A unique H2A histone variant occupies the ...

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A unique H2A histone variant occupies the transcriptional start site of active genes

© 2011 Nature America, Inc. All rights reserved.

Tatiana A Soboleva1, Maxim Nekrasov1, Anuj Pahwa1, Rohan Williams1,2, Gavin A Huttley1 & David J Tremethick1 Transcriptional activation is controlled by chromatin, which needs to be unfolded and remodeled to ensure access to the transcription start site (TSS). However, the mechanisms that yield such an ‘open’ chromatin structure, and how these processes are coordinately regulated during differentiation, are poorly understood. We identify the mouse (Mus musculus) H2A histone variant H2A.Lap1 as a previously undescribed component of the TSS of active genes expressed during specific stages of spermatogenesis. This unique chromatin landscape also includes a second histone variant, H2A.Z. In the later stages of round spermatid development, H2A.Lap1 dynamically loads onto the inactive X chromosome, enabling the transcriptional activation of previously repressed genes. Mechanistically, we show that H2A.Lap1 imparts unique unfolding properties to chromatin. We therefore propose that H2A.Lap1 coordinately regulates gene expression by directly opening the chromatin structure of the TSS at genes regulated during spermatogenesis. The crystal structure of the nucleosome core particle reveals that the surface is a highly contoured landscape with an uneven and distinctive charge distribution1. Particularly notable is an acidic region, referred to as the acidic patch or pocket, formed from six acidic residues of histone H2A (refs. 1,2). A eukaryotic cell can alter the acidic patch to directly regulate the extent of chromatin compaction. For example, incorporation of the human H2A variant H2A.Bbd into nucleosomal arrays completely inhibits array folding3. This inhibition is due to the loss in H2A. Bbd of three acidic amino acid residues, which partially neutralizes the acidic patch. The function of histone variants of this type is unknown, but we hypothesized that they have a role in activating gene transcription, as the unfolding effect of H2A.Bbd can directly reverse chromatinmediated transcriptional repression in vitro3. Here we have used the mouse (Mus musculus) as a model system to address this hypothesis. RESULTS H2A.Lap1 is expressed during meiosis and post-meiotically We searched the mouse genome and identified an H2A variant with a partially neutralized acidic patch that was most similar to human H2A.Bbd (55% identical, 68% similar, ClustalW score 52). We designated the variant H2A.Lap1, for ‘lack of an acidic patch’, to more accurately reflect this important feature. (Three paralogs exist, described in Online Methods. We also identified three other mouse coding sequences similar to human H2A.Bbd, shown in Supplementary Fig. 1a.) To understand the biological function of H2A.Lap1, we determined its expression pattern. We isolated mRNA from a number of different mouse tissues and found that H2A.Lap1 is highly expressed in the testis, with a low level of expression detected in the brain (Supplementary Fig. 2a). In the testis, H2A.Lap1 mRNA expression

begins at the pachytene stage (day 19; Supplementary Fig. 2b). To confirm this, we raised polyclonal antibodies specific for H2A.Lap1 for indirect immunofluorescence studies (Supplementary Fig. 3) and carried out indirect immunofluorescence on testis sections showing all 12 stages of the cell cycle of the seminiferous tubule (Fig. 1a, Supplementary Fig. 4 and data not shown). H2A.Lap1 was first observed in late-pachytene spermatocytes (compare tubules at stage VII–VIII with stage V–VI), and its enrichment in round spermatids is evident in the majority of tubular stages (Fig. 1a and Supplementary Fig. 4). By contrast, nuclear staining no longer appears in elongating spermatids (see stage IX–X in Supplementary Fig. 4). Indirect immunostaining of surface-spread preparations of the testis confirmed these observations and revealed that H2A.Lap1 is present in elongating spermatids but is located in the cytoplasm, indicating that it has been exported out of the nucleus (100% of cells examined, N = 500; Fig. 1b). This observation explains why H2A.Lap1 can be detected in elongating spermatids using a crude western-blotting approach4. Our results show that H2A.Lap1 is expressed in a temporally specific manner in the testis, between the late pachytene and the round spermatid stages of spermatogenesis, before it is exported out of the nucleus in elongating spermatids. This period of H2A.Lap1 expression mirrors the highest levels of transcriptional activity that occur during spermatogenesis. Moreover, in elongating spermatids transcription ceases. Notably, as H2A.Lap1 moves out of the elongating-spermatid nucleus, another H2A.Bbd-like histone variant seems to take its place (Supplementary Fig. 5). H2A.Lap1 avoids heterochromatin with one exception Supporting our hypothesis, H2A.Lap1 generally avoids heterochromatin (the intensely DAPI-stained regions). In the pachytene stage,

