Mouse BAZ1A (ACF1) Is Dispensable for Double-Strand Break Repair but Is Essential for Averting Improper Gene Expression during Spermatogenesis
[['The nucleosome , a complex of eight histone proteins wrapped by 146 bp of DNA , is a fundamental packaging unit for nuclear DNA , controlling access by proteins involved in transcription , replication , recombination and repair .', 'Granting or blocking DNA access can be effected through changes in histone-DNA contacts by action of chromatin remodelers , ATP-dependent multi-protein complexes that assemble , reposition , restructure and/or disassemble nucleosomes [1] , [2] .', "Each remodeler comprises an ATPase of the Swi2/Snf2 family of helicases/translocases and one or more accessory factors that confer biological specificity by modulating the ATPase's activity and/or targeting to particular genomic locations .", 'ACF and CHRAC are particularly well-studied examples of the widely conserved imitation switch ( ISWI ) family of remodelers , first purified from Drosophila [3] , [4] .', 'Drosophila ACF ( ATP-dependent chromatin assembly and remodeling factor ) is a two-subunit complex containing the ATPase ISWI bound to Acf1 [5] .', 'Its human counterpart contains the ISWI homolog SNF2H ( SMARCA5 ) and the Acf1 homolog BAZ1A ( also known as ACF1 ) [6] , [7] .', 'CHRAC ( chromatin accessibility complex ) is a larger version of ACF which , in addition to Acf1 and ISWI , contains two small histone-fold proteins: CHRAC14 and -16 in Drosophila [8] , [9] , CHRAC15 and -17 in human [10] ( Figure S1A ) .', 'BAZ1A is so named because it shares domain architecture with other BAZ family members: a bromodomain adjacent to a zinc finger , typically a plant homeo domain ( PHD ) [11] ( Figure S1B ) .', 'Drosophila and mammals have multiple Acf1 homologs , each of which associates with an ATPase with or without additional proteins to form a large family of distinct ISWI chromatin remodeling complexes ( Figure S1A ) .', 'Of the seven Acf1 homologs in mammals , BAZ1A is the one most similar to Drosophila Acf1 ( Figure S1C ) , so it is a defining subunit of mammalian ACF and CHRAC complexes .', 'A great deal is known about the enzymatic activities of ACF and CHRAC because the human and Drosophila proteins , and the equivalent ISW2 complex of budding yeast , have been paradigms for biochemical studies of ISWI complexes [reviewed in 12]–[14] .', 'In vitro , they catalyze movements of nucleosomes to form regularly spaced nucleosome arrays , consistent with a principal function in assembly and maintenance of properly ordered chromatin structures [3] , [4] .', 'Much is also known about the biochemical functions of the non-ATPase subunits .', 'Acf1/BAZ1A ( and/or their yeast homolog Itc1 ) directly contacts nucleosome-adjacent linker DNA , increases the affinity and processivity of the ATPase on nucleosomal substrates , and alters basal ISWI/SNF2H nucleosome spacing and positioning activities in vitro [5] , [8] , [15]–[18] .', 'The CHRAC proteins heterodimerize and directly bind both DNA and the N-terminus of Acf1/BAZ1A to further enhance ACF performance [15] , [19]–[21] .', 'Bulk biochemical , single-molecule , and structural studies have elucidated interactions of ACF and CHRAC with ATP , DNA , and nucleosomes , leading to detailed mechanistic models for their function in chromatin assembly and nucleosome movement [reviewed in 2] , [12]–[14] .', "In contrast , comparatively little is known about these complexes' physiological roles in metazoans [22] .", 'Iswi and Snf2h mutations cause embryonic lethality in flies and mice , respectively [23] , [24] , but these ATPases are the catalytic cores of multiple chromatin remodeling complexes with distinct biochemical properties and biological specificities [2] , [22] ( Figure S1A ) .', 'Thus far , Drosophila genetic studies provide the only in vivo data specific to ACF/CHRAC .', 'Null acf1 mutations cause substantial lethality at larval stage L3 , although about 25% of acf1 flies survive to adulthood and are fertile , suggesting the existence of compensatory factors [25] .', 'Mutants display less ordered nucleosome arrays , reduced position effect variegation , defects in establishment of pericentric heterochromatin and Polycomb-dependent repressive chromatin , and derepression of Wingless-regulated genes [25]–[27] .', 'These properties suggest that ACF and/or CHRAC promote proper chromatin assembly in vivo , also supported