moreover, the brain­ of species from yeast to humans, and many of these enriched hNURF complex interacts at the promoter of complexes show a high level of conservation in their two developmentally important genes to activate expres­ composition and function. In this regard, Drosophila sion. Importantly, we also demonstrate that this complex melanogaster has been shown to be a model organism to may induce neurite outgrowth, thereby suggesting a define these complexes. For example, three chromatin- developmental consequence of its activity. © European Molecular Biology Organization 6089 indicated. O.Barak et al. A B NE:f-SNF2L kDa: P11 p350 I I I I 0.1 0.3 0.5 1.0 250 - DEAE-Sephacel I I p140 120 - 0 . 1 0 . 5 .. ,�··· Superose6 0.5---r-----0.5 35- a-Flag Silver Stain I I FT B ! Peptide elution SDS-PAGE Fig. 1. Biochemical isolation of the hNURF SNF2L-associated complex. (A) Purification scheme. fSNF2L-HEK293 nuclear extract was fractionated by chromatography as described in Materials and methods. The horizontal and diagonal lines indicate stepwise and gradient elution, respectively. (B) Fractions immunoprecipitated from Superose 6 elutions using M2 anti-Flag beads were resolved on an SDS-polyacrylamide (4-12%) gel, and proteins were visualized by silver staining. Molecular masses of marker proteins (left) and polypeptide masses of associated subunits (right) are toma-associated protein 48 and 46 (RbAP48/46) gene Results products (Figure 2A). SNF2L exists in a multiprotein complex To facilitate the purification of SNF2L complexes, we The SNF2L-associated complex is the human generated HEK293 cell lines that expressed Flag epitope­ ortholog of dNURF tagged human SNF2L (f-SNF2L). This cell line was used BPTF and RbAP48/46 are the marnrnalian orthologs of due to its detectable but low endogenous levels of SNF2L Drosophila NURF301 and NURF55 polypeptides, and its propensity to express neuronal-specific genes (data respectively. Along with ISWI, these proteins represent not shown) (Shaw et al., 2002). We enriched for the three of the four components of the dNURF complex SNF2L-containing complex(es) by chromatographically (Martinez-Balbas et al., 1998; Xiao et al., 2001). fractionating nuclear extract derived from the f-SNF2L Therefore, we designated our SNF2L complex as cell line according to the scheme shown (Figure IA). The hNURF. The fourth component of dNURF, the NURF38 enriched fractions were subjected to affinity purification pyrophosphatase (Gdula et al., 1998), was not identified in using anti-Flag antibodies. The bound fraction was eluted our Flag-purified complex despite multiple attempts at with a peptide corresponding to the Flag epitope and purification (data not shown). analyzed by SDS-P AGE followed by silver stain. This BPTF is a known protein with two characterized analysis revealed three prominent polypeptides of ~350, alternatively spliced gene products. The full-length gene 140 and a 50 kDa doublet (Figure lB) as well as a few encodes a predicted 311 kDa protein containing a substoichiometric bands that proved to be breakdown bromodomain, two PHD fingers, three LXXLL putative products of the p350 protein (data not shown). nuclear receptor-binding motifs, a DDT DNA association Each subunit of the SNF2L complex was excised from domain, a BAZ domain present in many ISWI binding the gel, digested with trypsin, and subjected to sequencing partners, and a glutamine-rich region (Jones et al., 2000) by ion trap mass spectrometry. Forty-nine peptides (Figure 2A). Interestingly, the BPTF gene has been confirmed our predictions that the 140 kDa band consisted localized to chromosome 17q23, which is a hotspot for entirely of the SNF2L transgene. Forty-six peptides chromosomal changes in neuroblastomas and is a prog­ identified the 350 kDa band as the bromodomain PHD nostic factor for rapid disease progression (Lastowska finger transcription factor (BPTF) gene product, while 10 et al., 2001). A truncation of BPTF, called fetal peptides identified the 50 kDa doublet as the retinoblas- Alzheimer's clone 1 (FACl), contains the first 2643 6090 lgG u•2l lgG u•2l Isolation of human NURF ODTPHDBAZ Q-rich PHO Bromo BPTF(p350) co I 0 I i ■- SNF2 SANT SNF2L{p140) WD RbAP46/48(p46/48) ( 11118) B C In FT W B a-BPTF -I - ---I a-SN F2LI - -I �===� kDa: a-RbAP48_I ___ - BPTF - _. 191- -FAC1 97- 64- IP: FT 8 In 1B: a-BPTF! I �======. 39- a-SNF2L..._I -____ _ -__.I 28 . - 1 2 3 4 5 IP:a-Flag IB:a-BPTF-N Fxn .. : t � In 14 16 18 20 22 24 26 28 JO 32 34 ..., ~ � • (1-BPTF E] I - - - - - - - u-SNF2L EJI n-RbAP48 DI I Superose 6 Fig. 2. Composition of the novel SNF2L-associated complex. (A) Schematic of the SNF2L complex subunits. (B) Specificity of BPTF-N antibody for cDNAs expressing Facl (BPTF 1-2622) and full-length BPTF. (C) Immunoblot analysis of fractions from M2 anti-Flag beads resolved on an SDS­ polyacrylamide (4-12%) gel (I, input; FT, flow through; W, wash; B, bound). Antibodies used for immunoblot are indicated (left). (D) Endogenous hNURF immunoprecipitated from HEK293 partially purified nuclear extract. Immunoblot analysis of immunoprecipitation was resolved on an SDS­ polyacrylamide (4-12%) gel. Antibodies used for immunoprecipitation (IP) are indicated across the top, while antibodies used for immunoblot (IB) are on the left. Input represents 5% of IP. Flow through (FT) and bound (B) fractions are as indicated. (E) hNURF molecular mass is -1 MDa. Immunoblot of elutions from the Superose 6 gel filtration column from partially purified HEK293 nuclear extract. Fractions (fxn) as well as void (Vo) and thyroglobulin (670 kDa) molecular weight markers are indicated (top). nucleotides of BPTF and was isolated in a screen for ized using ectopically expressed Flag-BPTF as well as a proteins in Alzheimer' s disease senile plaques (Bowser truncated form of BPTF corresponding to the FACl et al., 1995). Subsequently, FACl was shown to be alternative splice form (Figure 2B). We confirmed the upregulated in motor neurons during development and in presence of BPTF and RbAP48 in the original SNF2L the neurodegenerative disease amyotrophic lateral scler­ immunoprecipitation experiment using the BPTF-N anti­ osis (Mu et al., 1997). FAC I was also shown to exhibit bodies and commercially available RbAP48 antibodies sequence-specific DNA binding activity and may function (Figure 2C). The absence of BPTF in the unbound fraction in transcriptional regulation (Jordan-Sciutto et al., 1999). of the immunoprecipitation suggested that the majority of To characterize hNURF further, we cloned the 8295 bp BPTF in the cell associated exclusively with SNF2L. A cDNA encoding BPTF. Antibodies were raised against an fraction of RbAP48 remaining in the unbound fraction N-terrninal peptide unique to BPTF, and were character- represented the RbAP48 in other known complexes such Vo hNURF 670kOa O.Barak et al. as NuRD (Zhang et al., 1999), E(Z) (Czermin et al., 2002), Sin3 (Zhang et al., 1998) and others. *� (j o '6 We isolated hNURF using an ectopic, highly expressed � '-: Flag-SNF2L. To confirm the presence of the endogenous hNURF complex, we subjected partially fractionated nai:ve -BPTF 250- [;:] HEK293 nuclear extract to immunoprecipitation of endogenous SNF2L followed by western analysis for the presence of BPTF. As predicted, SNF2L antibodies SNF2L [::::] immunoprecipitated a significant amount of endogenous SNF2L and BPTF as compared with control antibodies (Figure 2D, compare lanes 4 and 5). This experiment , .. -- 64- ✓RbAP48 allowed us to ask whether endogenous BPTF is primarily [i= ] associated with SNF2L, or if it is incorporated into other non-SNF2L complexes. We assayed the unbound fractions '-RbAP46 - - of the immunoprecipitations and observed that SNF2L immunodepleted BPTF from this fraction compared with IgG (Figure 2D, compare lanes 2 and 3). This suggests that the majority of cellular BPTF is associated with SNF2L. The predicted molecular weight of the hNURF complex Western assuming one copy of each subunit is ~500 kDa. To Silver Stain confirm this, we measured the molecular weight of the Fi . 3. hNURF purified with a one-step purification protocol. Mock and hNURF complex using gel filtration chromatography. We Flag-BPTF eluted fractions were fractionated by 8-16% SDS-PAGE showed that hNURF subunits co-eluted at ~ 1 MDa followed by silver staining and western analysis. BPTF, SNF2L, (Figure 2E), which is twice the predicted mass. This RbAP48 and RbAP46 poplypeptides are indicated on the right. Western blot analysis performed in parallel confirms the identification of the observation is in agreement with predicted versus meas­ polypeptides. Polypeptides marked with asterisks are contaminants also ured molecular weights for other ISWl-containing com­ present in the mock elution. Molecular weight markers are indicated on plexes including WCRF/hACF (Bochar et al., 2000), the left. huChRAC (Poot et al., 2000) and WICH (Bozhenok et al., 2002). precipitations from HEK293 nuclear extract. The chro­ One-step purification of hNURF matin-remodeling activity of hNURF was assessed using The purification scheme employed for the isolation of the restriction enzyme accessibility assay, which relies on hNURF (Figure IA) permitted the characterization of the the ability of DNA incorporated into chromatin to be subunit composition of the complex. However, we found refractory to restriction endonuclease digestion. Upon that enzymatic assays on the purified complex were addition of a chromatin-remodeling protein (or complex), difficult, mainly due to an inherent degradation of the mobilization of chromatin at the restriction endonuclease BPTF subunit, presumably from endogenous proteolytic site is facilitated, resulting in an ATP-dependent decrease activity. The !ability of BPTF was also seen in its in undigested array. Drosophila ortholog, NURF301 (Xiao et al., 2001), In the presence of an elution from a mock immunopre­ resulting in a smaller size (215 kDa) and difficulty in cipitation, we find that ~50% of the DNA remained uncut sequencing the peptide. This led us to develop a novel one­ (U) and thus refractory to restriction enzyme cleavage step protocol for the isolation of hNURF. (Figure 4A, lane 1). The cut (C) band represented the basal We generated an HEK293 stable cell line expressing a level of accessible DNA in the assay. Upon addition of Flag epitope-tagged BPTF cDNA. Nuclear extract from purified hNURF, we found a potent decrease in the level of the Flag-BPTF cell line was prepared, subjected to uncut (U) DNA along with the concomitant increase in cut immunopreciptation with Flag beads, washed stringently, (C) DNA (Figure 4B, lane 2). Consistent with the fact that and eluted with Flag peptide. SDS-P AGE fractionation of chromatin remodeling is coupled to release of energy from immunoprecipitated proteins followed by silver staining ATP hydrolysis, hNURF decreased the levels of the uncut demonstrates the presence of an electrophoretically pure (U) DNA in an ATP-dependent manner (Figure 4B, lane 2, hNURF complex (Figure 3) containing a BPTF subunit compare top and bottom panels). Densitometric analysis of with minimal proteolysis. We confirmed the identity of the restriction enzyme accessibility assays performed in subunits by western analysis using BPTF, SNF2L, triplicate demonstrated a significant ( ~ 2.5-fold) decrease RbAP48 and RbAP46 antibodies. As in our previous in the fraction of uncut DNA (Figure 4B). purification, both silver (Figure 3) and colloidal staining By titrating in increasing amounts of hNURF and (data not shown) failed to demonstrate the presence of the maintaining saturating amounts of A TP and nucleosomal human ortholog of one of the dNURF subunits, the p38 array, we found that as few as 1.25 fmol of hNURF pyrophosphatase. remodeled the nucleosomal array in an ATP-dependent manner (Figure 4C). We then measured the rate of hNURF remodels chromatin hNURF-mediated remodeling. At all time points, The hNURF complex, by virtue of its SNF2L subunit, is hNURF demonstrated ATP-dependent remodeling activity predicted to function as a chromatin-remodeling complex. indicated by the decrease in the fraction of uncut array To confirm this, we performed chromatin-remodeling compared with hNURF without A TP or with restriction assays comparing purified hNURF with mock immuno-enzyme alone (Figure 4D). We calculated the specific kDa: Isolation of human NURF remodeling complex, ySWI/SNF, using the identical assay A B (Logie and Peterson, 1997). 