REVIEW ARTICLE
The role of histones in chromatin remodelling during mammalian
spermiogenesis
Je
´
ro
ˆ
me Govin, Ce
´
cile Caron, Ce
´
cile Lestrat, Sophie Rousseaux and Saadi Khochbin
Laboratoire de Biologie Mole
´
culaire et Cellulaire de la Diffe
´
renciation, INSERM U309, E
´
quipe Chromatine et Expression des ge
`
nes,
Institut Albert Bonniot, Faculte
´
de me
´
decine, La Tronche, France
One of the most dramatic chromatin remodelling processes
takes place during mammalian spermatogenesis. Indeed,
during the postmeiotic maturation of male haploid germ
cells, o r s permiogenesis, histones are replaced by small basic
proteins, which in mammals are transition proteins and

utilizing complexes act directly on nucleosomes to modify
the a ccessibility of factors to limited DNA regions present
in a nucleosome [3]. Histone modifying enzymes dictate
combinations of post-translational modifications of
histones to create specific signals defining the Ôhistone codeÕ,
which in turn induces localized alterations of the c hromatin
structure and function. The histone code hypothesis postu-
lates that specific factors can act on chromatin by recog-
nizing and binding particular histone modifications [4–6].
This hypothesis is so far supported by the discovery of
chromatin interacting modules present in various factors,
specifically recognizing methylated or acetylated lysines of
histones [7].
Finally, variants of histones H2A, H2B, H3 and H1 have
been identified. Some of these variants have a lready been
shown to mediate specific functions such as DNA repair in
response to genotoxic treatments [8].
In somatic c ells, these three mechanisms act together to
locally induce alterations of the chromatin structure and to
maintain a region-dependent differentiation of chromatin
over generations of cells, although many questions remain
unanswered on the molecular basis of their action. An
extreme case of chromatin remodelling occurs during
spermatogenesis, where histones are massively removed
and replaced [9]. Although n othing is known o f the
underlying me chanisms, one can e xpect a major participa-
tion of the three chromatin modifying mechanisms already
known to a ct in somatic cells. Indeed, disparate data from
the literature suggest that histone removal during
spermiogenesis is preceded by a massive incorporation of

progression, whereas replacement histones can be produced
and incorporated throughout the cell c ycle. Testis specific
variants have been described [9], but many nontissue s pecific
histone variants are also e xpressed and incorporated into
chromatin during spermatogenesis (Fig. 1).
Linker histone variants
In mammals, at least six somatic subtypes (H1.1–H1.5 and
H1°), one oocyte-specific and two testis-specific linker
histones (H1t and HILS1) are expressed [2,10,11].
H1t contains the usual tripartite structure of linker
histones, but is highly divergent in its primary structure
compared to the other five members H1.1–H1.5 (Fig. 2). Its
expression has been characterized in the mouse [10] as well
as in the rat [12]. In situ hybridization detects the RNA in
mid-pachytene s permatocytes, a nd immunodetection indi-
cates the presence of the p rotein from the stage of pachytene
spermatocytes until round and elongating spermatids
[10,13]. At this stage, the H1t amount constitutes up to
55% of t he total linker histones. Mice bearing invalidated
H1t gene display no phenotype [14–16], but the analysis of
enriched populations of pachytene s permatocytes and
round spermatids in these mice has shown that its absence
is partially compensated by the other H1s, still permissive to
end maturation and fertilization [14,15]. Interestingly, other
groups have shown that the interaction of H1t with
nucleosomes leads to a less compact structure than that of
other H1 subtypes [17,18], suggesting that this variant may
help chromatin de-compaction, giving accessibility to other
chromatin remodelling factors.
Among the somatic linker histones, H1.1 (H1a) is