by studies of homologous protein complexes in budding yeast [28] .', 'RNAi depletion studies in cultured , tumor-derived human cells indicate that Baz1a facilitates replication through heterochromatic DNA [29] , contributes to repression of vitamin D3 receptor-regulated genes [30] , and promotes DNA double-strand break ( DSB ) repair [31] and the G2/M DNA damage checkpoint [32] .', 'These findings may also reflect chromatin assembly functions of ACF/CHRAC , but whether these cellular roles are essential in vivo has not been addressed .', 'Of necessity , chromatin assembly occurs on a large scale during DNA replication .', 'It has been suggested that ACF and/or CHRAC are especially critical at this time , in part because expression of Drosophila Acf1 is highest and its function most crucial during the rapid divisions of early embryonic development [25] , [26] .', 'However , chromatin assembly also occurs in non-replicative contexts .', 'For example , developmentally programmed , post-replicative chromatin changes are a hallmark of mammalian spermatogenesis , including altered histone modifications , substitution of canonical histones with histone variants ( many of which are testis-specific ) , and , late in spermiogenesis , replacement of most histones with transition proteins and then protamines [33] .', 'Some of these alterations occur genome-wide , while some are targeted to specific regions such as sex chromosomes or transposable elements .', 'Contemporaneously , gene expression is substantially rewired in successive waves to execute each spermatogenic stage [34] .', 'Many factors that implement these normal programs of chromatin alteration and gene expression changes have been discovered , e . g . , transcription factors CREM and MYBL1 and the histone H4 hyperacetylation reader BRDT [35]–[37] .', 'However , given the profound influence of chromatin structure on gene expression , it is an open question how the extensive chromatin remodeling during spermatogenesis affects transcription .', 'Here , we examine the physiological functions of mammalian ACF and CHRAC by generating mice with a targeted Baz1a mutation .', 'Surprisingly , Baz1a is dispensable for embryonic development , postnatal viability , and development of cells that experience programmed DSBs .', 'Instead , BAZ1A is essential for proper spermiogenesis and thus male fertility , with BAZ1A required ( directly or indirectly ) to prevent inappropriate gene expression during periods of large-scale chromatin restructuring in spermatogenesis .'], ['BAZ1A-containing complexes ( ACF and CHRAC ) have been purified from cultured human cells , but their distribution in vivo had not been addressed .', 'Human BAZ1A transcript levels are high in the testis compared to other tissues [11] .', 'The same is true at the protein level in mice: BAZ1A was readily detected by immunoblotting of testis extracts but was below detection in other adult tissues examined ( Figure 1A ) .', 'Conversely , its paralog BAZ1B was readily detected in all tissues ( Figure 1A ) .', 'SNF2H was highly expressed in testis and showed strong expression in other tissues ( Figure 1A ) .', 'BAZ1A is likely produced in tissues other than testis , however , as it could be detected using the same western blot conditions in more concentrated extracts of cultured human and mouse cells ( Figure 1B ) and by RT-PCR from a panel of mouse tissues ( data not shown ) .', 'To more precisely define BAZ1A expression and localization , we immunostained testis sections .', 'As detailed below , expression was first detected in germ cells late in meiotic prophase , and continued through early steps of postmeiotic spermatid differentiation .', 'BAZ1A abundance and sub-nuclear localization patterns were highly dynamic across this developmental timeline .', 'Spermatogenesis begins from spermatogonia , stem cells residing near the basal lamina of seminiferous tubules .', 'Differentiation to primary spermatocytes is accompanied by migration toward the tubule lumen and onset of meiosis , in which one round of DNA replication precedes two rounds of chromosome segregation to generate haploid cells .', 'Postmeiotic differentiation ( spermiogenesis ) converts these meiotic products into mature sperm .', 'The progression of spermatogenesis in semi-synchronous waves allows classification of histological sections of the seminiferous epithelium into 12 stages based on their cellular content , which