0.6 hNURF possesses intrinsic A TPase activity Recombinant monomeric ISWI proteins and ISWI­ containing complexes such as WCRF/hACF and ChRAC § 0.4 exhibit an intrinsic ATPase activity that is activated by addition of naked DNA and further potentiated by the addition of nucleosomes (Varga-Weisz, 1997; Bochar g 0.2 1... et al., 2000; Aalfs et al., 2001). Among ISWI complexes, LL. dNURF is unique in that its ATPase activity is exclusively nucleosome dependent (Xiao et al., 2001). We predicted Mock hNURF that hNURF would behave similarly to its Drosophila counterpart. In fact, we found that hNURF exhibited a slight but measurable DNA-dependent ATPase activity (Figure SA, compare lanes 5 and 4). However, the hNURF ATPase activity was far more pronounced upon addition •··-.. ••• • ATP of nucleosomes (Figure SA, compare lanes 6 and 5). --+ATP Indeed, upon titration of the two hNURF substrates, we found that nucleosomes consistently displayed a stronger potentiation of ATPase activity (Figure SB). These data, along with previously published ATPase assays using dNURF (Xiao et al., 2001), suggest that the physiological substrate of the NURF complex may indeed be nucleo­ 10 20 30 40 50 60 somes rather than free DNA. fmol hNURF Further characterization of hNURF revealed that its ATPase activity was dose dependent, with as little as 20 fmol of hNURF displaying potent nucleosome­ dependent ATPase activity (Figure SC). The specific - • - • Buffer+A TP 0.5 activity of the hNURF nucleosome-stimulated ATPase ............... NURF-ATP activity determined by measuring the rate of A TP a o. 4 --NURF+ATP :::i hydrolysis (Figure 5D) was calculated at 200 U/mg of C 0.3 SNF2L (1 U = 1 pmol of ATP hydrolyzed per rnin). :g 0.2 Consistent with the minimal responsiveness to DNA, the \! o.1 specific activity of the DNA-stimulated ATPase activity was found to be ~100-fold lower than with nucleosomes. 10 20 30 40 60 60 70 Time, minutes SNF2L and BPTF co-localize within adult mouse brain to hippocampus and cerebellum Fi g . 4. hNURF is a chromatin remodeler. (A) hNURF remodels Mouse SNF2L has been shown to exhibit a restricted chromatin. Autoradiograph of the restriction endonuclease-coupled remodeling assay demonstrating chromatin-remodeling activity. Mock expression pattern, which included higher expression IP failed to demonstrate any decrease in the uncut (U) array in an A TP­ levels in the central nervous system (Lazzaro and dependent manner (compare top and bottom panel of lane 1). Addition Picketts, 2001). Since the majority of BPTF appeared to of purified hNURF results in a qualitative decrease in the uncut (U) be associated exclusively with SNF2L in HEK293 cells, array compared with mock (compare lanes 1 and 2, top panel). This we postulated that the BPTF gene should have a similar activity is ATP dependent (compare lane 2 top and bottom panels). (B) hNURF addition results in an ~2.5-fold decrease in the fraction of expression profile to SNF2L in the brain. As such, adult the uncut array. Densitometric analysis of restriction endonuclease­ mouse brain was analyzed for expression of SNF2L and coupled remodeling. The graph represents the averages of the quanti­ BPTF by RNA in situ hybridization. Our results show that fied fraction of the uncut array (U/U + C) from three experiments. the BPTF transcript is highly expressed throughout the Standard errors for the experiments are as indicated in the graph. hippocampus and in the cerebellum, which is nearly (C) hNURF exhibits dose-dependent chromatin-remodeling activity. Graphical representation of densitometric analysis of assays over 0, identical to the SNF2L expression profile (Figure 6A and 1.25, 2.5, 5 and 50 frnol of hNURF with or without ATP. Points repre­ B) (Lazzaro and Picketts, 2001). The specificity of the sent the average of three reactions, with standard error as indicated. hybridization is demonstrated by the lack of signal with (D) Time course of hNURF-mediated chromatin remodeling. sense SNF2L and BPTF probes (Figure 6C and D, Restriction enzyme accessibility for buffer + ATP, 50 fmol hNURF­ ATP or 50 fmol hNURF + ATP. Points represent the average of three respectively). Higher magnification of cresyl violet­ reactions, with standard error as indicated. counterstained sections showed that the SNF2L and BPTF transcripts are both localized within the granule and Purkinje cell layer of the cerebellum (Figure 6E and F) activity of hNURF in the remodeling assay at 15 U/mg and in the pyramidal neurons throughout the hippocampus of SNF2L (1 U = 1 pmol of array digested/ruin). This (Figure 6G and H). The BPTF and SNF2L expression translates to ~ 1 pmol of hNURF remodeling 2 fmol profiles suggest that the hNURF complex is involved in the of array per min. This activity is of the same order as regulation of cerebellar and hippocampal function. In the published measured activity of another chromatin- addition, analysis of BPTF and SNF2L reveals a similar 6093 O.Barak et al. A B Buff er hNU RF DNA - + - • + - Nucs. • • + . . + Pi -· -- 50 100 150 2.00 250 300 DNA, ng AT P _ _,h NUR F ... ......... .B uff er +Nucs +Nucs __ _hNUR F Bu ffe r 1 2 3 4 5 6 +Free ONA +Free DNA C D •�t ··· ··· - I ......, _ -:_ -- ... 10 20 30 40 50 60 70 20 40 60 80 100 120 fm ol hNURF Time, min utes - - - Buff er - - - BUFFER ·· · ········ ··· ········ DN A oNA -- NUCS -N ucs Fig. 5. hNURF exhibits intrinsic nucleosome·dependent ATPase activity. (A) hNURF exhibits DNA· and nucleosome·stimulated ATPase activity. ATPase activity assays performed on [y. P]ATP followed by separation of free phosphate (Pi) from ATP by PEl•cellulose TLC and autoradiography of TLC plates. hNURF activity is stimulated by supplementing the reaction with DNA (50 ng) and is potentiated further by nucleosomes (50 ng) ; compare lanes 4, 5 and 6. (B) hNURF activity responds to increasing amounts of nucleosomes. A 25 fmol concentration of hNURF or buffer was incu• bated with increasing amounts (0, 50, 100 or 250 ng) of either free DNA or free nucleosomes. Points represent the average of three reactions, with standard error as indicated. (C) hNURF exhibits a dose•dependent ATPase activity in the presence of nucleosomes. ATPase activity of three different hNURF concentrations (20, 50 and 100 frnol) with buffer, free DNA or nucleosomal DNA. Points represent the average of three reactions, with stand• ard error as indicated. (D) Time course of hNURF ATPase activity. hNURF (100 fmol) was incubated with buffer, free DNA or nucleosomal DNA, and reactions were assayed for ATPase activity at the indicted times. Points represent the average of three reactions, with standard error as indicated. pattern of expression during embryonic development observed in the cerebellum prompted us to examine the (Supplementary figure 1 available at The EMBO Jo urnal human en-1 and en-2 genes as possible targets of the Online). hNURF complex. To this end, we employed chromatin immunoprecipitation (ChIP) assays using antibodies against two components of the hNURF complex, SNF2L In vivo localization of hNURF to human engrailed and BPTF. To demonstrate specificity, we assayed for promoters their presence at other promoters important in various The mammalian homologs of the Drosophila segment biological processes including neuronal development polarity genes engrailed-1 (e n-1) , engrailed-2 (en-2), wnt- (Johnston et al., 2001; Tanzi and Bertram, 2001). While 7b and Pax-2 have been shown to have a crucial role in our antibodies failed to show an interaction between cerebellar development (Millen et al., 1995). Mice lacking hNURF and presenilin -1, zn j261, uxt, /JA PP or Mecp2 either en -1 or en-2 demonstrate mid-hindbrain malforma­ promoters, we successfully demonstrated a specific inter­ tions and cerebellar dysgenesis, respectively (Wurst et al., action with the promoters of both En -1 and En-2 1994; Millen et al., 1995). Interestingly, expression of (Figure 7 A). The regions demonstrating the strongest Drosophila engrailed was severely compromised in flies hNURF binding correspond to -1 kb upstream on the en-1 mutated for ISWI or NURF301 expression (Deuring et al., and en-2 transcription start sites. Interestingly, while the 2000; Badenhorst et al., 2002). These results and the overlapping expression of SNF2L and BPTF that we en-1 promoter is still relatively uncharacterized, the en-2 6094 Isolation of human NURF levels show a significant reduction (2-fold) in response to SNF2L BPTF depletion of SNF2L (Figure 7C). We attempted similar experiments using siRNAs against BPTF, and demon­ strated a significant decrease in en -1 transcript level (Figure7D). However, we were unable to affect en-2 transcript levels. This may be due to the fact that the siRNAs against BPTF reduced its concentration by only 2- fold (Figure 7D), which may not be sufficient to alter en-2 expression. Supporting this notion are reports in the literature demonstrating substantial differences in en-1 and en-2 regulation (Hanks et al., 1995). Taken together, our data reveal that hNURF associates with the promoters of En-1 and En-2 and can modulate expression of en-1 and probably en-2. Furthermore, since loss of En-1 and En-2 causes defects in the formation of the cerebellum (Joyner and Martin, 1987; Wurst et al., 1994; Millen et al., 1995), these data suggest a role for hNURF in the regulation of genes required for normal brain development. SNF2L promotes neurite outgrowth The functional maturation of the developing nervous system relies, in part, on the formation of axonal and dendritic processes that extend from neuronal cell bodies, and on the ability of these processes to form proper Fig. 6. SNF2L and BPTF show overlapping expression in adult mouse synaptic connections. Several factors, including the brain. Adjacent sections from an adult mouse brain were hybridized Engrailed homeodomain protein (Cosgaya et al., 1998), with SNF2L (A, E and G) and BPTF (B, F and H) antisense RNA are known to promote outgrowth of neuritic processes. probes. Both SNF2L and BPTF were highly expressed throughout all Since the two human orthologs of Engrailed are regulated regions of the hippocampus (CAI-4) and in the granule cell layer of by hNURF, we asked whether SNF2L would promote the cerebellum (A and B dark field). The specificity of SNF2L and BPTF antisense probes is demonstrated by the lack of hybridization neurite outgrowth. of corresponding sense probes (C and D). Higher magnifications of To investigate this, we utilized the NlE-115 mouse sections shown in (A) and (B) counterstained with cresyl violet demon­ neuroblastoma cell line, which can be induced to differ­ strate the expression of SNF2L and BPTF in the granule and Purkinje entiate morphologically to a neuronal phenotype and cell layer of the cerebellum (E and F) and in the pyramidal neurons of the hippocampus (G and H). Scale bars, 2 mm in (B) ; 50 µm in (F) extend neurites when cultured in conditions of reduced and (H); gc, granule cells; p, Purkinje neurons; CAI pyr, CAI serum (Hirose et al., 1998). We asked whether uninduced pyramidal neurons. NlE-115 cells undergo differentiation following transfec­ tion with SNF2L. Cells were transfected with empty vector, SNF2L-WT, or SNF2L-K213R, which contains a promoter was found to have an upstream activation point mutation in the nucleotide-binding P-loop motif, element proximal to the hNURF-binding sites (McGrew abrogating all ATPase activity. Co-transfection of a et al., 1999). �-galactosidase vector permitted visualization of neurites by immunofluorescence microscopy. At 30 h post­ hNURF regulates expression of endogenous transfection, a comparison of the ability of each trans­ human engrailed fected plasmid to promote morphological differentiation The localization of the hNURF complex at the en-1 and and neurite outgrowth was assessed by counting cells with en-2 promoters prompted us to assess the role of hNURF in neurite extensions measuring more than twice the length of regulating their expression. Small interfering (si)RNAs the cell body. that are specific for SNF2L were generated and transfected Cells transfected with empty vector exhibited a very into HEK293 cells, and expression of SNF2L was checked low, but measurable, rate of uninduced neurite outgrowth by western blot. The SNF2L siRNAs reduced expression (Figure 8A and D). However, transfection of wild-type of SNF2L to below detectable levels, while control SNF2L resulted in a significant potentiation of neurite siRNAs had no effect (Figure 7B). outgrowth (Figure 8, compare B and E with A and D). This Using the siRNAs for SNF2L, we then assayed for ability to promote neurite outgrowth was dependent on the transcript levels of endogenous en-1 and en-2 in the enzymatic activity of SNF2L since the SNF2L-K213R presence or absence of hNURF. Total RNA was extracted ATPase-dead mutant virtually abolished all neuronal from SNF2L siRNA- and control siRNA-transfected cells, outgrowth (Figure 8, compare C and F with B and E). reverse transcribed to generate cDNAs, and subjected to Similarly, the specificity for SNF2L was demonstrated real-time PCR. Transcript levels were quantified and further by a failure of SNF2H to induce neurite extension normalized to �-actin levels using the ABI7000 sequence (data not shown). Quantification of these results demon­ detection software. Transcript levels of PSI and MECP2, strates that, indeed, transfection of SNF2L-WT promotes which lack hNURF at their promoters, are unaffected by neurite outgrowth while SNF2L-K213R renders outgrowth depletion of SNF2L. Importantly, en -1 and en-2 transcript undetectable (Figure 8F). The promotion of neurite 609 5 O.Barak et al. A B In lgG BPTF SNF2L siRNA Prese nilin- 1 En graile d-1 a- SNF 2 L I - Engr ailed-2 - I !