1The

John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia. 2Present address: Singapore Centre on Environmental Life Sciences Engineering, Environmental National University of Singapore, Singapore. Correspondence should be addressed to D.J.T. ([email protected]). Received 2 June; accepted 21 September; published online 4 December 2011; doi:10.1038/nsmb.2161

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H2A.Lap1 is excluded from constitutive heterochromatic regions as well as from the heterochromatic XY body enriched with γH2A.X (Fig. 1b, green spot marked by filled white arrow). Similarly, in contrast to the H2A variant macroH2A, H2A.Lap1 avoids the large constitutive heterochromatic chromocenter in round spermatids (Fig. 1b). Therefore, we conclude that H2A.Lap1 is mainly euchromatic. Notably, however, we did observe one domain of heterochromatin enriched with H2A.Lap1 immediately adjacent to the chromocenter in round spermatids (Fig. 1b, unfilled white arrow). We and others have previously demonstrated that this distinctive DAPI-intense region is a sex chromosome (the X or Y chromosome5–7; Fig. 1c). H2A.Lap1 associates with both the heterochromatic X and Y chromosomes. The X and Y chromosomes can readily be distinguished because the variant macroH2A1.2 is a component of constitutive heterochromatin of the Y chromosome but not the X chromosome8. Consistent with this, about half of the round spermatids analyzed (N = 117) contained a small focus of macroH2A adjacent to the heterochromatic H2A. Lap1-containing domain (Fig. 1b). H2A.Lap1 dynamically loads onto the sex chromosomes There are eight stages of round spermatids before they develop into elongating spermatids9. We therefore wondered whether the incorporation of H2A.Lap1 into the sex chromosome depends on the stage

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Figure 1 H2A.Lap1 dynamically loads onto the sex chromosomes in late round spermatids. (a) Seminiferous tubule sections were indirectly immunostained with H2A.Lap1 antibodies (red) and stained with peanut agglutinin Alexa Fluor 488 conjugate (LectinPNA, green) to allow identification of the various spermatid stages on the basis of acrosome maturation9. DNA was costained with DAPI (blue). P, mid-pachytene spermatocytes; LP, late-pachytene spermatocytes; RS, round spermatids. Arrow marks residual bodies (RB; Supplementary Fig. 8). Scale bars, 25 µm. (b) Surface spreads of leptotene and zygotene cells were prepared from pre-pachytene testes of 12-day-old mice. Surface spreads of pachytene spermatocytes, round spermatids and elongating spermatids were prepared from adult mice. Different cell types were immunostained with SCP3 (a marker for the progression of chromosome synapsis), γH2A.X (a component of the XY body in pachytene spermatocytes) or macroH2A1.2 (enriched in centromeric heterochromatin of the Y chromosome in round spermatids). Filled white arrow, XY body. Unfilled white arrow, Y chromosome centromeric heterochromatin. Scale bar, 10 µm. (c) Fluorescence in situ hybridization analyses of round spermatids using specific chromosome-X or chromosome-Y paints (reproduced with permission from our previous published study5). White lines in merged images indicate the paths used to determine fluorescence intensity across the sex chromosomes and the chromocenter, graphed at right. C, hromocenter. Scale bars, 10 µm. (d) Round spermatids were immunostained with H2A.Lap1 antibodies and stained with DAPI. Round spermatids were also stained with LectinPNA to distinguish whether a round spermatid was at an early or late stage of development. White lines in merged images indicate the paths used to determine the fluorescence intensity of H2A.Lap1 across the DAPI stained sex chromosome, graphed at right. Scale bar, 10 µm.

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of round-spermatid development. To test this, we divided samples into early or late stages of round-spermatid development according to the size of the maturating acrosome. We observed no enrichment of H2A.Lap1 on the DAPI-intense sex-chromosome domain next to the chromocenter in early round spermatids. However, in late round spermatids 100% of all sex chromosomes examined contained H2A. Lap1 (N = 120; Fig. 1d). Therefore, in what must be a highly regulated temporal and spatial process, H2A.Lap1 loads onto the inactive sex chromosomes in the later stages of round-spermatid development. H2A.Lap1 is incorporated into active chromatin To directly determine whether H2A.Lap1 is incorporated into active chromatin, we carried out chromatin immunoprecipitation with sequencing (ChIP-seq) using affinity-purified H2A.Lap1 antibodies and mononucleosomes obtained from testes of 30-day-old mice (Fig. 2; 82% of tubules contained round spermatids, as described in Online Methods; N = 109). First, we investigated whether H2A.Lap1 is present on genes that escape the X-chromosome inactivation process (Fig. 2a). To analyze this, we used published gene expression data6 together with our ChIPseq results. These published data indicate that ~87% of X-chromosome genes are transcriptionally suppressed in round spermatids, and the X-chromosome genes active in round spermatids fall into two