in turn allows the developmental timing of germ cells to be inferred ( Figure S2A ) .', 'BAZ1A was not detected in leptotene/zygotene spermatocytes ( e . g . , in stage XI tubules; Figure 1C i′ ) or early pachytene spermatocytes ( e . g . , in stage II–III tubules; Figure 1C ii′ ) , but was detectable in mid-pachytene spermatocytes starting in stage V ( data not shown ) , becoming strongly stained in later-stage pachytene spermatocytes in stage VIII , IX , and X tubules where it was particularly enriched in DAPI-dense pericentric heterochromatin ( Figure 1C iii′ , Figure S2B i , S2B ii , and data not shown ) and the sex body ( see below ) .', 'It was also enriched in pericentric heterochromatin but not sex bodies of diplotene spermatocytes in stage XI tubules ( Figure 1C i′ ) , in the chromocenters of round spermatids at stage II–III ( Figure 1C ii′ ) , and weakly in the further developed spermatids at stages VIII and IX , immediately prior to and just beginning to elongate , respectively ( Figure 1C iii′ , Figure S2B i′ , and data not shown ) .', 'BAZ1A partially overlapped with HP1β , confirming enrichment in heterochromatin ( Figure 1D and Figure S2B ) , consistent with localization in cultured cells [29] , [38] .', 'BAZ1A was not detected in somatic Sertoli and peritubular myoid cells present in all tubule stages ( Figure 1C i′–iii′ ) .', 'Figure 1D summarizes BAZ1A expression during spermatogenesis: it was first detected in mid-pachytene spermatocytes and continued through step 9 spermatids; it was not detectable in elongating step 10–11 spermatids ( Figure S2B ii′ and iii′ , insets ) , but was seen again later , in step 15 spermatids ( Figure S2B iv′ , inset ) .', 'By contrast , BAZ1A staining was only weakly detectable in pachytene and diplotene/dictyate oocytes in sections of embryonic or early postnatal ovaries ( Figure S2C ) .', 'Thus , high-level BAZ1A expression in germ cells is specific for males .', 'BAZ1A and SNF2H showed reciprocal coimmunoprecipitation from testis extracts ( Figure 1E; see also below ) , and displayed essentially complete overlap on the pericentric heterochromatin in pachytene spermatocytes ( Figure 1F , top magnification ) and in the chromocenters in round spermatids ( data not shown ) .', 'Thus , the ACF and/or CHRAC complexes form in these cells .', 'SNF2H was also detected in spermatogonia , where BAZ1A was absent ( Figure 1F , bottom magnification ) , consistent with presence of BAZ1A-independent ISWI complexes such as those containing SNF2H and CECR2 [39] ( Figure S1A ) .', 'To provide a more detailed evaluation of chromosomal distribution during meiosis , we examined BAZ1A staining on squash preparations of spermatocyte nuclei , using anti-SYCP3 staining to evaluate stages of synaptonemal complex assembly .', 'BAZ1A was not detectable above background until after mid-pachynema ( Figure 1G ) , whereupon it was present diffusely on chromatin with weak enrichment in pericentric heterochromatin and strong accumulation in the sex body , a subnuclear domain containing the heterochromatinized sex chromosomes [40] .', 'At diplonema , BAZ1A was no longer detectable in the sex body , but remained diffuse along chromatin and was now highly enriched in pericentric heterochromatin ( Figure 1G ) .', 'These results agree with analysis of testis sections co-stained for γH2AX , which is enriched in the sex body ( Figure 1C , arrows ) : sex body accumulation of BAZ1A was observed in pachytene spermatocytes as early as stage V tubules ( mid pachynema ) and as late as stage X ( late pachynema ) , but not diplotene spermatocytes in stage XI ( Figure 1C , Figure S2B , and data not shown ) .', 'We also examined spread chromosome preparations , in which proteins are more stringently extracted from chromatin .', 'Throughout pachynema and diplonema , BAZ1A staining displayed a punctate pattern dispersed across chromatin , with little of the enrichment in pericentric heterochromatin seen with squashes ( Figure S2D ) .', 'This difference suggests that BAZ1A association with pericentric heterochromatin is less robust than its binding to more dispersed sites on euchromatin .', 'BAZ1A showed enrichment in the sex body in late pachynema , but was largely excluded there in diplonema .', 'The high level of BAZ1A expression in testicular germ cells and its dynamic localization pattern on chromatin led us to suspect that ACF and/or CHRAC might have prominent roles in