pachytene and diplotene stages of the first meiotic division prophase. Meiotic I division yields secondary spermatocytes which then rapidly go
through m eiotic II division, generating haploid round spermatids. During its postmeiotic maturation, the spermatid undergoes a global remodelling
of its nucleus, which e longates and compacts into the very un ique nucleus structure of the sperm atozoa.
3460 J. Govin et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Fig. 2. Sequence analysis of known histone variants expressed during spermatogenesis. The sequences of conventional histones and their sperma-
togenic variants are aligned in (A), (B), (C) and (D). All sequences are murine, as most of the sequence data are available for this species, except for
TH2A (rat), and hTH2B and H3t (human sequences). Con ventional histone sequences were chosen on the basis of work by M arzluff and colleagues
[94]. Alignments were perf ormedwiththealgorithm
CLUSTALW
on the web interface o f the PBIL at http://npsa-pbil.ibcp.fr/[95] and coloured with
ESPRIT
at http://prodes.toulouse.inra.fr/ESPript/ES Pript/[96]. Some of the histone modifications d iscussed are indicated [67,91]. Modification
cassettes (amino acids Thr/Ser-Lys or Lys-Thr/Ser) [91] were searched in conventional histones and variants, and are rep resented by small
rectangles, underneath the corresponding sequenc es. Black r ectan gles underline cassettes p resent in conventional histones. Some cassettes are not
conserved in variants and arrows indicate changes leading to the cassette disappearance in the variants. Open rectangles underline new cassettes
specific t o a variant and absent in conventional his tones. Original crystallographic data were used for the representat ion of the secondary structu res
[1,97]. Sequence accession numbers: H3.1 (P16106); H3.3 (P06351); H3t (Q16695); H2A (NP_783591); TH2A (Q00728); H2B (NP_835502); TH2B
(Q00729); hTSH2B (NP_733759); H1.1 (P43275); H1t ( Q07133); HILS1 (Q9QYL0).
Ó FEBS 2004 Chromatin code and spermatogenesis (Eur. J. Biochem. 271) 3461
A testis-specific H3 variant, only detected in the human,
has been isolated in 1996 [24,25]. This variant, named H3t,
differs from the canonical H3 by only four residues
(Fig. 2A). The RNA of this variant was only detected in
primary spermatocytes. T he experimental sequencing
helped to identify another t estis specific variant, n amed
TH3 in rat [26]. However, no gene or sequen ce information
is available on this putative histone variant and no
corresponding genes have b een found in known mammalian
genomes [25].
More data is available on nontestis specific H3 variants.

to be enriched in H3.3 [32], suggesting that the replacement
of H3 by H3.3 in spermatocytes could also be linked to the
very active transcription that takes place during meiosis [9].
H2B variants
Rat, mouse and human TH2B have been cloned, showing
very high levels of conservation [ 33–35]. The main differ-
ences between H2B and TH2B a re in the N -terminal, and to
a lesser extent, the histone fold domain (Fig. 2C). Most of
these differences are c onserved between the three species,
suggesting a conserved role for this variant during sperma-
togenesis (see below).
In rat, TH2B is actively expressed in early primary
spermatocytes until mid–late pachytene [19] and then
remains the major form o f H2B in round and elongating
spermatids. Using an antibody that, luckily, cross-reacts
with TH2B, it has been shown in human testis that TH2B
first appears in spermatogonia, is maximal in round
spermatids, and then gradually disappears during the
elongation of spermatids [36]. In contrast, the human
TH2B, hTSH2B, was retained in mature sperms and
presented a specific nuclear localization only in 20% of
sperm populations [35].
There is also apparently a nonchromatin function for
histones during spermatogenesis. Indeed, recently, in bull
somatic type core histones h ave been foun d associated with
the perinuclear theca, which is a layer surrounding the
nucleus of mammalian sperms [37]. A h istone H2B variant,
named SubH2Bv, has also been found associated with the
theca in bull sperm [38]. The function of these non-nuclear
histones has not been defined.

to as the macrochromatin body. MacroH2A1.2 is found
at high concentrations in mice testis [47,48]. During
spermatogenesis, it has been observed in the nuclei of
germ cells, with a localization that is largely to the
developing XY-body in early pachytene spermatocytes
[49,50]. Hence, the process of X-inactivation in XX
somatic cells [51] and that in XY spermatocytes show
some similarities, including a heterochromatinization of
the region which is densely stained (forming, respectively,
the Barr Body or the S ex Vesicle) and a coating of the
X with the Xist RNA, a non coding RNA specifically
associated w ith the inactive X chromosome [52]. Interest-
ingly, a potential relationship has been discovered
between macroH2A1.2 and the mammalian HP1-like
heterochromatin p rotein M31 (HP1beta or MOD1)
during meiosis. The HP1-like protein M31 was found
initially to colocalize with heterochromatic regions in
Sertoli cells, in mid-stage pachytene spermatocytes, a s well
as in round spermatids (where it localized with the
3462 J. Govin et al.(Eur. J. Biochem. 271) Ó FEBS 2004
centromeric chromocenter) [53]. Both macroH2A1.2 and
M31 were found to colocalize in a time-dependent
manner at specific nuclear regions, including the pseudo-
autosomal region (PAR) of the sex body [50], suggesting
a role for this heterochromatic region in preventing
precocious desynapsis of the terminally associated X and
Y chromosomes prior to a naphase I. According to the
data described above, the large histone H2A variant,
macroH2A1.2, along with the HP1-like protein M31,
could be involved in the partial pairing of X and Y

