Chd5 orchestrates chromatin remodelling during sperm development

ARTICLE Received 20 Feb 2014 | Accepted 4 Apr 2014 | Published 13 May 2014 DOI: 10.1038/ncomms4812 Chd5 orchestrates chromatin remodelling during sperm development Wangzhi Li1,2, Jie Wu1,3, Sang-Yong Kim1, Ming Zhao4, Stephen A. Hearn1, Michael Q. Zhang5,6, Marvin L. Meistrich4 & Alea A. Mills1 One of the most remarkable chromatin remodelling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodelling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orches- trates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone- to-protamine replacement and depict the cascade of molecular events underlying this process of extensive chromatin remodelling. 1 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA. 2 Molecular and Cellular Biology Program, Stony Brook University, Stony Brook, New York 11794, USA. 3 Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, New York 11794, USA. 4 Department of Experimental Radiation Oncology, MD Anderson Cancer Center, Houston, Texas 77030, USA. 5 Department of Molecular and Cell Biology, Center for Systems Biology, The University of Texas at Dallas, Richardson, Texas 75080, USA. 6 MOE Key Laboratory of Bioinformatics and Bioinformatics Division, Center for Synthetic and System Biology, TNLIST/Department of Automation, Tsinghua University, Beijing 100084, China. Correspondence and requests for materials should be addressed to A.A.M. (email: [email protected]). NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 S permatogenesis is an intricate biological process that Results transforms diploid spermatogonial stem cells into haploid Chd5 is expressed in spermatids during spermiogenesis. Using spermatozoa in seminiferous tubules of testis. It consists of immunofluorescence analyses with a previously validated anti- three major phases: mitosis, meiosis and spermiogenesis1. body specific for Chd5 (refs 10,12) we found that Chd5 is Spermatogonial stem cells first multiply by repeated rounds of expressed in mouse testes specifically during spermiogenesis mitosis and differentiate into primary spermatocytes, which (Fig. 1, Supplementary Fig. 1). Chd5 was first detectable after subsequently undergo meiosis and become haploid round meiosis, when it was expressed within nuclei of step 4 spermatids spermatids. Round spermatids then mature into highly (Fig. 1). At this phase, Chd5 was weakly expressed throughout the specialized spermatozoa through spermiogenesis, the final phase nucleus but was highly expressed in an intense focal spot near the of spermatogenesis1. During spermiogenesis, round spermatids chromocentre, a cluster of centromeres and pericentromeric undergo a number of characteristic changes including elongation heterochromatin13. Chd5 expression peaked at steps 7–8, when it and condensation of the nucleus, formation of the acrosome and was expressed robustly throughout the nucleus and was enriched flagellum, and removal of cytoplasm2. In mouse, spermatogenesis in the chromocentre, where Chd5 colocalized with the hetero- is subdivided into twelve stages (stages I–XII), whereas chromatin mark H3K9me3, and was expressed in a pattern spermiogenesis is further divided into 16 steps (steps 1–16) similar to that of the repressive histone mark H3K27me3 (Fig. 1b, mainly defined by changes in acrosome structure and nuclear Supplementary Fig. 2). Chd5 expression decreased after step 9, morphology of the maturing spermatids1,3–5. when it remained enriched in heterochromatin, and was not Extensive chromatin remodelling occurs during spermiogen- detectable after step 10 (Fig. 1). The Chd5-intense focal spot esis, which results in the majority of nucleosomal histones being juxtaposed to the edge of the chromocentre was present in round replaced by sperm-specific basic proteins, initially transition spermatids from steps 4–8, and was positioned at the junction proteins and ultimately protamines6. Protamines are distinct between the chromocentre and the post-meiotic sex chromosome, from histones, packaging the sperm genome into a distinct toroid both of which are DAPI-intense sub-nuclear structures within chromatin structure7. This dramatic histone-to-protamine spermatids (Fig. 1, Supplementary Fig. 3a)14. In contrast to the remodelling repackages the sperm genome into a chromatin chromocentre, the Chd5-intense focal spot was DAPI-weak and structure that is sixfold or more compact than that of was negative for H3K9me3 (Fig. 1b, Supplementary Fig. 3a), somatic cells, and is essential for