spermatogenesis .', 'To determine the in vivo function ( s ) of these complexes , we chose a conditional targeting strategy to generate a mouse Baz1a mutant because of the semi-lethality of acf1 mutation in flies [25] .', 'loxP sites were inserted on either side of exon six ( Figure 2A ) , deletion of which creates a frame shift ( codon 256 of 1552 total ) and introduces a premature termination codon 25 nucleotides downstream .', 'Successful targeting in ES cells was confirmed by Southern blot ( Figure S3A , B ) .', 'To delete exon six , we crossed Baz1aflox mice to mice expressing Cre recombinase under the control of the male germ line-specific Stra8 promoter [41] .', 'Recombination of loxP sites was confirmed by PCR of tail DNA ( Figure 2B ) .', 'Males heterozygous for germ line deletion were fertile and were bred to generate mice homozygous for exon six deletion ( hereafter , Baz1a−/− ) .', 'Contrary to initial expectation , Baz1a−/− mice were viable and Mendelian ratios of wild-type ( 95/363 , 26% ) , heterozygous ( 186/363 , 51% ) and homozygous mutant ( 82/363 , 23% ) offspring were recovered from heterozygous crosses .', 'Histo-pathological examination of all major organs revealed no abnormalities in Baz1a−/− mice ( data not shown ) with the exception of the testis ( discussed further below ) .', 'BAZ1A protein was undetectable in Baz1a−/− animals by immunoprecipitation/western blot of whole testis extracts ( Figure 2C ) , western blot of extracts of ear fibroblasts ( EFs ) derived from these mice ( Figure 1B ) , or immunostaining of testis sections ( Figure 2D ) or spermatocyte squashes ( Figure 2E ) , indicating that this is a null or severely hypomorphic allele .', 'In wild-type diplotene cells , both SNF2H and CHRAC17 showed localization similar to BAZ1A , i . e . , diffuse distribution on chromatin with enrichment in pericentric heterochromatin ( Figure 2E , F , G ) .', 'In Baz1a−/− spermatocytes , diffuse chromatin staining for both SNF2H and CHRAC17 was present but heterochromatin enrichment was lost ( Figure 2F , G ) .', 'CHRAC17 levels were also greatly reduced on Baz1a−/− chromosome spreads ( Figure 2G ) , but total CHRAC17 levels were reduced to a lesser degree in immunoprecipitates/western blots from whole testis extracts ( Figure S3C ) .', 'Thus , CHRAC17 appears to be less stably bound to chromatin in the absence of BAZ1A .', '( Various commercial antibodies directed against CHRAC15 failed to detect a signal ( data not shown ) ) .', 'CHRAC17 antibodies coimmunoprecipitated both BAZ1A and SNF2H from wild-type but not Baz1a−/− testis extracts ( Figure S3C ) .', 'These results confirm that the mutant is defective for BAZ1A function .', 'More importantly , these findings demonstrate that BAZ1A is required for interaction between CHRAC17 and SNF2H and that a molecular function of BAZ1A is to target ACF and/or CHRAC to specific subnuclear locations .', 'RNAi directed against BAZ1A transcripts in cultured , tumor-derived human cells caused defects in DSB repair via non-homologous end-joining ( NHEJ ) and homologous recombination ( HR ) [31] .', 'Moreover , GFP-tagged constructs of BAZ1A and SNF2H accumulate at sites of DNA damage in cultured cells , consistent with a possible direct role in repair of DNA damage [31] , [42] .', 'Several cell populations in vivo experience developmentally programmed DSBs that must be repaired for further differentiation to proceed: spermatocytes and oocytes ( meiotic recombination ) , T-cells ( V ( D ) J recombination ) and B-cells ( V ( D ) J and class switch recombination ( CSR ) ) .', 'We asked whether Baz1a is essential in these cells .', 'Meiotic recombination is initiated by formation of DSBs by SPO11 during leptonema [43] .', 'Break sites are decorated by γH2AX and DMC1 foci , which dissipate as DSBs are progressively repaired during zygonema and pachynema ( Figure 3A , C ) .', 'In spermatocyte squash preparations , both γH2AX staining and DMC1 foci appeared and disappeared in the Baz1a−/− mutant with timing comparable to wild type , although with a slightly elevated frequency of DMC1 foci in early zygonema ( Figure 3A–C ) .', 'This pattern contrasts with mutants with DSB repair defects , which display persistent staining of both markers into pachynema or beyond [e . g . , 44] .', 'The large domain of γH2AX staining in both wild-type and mutant cells during pachynema and diplonema is the sex body ( Figure 1C , 3A , B ) , which appears to form normally in the mutant unlike in severely recombination-defective mutants [44] .', 'We did not observe any impairment in synaptonemal complex formation , which is also hampered by DSB repair defects [45] ( Figure 3B and data not shown ) .', 'MLH1 foci , which mark nascent crossover sites , formed normally but at slightly increased frequency ( Figure 3D , E ) .', 'Finally , we note that mutant females were fertile ( n\u200a=\u200a4; average litter size 8 . 