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Figure 2 H2A.Lap1 is located at the TSS of active genes. (a) H2A.Lap1 ChIP profiles on genes active in round-spermatid X-chromosomes. Each line represents 50 genes, grouped by expression level using published gene expression data 6; coloring indicates average gene expression rank in the group. The sum of frequency tag counts in the group is plotted at each base pair relative to the TSS. (b) H2A.Lap1 ChIP profile showing sum of frequency tag counts on 44 X-linked Group C genes, aligned between −1 kb and +1 kb from the TSS. (c,d) H2A.Lap1 ChIP profiles for genes on chromosome 1 (c) and for the whole genome (d), grouped by expression level using global expression of all mouse genes in the 30-day-old testis. We separated 23 groups of 50 genes on chromosome 1 and 201 groups of 100 genes in the whole genome; coloring is as in a. Note that for the whole-genome plot, the sum of all shown frequency tag counts equals 1. (e) H2A.Lap1 ChIP profile for the 1,000 most highly expressed mouse genes in the 30-day-old testis, aligned between −5 and +5 kb from the TSS. (f) H2A.Lap1 ChIP profiles for all mouse genes expressed at the pachytene stage, grouped by expression level (164 groups of 100 genes) using published pachytene expression data 6. Coloring is as in a.

classes: those that are reactivated (Group B) and those that are specifically expressed only in round spermatids (Group C, which comprises 44 genes that we could uniquely map to a ENSEMBL stable identifier). We separated 550 X-chromosome genes into 11 groups of 50 genes according to their expression levels. For each group of genes, a single line in Figure 2a represents the frequency tag counts at each base pair relative to the TSS normalized to the whole genome plot (see Supplementary Methods). ‘Heat map’ coloring indicates the gene expression rank of each group. Notably, H2A.Lap1 is selectively enriched at the promoters of active X-chromosome genes, specifically at the TSS; there is a peak in H2A.Lap1 tags on highly expressed genes at about −50 base pairs (bp) relative to the TSS (see below). Group C genes also have H2A.Lap1 located at their TSS (Fig. 2b), as do Group B genes (Supplementary Fig. 6a). A complementary heat map for input mononucleosomes is shown in Supplementary Figure 6b.

Is H2A.Lap1 associated only with active genes on the X chromosome? To answer this question, we examined global gene expression in the 30-day-old testis using whole-mouse expression microarrays. A positive correlation between the abundance of H2A.Lap1 at the TSS and the expression level was evident both for genes on chromosome 1 (Fig. 2c) and for all mouse genes (Fig. 2d). For both profiles, a marked single peak of H2A.Lap1 signal occurred at about −50 bp relative to the TSS (we designate this position as nucleosome −1). To find out whether there H2A.Lap1 increases in the coding region, we created a single group of the 1,000 most highly expressed genes in the 30-day-old mouse testis and examined the H2A.Lap1 mononucleo some profile between −5 kb to +5 kb relative to the TSS (Fig. 2e). Although there was a modest increase in the H2A.Lap1 signal 1–2 kilobases (kb) downstream from the TSS, this signal gradually declined further into the coding region.


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H2A.Z and H2A.Lap1 create an unusual active landscape Many studies using various cell types have shown that two major H2A.Z-containing nucleosomes are positioned on either side of the TSS (at nucleosome positions −2 and +1) of a poised or active RNA polymerase II promoter10,11,13–15. To investigate whether H2A.Z is also a component of active promoters when H2A.Lap1 is present at an active TSS, we conducted ChIP-seq experiments using affinity-purified H2A.Z antibodies. Similarly to H2A.Lap1, H2A.Z’s abundance was positively correlated with the level of gene expression (Fig. 3c). To our surprise, however, the marked H2A.Z signal observed at nucleosome position +1 was absent, with only one major peak of H2A.Z observed ~200 bp upstream from the TSS (nucleosome position −2). To examine these chromatin features more precisely, we created a single group of the 1,000 most highly expressed genes and examined the TSS profile for both H2A.Lap1 and H2A.Z on the same plot (Fig. 3d,e). On the basis of these data, we propose that the gene activation process in the testis requires a specialized chromatin landscape involving the action of two different histone variant– containing nucleosomes positioned next to each other.