=::==::::: ZN F261 a-Rb AP48 _I ___ I UXT 2 3 bAPP MECP2 C D 1.2 1.2 l l .I L a 1 "i; .: � 0.8 � 0.8 I: � 0 .6 � 0.6 'C o 0,4 � 0.4 0. 2 0,2 .. - 0 Ps1 ME CP2 En-1 En-2 Ps1 MECP2 En-1 En-2 BPTF Co ntro l D Co ntrol ■ SN F2L ■ BPTF Fig. 7. hNURF localizes to and regulates human Engrailed. (A) ChIP assays localize hNURF specifically to engrailed-I (e n-I) and engrailed-2 (en-2) promoters. Chromatin from HEK293 cells was subjected to formaldehyde cross-linking followed by sonication and immunoprecipitation with non­ specific, BPTF and SNF2L antibodies. After extensive washes, bound DNA was eluted and sub jected to PCR with the indicated promoter primers. A specific and reproducible signal at the engrailed promoters comparable with input (In) was detected in the BPTF and SNF2L IPs but not in the non­ specific IgG. Input fractions represent 1 % of the total IP. Multiple primer pairs from each promoter were employed. Non-specific promoters include presenilin-1, znf261, UXT, APP and MECP2 as indicated. A representative set is displayed. (B) siRNA-mediated depletion of SNF2L. HEK293 cells transfected with either mock, SNF2L-specific siRNAs or non-specific siRNAs. Extract from cells 72 h after transfection were prepared and separated by SDS-PAGE followed by western analysis for SNF2L levels. The SNF2L-specific siRNA depletes SNF2L to undetectable levels compared with mock and non-specific siRNAs (compare lane 2 with lanes I and 3, top panel). Lower panel: loading control for protein levels determined by RbAP48 levels. (C) Depletion of SNF2L leads to a significant decrease in en-I and en-2 transcripts. Total RNA was extracted from SNF2L-depleted and control HEK293 cells and subjected to reverse transcription followed by real-time PCR. Levels of transcripts were quantified and normalized to �-actin levels using the ABl7000 sequence detection system. Genes lacking hNURF at their promoters as determined by ChIP were used as controls. The bar graph depicts the average of three independent experiments. Standard errors are indicated. (D) Depletion of BPTF leads to a significant decrease in en-I transcript. Experiments were performed as in (C). The bar graph depicts the averages of three independent experiments. Standard errors are indicated. extension that we observed following transfection of Discussi on SNF2L is similar to that observed for other factors that modulate neurite outgrowth (Kobayashi et al., 2002; Li The Drosophila ISWI protein exists in three multiprotein complexes, namely, ACF, CHRAC and NURF et al ., 2002; Abe et al., 2003). We believe that the (Tsukiyama et al ., 1995; Varga-Weisz et al ., 1997; Ito ectopically expressed SNF2L acts via incorporation into the NURF complex; however, we cannot rule out the et al., 1999). Mammalian complexes corresponding to possibility that the neurite outgrowth phenotype could ACF and CHRAC have been purified and contain the have resulted from unincorporated SNF2L. While these SNF2H protein (Bochar et al., 2000; Poot et al., 2000). data were derived from a tissue culture-based neurite Additional unique mammalian ISWI complexes have also outgrowth assay, they lend further support to the hypoth­ been purified, including RSF, WICH, NoRC and SNF2H­ esis that SNF2L may play a role in the differentiation and cohesin (LeRoy et al ., 2000; Strohner et al ., 2001; maturation of neurons during development. Bozhenok et al ., 2002; Hakimi et al., 2002), and these 609 6 2.5 Isolation of human NURF SNF2L SNF2L Vector WT K2 13 R Low Mag . High Mag . 7.5 ."!:'. '- :::, Q,) C: .!: .!: "'O LL Vector SNF2L SNF2L WT K213 R Fig. 8. SNF2L promotes neurite outgrowth. NlE-115 cells were co-transfected transiently with vector (A and D), f-SNF2L-WT (B and E) or f-SNF2L-K213R (C and F) along with a �-galactosidase expression plasmids in a 10:1 ratio. After transfection, cells were cultured for 30 h in normal (non-inducing) growth medium and then fixed for immunofluorescence staining (Flag = green, �-galactosidase = red). Differentiated cells were identified as cells in which the length of the neurite extensions was at least twice the diameter of the cell body. The fold increase in neurite outgrowth in f- SNF2L-transfected cells compared with vector-transfected cells was calculated from cell counts (600 cells) obtained from three different experiments (G) and is statistically significant at P < 0.005. Scale bars, 50 µm. all comprise the SNF2H protein. Despite the growing list hNURF is similar as it displayed predominantly nucleo­ of mammalian ISWI complexes, a NURF equivalent or some-stimulated ATPase activity, as well as potent complexes containing the related protein SNF2L are chromatin-remodeling activity on oligonucleosomal notably absent. Here, we describe the purification of the arrays. first human SNF2L complex. The subunit composition The brain-enriched expression profile of SNF2L suggests that it represents the human ortholog of the prompted us to examine a role for hNURF in neuronal dNURF complex. In this regard, the hNURF complex also physiology. We showed that SNF2L chromatin-remodel­ contains BPTF and RbAP46/48. Surprisingly, hNURF ing activity could induce neurite outgrowth in a tissue does not contain the inorganic pyrophosphatase protein culture-based assay, and that this was specific to SNF2L­ NURF38. Nonetheless, the biochemical activity of containing ISWI complexes since SNF2H expression did 6097 O. Barak et al. staining and immunoblot. Alternatively, nuclear extract was prepared not result in a similar induction (data not shown). The from Flag-BPTF-expressing cells and sub jected to immunoprecipitation conversion of a neuroblast to a differentiated neuron will as indicated. Beads were washed with 0.5 M KCI and 0.5% NP-40 for require the modification of chromatin structure at numer­ 10 min, followed by 0.75 M KCI and 0.1 % NP-40 in IP buffer for 10 min, ous genes, for both activation and repression, and it is not followed by 1.0 M KCI with 0.1 % NP-40 in IP buffer for 10 min. Beads were sub jected to elution as described for the previous purification. likely to be restricted to the NURF complex. Nonetheless, our studies suggest that hNURF has a role in this process Mass spectrometric peptide sequencing and, thus, identification of target genes will help elucidate Excised bands were subj ected to N-terminal sequence analysis as the molecular pathways. In this regard, we clearly described (Bachar et al., 2000). demonstrate that hNURF can regulate the mammalian engrailed genes, through a direct interaction at the In situ hyb ridization promoters of these two homeotic loci. The murine For in situ hybridization experiments, an adult mouse brain cDNA was used to amplify by PCR a 475 bp fragment corresponding to a segment of engrailed genes are critical regulators of mid-hindbrain the 3'-coding region and untranslated region of human BPTF. The mouse development, as ablation leads to animals that are missing BPTF fragment was cloned into pBluescript KS as a template for RNA most of the colliculi and cerebellum (Wurst et al., 1994). probes. Brains dissected from adult mice perfused with 4% paraformal­ Although engrailed was identified previously as a NURF dehyde in 100 mM NaP0pH 7.4 were processed for cyosectioning. Antisense and sense Snt'21 and Bptf RNA probes were labeled with target gene through the characterization of flies harboring [ P]UTP (2000 Ci/mmol) (Amersham Biosciences, Sweden) by in vitro mutant ISWI or NURF301 genes (Deuring et al., 2000; transcription from plasmid templates using T3 and T7 RNA polymerases Badenhorst et al., 2002), a neural defect was not appre­ (Promega, USA). Cryosections were processed for hybridization, ciated due to the early lethality of these animals. As such, hybridized with probes and exposed to autoradiographic emulsion as this may represent a novel function for the NURF described previously. complex. Restriction endonuclease-coupled remodeling assay The effect of chromatin-remodeling complexes on Reconstitution of nucleosomal arrays and chromatin-remodeling assays development is a well-established phenomenon. were performed as described (Logie and Peterson, 1997). More details are Linkages between chromatin remodeling and develop­ provided in the Supplementary data. mental disorders include ATRX and mental retardation (Picketts et al., 1996), SMARCALl and Schimke Purification of nucleosomes Nucleosomes were purified from HeLa cell nuclear pellet as described immuno-osseous dysplasia (Boerkoel et al., 2002), CSB (Utley et al., 1996). More details can be found in the Supplementary data. and Cockayne syndrome (Citterio et al., 2000), and SNF2H and William's syndrome (Bachar et al., 2000). ATPase assays Based on our results, we hypothesize that the hNURF Measurement of ATPase activity was performed in 10 µI reactions under complex may represent another connection of a chroma­ the following conditions: 20 mM Tris-HCI, 60 mM KCI, 4% glycerol, tin-remodeling protein to disorders of development, and 4 mM MgC1 , 1 mM cold ATP, 1 µCi of [y-P]ATP and 2 nmol of hNURF or an equivalent volume of elution buffer. Where indicated, the we are confident that the hNURF complex regulates other reaction was supplemented with 50 ng of naked DNA or nucleosomes. developmentally important genes. In this regard, the Reactions were performed at 30 C for 1 h. Free phosphate and ATP were analysis of flies ablated for the NURF complex also separated by TLC on PEI-cellulose plates (J.T.Baker, USA). A 1 µ1 suggests a role for the hNURF complex in hematopoietic aliquot of the reaction was spotted onto a plate and TLC was carried out in 1 M formic acid, 0.5 M LiCL Plates were then allowed to dry and were development and the regulation of chromosome structure visualized by exposure to a phosporimager cassette (Molecular (Badenhorst et al., 2002). However, such studies in Dynamics, Sweden) for densitometric analysis, or film (Kodak, USA). mammals must await further dissection using in vivo For rate assays, reaction mix was pre-warmed to 30 C prior to addition of model systems. hNURF. Chromatin immunoprecipitation (ChlPJ Materials and meth ods ChIP experiments and buffers were performed as described in the Upstate ChIP protocol. More details are available in the Supplementary data. Purification of the hNURF complex hNURF was purified from 350 mg of t'SNF2L-HEK293 nuclear extract. Quantitative PCR Nuclear extract was loaded on a 100 ml column of phosphocellulose (PI 1, Transcript levels were determined by quantitative PCR using an ABI7000 Whatman, USA) and fractionated stepwise by the indicated KCI (Applied Biosystems, USA). PCR was performed using the Sybr Green concentrations in buffer A [20 mM Tris-HCI pH 7.9, 0.2 mM 2x Master Mix (Applied Biosystems, USA) as per the manufacturer's EDTA,10 mM �-mercaptoethanol, 10% glycerol, 0.2 mM phenylmethyl­ protocol. Unknowns were amplified in triplicate. Positive control sulfonyl fluoride (PMSF)]. The 1.0 M KCI fraction (25 mg) of Pll was reactions were performed to establish a standard curve for each primer loaded on a 5 ml DEAE-Sephacel column (Amersham Biosciences, pair for transcript level normalization. �-actin served as an internal Sweden) and eluted with 0.5 M KCL The 0.5 M KCI elution (12.5 mg) control. was dialyzed to 0.1 M KCI in buffer A containing I µg/ml aprotinin, leupeptin and pepstatin, and loaded on a Mono Q HR 5/5 column Neurite outgrowth assay (Amersham Biosciences, Sweden). The column was resolved by using a NlE-115 cells were seeded at 50% confluency 24 h prior to transfection. linear 10 column volume gradient of 100-500 mM KCI in buffer A Cells were transfected with pCDNA3 vector, pCDNA3-f-SNF2L-WT or containing 1 µg/ml aprotinin, leupeptin and pepstatin. SNF2L-containing pCDNA3-f-SNF2L-K213R and �-galactosidase expression plasmids in fractions were fractionated on a Superose 6 HR 10/30 column (Amersham a 10: 1 ratio. Cells were maintained in normal growth medium for Biosciences, Sweden) equilibrated in 0.5 M KCI in buffer A containing 30 h post-transfection. Cells were then fixed for immunofl.uorescent 0.1 % NP-40, 1 µg/ml aprotinin, leupeptin and pepstatin. Fractions 18-20 staining. Transfected cells and neurites were visualized by Flag and were subj ected to immunoaffinity purification using M2 anti-Flag �-galactosidase antibodies. Differentiated cells were identified as cells in antibody-conjugated agarose beads (Sigma, USA). Beads were washed which the length of neurite extensions was at least twice the diameter of with 0.5 M KCI in IP buffer [20 mM Tris-HCI pH 7.9, 0.2 mM EDTA, the cell body. For quantification experiments, 600 cells were counted. 