support of this hypothesis [7,55].
The histone code is probably in a ction in spermatogenic
cells as stage-specific histone modifications have been
reported to occur during the postmeiotic genome reorgan-
ization phase. However, despite detailed descriptions of
some histone modifications [9], nothing is known about
their potential function in chromatin reorganization and
histone replacement in elongating spermatids (Fig. 1).
Histone acetylation
Acetylated forms of histones have been found during
spermatogenesis in various species including, trout [56], rat
[57] and rooster [58]. The use of antibodies, specifically
recognizing individual acetylated residues, has allowed a
more precise characterization of histone acetylation pattern
during spermatogenesis [59]. Spermatogonia and prelepto-
tene spermatocytes contain acetylated H2A H2B and H4,
whereas histones are underacetylated during meiosis and in
round spermatids. The replication-dependent acetylation of
H4 and H 3 [60] can partially explain the acetylation signal
detected in DNA replicating cells.
Interestingly, these data also showed that in elon gating
spermatids, histones become hyperacetylated in the total
absence of DNA replication. In the case of histone H4, this
acetylation was shown to follow a stage-specific distribution
[59,61]. Indeed, the H4 hyperacetylation observed in the
early elongating spermatids affects the nucleus in a g lobal
manner. This distribution then changes during the elonga-
tion and condensation s tages and finally acetylated H4
disappears following an antero-caudal movement in con-
densing spermatids.

has also been shown to occur during meiosis very probably
associated with chromosome condensation [70]. However,
no information is available a bout the phosphorylation of
Ser28 during spermatogenesis.
Site-specific phosphorylations of H2A [71], H2AX [40]
and H2B [72] have also been reported. While nothing is
known a bout the phosphorylation of H2A and H2B
during spermatogenesis, that of H2AX may play a c rucial
role as it is tightly linked to the function of H2AX in
DNA double strands breaks repair [40]. Indeed, a
transient phosphorylation of H2AX on Ser139 accom-
panies double strand break damage repair, as well as
DNA cleavage events such as those associated with
meiotic recombination [73].
Ó FEBS 2004 Chromatin code and spermatogenesis (Eur. J. Biochem. 271) 3463
Histone ubiquitination
Ubiquitination is a modification known to b e a mark for
protein degradation via the proteasome pathway. How-
ever, the function of protein ubiquitination is not
restricted to degradation, and data from the literature
suggest its involve ment in DNA repair, cell cyc le control,
cellular response to stress, as well as in the histone code
[74].
H1 and H 3 have been found occasionally ubiquitinated
in vivo, but H2A and H2B appear to be the predominant
forms of ubiquitinated histones i n e ukaryotes, encompas-
sing 5–15% of H2A and 1–2% of H2B [75].
Histone ubiquitination has been described du ring sper-
matogenesis in many species, including rat, mouse, trout
and rooster [75]. In the mouse, a high proportion of

formation of nucleosomes with altered structure and
modified properties.
Histone variants incorporated during spermatogenesis,
although showing only small changes in their primary
structures, could therefore bring major changes in the
nucleosome function and stability.
A detailed analysis of testis-specific histone variants
shows that the histone fold is usually well conserved
between variants (Fig. 2A,B,C). The N-terminal region of
H3 is very similar between the variants, including H3.3 and
H3t, whereas the N-terminal regions of TH2A and TH2B
present several differences with their somatic c ounterparts,
which m ay potentially affect residues modified by known
histone post-translational modifications (Fig. 2).
Interestingly, the comparison of H2A/TH2A sequence
shows three amino acid changes in a region covering the end
of alpha1, loop1 and the beginning of alpha2. As a
structural analysi s has a lready shown that H2A Loop1 is
the only area of contact between the two (H2A–H2B)
dimers within the nucleosome core particle [1], the m inor
sequence changes observed in TH2A could have important
functional consequences, as a lready established in t he case
of H2A.Z by crystallographic data [79]. The structural
analysis also showed that the incorporation of two
heterodimers of H2A–H2B and H2A.Z–H2B within the
same nucleosome is unlikely, suggesting that the incorpor-
ation of the first (H2A.Z–H2B) dimer could facilitate t he
recruitment o f another H2A.Z-containing dimer [79,80].
Similarly, the i ncorporation of given testis-specific histone
variants might facilitate the incorporation of other variants,