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Figure 3 Targeting of H2A.Lap1 to X chromosome– linked genes occurs in late round spermatids. Six round spermatid–specific X-linked genes were chosen for H2A.Lap1 ChIP and gene expression analyses. (a) H2A.Lap1 enrichment for each gene in mouse testes at 18, 24, or 30 d of development, relative to Dusp21 at 18 d. H2A.Lap1 signal was assayed by ChIP. ChIP-seq libraries, normalized to the same DNA concentration, were analyzed by quantitative PCR using gene-specific primers that target the TSS. Data shown are means and s.d. of three repeats. (b) The mRNA level of each gene at each stage of testes development, determined by real-time quantitative PCR, relative to β-actin. Data shown are means and s.d. of three repeats. (c) H2A.Z ChIP profiles at TSS of all mouse genes expressed in the 30-day-old testis; genes are grouped by average gene expression rank as in Figure 2d (201 groups of 100 genes). (d) Normalized ChIP profiles of H2A.Z and H2A. Lap1 (with confidence intervals estimated by resampling) for the 1,000 most highly expressed genes in the 30-day-old mouse testis. (e) Cartoon depicting the location of H2A.Z- and H2A. Lap1-containing nucleosomes at the −2 and −1 positions, respectively, relative to the TSS.

and day 30. Similarly, between these stages of testes development there was a ~40-fold increase in the expression of 4930557A04Rik, with a corresponding approximately four-fold increase of H2A.Lap1 signal at its TSS. This targeting of H2A.Lap1 to the TSS was not due to an increase in H2A.Lap1 expression because H2A.Lap1 expression peaks around day 24 (Supplementary Fig. 2d). These observations indicate that a specific H2A.Lap1 loading complex, analogous to the complexes associated with other histone variants, is responsible for the H2A.Lap1 signal at the TSS. We conclude that these Group C genes are maximally expressed in late-stage round spermatids, which coincides with the highest H2A. Lap1 signal detected at their TSS. Together, these results strongly suggest that the X-chromosome enrichment of H2A.Lap1 that occurs in late round spermatids (Fig. 1d) has a role in the coordinated transcriptional activation of round spermatid–specific genes. Our data do not show whether H2A.Lap1 causes the gene activation process, but future mouse gene knockout studies may answer this question.

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X chromosome genes activated in late round spermatids To investigate this proposal, we determined whether the dynamic targeting of H2A.Lap1 to the X chromosome in late-stage round spermatids (Fig. 2d) is correlated with the activation of round spermatid– specific gene transcription. H2A.Lap1 ChIP assays were done on individual X-chromosome genes (see Supplementary Methods). Mononucleosomes were isolated from mice testes at different stages of development: 18 d (0% of tubules contained round spermatids; N = 81), 24 d (50% and 0% of tubules contained early and late round spermatids, respectively; N = 133) and 30 d (49% and 33% of tubules contained early and late round spermatids, respectively; N = 109). Six individual genes from Group C in ref. 6 were analyzed by quanti tative PCR using specific primer pairs designed to detect the H2A. Lap1-containing mononucleosome located at the TSS (Fig. 3a). The expression levels of these genes were also analyzed by real-time quanti tative PCR (Fig. 3b). Eighteen-day-old mice had very little H2A.Lap1 at the TSS of these Group C genes (Fig. 3a), which is consistent with the testes of these mice having no round spermatids. As expected, these six genes showed very little expression at this stage of testes development. For all six genes, the H2A.Lap1 signal at their TSS was highest at day 30, when late round spermatids appeared; the six genes were also most highly expressed at this time. For example, the enrichment of H2A.Lap1 at the TSS of Dusp21 and the expression of Dusp21 increased about six-fold and ten-fold, respectively, between day 24

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Next, given that H2A.Lap1 is first expressed in the late pachytene stage, we wondered whether H2A.Lap1 is also involved in the activation of transcription in these meiotic cells. As above, we used the published data on pachytene gene expression6 together with ChIP-seq results. Indeed, H2A.Lap1 was enriched at the TSS of genes expressed at the pachytene stage (Fig. 2f). We conclude that, whereas in other organisms and cell systems the TSS lacks active marks and is nucleosome depleted10–12, H2A.Lap1 is a previously undescribed histone variant found at the TSS of RNA polymerase II promoters active during meiosis and post-meiotically. Moreover, its specific expression pattern during spermatogenesis combined with its precise targeting to the TSS could provide a mechanism for coordinated gene activation.