1 mM dithiothreitol (DTI), 10% glycerol, 0.2 mM PMSF] containing 1 µg/ml aprotinin, leupeptin and pepstatin, and bound fractions were eluted with 400 µg/ml of Flag peptide (Sigma, USA) in 0.1 M KCI in IP Supplementary data buffer. Purified hNURF was sub jected to SDS-PAGE followed by silver Supplementary data are available at The EMBO Journal Online. 6098 Isolation of human NURF protein kinase (pl 60ROCK)-regulated neurite remodeling in Ackn owl edgements neuroblastoma Nl E-115 cells. J. Cell Biol., 141, 1625-1636. Ito,T., Bulger,M., Pazin,M.J., Kobayashi,R. and Kadonaga,J.T. (1997) We thank D.Bochar for comments and experimental advice, J.Neveu, ACF, an ISWI-containing and ATP-utilizing chromatin assembly and J.Asara and R.Robinson for their expertise in HPLC and mass remodeling factor. Cell, 90, 145-155. spectrometry, Dr Robert Bowser for Flag-FACl cDNA, the National Ito,T., Levenstein,M.E., Fyodorov,D.V., Kutach,A.K., Kobayashi,R. and Cell Culture Center (Minneapolis, MN) for propagation of fSNF2L-293 Kadonaga,J.T. (1999) ACF consists of two subunits, Acfl and ISWI, cells, and the Wistar Institute protein microchemistry/mass spectrometry that function cooperatively in the A TP-dependent catalysis of facility. R.S. was supported by grants from the NIH (GM61204) and the chromatin assembly. Genes Dev., 13, 1529-1539. American Cancer Society. O.G.B. was supported by the NIH cancer grant Johnston,M.V., Jeon,O.H., Pevsner,J., Blue,M.E. and Naidu,S. (2001) (CA09171-28). M.A.L. was supported by the Ontario Mental Health Neurobiology of Rett syndrome: a genetic disorder of synapse Foundation. D.J.P. is a CIHR New Investigator with work supported by a development. Brain Dev., 23, S206--S213. CIHR grant (MOP53224). Jones,M.H., Hamana,N. and Shimane,M. (2000) Identification and characterization of BPTF, a novel bromodomain transcription factor. Genomics, 63, 35-39. Jordan-Sciutt o,K.L., Dragich,J.M. and Bowser,R. (1999) DNA binding Referen ces activity of the fetal Alz-50 clone 1 (FACl) protein is enhanced by phosphorylation. Biochem. Biophys. Res. Commun. , 260, 785-789. Aalfs,J.D., Narlikar,G.J. and Kingston,R.E. (2001) Functional Joyner,A.L. and Martin,G.R. (1987) En-1 and En-2, two mouse genes differences between the human ATP-dependent nucleosome with sequence homology to the Drosophila engrailed gene: expression remodeling proteins BRGl and SNF2H. J. Biol. Chem., 276, 34270 -- during embryogenesis. Genes Dev., 1, 29-38. Kobayashi,M., Taniura,H. and Y oshikawa,K. (2002) Ectopic expression Abe,T., Kato,M., Miki,H., Takenawa,T. and Endo,T. (2003) Small of necdin induces differentiation of mouse neuroblastoma cells. J. Biol. GTPase TclO and its homologue RhoT induce N-WASP-mediated Chem., 277, 42128-42135. long process formation and neurite outgrowth. J. Cell Sci., 116, 155- Lastowska,M., Van Roy,N., Bown,N., Speleman,F., Roberts,P., Lunec,J., Strachan,T., Pearson,A.D. and Jackson,M.S. (2001) Molecular Aihara,T., Miyoshi,Y., Koyama,K., Suzuki,M., Takahashi,E., cytogenetic definition of 17 q translocation breakpoints in Monden,M. and Nakamura,Y. (1998) Cloning and mapping of neuroblastoma. Med. Pedia tr. Oneal., 36, 20--23. SMARCAS encoding hSNF2H, a novel human homologue of Lazzaro,M.A. and Picketts,D.J. (2001) Cloning and characterization of Drosophila ISWI. Cytogenet. Cell Genet., 81, 191-193. the murine imitation switch (ISWI) genes: differential expression Badenhorst,P., Voas,M., Rebay,I. and Wu,C. (2002) Biological functions patterns suggest distinct developmental roles for Snf2h and Snf21. J. of the ISWI chromatin remodeling complex NURF. Genes Dev., 16, Neurochem., 77, 1145-1156. 3186--3198. LeRoy,G., Orphanides,G., Lane,W.S. and Reinberg,D. (1998) Bochar,D.A., Savard,J., Wang,W., Lafleur,D.W., Moore,P., Cote,J. and Requirement of RSF and FACT for transcription of chromatin Shiekhattar,R. (2000) A family of chromatin remodeling factors templates in vitro. Science, 282, 1900--1904. related to Williams syndrome transcription factor. Proc. Natl Acad. LeRoy,G., Loyola,A., Lane,W.S. and Reinberg,D. (2000) Purification Sci. USA, 97, 1038---1043. and characterization of a human factor that assembles and remodels Boerkoel,C.F. et al. (2002) Mutant chromatin remodeling protein chromatin. J. Biol. Chem., 275, 14787-14790. SMARCALl causes Schimke immuno-osseous dysplasia. Nat. Li,X., Meriane,M., Triki,I., Shekarabi,M., Kennedy,T.E., Larose,L. and Genet., 30, 215-220. Lamarche-Vane,N. (2002) The adaptor protein Nck-1 couples the Bowser,R., Giambrone,A. and Davies,P. (1995) FACl, a novel gene netrin-1 receptor DCC ( deleted in colorectal cancer) to the activation identified with the monoclonal antibody Alz50, is developmentally of the small GTPase Rae l through an atypical mechanism. J. Biol. regulated in human brain. Dev. Neurosci., 17, 20--37. Chem. , 277, 3778 8-37797. Bozhenok,L., Wade,P.A. and Varga-Weisz,P. (2002) WSTF-ISWI Logie,C. and Peterson,C.L. (1997) Catalytic activity of the yeast SWI/ chromatin remodeling complex targets heterochromatic replication SNF complex on reconstituted nucleosome arrays. EMBO J. , 16, foci. EMB O J. , 21, 2231-2241. 6772--6782. Citterio,E., Van Den Boom,V., Schnitzler,G., Kanaar,R., Bonte,E., Martinez-Balbas,M.A., Tsukiyama,T., Gdula,D. and Wu,C. (1998) Kingston,R.E., Hoeijmakers,J.H. and Vermeulen,W. (2000) ATP­ Drosophila NURF-55, a WD repeat protein involved in histone dependent chromatin remodeling by the Cockayne syndrome B DNA metabolism. Proc. Natl Acad. Sci. USA, 95, 132-137. repair-transcription-coupling factor. Mol. Cell. Biol., 20, 7643-7653. McGrew,L.L., Takemaru,K., Bates,R. and Moon,R.T. (1999) Direct Cosgaya,J.M., Aranda,A., Cruces,J. and Martin-Blanco,E. (1998) regulation of the Xenopus engrailed-2 promoter by the Wnt signaling Neuronal differentiation of PC12 cells induced by engrailed pathway and a molecular screen for Wnt-responsive genes, confirm a homeodomain is DNA-binding specific and independent of MAP role for Wnt signaling during neural patterning in Xenopus. Mech. kinases. J. Cell Sci., 111, 2377-2384. Dev., 87, 21-32. Czermin,B., Melfi,R., McCabe,D., Seitz,V., Imhof,A. and Pirrotta,V. Millen,K.J., Hui,C.C. and Joyner,A.L. (1995) A role for En-2 and other (2002) Drosophila enhancer of Zeste/ESC complexes have a histone murine homologues of Drosophila segment polarity genes in H3 methyltransferase activity that marks chromosomal Polycomb regulating positional information in the developing cerebellum. sites. Cell, 111, 185-196. Devel opment, 121, 3935-3945. Deuring,R. et al. (2000) The ISWI chromatin-remodeling protein is Mu,X., Springer,J.E. and Bowser,R. (1997) FAC l expression and required for gene expression and the maintenance of higher order localization in motor neurons of developing, adult and amyotrophic chromatin structure in viva. Mol. Cell, 5, 355 -365. lateral sclerosis spinal cord. Exp. Neural., 146, 17-24. Gdula,D.A., Sandaltzopoulos,R., Tsukiyama,T., Ossipow,V. and Wu,C. Okabe,I., Bailey,L.C., Attree,O., Srinivasan,S., Perkel,J.M., (1998) Inorganic pyrophosphatase is a component of the Drosophila Laurent,B.C., Carlson,M., Nelson,D.L. and Nussbaum,R.L. (1992) nucleosome remodeling factor complex. Genes Dev., 12, 3206--3216. Cloning of human and bovine homologs of SNF2/SWI2: a global Hakimi,M.A., Bochar,D.A., Schrniesing,J.A., Dong,Y., Barak,O.G., activator of transcription in yeast S.cerevisiae. Nucleic Acids Res., 20, Speicher,D.W., Yokomori,K. and Shiekhattar,R. (2002) A chromatin 4649-4655. remodelling complex that loads cohesin onto human chromosomes. Picketts,D.J., Higgs,D.R., Bachoo,S., Blake,D.J., Quarrell,O.W. and Na ture, 418, 994-998. Gibbons,R.J. (1996) ATRX encodes a novel member of the SNF2 Hanks,M., Wurst,W., Anson-Cartwright,L., Auerbach,A.B. and family of proteins: mutations point to a common mechanism Joyner,A.L. (1995) Rescue of the En-1 mutant phenotype by underlying the ATR-X syndrome. Hum. Mol. Genet., 5, 1899-1907. replacement of En-1 with En-2. Science, 269, 679--6 82. Poot,R.A., Dellaire,G., Hulsmann,B.B., Grimaldi,M.A., Corona,D.F., Harlow,E. and Lane,D. (1988) Antibodies: A Laboratory Manual. Cold Becker,P.B., Bickmore,W.A. and Varga-Weisz,P.D. (2000) Spring Harbor Laboratory Press, Cold Spring Harbor, NY. HuCHRAC, a human ISWI chromatin remodelling complex contains Hendrich,B. and Bickmore,W. (2001) Human diseases with underlying hACFl and two novel histone-fold proteins. EMB O J. , 19, 3377- 3387. defects in chromatin structure and modification. Hum. Mol. Genet., 10, Shaw,G., Morse,S., Ararat,M. and Graham,F.L. (2002) Preferential 2233-2242. transformation of human neuronal cells by human adenoviruses and Hirose,M. et al. (1998) Molecular dissection of the Rho-associated the origin of HEK 293 cells. FASEB J. , 16, 869-871. 6099 O.Barak et al. Strohner,R., Nemeth,A., Jansa,P., Hofmann-Rohrer,U., Santoro,R., Langst,G. and Grummt,I. (2001) NoRC-a novel member of mammalian ISWI-containing chromatin remodeling machines. EMB O J. , 20, 4892-4900. Tanzi,R.E. and Bertram,L. (2001) New frontiers in Alzheimer's disease genetics. Neuron, 32, 181-184. Tsukiyama,T., Daniel,C., Tamkun,J. and Wu,C. (1995) ISWI, a member of the SWI2/SNF2 A TPase family, encodes the 140 kDa subunit of the nucleosome remodeling factor. Cell, 83, 1021-1026. Utley,R.T., Owen-Hugbes,T.A., Juan,L.J., Cote,J., Adams,C.C. and Workman,J.L. (1996) In vitro analysis of transcription factor binding to nucleosomes and nucleosome disruption/displacement. Methods Enzymol., 274, 276--291. Varga-Weis z,P.D., Wilm,M., Bonte,E., Dumas,K., Mann,M. and Becker,P.B. (1997) Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Na ture, 388, 598---602. Wurst,W., Auerbach,A.B. and Joyner,A.L. (1994) Multiple developmental defects in Engrailed-1 mutant mice: an early mid­ hindbrain deletion and patterning defects in forelimbs and sternum. Devel opment, 120, 2065-2075. Xiao,H., Sandaltzopoulos,R., Wang,H.M., Hamiche,A., Ranallo,R., Lee,K.M., Fu,D. and Wu,C. (2001) Dual functions of largest NURF subunit NURF301 in nucleosome sliding and transcription factor interactions. Mol. Cell, 8, 531-543. Zhang,Y., Sun,Z.W., Iratni,R., Erdjument-Bromage,H., Tempst,P., Hampsey,M. and Reinberg,D. (1998) SAP30, a novel protein conserved between human and yeast, is a component of a histone deacetylase complex. Mol. Cell, 1, 1021-1031. Zhang,Y., Ng,H.H., Erdjument-Bromage,H., Tempst,P., Bird,A. and Reinberg,D. (1999) Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev., 13, 1924-1935. Received May 23, 2003; revised Se ptember 3, 2003; accepted Se ptember 26, 2003 61 00 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Isolation of human NURF: a regulator of Engrailed gene expression

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The EMBO Journal Vol. 22 No. 22 pp. 6089-6100, 2003 Isolation of human NURF: a regulator of Engrailed gene expression 1,2 remodeling complexes containing the ISWI protein as the Orr Barak , Maribeth A.Lazzaro , ATPase catalytic subunit were identified initially in William S.Lane , David W.Speicher, 1 4 5 Drosophila and include NURF (nucleosome-remodeling David J.Picketts • and Ramin Shiekhattar factor), ACF (ATP-utilizing chromatin assembly and The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, remodeling factor) and CHRAC (chromatin assembly Harvard Microchemistry and Proteomics Analysis Facility, Harvard complex), and it is likely that each complex performs University, Cambridge, MA 02138, USA, The Ottawa Health unique functions (Tsukiyama et al., 1995; Ito et al., 1997; Research Institute, 501 Smyth Road, Ottawa, Ontario KlH 8L6 and 4university of Ottawa, Centre for Neuromuscular Disease and Varga-Weisz et al., 1997). Departments of Medicine, Biochemistry, Microbiology and Mammalian genomes encode two genes with close Immunology, Ottawa, Ontario, Canada homology to Drosophila ISWI, SNF2H and SNF2L Present address: Health Canada, Therapeutic Products Directorate, (Okabe et al., 1992; Aihara et al., 1998; Lazzaro and Bureau of Cardiology, Allergy and Neurological Sciences, Tunney's Picketts, 2001). In vivo, SNF2H is ubiquitously expressed, Pasteur, Ottawa, Ontario KIA 1B9, Canada with highest expression in populations of actively dividing Corresponding author cells during development and in adults (Lazzaro and e-mail: [email protected] Picketts, 2001). Biochemically, SNF2H is the ATPase O.Barak and M.A.Lazzaro contributed equally to this work catalytic core subunit of a variety of complexes including WCRF (WSTF-related chromatin-remodeling factor)/ The modification of chromatin structure is an import­ hACF (Bachar et al., 2000; LeRoy et al., 2000), RSF ant regulatory mechanism for developmental gene (remodeling and spacing factor) (LeRoy et al., 1998), expression. Differential expression of the mammalian WICH (WSTF-ISWI chromatin-remodeling complex) ISWI genes, SNF2H and SNF2L, has suggested that (Bozhenok et al., 2002), NoRC (nucleosome-remodeling they possess distinct developmental roles. Here we complex) (Strohner et al., 2001), huCHRAC (Poot et al., describe the purification and characterization of the 2000) and SNF2H--cohesin (Hakimi et al., 2002). The first human SNF2L-containing complex. The