8±1 . 9 ) and had abundant oocytes in primordial and growing follicles at 3 months of age , albeit slightly reduced in number compared to littermate controls ( Figure 3F–H ) .', 'Follicle formation is highly sensitive to persistent SPO11-generated DSBs , such that repair-deficient mutants generally lack follicles entirely by 3–4 wks of age [46] , [47] .', 'These findings indicate that Baz1a is dispensable for meiotic recombination .', 'During T cell development , RAG recombinases create DSBs to induce V ( D ) J recombination ( rearrangement of the variable ( V ) , diversity ( D ) and joining ( J ) gene segments of the T cell receptor ( TCR ) locus ) to diversify the TCR repertoire [48] .', 'Failure to repair these DSBs by NHEJ prevents normal T cell development in the thymus and reduces T cell numbers in secondary lymphoid organs , such as the spleen , which can be measured by quantifying cells expressing CD4 and CD8 co-receptors .', 'Baz1a−/− thymus and spleen had numbers of CD4 and CD8 single- and double-positive thymocytes and T cells comparable to wild-type littermates ( Figure 4A , B ) .', 'Because T cells undergo selection and clonal expansion during their development in the thymus , small quantitative defects in DSB repair would not be apparent in this assay , but gross NHEJ defects can be ruled out .', 'In B cells , CSR is initiated by formation of DSBs in the immunoglobulin ( Ig ) heavy-chain locus by activation-induced deaminase ( AID ) and other enzymes .', 'These DSBs are repaired by NHEJ to change the class of antibody produced [49] .', 'Mature B cells were stimulated to undergo CSR in vitro by treating with anti-CD40 antibody plus IL4 to induce a switch to IgG1 , or lipopolysaccharide ( LPS ) to induce a switch to IgG3 .', 'Indistinguishable numbers of IgG1- or IgG3-positive cells were recovered from wild-type and Baz1a−/− cells , indicating that CSR occurs efficiently ( Figure 4C , D ) .', 'Moreover , Ig variable regions are assembled from V , D and J gene segments by the same mechanisms involved in assembly of the TCR variable regions [48] , and failure in this process results in developmental arrest at the CD43+ progenitor stage without functional Ig expression [50] .', 'Thus , the ability to purify mature ( CSR-competent , Ig+ , CD43− ) B cells from spleens of Baz1a−/− animals showed that V ( D ) J recombination is also successful during B cell development .', 'To further test whether BAZ1A plays a role in DSB repair by HR in somatic cells , Baz1a mice were crossed with mice containing the DR-GFP reporter targeted to the Pim1 locus [51] .', 'DR-GFP is a direct repeat of defective GFP genes; a DSB introduced by the I-SceI endonuclease in the upstream copy of the repeat , followed by gene conversion with the downstream copy , gives rise to GFP+ cells ( Figure 4E ) .', 'Early passage mouse embryonic fibroblasts ( MEFs ) from Baz1a−/−Pim1+/Drgfp embryos and control ( Baz1a+/+Pim1+/Drgfp or Baz1a+/−Pim1+/Drgfp ) embryos were transiently transfected with an I-SceI expression vector and analyzed by flow cytometry 48 h after transfection .', 'No significant difference was observed in HR between Baz1a−/− and control MEFs ( Figure 4F ) ( p\u200a=\u200a0 . 885 , two-tailed t test ) .', 'Similarly , no significant difference in HR was observed in primary ear fibroblasts from Baz1a−/−Pim1+/Drgfp and control mice after transfection with the I-SceI expression vector ( Figure 4G ) ( p\u200a=\u200a0 . 