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Figure 4 H2A.Lap1 has gained a single acidic amino acid residue, which enables nucleosome arrays to partially fold. (a) Sedimentation coefficient distribution plots of arrays containing human H2A.Bbd in the absence or presence of 0.3 mM MgCl 2 (reproduced from our previous published study3). (b,c) Sedimentation coefficient distribution plots of arrays containing wild-type (WT) H2A or H2A.Lap1 in the absence or presence of 1.2 mM MgCl 2. (d,e) Sedimentation coefficient distribution plots of arrays containing WT H2A, H2A.Lap1 or mutant H2A.Lap1 (D100T) in the absence or presence of 0.4 mM MgCl2.

H2A.Lap1 allows partial folding of a nucleosomal array Next, we explored the mechanism underpinning the transcriptional function of H2A.Lap1 by determining whether H2A.Lap1 inhibits the chromatin folding process analogously to human H2A.Bbd (ref. 3) (Fig. 4a; the partially neutralized acidic patch of H2A.Bbd and H2A.Lap1, compared to H2A, is shown in Supplementary Fig. 1a). We assembled control and H2A.Lap1-containing nucleosome arrays using 601-200-12 DNA as template DNA, as described previously for human H2A.Bbd (ref. 3). In all experiments described here, the assembled arrays had >95% of their DNA repeats occupied by histone octamers3. We analyzed the conformations of the arrays by sedimentation velocity experiments. In low-salt conditions, wild-type nucleosomal arrays show a 32S to 33S sedimentation profile, which corresponds to the unfolded 10-nm ‘beads-on-a-string’ fiber structure3,16 (Fig. 4b). By contrast, H2A.Bbd arrays show a 25S to 26S conformation, which is even more open than the 32S (10-nm fiber) conformation3 (Fig. 4a). As the concentration of divalent cations increases (up to ~1.5 mM MgCl2), the wild-type nucleosomal array folds gradually until it reaches the 55S conformation, the most locally compacted form, equivalent to the 30-nm fiber3,16 (Fig. 4c). By contrast, H2A.Bbd arrays cannot fold17 beyond the 32S conformation before they precipitate3. To our surprise, rather than adopting a 26S conformation, H2A. Lap1 arrays showed a 32S conformation identical to that of control arrays in low-salt conditions (Fig. 4b). Moreover, in the presence of 1.2 mM MgCl2, these variant arrays had an save of 41S (save is defined as the sedimentation coefficient at the boundary fraction equivalent to 50%; Fig. 4c). Control arrays had an save of 50S. In other words, in contrast to H2A.Bbd, H2A.Lap1-containing arrays can partially fold to a moderately compacted 40S structure. This unexpected discovery led us to re-examine the amino acid sequence of H2A.Lap1 to determine whether this variant has gained any new acidic amino acid residues in the vicinity of the acidic patch, compared to human H2A.Bbd, that could explain these results. Indeed, an aspartate residue (Asp100) is present in H2A.Lap1 but absent in human H2A.Bbd (and H2A; Supplementary Fig. 1a). To test whether this single acidic amino acid residue is responsible for the ability of H2A.Lap1 arrays to partially fold, we assembled mutant arrays in which this single residue was changed to the equivalently positioned residue found in human H2A.Bbd (D100T). These mutant arrays behaved identically to human H2A.Bbd-containing arrays. Mutant H2A.Lap1 D100T arrays adopted a more open 26S conformation in low-salt

conditions (Fig. 4d) and had an save of 29S in the presence of 0.4 mM MgCl2 (Fig. 4e). At this divalent cation concentration, H2A.Lap1 and control arrays had an save of 36S and 40S, respectively (Fig. 4e). Also, similarly to human H2A.Bbd arrays3, folding experiments could not be carried out at higher MgCl2 concentrations because the mutant H2A.Lap1 D100T arrays precipitated. DISCUSSION In this study we have identified the histone variant H2A.Lap1 as a previously undescribed component of chromatin on active genes that are expressed at specific stages during meiosis and post-meiotically. Although both H2A.Lap1 (Supplementary Fig. 1b,c) and human H2A.Bbd (ref. 17) yield unstable nucleosome core particles that protect only ~120 bp of DNA from micrococcal nuclease digestion, we conclude that they are functionally distinct histone variants as they impart different and unique folding properties to an array. Notably, this difference can be attributed to the gain of just a single acidic amino acid residue by H2A.Lap1. This Asp100 residue (Lys96 in mouse H2A) is located in the H2A C-terminal tail α-helix (Supplementary Fig. 7), which we have previously demonstrated is responsible for the ability of H2A.Z to promote the formation of the 30-nm fiber3. Together, these findings identify the C-terminal tail α-helix of H2A as an important modulator of chromatin compaction and show how the multifunctional nucleosome can be regulated by minor changes in amino acid residues (and in the absence of histone modifications). H2A.Lap1 allows only partial folding of a nucleosome array, thereby inhibiting the formation of the highly condensed 30-nm fiber. This degree of chromatin unfolding mediated by H2A.Lap1 is sufficient to directly overcome chromatin-mediated transcriptional repression in vitro3. These unfolding properties in the presence of H2A.Lap1 are essentially identical to those of arrays assembled with acetylated H3 and H4, which define active chromatin18,19. In other words, incorporation of this histone variant bypasses the initial need to engage histone acetyltransferases for acetylation of nucleosomes to yield an open chromatin structure, which may be advantageous during the rapid remodel ing of chromatin that occurs during spermatogenesis. Subsequently, analogously to human H2A.Bbd chromatin17,20, this open and unstable structure may both promote the incorporation of other active marks and prevent the binding of repressor proteins such as histone H1 to initiate high levels of transcription. In conclusion, we have identified a previously undescribed strategy for transcriptional activation in the mouse testis, which involves the unique histone variant H2A.Lap1.