subunit SNF2H complexes all possess nucleosome spacing composition suggests that it represents the human activity that results in the formation of regular ordered ortholog of the Drosophila nucleosome-remodeling arrays. Moreover, several of these complexes associate factor (NURF) complex. Human NURF (hNURF) is with heterochromatin and facilitate its replication. Taken enriched in brain, and we demonstrate that it regu­ together, it is suggestive of a role for SNF2H complexes in lates human Engrailed, a homeodomain protein that the establishment and maintenance of heterochromatin regulates neuronal development in the mid-hindbrain. and, by extension, gene repression. Furthermore, we show that hNURF potentiates neur­ Despite a growing number of SNF2H-containing com­ ite outgrowth in cell culture. Taken together, our data plexes, relatively little is known about the closely related suggess a role for an ISWI complex in neuronal SNF2L protein. Specific antibodies to SNF2L suggest that growth. a small amount of HuCHRAC or WCRF/hACF comprises Keywords: BPTF/Engrailed/ISWI/NURF/SNF2L SNF2L rather than SNF2H, although an SNF2L-contain­ ing complex has not been purified (Bozhenok et al., 2002). The absence of a mammalian NURF complex has led to speculation that this may be an SNF2L-specific complex Introduction involved in transcriptional activation, yet this remains to Modification of histone tails or altering the placement of be determined. Furthermore, the brain-enriched expression nucleosomes within the context of a promoter can lead to pattern of SNF2L suggests a neurocentric role for this profound effects on gene expression and, ultimately, on putative complex (Lazzaro and Picketts, 2001). cell function and/or differentiation. As such, mutations in To address the physiological function of SNF2L, here genes encoding chromatin-modifying proteins cause a we report the purification of an SNF2L complex to wide range of human developmental disorders. Such a homogeneity, which corresponds to the human ortholog of wide range of defects probably reflects the specificity of Drosophila NURF (dNURF). We demonstrate that human the individual complexes that these proteins form NURF (hNURF) has nucleosome-, and to a lesser extent, (Hendrich and Bickmore, 2001). Indeed, chromatin­ DNA-stimulated ATPase activity, and that it can remodel remodeling complexes have been identified in a number a chromatin template in vitro. Moreover, the brain­ of species from yeast to humans, and many of these enriched hNURF complex interacts at the promoter of complexes show a high level of conservation in their two developmentally important genes to activate expres­ composition and function. In this regard, Drosophila sion. Importantly, we also demonstrate that this complex melanogaster has been shown to be a model organism to may induce neurite outgrowth, thereby suggesting a define these complexes. For example, three chromatin- developmental consequence of its activity. © European Molecular Biology Organization 6089 indicated. O.Barak et al. A B NE:f-SNF2L kDa: P11 p350 I I I I 0.1 0.3 0.5 1.0 250 - DEAE-Sephacel I I p140 120 - 0 . 1 0 . 5 .. ,�··· Superose6 0.5---r-----0.5 35- a-Flag Silver Stain I I FT B ! Peptide elution SDS-PAGE Fig. 1. Biochemical isolation of the hNURF SNF2L-associated complex. (A) Purification scheme. fSNF2L-HEK293 nuclear extract was fractionated by chromatography as described in Materials and methods. The horizontal and diagonal lines indicate stepwise and gradient elution, respectively. (B) Fractions immunoprecipitated from Superose 6 elutions using M2 anti-Flag beads were resolved on an SDS-polyacrylamide (4-12%) gel, and proteins were visualized by silver staining. Molecular masses of marker proteins (left) and polypeptide masses of associated subunits (right) are toma-associated protein 48 and 46 (RbAP48/46) gene Results products (Figure 2A). SNF2L exists in a multiprotein complex To facilitate the purification of SNF2L complexes, we The SNF2L-associated complex is the human generated HEK293 cell lines that expressed Flag epitope­ ortholog of dNURF tagged human SNF2L (f-SNF2L). This cell line was used BPTF and RbAP48/46 are the marnrnalian orthologs of due to its detectable but low endogenous levels of SNF2L Drosophila NURF301 and NURF55 polypeptides, and its propensity to express neuronal-specific genes (data respectively. Along with ISWI, these proteins represent not shown) (Shaw et al., 2002). We enriched for the three of the four components of the dNURF complex SNF2L-containing complex(es) by chromatographically (Martinez-Balbas et al., 1998; Xiao et al., 2001). fractionating nuclear extract derived from the f-SNF2L Therefore, we designated our SNF2L complex as cell line according to the scheme shown (Figure IA). The hNURF. The fourth component of dNURF, the NURF38 enriched fractions were subjected to affinity purification pyrophosphatase (Gdula et al., 1998), was not identified in using anti-Flag antibodies. The bound fraction was eluted our Flag-purified complex despite multiple attempts at with a peptide corresponding to the Flag epitope and purification (data not shown). analyzed by SDS-P AGE followed by silver stain. This BPTF is a known protein with two characterized analysis revealed three prominent polypeptides of ~350, alternatively spliced gene products. The full-length gene 140 and a 50 kDa doublet (Figure lB) as well as a few encodes a predicted 311 kDa protein containing a substoichiometric bands that proved to be breakdown bromodomain, two PHD fingers, three LXXLL putative products of the p350 protein (data not shown). nuclear receptor-binding motifs, a DDT DNA association Each subunit of the SNF2L complex was excised from domain, a BAZ domain present in many ISWI binding the gel, digested with trypsin, and subjected to sequencing partners, and a glutamine-rich region (Jones et al., 2000) by ion trap mass spectrometry. Forty-nine peptides (Figure 2A). Interestingly, the BPTF gene has been confirmed our predictions that the 140 kDa band consisted localized to chromosome 17q23, which is a hotspot for entirely of the SNF2L transgene. Forty-six peptides chromosomal changes in neuroblastomas and is a prog­ identified the 350 kDa band as the bromodomain PHD nostic factor for rapid disease progression (Lastowska finger transcription factor (BPTF) gene product, while 10 et al., 2001). A truncation of BPTF, called fetal peptides identified the 50 kDa doublet as the retinoblas- Alzheimer's clone 1 (FACl), contains the first 2643 6090 lgG u•2l lgG u•2l Isolation of human NURF ODTPHDBAZ Q-rich PHO Bromo BPTF(p350) co I 0 I i ■- SNF2 SANT SNF2L{p140) WD RbAP46/48(p46/48) ( 11118) B C In FT W B a-BPTF -I - ---I a-SN F2LI - -I �===� kDa: a-RbAP48_I ___ - BPTF - _. 191- -FAC1 97- 64- IP: FT 8 In 1B: a-BPTF! I �======. 39- a-SNF2L..._I -____ _ -__.I 28 . - 1 2 3 4 5 IP:a-Flag IB:a-BPTF-N Fxn .. : t � In 14 16 18 20 22 24 26 28 JO 32 34 ..., ~ � • (1-BPTF E] I - - - - - - - u-SNF2L EJI n-RbAP48 DI I Superose 6 Fig. 2. Composition of the novel SNF2L-associated complex. (A) Schematic of the SNF2L complex subunits. (B) Specificity of BPTF-N antibody for cDNAs expressing Facl (BPTF 1-2622) and full-length BPTF. (C) Immunoblot analysis of fractions from M2 anti-Flag beads resolved on an SDS­ polyacrylamide (4-12%) gel (I, input; FT, flow through; W, wash; B, bound). Antibodies used for immunoblot are indicated (left). (D) Endogenous hNURF immunoprecipitated from HEK293 partially purified nuclear extract. Immunoblot analysis of immunoprecipitation was resolved on an SDS­ polyacrylamide (4-12%) gel. Antibodies used for immunoprecipitation (IP) are indicated across the top, while antibodies used for immunoblot (IB) are on the left. Input represents 5% of IP. Flow through (FT) and bound (B) fractions are as indicated. (E) hNURF molecular mass is -1 MDa. Immunoblot of elutions from the Superose 6 gel filtration column from partially purified HEK293 nuclear extract. Fractions (fxn) as well as void (Vo) and thyroglobulin (670 kDa) molecular weight markers are indicated (top). nucleotides of BPTF and was isolated in a screen for ized using ectopically expressed Flag-BPTF as well as a proteins in Alzheimer' s disease senile plaques (Bowser truncated form of BPTF corresponding to the FACl et al., 1995). Subsequently, FACl was shown to be alternative splice form (Figure 2B). We confirmed the upregulated in motor neurons during development and in presence of BPTF and RbAP48 in the original SNF2L the neurodegenerative disease amyotrophic lateral scler­ immunoprecipitation experiment using the BPTF-N anti­ osis (Mu et al., 1997). FAC I was also shown to exhibit bodies and commercially available RbAP48 antibodies sequence-specific DNA binding activity and may function (Figure 2C). The absence of BPTF in the unbound fraction in transcriptional regulation (Jordan-Sciutto et al., 1999). of the immunoprecipitation suggested that the majority of To characterize hNURF further, we cloned the 8295 bp BPTF in the cell associated exclusively with SNF2L. A cDNA encoding BPTF. Antibodies were raised against an fraction of RbAP48 remaining in the unbound fraction N-terrninal peptide unique to BPTF, and were character- represented the RbAP48 in other known complexes such Vo hNURF 670kOa O.Barak et al. as NuRD (Zhang et al., 1999), E(Z) (Czermin et al., 2002), Sin3 (Zhang et al., 1998) and others. *� (j o '6 We isolated hNURF using an ectopic, highly expressed � '-: Flag-SNF2L. To confirm the presence of the endogenous hNURF complex, we subjected partially fractionated nai:ve -BPTF 250- [;:] HEK293 nuclear extract to immunoprecipitation of endogenous SNF2L followed by western analysis for the presence of BPTF. As predicted, SNF2L antibodies SNF2L [::::] immunoprecipitated a significant amount of endogenous SNF2L and BPTF as compared with control antibodies (Figure 2D, compare lanes 4 and 5). This experiment , .. -- 64- ✓RbAP48 allowed us to ask whether endogenous BPTF is primarily [i= ] associated with SNF2L, or if it is incorporated into other non-SNF2L complexes. We assayed the unbound fractions '-RbAP46 - - of the immunoprecipitations and observed that SNF2L immunodepleted BPTF from this fraction compared with IgG (Figure 2D, compare lanes 2 and 3). This suggests that the majority of cellular BPTF is associated with SNF2L. The predicted molecular weight of the hNURF complex Western assuming one copy of each subunit is ~500 kDa. To Silver Stain confirm this, we measured the molecular weight of the Fi . 3. hNURF purified with a one-step purification protocol. Mock and hNURF complex using gel filtration chromatography. We Flag-BPTF eluted fractions were fractionated by 8-16% SDS-PAGE showed that hNURF subunits co-eluted at ~ 1 MDa followed by silver staining and western analysis. BPTF, SNF2L, (Figure 2E), which is twice the predicted mass. This RbAP48 and RbAP46 poplypeptides are indicated on the right. Western blot analysis performed in parallel confirms the identification of the observation is in agreement with predicted versus meas­ polypeptides. Polypeptides marked with asterisks are contaminants also ured molecular weights for other ISWl-containing com­ present in the mock elution. Molecular weight markers are indicated on plexes including WCRF/hACF (Bochar et al., 2000), the left. huChRAC (Poot et al., 2000) and WICH (Bozhenok et al., 2002). precipitations from HEK293 nuclear extract. The chro­ One-step purification of hNURF matin-remodeling activity of hNURF was assessed using The purification scheme employed for the isolation of the restriction enzyme accessibility assay, which relies on hNURF (Figure IA) permitted the characterization of the the ability of DNA incorporated into chromatin to be subunit composition of the complex. However, we found refractory to restriction endonuclease digestion. Upon that enzymatic assays on the purified complex were addition of a chromatin-remodeling protein (or complex), difficult, mainly due to an inherent degradation of the mobilization of chromatin at the restriction endonuclease BPTF subunit, presumably from endogenous proteolytic site is facilitated, resulting in an ATP-dependent decrease activity. The !ability of BPTF was also seen in its in undigested array. Drosophila ortholog, NURF301 (Xiao et al., 2001), In the presence of an elution from a mock immunopre­ resulting in a smaller size (215 kDa) and difficulty in cipitation, we find that ~50% of the DNA remained uncut sequencing the peptide. This led us to develop a novel one­ (U) and thus refractory to restriction enzyme cleavage step protocol for the isolation of hNURF. (Figure 4A, lane 1). The cut (C) band represented the basal We generated an HEK293 stable cell line expressing a level of accessible DNA in the assay. Upon addition of Flag epitope-tagged BPTF cDNA. Nuclear extract from purified hNURF, we found a potent decrease in the level of the Flag-BPTF cell line was prepared, subjected to uncut (U) DNA along with the concomitant increase in cut immunopreciptation with Flag beads, washed stringently, (C) DNA (Figure 4B, lane 2). Consistent with the fact that and eluted with Flag peptide. SDS-P AGE fractionation of chromatin remodeling is coupled to release of energy from immunoprecipitated proteins followed by silver staining ATP hydrolysis, hNURF decreased the levels of the uncut demonstrates the presence of an electrophoretically pure (U) DNA in an ATP-dependent manner (Figure 4B, lane 2, hNURF complex (Figure 3) containing a BPTF subunit compare top and bottom panels). Densitometric analysis of with minimal proteolysis. We confirmed the identity of the restriction enzyme accessibility assays performed in subunits by western analysis using BPTF, SNF2L, triplicate demonstrated a significant ( ~ 2.5-fold) decrease RbAP48 and RbAP46 antibodies. As in our previous in the fraction of uncut DNA (Figure 4B). purification, both silver (Figure 3) and colloidal staining By titrating in increasing amounts of hNURF and (data not shown) failed to demonstrate the presence of the maintaining saturating amounts of A TP and nucleosomal human ortholog of one of the dNURF subunits, the p38 array, we found that as few as 1.25 fmol of hNURF pyrophosphatase. remodeled the nucleosomal array in an ATP-dependent manner (Figure 4C). We then measured the rate of hNURF remodels chromatin hNURF-mediated remodeling. At all time points, The hNURF complex, by virtue of its SNF2L subunit, is hNURF demonstrated ATP-dependent remodeling activity predicted to function as a chromatin-remodeling complex. indicated by the decrease in the fraction of uncut array To confirm this, we performed chromatin-remodeling compared with hNURF without A TP or with restriction assays comparing purified hNURF with mock immuno-enzyme alone (Figure 4D). We calculated the specific kDa: Isolation of human NURF remodeling complex, ySWI/SNF, using the identical assay A B (Logie and Peterson, 1997). 