110 ) .', 'HR mutants , such as those defective in Brca1 , are particularly sensitive to DNA crosslinking agents such as mitomycin C ( MMC ) .', 'To test for MMC sensitivity , 6 Baz1a−/− adult mice and age-matched controls were injected into the peritoneal cavity with 5 mg MMC per kg body weight .', 'All of the Baz1a−/− mice and controls survived 21 days after treatment .', 'By contrast , Brca1 hypomorphic mice succumb to even lower MMC doses [51] .', 'We conclude that in the absence of Baz1a , NHEJ and HR repair of developmentally programmed DSBs in the immune system and germ line , respectively , occurs relatively normally , as does HR repair of DSBs and crosslinks from exogenous sources .', 'Fifteen Baz1a−/− males bred with wild-type females for eight weeks produced no pups despite the presence of copulatory plugs , indicative of male infertility .', 'Sperm from total epididymides of Baz1a−/− mice displayed an array of aberrant head morphologies ( teratospermia ) , all of which lacked a normal hook characteristic of wild-type sperm heads ( Figure 5A ) .', 'Mutant sperm also had numerous tail abnormalities ( Figure 5B ) including frequent narrowing of the annulus , a domain separating the principal piece of the tail from the mid-piece ( arrows in Figure 5B ii &', 'v ) .', 'Other defects included heads folded back against the tail', '( iii ) , two tails', '( iv ) , midpieces folded back against the tail', '( v ) , and coiling of the tail around the head', '( vi ) .', 'There was a 5-fold reduction in total sperm ( oligospermia ) and an almost complete absence of motile sperm ( asthenospermia ) ( Figure 5C , D ) , with the exception of a few twitching movements that did not support forward progression ( data not shown ) .', 'Caput epididymides were devoid of sperm in the mutant in contrast to wild type , which displayed lumens packed with mature sperm ( Figure 5E , F ) .', 'Unlike wild-type cauda epididymides , where darkly stained sperm heads and tangles of lightly stained tails were visible ( Figure 5G ) , those from the mutant contained only debris and degenerating , mostly round cells that likely sloughed from the seminiferous tubules ( Figure 5H ) .', 'Injection of nuclei from Baz1a−/− round spermatids isolated from testes into wild-type oocytes , followed by artificial activation , supported formation of embryos competent to reach at least the blastocyst stage ( 6 of 19 successful injections , 32% ) .', 'In contrast , intracytoplasmic sperm injection ( ICSI ) using Baz1a−/− epididymal sperm failed to spontaneously activate oocytes , and even after artificial activation , only rarely yielded embryos reaching the blastocyst stage ( 1 of 23 successful injections ) .', 'In a control experiment using sperm from unrelated wild-type mice , 21 of 47 successfully injected embryos ( 45% ) developed to the blastocyst stage .', 'Therefore , whereas Baz1a−/− round spermatids are competent to support early embryonic development , mutant sperm are compromised in both oocyte activation and preimplantation development .', 'Seminiferous tubule sections showed multiple defects in Baz1a mutants .', 'Some stage IX tubules displayed spermiation failure as indicated by the presence of mature sperm , which should have been fully released into the lumen at this stage ( compare Figure 6A and B ) .', 'There was also a high frequency of multi-nucleate cells in tubules of various stages ( Figure 6C , D ) .', 'Electron microscopy on testis sections revealed frequent bi-nucleate round spermatids ( Figure 6E ) and bi-nucleate elongating spermatids , often with a single acrosome stretching over both nuclei ( Figure 6F ) .', 'Thus , the highly ordered spermiogenic program is thrown off course in the absence of Baz1a , resulting in a range of aberrations .', 'Testis sections from Baz1a−/− mice also displayed a modest but significant increase in the average number of apoptotic cells per tubule and a three-fold increase in the percentage of mutant tubule sections with one or more apoptotic cells ( Figure 6G–J ) .', 'Apoptosis did not appear to be restricted to any particular cell type because TUNEL-positive cells were found scattered throughout tubule sections irrespective of stage ( Figure 6H and data not shown ) .', 'Moreover , apoptosis was too infrequent to account quantitatively for the decrease in mature spermatozoa or the morphological defects .', 'Since BAZ1A is a chromatin-remodeling factor , we considered that it may be required for the large-scale replacement of chromatin proteins that occurs during spermatogenesis [33] .', 'To test this , we looked at the exchange of somatic histones for testis-specific histone variants by sub-cellular fractionation of whole testes followed by immunoblotting for various histone variants .', 'All of the variants examined were expressed and present in the chromatin-enriched fraction ( Figure 7A ) .', 'These findings indicate that Baz1a-deficiency does not globally disrupt this process , although