articles Methods Methods and any associated references are available in the online version of the paper at http://www.nature.com/nsmb/. Note: Supplementary information is available on the Nature Structural & Molecular Biology website.


Acknowledgments We thank J. Fan (The Australian National University) for initial protein preparations of H2A.Lap1, J. Pehrson (University of Pennsylvania) for macroH2A antibodies, K. Luger (Colorado State University) for core histone recombinant proteins, S. McBryant and J. Hansen for helping M.N. to set up sedimentation velocity experiments, S. Grigoryev and R. Reeves for reading the manuscript, our in-house Biomolecular Research Service, headed by S. Palmer, for high-throughput DNA sequencing, and A. Prins and C. Gillespie for help in histological sample preparation and microscopy. This work was supported by Australian National Health and Medical Research Council project grants to T.A.S. and D.J.T., and to M.N. and D.J.T. AUTHOR CONTRIBUTIONS T.A.S. helped design the experiments, cloned H2A.Lap, conducted all spermatogenesis experiments, prepared chromatin for ChIP-seq experiments and conducted the gene expression and ChIP experiments on individual X-chromosome genes. M.N. conducted the biochemical and biophysical experiments on the nucleosome arrays and prepared DNA ChIP libraries for high-throughput sequencing. R.W. developed and did data analysis of global mouse gene expression data. G.A.H. designed and executed the analysis of the Illumina short-read data. A.P. assisted with the analyses of Illumina short-read data. D.J.T. conceived the project, helped design the experiments and wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/nsmb/. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F. & Richmond, T.J. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389, 251–260 (1997). 2. Caterino, T.L. & Hayes, J.J. Chromatin structure depends on what’s in the nucleosome’s pocket. Nat. Struct. Mol. Biol. 14, 1056–1058 (2007).

3. Zhou, J., Fan, J.Y., Rangasamy, D. & Tremethick, D.J. The nucleosome surface regulates chromatin compaction and couples it with transcriptional repression. Nat. Struct. Mol. Biol. 14, 1070–1076 (2007). 4. Ishibashi, T. et al. H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis. Nucleic Acids Res. 38, 1780–1789 (2009). 5. Greaves, I.K., Rangasamy, D., Devoy, M., Marshall Graves, J.A. & Tremethick, D.J. The X and Y chromosomes assemble into H2A.Z-containing facultative heterochromatin following meiosis. Mol. Cell. Biol. 26, 5394–5405 (2006). 6. Namekawa, S.H. et al. Postmeiotic sex chromatin in the male germline of mice. Curr. Biol. 16, 660–667 (2006). 7. Turner, J.M., Mahadevaiah, S.K., Ellis, P.J., Mitchell, M.J. & Burgoyne, P.S. Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids. Dev. Cell 10, 521–529 (2006). 8. Turner, J.M., Burgoyne, P.S. & Singh, P.B. M31 and macroH2A1.2 colocalise at the pseudoautosomal region during mouse meiosis. J. Cell Sci. 114, 3367–3375 (2001). 9. Oakberg, E.F. A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat. 99, 391–413 (1956). 10. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007). 11. Schones, D.E. et al. Dynamic regulation of nucleosome positioning in the human genome. Cell 132, 887–898 (2008). 12. Wang, Z. et al. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat. Genet. 40, 897–903 (2008). 13. Conerly, M.L. et al. Changes in H2A.Z occupancy and DNA methylation during B-cell lymphomagenesis. Genome Res. 20, 1383–1390 (2010). 14. Henikoff, S., Henikoff, J.G., Sakai, A., Loeb, G.B. & Ahmad, K. Genome-wide profiling of salt fractions maps physical properties of chromatin. Genome Res. 19, 460–469 (2009). 15. Jin, C. et al. H3.3/H2A.Z double variant-containing nucleosomes mark ‘nucleosomefree regions’ of active promoters and other regulatory regions. Nat. Genet. 41, 941–945 (2009). 16. Hansen, J.C. Conformational dynamics of the chromatin fiber in solution: determinants, mechanisms, and functions. Annu. Rev. Biophys. Biomol. Struct. 31, 361–392 (2002). 17. Montel, F. et al. The dynamics of individual nucleosomes controls the chromatin condensation pathway: direct atomic force microscopy visualization of variant chromatin. Biophys. J. 97, 544–553 (2009). 18. Allahverdi, A. et al. The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association. Nucleic Acids Res. 39, 1680–1691 (2011). 19. Shogren-Knaak, M. et al. Histone H4–K16 acetylation controls chromatin structure and protein interactions. Science 311, 844–847 (2006). 20. Angelov, D. et al. SWI/SNF remodeling and p300-dependent transcription of histone variant H2ABbd nucleosomal arrays. EMBO J. 23, 3815–3824 (2004).