0.6 hNURF possesses intrinsic A TPase activity Recombinant monomeric ISWI proteins and ISWI­ containing complexes such as WCRF/hACF and ChRAC § 0.4 exhibit an intrinsic ATPase activity that is activated by addition of naked DNA and further potentiated by the addition of nucleosomes (Varga-Weisz, 1997; Bochar g 0.2 1... et al., 2000; Aalfs et al., 2001). Among ISWI complexes, LL. dNURF is unique in that its ATPase activity is exclusively nucleosome dependent (Xiao et al., 2001). We predicted Mock hNURF that hNURF would behave similarly to its Drosophila counterpart. In fact, we found that hNURF exhibited a slight but measurable DNA-dependent ATPase activity (Figure SA, compare lanes 5 and 4). However, the hNURF ATPase activity was far more pronounced upon addition •··-.. ••• • ATP of nucleosomes (Figure SA, compare lanes 6 and 5). --+ATP Indeed, upon titration of the two hNURF substrates, we found that nucleosomes consistently displayed a stronger potentiation of ATPase activity (Figure SB). These data, along with previously published ATPase assays using dNURF (Xiao et al., 2001), suggest that the physiological substrate of the NURF complex may indeed be nucleo­ 10 20 30 40 50 60 somes rather than free DNA. fmol hNURF Further characterization of hNURF revealed that its ATPase activity was dose dependent, with as little as 20 fmol of hNURF displaying potent nucleosome­ dependent ATPase activity (Figure SC). The specific - • - • Buffer+A TP 0.5 activity of the hNURF nucleosome-stimulated ATPase ............... NURF-ATP activity determined by measuring the rate of A TP a o. 4 --NURF+ATP :::i hydrolysis (Figure 5D) was calculated at 200 U/mg of C 0.3 SNF2L (1 U = 1 pmol of ATP hydrolyzed per rnin). :g 0.2 Consistent with the minimal responsiveness to DNA, the \! o.1 specific activity of the DNA-stimulated ATPase activity was found to be ~100-fold lower than with nucleosomes. 10 20 30 40 60 60 70 Time, minutes SNF2L and BPTF co-localize within adult mouse brain to hippocampus and cerebellum Fi g . 4. hNURF is a chromatin remodeler. (A) hNURF remodels Mouse SNF2L has been shown to exhibit a restricted chromatin. Autoradiograph of the restriction endonuclease-coupled remodeling assay demonstrating chromatin-remodeling activity. Mock expression pattern, which included higher expression IP failed to demonstrate any decrease in the uncut (U) array in an A TP­ levels in the central nervous system (Lazzaro and dependent manner (compare top and bottom panel of lane 1). Addition Picketts, 2001). Since the majority of BPTF appeared to of purified hNURF results in a qualitative decrease in the uncut (U) be associated exclusively with SNF2L in HEK293 cells, array compared with mock (compare lanes 1 and 2, top panel). This we postulated that the BPTF gene should have a similar activity is ATP dependent (compare lane 2 top and bottom panels). (B) hNURF addition results in an ~2.5-fold decrease in the fraction of expression profile to SNF2L in the brain. As such, adult the uncut array. Densitometric analysis of restriction endonuclease­ mouse brain was analyzed for expression of SNF2L and coupled remodeling. The graph represents the averages of the quanti­ BPTF by RNA in situ hybridization. Our results show that fied fraction of the uncut array (U/U + C) from three experiments. the BPTF transcript is highly expressed throughout the Standard errors for the experiments are as indicated in the graph. hippocampus and in the cerebellum, which is nearly (C) hNURF exhibits dose-dependent chromatin-remodeling activity. Graphical representation of densitometric analysis of assays over 0, identical to the SNF2L expression profile (Figure 6A and 1.25, 2.5, 5 and 50 frnol of hNURF with or without ATP. Points repre­ B) (Lazzaro and Picketts, 2001). The specificity of the sent the average of three reactions, with standard error as indicated. hybridization is demonstrated by the lack of signal with (D) Time course of hNURF-mediated chromatin remodeling. sense SNF2L and BPTF probes (Figure 6C and D, Restriction enzyme accessibility for buffer + ATP, 50 fmol hNURF­ ATP or 50 fmol hNURF + ATP. Points represent the average of three respectively). Higher magnification of cresyl violet­ reactions, with standard error as indicated. counterstained sections showed that the SNF2L and BPTF transcripts are both localized within the granule and Purkinje cell layer of the cerebellum (Figure 6E and F) activity of hNURF in the remodeling assay at 15 U/mg and in the pyramidal neurons throughout the hippocampus of SNF2L (1 U = 1 pmol of array digested/ruin). This (Figure 6G and H). The BPTF and SNF2L expression translates to ~ 1 pmol of hNURF remodeling 2 fmol profiles suggest that the hNURF complex is involved in the of array per min. This activity is of the same order as regulation of cerebellar and hippocampal function. In the published measured activity of another chromatin- addition, analysis of BPTF and SNF2L reveals a similar 6093 O.Barak et al. A B Buff er hNU RF DNA - + - • + - Nucs. • • + . . + Pi -· -- 50 100 150 2.00 250 300 DNA, ng AT P _ _,h NUR F ... ......... .B uff er +Nucs +Nucs __ _hNUR F Bu ffe r 1 2 3 4 5 6 +Free ONA +Free DNA C D •�t ··· ··· - I ......, _ -:_ -- ... 10 20 30 40 50 60 70 20 40 60 80 100 120 fm ol hNURF Time, min utes - - - Buff er - - - BUFFER ·· · ········ ··· ········ DN A oNA -- NUCS -N ucs Fig. 5. hNURF exhibits intrinsic nucleosome·dependent ATPase activity. (A) hNURF exhibits DNA· and nucleosome·stimulated ATPase activity. ATPase activity assays performed on [y. P]ATP followed by separation of free phosphate (Pi) from ATP by PEl•cellulose TLC and autoradiography of TLC plates. hNURF activity is stimulated by supplementing the reaction with DNA (50 ng) and is potentiated further by nucleosomes (50 ng) ; compare lanes 4, 5 and 6. (B) hNURF activity responds to increasing amounts of nucleosomes. A 25 fmol concentration of hNURF or buffer was incu• bated with increasing amounts (0, 50, 100 or 250 ng) of either free DNA or free nucleosomes. Points represent the average of three reactions, with standard error as indicated. (C) hNURF exhibits a dose•dependent ATPase activity in the presence of nucleosomes. ATPase activity of three different hNURF concentrations (20, 50 and 100 frnol) with buffer, free DNA or nucleosomal DNA. Points represent the average of three reactions, with stand• ard error as indicated. (D) Time course of hNURF ATPase activity. hNURF (100 fmol) was incubated with buffer, free DNA or nucleosomal DNA, and reactions were assayed for ATPase activity at the indicted times. Points represent the average of three reactions, with standard error as indicated. pattern of expression during embryonic development observed in the cerebellum prompted us to examine the (Supplementary figure 1 available at The EMBO Jo urnal human en-1 and en-2 genes as possible targets of the Online). hNURF complex. To this end, we employed chromatin immunoprecipitation (ChIP) assays using antibodies against two components of the hNURF complex, SNF2L In vivo localization of hNURF to human engrailed and BPTF. To demonstrate specificity, we assayed for promoters their presence at other promoters important in various The mammalian homologs of the Drosophila segment biological processes including neuronal development polarity genes engrailed-1 (e n-1) , engrailed-2 (en-2), wnt- (Johnston et al., 2001; Tanzi and Bertram, 2001). While 7b and Pax-2 have been shown to have a crucial role in our antibodies failed to show an interaction between cerebellar development (Millen et al., 1995). Mice lacking hNURF and presenilin -1, zn j261, uxt, /JA PP or Mecp2 either en -1 or en-2 demonstrate mid-hindbrain malforma­ promoters, we successfully demonstrated a specific inter­ tions and cerebellar dysgenesis, respectively (Wurst et al., action with the promoters of both En -1 and En-2 1994; Millen et al., 1995). Interestingly, expression of (Figure 7 A). The regions demonstrating the strongest Drosophila engrailed was severely compromised in flies hNURF binding correspond to -1 kb upstream on the en-1 mutated for ISWI or NURF301 expression (Deuring et al., and en-2 transcription start sites. Interestingly, while the 2000; Badenhorst et al., 2002). These results and the overlapping expression of SNF2L and BPTF that we en-1 promoter is still relatively uncharacterized, the en-2 6094 Isolation of human NURF levels show a significant reduction (2-fold) in response to SNF2L BPTF depletion of SNF2L (Figure 7C). We attempted similar experiments using siRNAs against BPTF, and demon­ strated a significant decrease in en -1 transcript level (Figure7D). However, we were unable to affect en-2 transcript levels. This may be due to the fact that the siRNAs against BPTF reduced its concentration by only 2- fold (Figure 7D), which may not be sufficient to alter en-2 expression. Supporting this notion are reports in the literature demonstrating substantial differences in en-1 and en-2 regulation (Hanks et al., 1995). Taken together, our data reveal that hNURF associates with the promoters of En-1 and En-2 and can modulate expression of en-1 and probably en-2. Furthermore, since loss of En-1 and En-2 causes defects in the formation of the cerebellum (Joyner and Martin, 1987; Wurst et al., 1994; Millen et al., 1995), these data suggest a role for hNURF in the regulation of genes required for normal brain development. SNF2L promotes neurite outgrowth The functional maturation of the developing nervous system relies, in part, on the formation of axonal and dendritic processes that extend from neuronal cell bodies, and on the ability of these processes to form proper Fig. 6. SNF2L and BPTF show overlapping expression in adult mouse synaptic connections. Several factors, including the brain. Adjacent sections from an adult mouse brain were hybridized Engrailed homeodomain protein (Cosgaya et al., 1998), with SNF2L (A, E and G) and BPTF (B, F and H) antisense RNA are known to promote outgrowth of neuritic processes. probes. Both SNF2L and BPTF were highly expressed throughout all Since the two human orthologs of Engrailed are regulated regions of the hippocampus (CAI-4) and in the granule cell layer of by hNURF, we asked whether SNF2L would promote the cerebellum (A and B dark field). The specificity of SNF2L and BPTF antisense probes is demonstrated by the lack of hybridization neurite outgrowth. of corresponding sense probes (C and D). Higher magnifications of To investigate this, we utilized the NlE-115 mouse sections shown in (A) and (B) counterstained with cresyl violet demon­ neuroblastoma cell line, which can be induced to differ­ strate the expression of SNF2L and BPTF in the granule and Purkinje entiate morphologically to a neuronal phenotype and cell layer of the cerebellum (E and F) and in the pyramidal neurons of the hippocampus (G and H). Scale bars, 2 mm in (B) ; 50 µm in (F) extend neurites when cultured in conditions of reduced and (H); gc, granule cells; p, Purkinje neurons; CAI pyr, CAI serum (Hirose et al., 1998). We asked whether uninduced pyramidal neurons. NlE-115 