The genomic organization of the histone clusters on human 6p21.3
Jung Ahn, Jeffrey R. Gruen
Yale Child Health Research Center, Department of Pediatrics, Yale University School of Medicine, 464 Congress Avenue,
New Haven, Connecticut 06520-8081, USA
Received: 22 December 1998 / Accepted: 26 February 1999
Histone proteins are essential for stabilizing and folding DNA in
the cell nucleus. In higher eukaryotes, four core histone proteins,
H2A, H2B, H3 and H4, form the octamer of the nucleosome, the
smallest basic unit of chromatin structure (Stein et al. 1984). An-
other histone, H1, interacts with linker DNA between nucleosomes
and is involved in making compact structures of chromatin. While
all the histone genes are defined by their highly conserved coding
sequences, expression pattern and genomic structure can further
classify them. Cell cycle-independent histone genes, or replace-
ment histone genes, are constitutively expressed in most tissues
and cells and especially in terminally differentiated or quiescent
cells; they have introns and polyadenylation signals. In contrast,
the expression of cell cycle-dependent histone genes is tightly
linked to DNA synthesis and abundant in rapidly dividing cells;
they lack polyadenylation signals and introns.
In humans, most cell cycle-dependent histone genes are located
on the short arm of Chromosome (Chr) 6 (6p21.3-22), with a few
mapping to Chr 1 (Tripputi et al. 1986). They are grouped into two
clusters separated by 2 Mb. According to Albig and associates, the
telomeric cluster near marker D6S2239 contains 34 genes, and the
centromeric cluster near D6S105 contains 18 genes. Both clusters
include two pseudogenes (Albig et al. 1997; Albig and Doenecke
The genomic organization of histone genes has been deter-
mined in only two other vertebrates: chicken and mouse. Chicken
has a single cluster of 39 histone genes. Mouse has three distinct
histone clusters totaling 45 genes distributed within 2 Mb on Chr
13 (syntenic to human Chr 6p). While total histone gene number
increases from chicken (39 genes) to mouse (45 genes) to human
(52 genes), the number of distinct clusters appears random at one,
three, and two respectively.
While developing a transcript map of the human 6p21.3-22
region, we identified additional histone genes that do not map to
either described cluster. Here, we report the results ofa2Mb
search for additional human histone genes and describe the geno-
mic organization of the histone clusters on 6p21.3-22, including a
new microcluster. The organization of the histone clusters is con-
trasted in human, mouse, and chicken.
We converted a YAC contig of human 6p21.3-22 (Bray-Ward
et al. 1996) into a contig of smaller bacterial clones consisting of
PACs, BACs, and cosmids. Human sequences extracted by inter-
IRS PCR (Stein 1994) from three overlapping YACs
905G1, and 947F6, were used to screen a human Chr 6 cosmid
library (LANL Life Science Division, Los Alamos, NM; http://
www-bio.llnl.gov/genome/html/cosmid-html) and a human PAC
library (Roswell Park Cancer Institute, Buffalo, N.Y.; http://
bacpac.med.buffalo.edu). Cosmid and PAC clones identified from
the screening were grouped into several bins by dot blot hybrid-
ization and by shared STSs that were obtained from public data-
and from clone end sequences. The bins were then con-
nected by rescreening the libraries with clone end fragments iso-
lated by the inverse PCR method (Silver 1991). This approach
yielded a contig of PACs and cosmids spanning 2 Mb, from
D6S2239 (pter) through D6S105 (cen), with one gap in the middle.
We filled the gap with BAC clones, 61E19 and 24O18, and ex-
tended the telomeric end of the contig with PAC clones described
by Lauer and colleagues (1997; Fig. 1).
To eliminate any possible uncertainties in the contig, partial
and complete XbaI restriction digests of individual PAC, BAC,
and cosmid clones were compared to confirm overlaps. Both con-
ventional and field inversion agarose gel electrophoresis (FIGE)
were used to maximize the resolution of the 0.5–40 kb fragments
that were generated by the XbaI digests (Ota and Amemiya 1996).
The resulting restriction patterns eliminated false positives and
provided an accurate estimate of the physical distances between
clones, genes, and markers (Fig. 1). We also searched the entire
minimum tiling path consisting of 35 overlapping PACs, two
BACs, and five cosmids for additional histone genes. These clones
were sent to the Sanger Centre (Hinxton, Cambridge, CB10 1SA,
UK) for sequencing as a part of the Chr 6 sequencing project.
Histone genes were detected by hybridizing five unique his-
tone probes from H1, H2A, H2B, H3, and H4 genes to Southern
blot filters made from PACs, BACs, and cosmids of our contig.
These hybridizations detected clusters of histone genes in three
unique locations (Fig. 1). The centromeric cluster contained 18
genes (one H1, five H2A and H2B, three H3, and four H4) dis-
tributed over 120 kb near D6S105. The telomeric cluster contained
34 genes (five H1, six H2A, eight H2B, seven H3, and eight H4)
distributed over 300 kb around D6S2239. These results were con-
sistent with previous reports by Albig and coworkers (Albig et al.
1997; Albig and Doenecke 1997).
Between the two known clusters, a new microcluster was de-
tected containing five histone genes mapping within three XbaI
fragments from four overlapping clones: p13H15, p86C11,
p48N19, and b61E19 (Fig. 1). Two fragments were positive for
both H2A and H2B, while the third contained only H4. All four
clones contained D6S2252, which we mapped between D6S1260
(cen) and D6S1558 (pter). The relative order and orientation of the
five histone genes were determined from the sequence of PAC
clone p86C11 (AL021807). p86C11 is an 89,016-bp clone identi-
fied by several groups (Lauer et al. 1997; Volz and Ziegler 1996)
including our own and sequenced by the Chr 6 group at the Sanger
Centre (www.sanger.ac.uk/HGP/Chr6). Three genes in this new
middle cluster were previously reported and named H4/m
(AB000905), H2A/p (L19778), and H2B/r (X00088; Albig and
Doenecke 1997). The other two genes, an H2A and an H2B, were
not previously described, and we name them H2A/s and H2B/s.
All five histones have adequate open reading frames and typical
Correspondence to: J.R. Gruen
From CEPH mega YAC library.
GDB: The Genome Database, http://gdbwww.gdb.org. Whitehead data-
base: Center for genome research at Whitehead Institute for Biomedical
Mammalian Genome 10, 768–770 (1999).
© Springer-Verlag New York Inc. 1999