we cannot rule out the possibility that other histone variants are improperly loaded , and these experiments do not address whether the spatial organization of the replaced histones is normal .', 'Following incorporation of histone variants , N-terminal lysines of histone H4 are hyper-acetylated , which is thought to aid in the histone-to-protamine exchange that follows [52] .', 'Immunostaining for pan-acetyl-H4 showed comparable signal in the lumen-proximal elongating spermatids from both genotypes ( Figure 7B ) , suggesting hyper-acetylation was also unperturbed in the mutant .', 'The highly basic transition proteins ( TP1 and 2 ) and protamines ( PRM1 and 2 ) that replace histones were extracted from late-stage spermatids , which are sonication-resistant because of their compact nature .', 'Coomassie staining of acid-urea polyacrylamide gels revealed bands with the expected migration of all four proteins and the higher molecular weight precursors of PRM2 ( pPRM2 ) , which are proteolytically cleaved to yield the mature form ( Figure 7C , top panel ) .', 'PRM1 is less soluble in TCA and was therefore detected in the 0–4% cut while the other proteins were detected in the 4–20% cut .', 'As expected , these bands were largely undetectable in the sonication-sensitive extracts .', 'Immunoblotting confirmed the identity of the bands ( Figure 7C , bottom panels ) .', 'Levels of mature PRM2 were modestly decreased in sonication-resistant mutant spermatids ( Figure 7C , bottom panels ) .', 'However , similar reductions have been observed in other spermiogenesis mutants [53]–[55] , so this may be a nonspecific consequence of developmental arrest rather than a causal factor .', 'Taken together , these findings indicate that BAZ1A is dispensable for global spermatogenesis-associated changes in chromatin protein composition .', 'This is consistent with findings in Drosophila , where Acf1 is dispensable for nucleosome deposition per se [25] .', 'However , whereas Acf1 promotes heterochromatinization in flies [26] , [56] , we found that Baz1a-deficient cells display normal staining patterns of heterochromatin markers HP1β , HP1γ and H3K9Me3 in primary spermatocytes and round spermatids ( Figure S4 ) , suggesting BAZ1A is not needed for heterochromatin formation in mouse .', 'Baz1a has been implicated in transcriptional regulation in other organisms [27] , [30] , so we hypothesized that altered gene expression might be the cause of defective spermiogenesis .', 'We therefore compared RNA profiles of pachytene/diplotene spermatocytes and round spermatids from Baz1a−/− and heterozygous littermates , using cells highly enriched by FACS of dissociated testes ( Table S1 ) .', 'Baz1a+/− mice displayed no overt phenotype , so they served as normal controls for these experiments .', 'Prior studies demonstrated large-scale expression changes accompanying the developmental switch from meiosis to postmeiotic differentiation [57] , [58] .', 'Accordingly , we found that 7573 microarray probes representing 5656 genes showed at least two-fold change in expression when normal control spermatocytes and round spermatids were compared ( false discovery rate ( FDR ) q\u200a=\u200a0 . 05 ) ( Figure 8A and Table S2 ) .', 'This accounts for 38 . 2% of all probes detectably expressed in at least one of the samples analyzed .', 'The probes with greatest change in expression were highly enriched for up-regulation in spermatids ( 83 . 2% of the top 1000 probes ) ( Figure 8A ) .', 'Thus , much of the postmeiotic gene expression program involves transcriptional activation of previously quiescent genes .', 'Baz1a−/− spermatocytes were grossly similar to controls using standard criteria for defining differential expression: only four probes scored as significantly different ( ≥2-fold , q\u200a=\u200a0 . 05 ) , all higher in the mutant ( Figure 8B i and Table S2 ) .', 'In contrast , round spermatids displayed widespread changes: 1069 probes ( 912 genes; 5 . 4% of expressed probes ) were altered two-fold or more in the mutant ( Figure 8B ii and Table S2 ) , of which 70 . 