cells undergo differentiation following transfec­ tion with SNF2L. Cells were transfected with empty vector, SNF2L-WT, or SNF2L-K213R, which contains a promoter was found to have an upstream activation point mutation in the nucleotide-binding P-loop motif, element proximal to the hNURF-binding sites (McGrew abrogating all ATPase activity. Co-transfection of a et al., 1999). �-galactosidase vector permitted visualization of neurites by immunofluorescence microscopy. At 30 h post­ hNURF regulates expression of endogenous transfection, a comparison of the ability of each trans­ human engrailed fected plasmid to promote morphological differentiation The localization of the hNURF complex at the en-1 and and neurite outgrowth was assessed by counting cells with en-2 promoters prompted us to assess the role of hNURF in neurite extensions measuring more than twice the length of regulating their expression. Small interfering (si)RNAs the cell body. that are specific for SNF2L were generated and transfected Cells transfected with empty vector exhibited a very into HEK293 cells, and expression of SNF2L was checked low, but measurable, rate of uninduced neurite outgrowth by western blot. The SNF2L siRNAs reduced expression (Figure 8A and D). However, transfection of wild-type of SNF2L to below detectable levels, while control SNF2L resulted in a significant potentiation of neurite siRNAs had no effect (Figure 7B). outgrowth (Figure 8, compare B and E with A and D). This Using the siRNAs for SNF2L, we then assayed for ability to promote neurite outgrowth was dependent on the transcript levels of endogenous en-1 and en-2 in the enzymatic activity of SNF2L since the SNF2L-K213R presence or absence of hNURF. Total RNA was extracted ATPase-dead mutant virtually abolished all neuronal from SNF2L siRNA- and control siRNA-transfected cells, outgrowth (Figure 8, compare C and F with B and E). reverse transcribed to generate cDNAs, and subjected to Similarly, the specificity for SNF2L was demonstrated real-time PCR. Transcript levels were quantified and further by a failure of SNF2H to induce neurite extension normalized to �-actin levels using the ABI7000 sequence (data not shown). Quantification of these results demon­ detection software. Transcript levels of PSI and MECP2, strates that, indeed, transfection of SNF2L-WT promotes which lack hNURF at their promoters, are unaffected by neurite outgrowth while SNF2L-K213R renders outgrowth depletion of SNF2L. Importantly, en -1 and en-2 transcript undetectable (Figure 8F). The promotion of neurite 609 5 O.Barak et al. A B In lgG BPTF SNF2L siRNA Prese nilin- 1 En graile d-1 a- SNF 2 L I - Engr ailed-2 - I !=::==::::: ZN F261 a-Rb AP48 _I ___ I UXT 2 3 bAPP MECP2 C D 1.2 1.2 l l .I L a 1 "i; .: � 0.8 � 0.8 I: � 0 .6 � 0.6 'C o 0,4 � 0.4 0. 2 0,2 .. - 0 Ps1 ME CP2 En-1 En-2 Ps1 MECP2 En-1 En-2 BPTF Co ntro l D Co ntrol ■ SN F2L ■ BPTF Fig. 7. hNURF localizes to and regulates human Engrailed. (A) ChIP assays localize hNURF specifically to engrailed-I (e n-I) and engrailed-2 (en-2) promoters. Chromatin from HEK293 cells was subjected to formaldehyde cross-linking followed by sonication and immunoprecipitation with non­ specific, BPTF and SNF2L antibodies. After extensive washes, bound DNA was eluted and sub jected to PCR with the indicated promoter primers. A specific and reproducible signal at the engrailed promoters comparable with input (In) was detected in the BPTF and SNF2L IPs but not in the non­ specific IgG. Input fractions represent 1 % of the total IP. Multiple primer pairs from each promoter were employed. Non-specific promoters include presenilin-1, znf261, UXT, APP and MECP2 as indicated. A representative set is displayed. (B) siRNA-mediated depletion of SNF2L. HEK293 cells transfected with either mock, SNF2L-specific siRNAs or non-specific siRNAs. Extract from cells 72 h after transfection were prepared and separated by SDS-PAGE followed by western analysis for SNF2L levels. The SNF2L-specific siRNA depletes SNF2L to undetectable levels compared with mock and non-specific siRNAs (compare lane 2 with lanes I and 3, top panel). Lower panel: loading control for protein levels determined by RbAP48 levels. (C) Depletion of SNF2L leads to a significant decrease in en-I and en-2 transcripts. Total RNA was extracted from SNF2L-depleted and control HEK293 cells and subjected to reverse transcription followed by real-time PCR. Levels of transcripts were quantified and normalized to �-actin levels using the ABl7000 sequence detection system. Genes lacking hNURF at their promoters as determined by ChIP were used as controls. The bar graph depicts the average of three independent experiments. Standard errors are indicated. (D) Depletion of BPTF leads to a significant decrease in en-I transcript. Experiments were performed as in (C). The bar graph depicts the averages of three independent experiments. Standard errors are indicated. extension that we observed following transfection of Discussi on SNF2L is similar to that observed for other factors that modulate neurite outgrowth (Kobayashi et al., 2002; Li The Drosophila ISWI protein exists in three multiprotein complexes, namely, ACF, CHRAC and NURF et al ., 2002; Abe et al., 2003). We believe that the (Tsukiyama et al ., 1995; Varga-Weisz et al ., 1997; Ito ectopically expressed SNF2L acts via incorporation into the NURF complex; however, we cannot rule out the et al., 1999). Mammalian complexes corresponding to possibility that the neurite outgrowth phenotype could ACF and CHRAC have been purified and contain the have resulted from unincorporated SNF2L. While these SNF2H protein (Bochar et al., 2000; Poot et al., 2000). data were derived from a tissue culture-based neurite Additional unique mammalian ISWI complexes have also outgrowth assay, they lend further support to the hypoth­ been purified, including RSF, WICH, NoRC and SNF2H­ esis that SNF2L may play a role in the differentiation and cohesin (LeRoy et al ., 2000; Strohner et al ., 2001; maturation of neurons during development. Bozhenok et al ., 2002; Hakimi et al., 2002), and these 609 6 2.5 Isolation of human NURF SNF2L SNF2L Vector WT K2 13 R Low Mag . High Mag . 7.5 ."!:'. '- :::, Q,) C: .!: .!: "'O LL Vector SNF2L SNF2L WT K213 R Fig. 8. SNF2L promotes neurite outgrowth. NlE-115 cells were co-transfected transiently with vector (A and D), f-SNF2L-WT (B and E) or f-SNF2L-K213R (C and F) along with a �-galactosidase expression plasmids in a 10:1 ratio. After transfection, cells were cultured for 30 h in normal (non-inducing) growth medium and then fixed for immunofluorescence staining (Flag = green, �-galactosidase = red). Differentiated cells were identified as cells in which the length of the neurite extensions was at least twice the diameter of the cell body. The fold increase in neurite outgrowth in f- SNF2L-transfected cells compared with vector-transfected cells was calculated from cell counts (600 cells) obtained from three different experiments (G) and is statistically significant at P < 0.005. Scale bars, 50 µm. all comprise the SNF2H protein. Despite the growing list hNURF is similar as it displayed predominantly nucleo­ of mammalian ISWI complexes, a NURF equivalent or some-stimulated ATPase activity, as well as potent complexes containing the related protein SNF2L are chromatin-remodeling activity on oligonucleosomal notably absent. Here, we describe the purification of the arrays. first human SNF2L complex. The subunit composition The brain-enriched expression profile of SNF2L suggests that it represents the human ortholog of the prompted us to examine a role for hNURF in neuronal dNURF complex. In this regard, the hNURF complex also physiology. We showed that SNF2L chromatin-remodel­ contains BPTF and RbAP46/48. Surprisingly, hNURF ing activity could induce neurite outgrowth in a tissue does not contain the inorganic pyrophosphatase protein culture-based assay, and that this was specific to SNF2L­ NURF38. Nonetheless, the biochemical activity of containing ISWI complexes since SNF2H expression did 6097 O. Barak et al. staining and immunoblot. Alternatively, nuclear extract was prepared not result in a similar induction (data not shown). The from Flag-BPTF-expressing cells and sub jected to immunoprecipitation conversion of a neuroblast to a differentiated neuron will as indicated. Beads were washed with 0.5 M KCI and 0.5% NP-40 for require the modification of chromatin structure at numer­ 10 min, followed by 0.75 M KCI and 0.1 % NP-40 in IP buffer for 10 min, ous genes, for both activation and repression, and it is not followed by 1.0 M KCI with 0.1 % NP-40 in IP buffer for 10 min. Beads were sub jected to elution as described for the previous purification. likely to be restricted to the NURF complex. Nonetheless, our studies suggest that hNURF has a role in this process Mass spectrometric peptide sequencing and, thus, identification of target genes will help elucidate Excised bands were subj ected to N-terminal sequence analysis as the molecular pathways. In this regard, we clearly described (Bachar et al., 2000). demonstrate that hNURF can regulate the mammalian engrailed genes, through a direct interaction at the In situ hyb ridization promoters of these two homeotic loci. The murine For in situ hybridization experiments, an adult mouse brain cDNA was used to amplify by PCR a 475 bp fragment corresponding to a segment of engrailed genes are critical regulators of mid-hindbrain the 3'-coding region and untranslated region of human BPTF. The mouse development, as ablation leads to animals that are missing BPTF fragment was cloned into pBluescript KS as a template for RNA most of the colliculi and cerebellum (Wurst et al., 1994). probes. Brains dissected from adult mice perfused with 4% paraformal­ Although engrailed was identified previously as a NURF dehyde in 100 mM NaP0pH 7.4 were processed for cyosectioning. Antisense and sense Snt'21 and Bptf RNA probes were labeled with target gene through the characterization of flies harboring [ P]UTP (2000 Ci/mmol) (Amersham Biosciences, Sweden) by in vitro mutant ISWI or NURF301 genes (Deuring et al., 2000; transcription from plasmid templates using T3 and T7 RNA polymerases Badenhorst et al., 2002), a neural defect was not appre­ (Promega, USA). Cryosections were processed for hybridization, ciated due to the early lethality of these animals. As such, hybridized with probes and exposed to autoradiographic emulsion as this may represent a novel function for the NURF described previously. complex. Restriction endonuclease-coupled remodeling assay The effect of chromatin-remodeling complexes on Reconstitution of nucleosomal arrays and chromatin-remodeling assays development is a well-established phenomenon. were performed as described (Logie and Peterson, 1997). More details are Linkages between chromatin remodeling and develop­ provided in the Supplementary data. mental disorders include ATRX and mental retardation (Picketts et al., 1996), SMARCALl and Schimke Purification of nucleosomes Nucleosomes were purified from HeLa cell nuclear pellet as described immuno-osseous dysplasia (Boerkoel et al., 2002), CSB (Utley et al., 1996). More details can be found in the Supplementary data. and Cockayne syndrome (Citterio et al., 2000), and SNF2H and William's syndrome (Bachar et al., 2000). ATPase assays Based on our results, we hypothesize that the hNURF Measurement of ATPase activity was performed in 10 µI reactions under complex may represent another connection of a chroma­ the following conditions: 20 mM Tris-HCI, 60 mM KCI, 4% glycerol, tin-remodeling protein to disorders of development, and 4 mM MgC1 , 1 mM cold ATP, 1 µCi of [y-P]ATP and 2 nmol of hNURF or an equivalent volume of elution buffer. Where indicated, the we are confident that the hNURF complex regulates other reaction was supplemented with 50 ng of naked DNA or nucleosomes. developmentally important genes. In this regard, the Reactions were performed at 30 C for 1 h. Free phosphate and ATP were analysis of flies ablated for the NURF complex also separated by TLC on PEI-cellulose plates (J.T.Baker, USA). A 1 µ1 suggests a role for the hNURF complex in hematopoietic aliquot of the reaction was spotted onto a plate and TLC was carried out in 1 M formic acid, 0.5 M LiCL Plates were then allowed to dry and were development and the regulation of chromosome structure visualized by exposure to a phosporimager cassette (Molecular (Badenhorst et al., 2002). However, such studies in Dynamics, Sweden) for densitometric analysis, or film (Kodak, USA). mammals must await further dissection using in vivo For rate assays, reaction mix was pre-warmed to 30 C prior to addition of model systems. hNURF. Chromatin immunoprecipitation (ChlPJ Materials and meth ods ChIP experiments and buffers were performed as described in the Upstate ChIP protocol. More details are available in the Supplementary data. Purification of the hNURF complex hNURF was purified from 350 mg of t'SNF2L-HEK293 