ONLINE METHODS


Cloning of H2A.Lap1 and antibody generation. Our mouse genome analysis indicated that H2A.Lap1 was the mouse ortholog of human H2A.Bbd (ENSEMBL release 57, March 2010). There is one major form of H2A.Lap1, which accounts for ~90% of total H2A.Lap1 expression in the mouse testis (ENSMUSG00000083616, X chromosome, minus strand, 117,426,357–117,426,705; data not shown), and two minor forms (ENSMUSG00000067441, X chromosome, plus strand, 114,128,366–114,128,665; and ENSMUSG00000082482, X chromosome, plus strand, 113,794,787–113,795,134). The major H2A.Lap1 gene was amplified from the Balb/c mouse genomic library using gene-specific primers; this was followed by a second round of amplification with primers containing NdeI and BamHI sites, respectively. The final PCR product was cloned into a pET-15b vector through NdeI and BamHI sites. His6-H2A.Lap1 was expressed in the Escherichia coli strain BL-21DE3pLysS. Protein was purified from inclusion bodies under denaturing conditions using nickel–nitrilotriacetic acid agarose (Qiagen). One sheep was injected 5 times at 2-week intervals with 2 mg of His6H2A.Lap1 per injection (IMVS Veterinary Services Division). Antibodies to H2A. Lap1 were affinity-purified using untagged H2A.Lap1 protein immobilized on a PVDF membrane. Nucleosome-array reconstitution and sedimentation-velocity analysis. H2A. Lap1, mutant H2A.Lap1 D100T and control nucleosome arrays were reconstituted from mouse recombinant proteins with >95% reconstitution efficiency as described3. Sedimentation-velocity studies using a Beckman XL-A analytical centrifuge and analysis of boundary fractions were done as described3. Immunohistochemistry, mouse testis sections and surface-spread preparations. The testes of Balb/c wild type mice were removed and submerged into 10% (v/v) neutral buffered formalin for 6 h, embedded in paraffin and sectioned at a thickness of 4 µm. Deparaffinized sections were treated for antigen retrieval with trypsin solution (0.05% (w/v) trypsin, 0.1% (w/v) calcium chloride, pH 7.8) for 15 min at 37 °C. We blocked nonspecific binding by incubating sections with 0.5% (w/v) blocking reagent (PerkinElmer no. FP10210) in TBS (0.2 M Tris-HCl pH 7.6, 0.14 M NaCl) for 30 min at 22 °C. Primary antibodies were diluted (1/30 anti-H2A.Lap1, 1/50 anti-H2A.Z) in 0.5% blocking reagent– TBS, and sections were incubated overnight at 4 °C in a humidity chamber. After three 5-min washes with TBS, 0.025% (v/v) Triton ×100, fluorophore-conjugated secondary antibody and LectinPNA Alexa Fluor 488 were added to the sections, which were then incubated in the dark for 1 h at 22 °C. After three 5-min washes in TBS and 0.025% (v/v) Triton ×100, slides were incubated in 1 µM DAPI for 2 min. Before coverslips were added, Vectashield (Vecta Laboratories) was applied to prevent photobleaching. Surface spreads of germ cells were prepared as described21 with some variations. Germ cells were resuspended in 200 µl of hypotonic solution (100 mM sucrose, protease inhibitor cocktail (Roche), pH 8.2) and left on ice for 2 min. Poly(l-lysine)–coated slides were placed in a humidified chamber and hydrophobic circles were drawn, covering approximately two-thirds of a slide. We spread 100 µl of 1–2% (w/v) paraformaldehyde and 0.05% (v/v) Triton X100 plus protease inhibitor cocktail (Roche), pH 9.2, to cover the entire area within the circle. The germ cell suspension was drawn carefully into a 200-µl pipette tip, and one or two drops of cells were placed into this solution on the slide. Cells were left to attach and fixed for 2 h at 22 °C in a humidity chamber, then washed with PBS. The fixed cells were then blocked for 1 h with 3% (w/v) BSA in PBS at 22 °C before the primary antibody (diluted with 1% (w/v) BSA, 0.1% (v/v) Tween 20, in PBS) was applied. Slides were then incubated overnight at 4 °C in a humidity chamber. After three 5-min washes with PBS, slides were incubated with fluorophore-conjugated secondary antibodies for 1 h at 22 °C.