3% were higher in Baz1a−/− .', 'Thirty-six probes differed by ≥8-fold , all but one higher in the mutant .', 'Thus , substantial disruption of gene expression accompanies progression from meiosis into spermiogenesis in the absence of BAZ1A , most often reflecting aberrantly high expression .', 'If the multi-nucleate spermatids frequently observed in the mutant represent a subset of cells with more severe transcription defects , it is possible that this analysis underestimates the degree of mis-regulation .', 'These cells would be largely absent from the haploid round spermatid population enriched by FACS .', 'We asked how mis-regulation in Baz1a−/− cells compares with the expression changes across the normal spermatocyte-to-spermatid transition .', 'Multiple lines of evidence show that BAZ1A deficiency affects a largely independent group of transcripts , leaving developmentally programmed expression changes essentially intact .', 'First , there was little overlap of the two probe sets beyond that expected by chance .', 'Of 1069 mis-regulated probes , 460 also scored as developmentally regulated in controls ( 43%; Figure 8C ) .', "This overlap is only slightly above the fraction expected from two random samples ( p\u200a=\u200a0 . 04 , Fisher's exact test ) , because the developmental set is so large ( 38 . 2% of expressed probes ) .", 'More importantly , only 39 mis-regulated probes ( 3 . 6% ) overlapped with the top 1000 developmentally regulated probes , significantly less than the 5 . 1% overlap expected by chance ( p\u200a=\u200a0 . 043 ) .', 'Additionally , the probes found on both lists showed poor correlation with respect to the magnitude and direction of expression changes observed ( data not shown ) .', 'Second , hierarchical clustering based on developmentally regulated probes clearly distinguished samples by cell type as expected , but did a poor job of stratifying spermatocyte or spermatid samples by genotype ( Figure 8D ) .', 'In contrast , clustering based on the mi
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['ATP-dependent chromatin remodelers control DNA access for transcription , recombination , and other processes .', 'Acf1 ( also known as BAZ1A in mammals ) is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC , first purified over 15 years ago from Drosophila melanogaster embryos .', 'Much is known about biochemical properties of ACF and CHRAC , which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays .', 'Genetic studies in yeast , flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures .', 'RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair .', 'However , their essential in vivo roles in mammals are unknown .', 'Here , we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line , I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination , and DNA damage from mitomycin C exposure in vivo .', 'However , Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells , where it displays dynamic , stage-specific patterns of chromosomal localization .', 'Sterility is caused by pronounced defects in sperm development , most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated , primarily reflecting inappropriate up-regulation .', 'We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes , with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations .']
['The eukaryotic genome is packaged into a periodic nucleoprotein complex known as chromatin .', 'Wrapping of DNA around nucleosomes , the basic repeat unit of chromatin , enables packing of long stretches of DNA into a compact nucleus but also impedes access by protein factors involved in essential cellular processes such as transcription , replication , recombination and repair .', 'Chromatin remodeling factors are multi-protein complexes that utilize the energy released during ATP-hydrolysis to assemble , reposition , restructure and disassemble nucleosomes .', 'These complexes disrupt histone-DNA contacts to ‘remodel’ the chromatin and grant access to the genome .', 'Alternatively , access can also be denied to repress transcription , for example .', 'Spermatogenesis , the developmental program that produces sperm , comprises a dramatic chromatin makeover and the induction of a transcriptional program that engages nearly one-third of the genome .', 'Here we provide evidence suggesting that these large-scale alterations leave the genomic material vulnerable to spurious transcriptional changes which are normally repressed by ACF1 ( BAZ1A in mammals ) , the defining member of the well-studied ACF/CHRAC chromatin remodeling complex .', 'These findings indicate that Baz1a plays a previously unrealized role in male fertility and may represent a novel target for male contraceptive development .']
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