nuclear extract. Quantitative PCR Nuclear extract was loaded on a 100 ml column of phosphocellulose (PI 1, Transcript levels were determined by quantitative PCR using an ABI7000 Whatman, USA) and fractionated stepwise by the indicated KCI (Applied Biosystems, USA). PCR was performed using the Sybr Green concentrations in buffer A [20 mM Tris-HCI pH 7.9, 0.2 mM 2x Master Mix (Applied Biosystems, USA) as per the manufacturer's EDTA,10 mM �-mercaptoethanol, 10% glycerol, 0.2 mM phenylmethyl­ protocol. Unknowns were amplified in triplicate. Positive control sulfonyl fluoride (PMSF)]. The 1.0 M KCI fraction (25 mg) of Pll was reactions were performed to establish a standard curve for each primer loaded on a 5 ml DEAE-Sephacel column (Amersham Biosciences, pair for transcript level normalization. �-actin served as an internal Sweden) and eluted with 0.5 M KCL The 0.5 M KCI elution (12.5 mg) control. was dialyzed to 0.1 M KCI in buffer A containing I µg/ml aprotinin, leupeptin and pepstatin, and loaded on a Mono Q HR 5/5 column Neurite outgrowth assay (Amersham Biosciences, Sweden). The column was resolved by using a NlE-115 cells were seeded at 50% confluency 24 h prior to transfection. linear 10 column volume gradient of 100-500 mM KCI in buffer A Cells were transfected with pCDNA3 vector, pCDNA3-f-SNF2L-WT or containing 1 µg/ml aprotinin, leupeptin and pepstatin. SNF2L-containing pCDNA3-f-SNF2L-K213R and �-galactosidase expression plasmids in fractions were fractionated on a Superose 6 HR 10/30 column (Amersham a 10: 1 ratio. Cells were maintained in normal growth medium for Biosciences, Sweden) equilibrated in 0.5 M KCI in buffer A containing 30 h post-transfection. Cells were then fixed for immunofl.uorescent 0.1 % NP-40, 1 µg/ml aprotinin, leupeptin and pepstatin. Fractions 18-20 staining. Transfected cells and neurites were visualized by Flag and were subj ected to immunoaffinity purification using M2 anti-Flag �-galactosidase antibodies. Differentiated cells were identified as cells in antibody-conjugated agarose beads (Sigma, USA). Beads were washed which the length of neurite extensions was at least twice the diameter of with 0.5 M KCI in IP buffer [20 mM Tris-HCI pH 7.9, 0.2 mM EDTA, the cell body. For quantification experiments, 600 cells were counted. 1 mM dithiothreitol (DTI), 10% glycerol, 0.2 mM PMSF] containing 1 µg/ml aprotinin, leupeptin and pepstatin, and bound fractions were eluted with 400 µg/ml of Flag peptide (Sigma, USA) in 0.1 M KCI in IP Supplementary data buffer. Purified hNURF was sub jected to SDS-PAGE followed by silver Supplementary data are available at The EMBO Journal Online. 6098 Isolation of human NURF protein kinase (pl 60ROCK)-regulated neurite remodeling in Ackn owl edgements neuroblastoma Nl E-115 cells. J. Cell Biol., 141, 1625-1636. Ito,T., Bulger,M., Pazin,M.J., Kobayashi,R. and Kadonaga,J.T. (1997) We thank D.Bochar for comments and experimental advice, J.Neveu, ACF, an ISWI-containing and ATP-utilizing chromatin assembly and J.Asara and R.Robinson for their expertise in HPLC and mass remodeling factor. Cell, 90, 145-155. spectrometry, Dr Robert Bowser for Flag-FACl cDNA, the National Ito,T., Levenstein,M.E., Fyodorov,D.V., Kutach,A.K., Kobayashi,R. and Cell Culture Center (Minneapolis, MN) for propagation of fSNF2L-293 Kadonaga,J.T. (1999) ACF consists of two subunits, Acfl and ISWI, cells, and the Wistar Institute protein microchemistry/mass spectrometry that function cooperatively in the A TP-dependent catalysis of facility. R.S. was supported by grants from the NIH (GM61204) and the chromatin assembly. Genes Dev., 13, 1529-1539. American Cancer Society. O.G.B. was supported by the NIH cancer grant Johnston,M.V., Jeon,O.H., Pevsner,J., Blue,M.E. and Naidu,S. (2001) (CA09171-28). M.A.L. was supported by the Ontario Mental Health Neurobiology of Rett syndrome: a genetic disorder of synapse Foundation. D.J.P. is a CIHR New Investigator with work supported by a development. Brain Dev., 23, S206--S213. CIHR grant (MOP53224). Jones,M.H., Hamana,N. and Shimane,M. (2000) Identification and characterization of BPTF, a novel bromodomain transcription factor. Genomics, 63, 35-39. Jordan-Sciutt o,K.L., Dragich,J.M. and Bowser,R. (1999) DNA binding Referen ces activity of the fetal Alz-50 clone 1 (FACl) protein is enhanced by phosphorylation. Biochem. Biophys. Res. Commun. , 260, 785-789. Aalfs,J.D., Narlikar,G.J. and Kingston,R.E. (2001) Functional Joyner,A.L. and Martin,G.R. (1987) En-1 and En-2, two mouse genes differences between the human ATP-dependent nucleosome with sequence homology to the Drosophila engrailed gene: expression remodeling proteins BRGl and SNF2H. J. Biol. Chem., 276, 34270 -- during embryogenesis. Genes Dev., 1, 29-38. Kobayashi,M., Taniura,H. and Y oshikawa,K. (2002) Ectopic expression Abe,T., Kato,M., Miki,H., Takenawa,T. and Endo,T. (2003) Small of necdin induces differentiation of mouse neuroblastoma cells. J. Biol. GTPase TclO and its homologue RhoT induce N-WASP-mediated Chem., 277, 42128-42135. long process formation and neurite outgrowth. J. Cell Sci., 116, 155- Lastowska,M., Van Roy,N., Bown,N., Speleman,F., Roberts,P., Lunec,J., Strachan,T., Pearson,A.D. and Jackson,M.S. (2001) Molecular Aihara,T., Miyoshi,Y., Koyama,K., Suzuki,M., Takahashi,E., cytogenetic definition of 17 q translocation breakpoints in Monden,M. and Nakamura,Y. (1998) Cloning and mapping of neuroblastoma. Med. Pedia tr. Oneal., 36, 20--23. SMARCAS encoding hSNF2H, a novel human homologue of Lazzaro,M.A. and Picketts,D.J. (2001) Cloning and characterization of Drosophila ISWI. Cytogenet. Cell Genet., 81, 191-193. the murine imitation switch (ISWI) genes: differential expression Badenhorst,P., Voas,M., Rebay,I. and Wu,C. (2002) Biological functions patterns suggest distinct developmental roles for Snf2h and Snf21. J. of the ISWI chromatin remodeling complex NURF. Genes Dev., 16, Neurochem., 77, 1145-1156. 3186--3198. LeRoy,G., Orphanides,G., Lane,W.S. and Reinberg,D. (1998) Bochar,D.A., Savard,J., Wang,W., Lafleur,D.W., Moore,P., Cote,J. and Requirement of RSF and FACT for transcription of chromatin Shiekhattar,R. (2000) A family of chromatin remodeling factors templates in vitro. Science, 282, 1900--1904. related to Williams syndrome transcription factor. Proc. Natl Acad. LeRoy,G., Loyola,A., Lane,W.S. and Reinberg,D. (2000) Purification Sci. USA, 97, 1038---1043. and characterization of a human factor that assembles and remodels Boerkoel,C.F. et al. (2002) Mutant chromatin remodeling protein chromatin. J. Biol. Chem., 275, 14787-14790. SMARCALl causes Schimke immuno-osseous dysplasia. Nat. Li,X., Meriane,M., Triki,I., Shekarabi,M., Kennedy,T.E., Larose,L. and Genet., 30, 215-220. Lamarche-Vane,N. (2002) The adaptor protein Nck-1 couples the Bowser,R., Giambrone,A. and Davies,P. (1995) FACl, a novel gene netrin-1 receptor DCC ( deleted in colorectal cancer) to the activation identified with the monoclonal antibody Alz50, is developmentally of the small GTPase Rae l through an atypical mechanism. J. Biol. regulated in human brain. Dev. Neurosci., 17, 20--37. Chem. , 277, 3778 8-37797. Bozhenok,L., Wade,P.A. and Varga-Weisz,P. (2002) WSTF-ISWI Logie,C. and Peterson,C.L. (1997) Catalytic activity of the yeast SWI/ chromatin remodeling complex targets heterochromatic replication SNF complex on reconstituted nucleosome arrays. EMBO J. , 16, foci. EMB O J. , 21, 2231-2241. 6772--6782. Citterio,E., Van Den Boom,V., Schnitzler,G., Kanaar,R., Bonte,E., Martinez-Balbas,M.A., Tsukiyama,T., Gdula,D. and Wu,C. (1998) Kingston,R.E., Hoeijmakers,J.H. and Vermeulen,W. (2000) ATP­ Drosophila NURF-55, a WD repeat protein involved in histone dependent chromatin remodeling by the Cockayne syndrome B DNA metabolism. Proc. Natl Acad. Sci. USA, 95, 132-137. repair-transcription-coupling factor. Mol. Cell. Biol., 20, 7643-7653. McGrew,L.L., Takemaru,K., Bates,R. and Moon,R.T. (1999) Direct Cosgaya,J.M., Aranda,A., Cruces,J. and Martin-Blanco,E. (1998) regulation of the Xenopus engrailed-2 promoter by the Wnt signaling Neuronal differentiation of PC12 cells induced by engrailed pathway and a molecular screen for Wnt-responsive genes, confirm a homeodomain is DNA-binding specific and independent of MAP role for Wnt signaling during neural patterning in Xenopus. Mech. kinases. J. Cell Sci., 111, 2377-2384. Dev., 87, 21-32. Czermin,B., Melfi,R., McCabe,D., Seitz,V., Imhof,A. and Pirrotta,V. Millen,K.J., Hui,C.C. and Joyner,A.L. (1995) A role for En-2 and other (2002) Drosophila enhancer of Zeste/ESC complexes have a histone murine homologues of Drosophila segment polarity genes in H3 methyltransferase activity that marks chromosomal Polycomb regulating positional information in the developing cerebellum. sites. Cell, 111, 185-196. Devel opment, 121, 3935-3945. Deuring,R. et al. (2000) The ISWI chromatin-remodeling protein is Mu,X., Springer,J.E. and Bowser,R. (1997) FAC l expression and required for gene expression and the maintenance of higher order localization in motor neurons of developing, adult and amyotrophic chromatin structure in viva. Mol. Cell, 5, 355 -365. lateral sclerosis spinal cord. Exp. Neural., 146, 17-24. Gdula,D.A., Sandaltzopoulos,R., Tsukiyama,T., Ossipow,V. and Wu,C. Okabe,I., Bailey,L.C., Attree,O., Srinivasan,S., Perkel,J.M., (1998) Inorganic pyrophosphatase is a component of the Drosophila Laurent,B.C., Carlson,M., Nelson,D.L. and Nussbaum,R.L. (1992) nucleosome remodeling factor complex. Genes Dev., 12, 3206--3216. Cloning of human and bovine homologs of SNF2/SWI2: a global Hakimi,M.A., Bochar,D.A., Schrniesing,J.A., Dong,Y., Barak,O.G., activator of transcription in yeast S.cerevisiae. Nucleic Acids Res., 20, Speicher,D.W., Yokomori,K. and Shiekhattar,R. (2002) A chromatin 4649-4655. remodelling complex that loads cohesin onto human chromosomes. Picketts,D.J., Higgs,D.R., Bachoo,S., Blake,D.J., Quarrell,O.W. and Na ture, 418, 994-998. Gibbons,R.J. (1996) ATRX encodes a novel member of the SNF2 Hanks,M., Wurst,W., Anson-Cartwright,L., Auerbach,A.B. and family of proteins: mutations point to a common mechanism Joyner,A.L. (1995) Rescue of the En-1 mutant phenotype by underlying the ATR-X syndrome. Hum. Mol. Genet., 5, 1899-1907. replacement of En-1 with En-2. Science, 269, 679--6 82. Poot,R.A., Dellaire,G., Hulsmann,B.B., Grimaldi,M.A., Corona,D.F., Harlow,E. and Lane,D. (1988) Antibodies: A Laboratory Manual. Cold Becker,P.B., Bickmore,W.A. and Varga-Weisz,P.D. (2000) Spring Harbor Laboratory Press, Cold Spring Harbor, NY. HuCHRAC, a human ISWI chromatin remodelling complex contains Hendrich,B. and Bickmore,W. (2001) Human diseases with underlying hACFl and two novel histone-fold proteins. EMB O J. , 19, 3377- 3387. defects in chromatin structure and modification. Hum. Mol. Genet., 10, Shaw,G., Morse,S., Ararat,M. and Graham,F.L. (2002) Preferential 2233-2242. transformation of human neuronal cells by human adenoviruses and Hirose,M. et al. (1998) Molecular dissection of the Rho-associated the origin of HEK 293 cells. FASEB J. , 16, 869-871. 6099 O.Barak et al. Strohner,R., Nemeth,A., Jansa,P., Hofmann-Rohrer,U., Santoro,R., Langst,G. and Grummt,I. (2001) NoRC-a novel member of mammalian ISWI-containing chromatin remodeling machines. EMB O J. , 20, 4892-4900. Tanzi,R.E. and Bertram,L. (2001) New frontiers in Alzheimer's disease genetics. Neuron, 32, 181-184. Tsukiyama,T., Daniel,C., Tamkun,J. and Wu,C. (1995) ISWI, a member of the SWI2/SNF2 A TPase family, encodes the 140 kDa subunit of the nucleosome remodeling factor. Cell, 83, 1021-1026. Utley,R.T., Owen-Hugbes,T.A., Juan,L.J., Cote,J., Adams,C.C. and Workman,J.L. (1996) In vitro analysis of transcription factor binding to nucleosomes and nucleosome disruption/displacement. Methods Enzymol., 274, 276--291. Varga-Weis z,P.D., Wilm,M., Bonte,E., Dumas,K., Mann,M. and Becker,P.B. (1997) Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Na ture, 388, 598---602. Wurst,W., Auerbach,A.B. and Joyner,A.L. (1994) Multiple developmental defects in Engrailed-1 mutant mice: an early mid­ hindbrain deletion and patterning defects in forelimbs and sternum. Devel opment, 120, 2065-2075. Xiao,H., Sandaltzopoulos,R., Wang,H.M., Hamiche,A., Ranallo,R., Lee,K.M., Fu,D. and Wu,C. (2001) Dual functions of largest NURF subunit NURF301 in nucleosome sliding and transcription factor interactions. Mol. Cell, 8, 531-543. Zhang,Y., Sun,Z.W., Iratni,R., Erdjument-Bromage,H., Tempst,P., Hampsey,M. and Reinberg,D. (1998) SAP30, a novel protein conserved between human and yeast, is a component of a histone deacetylase complex. Mol. Cell, 1, 1021-1031. Zhang,Y., Ng,H.H., Erdjument-Bromage,H., Tempst,P., Bird,A. and Reinberg,D. (1999) Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev., 13, 1924-1935. Received May 23, 2003; revised Se ptember 3, 2003; accepted Se ptember 26, 2003 61 00

Journal

The EMBO Journal – Springer Journals

Published: Nov 17, 2003

Keywords: BPTF; Engrailed; ISWI; NURF; SNF2L

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