HIRA, or m aybe other spermatid-specific factors, could
recognize H3.3 and dismantle the nucleosomes. Histone
removal by HIRA may also occur in somatic cells but to a
much lesser extent than in spermatids. Therefore the
identification of HIRA partners in spermatids w ould be of
great interest in understanding the molecular b asis of
histone replacement during spermiogenesis and furthermore
in that of nucleosome disassembly in gene ral.
Recently, a histone variant exchanger that specifically
replaces co nventional H2A by H2A.Z has been identified in
yeast [ 84,85] showing that H3 and also H2A variants c an be
deposited by specific factors.
Recent work showed that in yeast, a protein identified as
Hif1p is a histone H3 and H4 chaperone involved in
chromatin assembly [86]. Interestingly, Hif1p is the
3464 J. Govin et al.(Eur. J. Biochem. 271) Ó FEBS 2004
homologue of a H1 chaperone, known as NASP, which has
a testis-specific variant expressed in different species of
mammals and is present all through spermiogenesis [87]. It
has been proposed that tNASP may bind and t ranslocate
testicular histone variants to nucleosomes [87]. Its presence
during late spermiogenesis suggests that the protein may
also function as a histone remover, as no chromatin
assembly occurs during these stages.
It is therefore very possible that the enrichment of
spermatid chromatin with different histone variants would
first increase the accessibility of chromatin to various factors
(such as those involved in recombination in pachytene cells
or histone modifying enzymes in spermatids) and then
facilitate histone replacement. Moreover, histone modifica-

Bromodomains are acetyl-lysine binding modules present
in ATP-dependent chromatin remodelling factors as well as
in some HATs and other nuclear protein s of unknown
function [89]. B romodomain-containing proteins therefore
appear to be excellent candidates to i nterpret the signal
generated by the global histone acetylation taking place
during spermiogenesis. Recently, a testis-specific double
bromodomain-containing protein, named BRDT, was
shown to be capable of inducin g a dramatic condensation
of chromatin strictly dependent o n histone hyp eracetylation
[66]. These data present a new scenario regarding the
significance of histone acetylation during spermiogenesis: it
could primarily act as a signal for chromatin condensation.
In support of this hypothesis, nuclear domains containing
condensed chromatin in elongating spermatids also corres-
pond to regions enriched in acetylated histone H4 (J. Go vin,
C. Caron, C. Lestrat, S. Ro usseaux and S. Khochbin,
unpublished results).
Bromodomain-containing factors, such as BRDT,
upon their interaction with acetylated histones, could
also recruit testis-specific chaperones t o mediate histone
removal. In fact, a new bromodomain-interacting chap-
erone, CIA-II, highly expressed in t he testis, also interacts
with histon e H3 in vivo andwithhistonesH3/H4in vitro
[90]. Such fac tors may establish a link between an
acetylation-dependent chromatin compaction mediated by
bromodomain p roteins and histone displacement. More-
over, it has recently been shown in yeast that Hat1p/
Hat2p/Hif1p specifically binds acetylated histones H4 and
H3 [86]. A s mentioned a bove, the testis-specific homo-

some of these proteins, such as heterochromatin protein 1
(HP1), the chromodomain has been shown to specifically
interact with histone tails bearing methylated lysines [7].
In order to assess the potential function of these binary
switches during spermatogenesis, they were searched for
on the primary sequences of the different histone
variants. Among the three testis-specific core histones,
TH2B seems t o be the on ly variant which presents
significantly divergent binary cassettes compared to its
somatic counterpart. Indeed, in testis-specific H2Bs, in
three cases the Thr/Ser residues occurring in somatic type
H2B next to a Lys residue were replaced by nonphos-
pho-acceptor residues, and three new binary cassettes
were created (Fig. 2C).
These analyses show that, on top of a structural role,
sequence divergence in testis-specific histone variants
may participate in increasing the complexity of the histone
code.
Ó FEBS 2004 Chromatin code and spermatogenesis (Eur. J. Biochem. 271) 3465
Concluding remarks
After analysing all the available data i t clearly appears that a
massive chromatin alteration occurs before histone replace-
ment due to an extensive incorporation of histone variants
as well as to globally specific histone modifications.
Recruitment of histone variants in nucleosomes may have
two general effects on chromatin structure and function.
First, subtle sequence divergences can have important
consequences on the stability of the nucleosome. Second,
these sequence divergences may change t he potential of core
histones to b e modified. A testis-specific histone code can

information on the yet unkn own mechanism of nucleosome
disassembly.
Acknowledgements
This work was supported by ‘‘Re
´
gion Rhoˆ ne-Alpes’’ emergence pro-
gram. C.L. i s supported by ‘‘Re
´
gion Rhoˆ ne-Alpes’’ PhD fellowship.
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