doi:10.1038/nsmb.2161

Preparation of mononucleosomes. Testis tubules were placed in 1–2 ml of icecold PBS-GL buffer (PBS supplemented with 5.6 mM d-glucose, 5.4 mM dllactate, 0.5 mM DTT, 0.2 mM PMSF, protease inhibitor cocktail (Roche)) and dissected with scissors, then homogenized with three or four strokes in a Dounce homogenizer (pestle type A, cell integrity monitored by microscopy). The cellular mixture was filtered through a 70-µm cell strainer to obtain a single-cell suspension. Cross-linking was carried out in 1% (v/v) formalin for 15 min at 22 °C with rotation and stopped with glycine (final concentration of 125 mM). The cross-linked cells were washed three times with PBS and resuspended in 2 ml of swelling buffer 1 (25 mM HEPES, 15 mM NaCl, 10 mM KCl, 2 mM MgCl2, 0.2% (v/v) NP-40, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, 0.2 mM PMSF, supplemented with protease inhibitor cocktail, pH 7.6) and left on ice for 10 min. The swelled cells were mixed with 2 ml of sucrose buffer (0.6 M sucrose, 120 mM KCl, 15 mM HEPES, 15 mM NaCl, 2 mM MgCl2, 0.2% (v/v) NP-40, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, 0.2 mM PMSF and protease inhibitor cocktail, pH 7.6) and the nuclei were released by 16–20 strokes in a Dounce homogenizer (pestle type B, monitored by light microscopy). Nuclei were pelleted by centrifugation for 40 min at 4,000g through an 8-ml sucrose cushion (1.2 M sucrose, 60 mM KCL, 15 mM HEPES, 15 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, 0.2 mM PMSF and protease inhibitor cocktail, pH 7.6). The supernatant was carefully removed, and pelleted nuclei were resuspended in 1 ml of micrococcal digestion buffer (50 mM Tris-HCl pH 7.6, 3 mM CaCl2) and passed five or six times through a size 29–gauge needle. The nuclear extract was digested for 55–65 min at 37 °C with micrococcal nuclease (New England Biolabs; 30 units per 220 µl of extract) to obtain ~80% mononucleosomes. After centrifugation at 10,000g for 5 min, the supernatant (S1) and the resuspended pellet (S2) were dialyzed against 10 mM Tris-HCl pH 7.6, 1 mM EDTA and 0.5 mM EGTA overnight at 4 °C. The S1 and S2 supernatants were then combined and dialyzed against 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 4% (v/v) glycerol, divided into aliquots and stored at −80 °C. Chromatin immunoprecipitation, preparation of ChIP-seq libraries and DNA sequencing analysis. Chromatin immunoprecipitation assays were done as described22, with modifications as described in Supplementary Methods. Real-time PCR primer sequences for ChIP assays are described in Supplementary Table 1. Expression and microarray data analysis. Total RNA was isolated using TRIzol (Invitrogen) and the RNeasy Mini Kit (Qiagen). DNA was either analyzed by real-time quantitative PCR using SYBR Green PCR master mix (Applied Biosystems) or analyzed using DNA microarray analysis (in triplicate) with the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Robust Multichip Average (RMA) correction and probe-set summaries were obtained using Affymetrix Power Tools software annotation (Affymetrix) based on the mm9 mouse genome. Subsequent expression analyses (see Supplementary Methods) were done in the R statistical computing environment (version 2.11.1). Realtime PCR primer sequences for gene expression assays are described in Supplementary Table 1. Analysis of short-read sequence data. A custom pipeline for processing Illumina sequence data was implemented as described in Supplementary Methods. 21. Peters, A.H., Plug, A.W., van Vugt, M.J. & de Boer, P. A drying-down technique for the spreading of mammalian meiocytes from the male and female germline. Chromosome Res. 5, 66–68 (1997). 22. Orlando, V., Strutt, H. & Paro, R. Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11, 205–214 (1997).

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