normal sperm development6,8. suggesting that it marks transcriptionally active chromatin. We Given its extensive degree of chromatin remodelling, speculated that the Chd5-intense foci may be within nucleoli. Co- spermiogenesis offers a unique process to study mechanisms of immunostaining of Chd5 with the nucleolar marker fibrillarin chromatin remodelling. However, this process is currently showed that the Chd5-intense spot was near the nucleolus in understudied and still poorly understood, mainly due to the many spermatid nuclei, but was clearly separated in others complexity of the process itself and lack of in vitro experimental (Supplementary Fig. 3b), raising the possibility that Chd5 systems for studying it. In particular, chromatin remodellers are transiently associates with the nucleolus. These findings indicate believed to be essential for facilitating the extensive degree of that Chd5 is expressed specifically in nuclei of round and early chromatin remodelling during spermiogenesis, but their roles in elongating spermatids during spermiogenesis where it is primarily this process are not well elucidated. In this study, we discovered enriched in heterochromatic regions, and that Chd5 is also that Chromodomain helicase DNA-binding protein 5 (Chd5) expressed in an intense focal spot in a non-heterochromatic plays an orchestrating role in the histone-to-protamine region juxtaposed to the chromocentre. remodelling process during spermiogenesis. Chd5 is a member of the CHD family of chromatin remodellers, which we identified Chd5 deficiency impairs sperm development and fertility. The as a dosage-sensitive tumour suppressor9. While recent studies dynamics of Chd5 expression during spermiogenesis indicated reveal that Chd5 binds unmodified histone 3 (H3) via its dual that it might play a functional role in chromatin remodelling plant homeodomains10,11 and that this interaction is essential for during spermatid maturation. To explore this possibility, we tumour suppression10, the ability of Chd5 to mediate chromatin generated Chd5-deficient mice carrying the Chd5Aam1 null allele dynamics in the context of normal cells is not well understood. (Supplementary Fig. 4). Western blot analyses using a validated We find that Chd5 is highly expressed during spermiogenesis and antibody10 recognizing a part of Chd5 protein not disrupted by plays essential roles during sperm development. Inactivation of gene targeting demonstrated that the Chd5 protein was not Chd5 in mice leads to sperm chromatin compaction defects and detected in testes of Chd5Aam1 homozygotes (Fig. 2a). male infertility. We reveal that Chd5 both mediates a cascade of Chd5Aam1  /  mice were viable and grossly normal. Mating molecular events for histone removal and modulates the tests revealed that whereas Chd5Aam1  /  female mice and homeostasis of transition proteins and protamines, identifying Chd5Aam1 þ /  mice of both genders were fertile, Chd5Aam1  /  Chd5 as a master regulator of the histone-to-protamine males were either sub-fertile or sterile (Supplementary Table 1). chromatin remodelling process during spermiogenesis. Chd5Aam1  /  mice had significantly lower sperm counts, and Figure 1 | Chd5 is expressed in step 4–10 spermatids and is enriched in heterochromatin during spermiogenesis. Roman numerals indicate the spermatogenic stages of the tubules in wild-type testes sections. (a) Blue, DAPI; green, Chd5; RS, round spermatid; ES, elongating spermatid; ECS, elongating and condensing spermatid; CS, condensed spermatid; P, pachytene spermatocyte; Mi, meiotic division. Arrow heads mark the chromocentre. Scale bar, 10 mm. (b) Chd5 is enriched in DAPI-intense heterochromatic regions, and colocalizes with heterochromatin marker H3K9me3. Top panel, step 7–8 round spermatids; bottom panel, step 9–10 elongating spermatids. Arrow heads mark the chromocentre. Scale bar, 5 mm. (c) Schematic of Chd5 expression during spermatogenesis. Spermatogenesis is divided into twelve stages (stage I–XII) in mouse and each stage has a distinct cellular composition. Spermiogenesis, the maturation process of haploid spermatids, is divided into 16 steps (steps 1–16). Green marks Chd5 protein expression. Chd5 is specifically expressed in spermatids from steps 4–10, with peak expression in step 7–8 round spermatids, where Chd5 is enriched in the heterochromatic chromocentre. A focus of intense Chd5 protein expression is located adjacent to the junction of the chromocentre and the post-meiotic sex chromosomes in steps 4–8 spermatids. Spermatogonia (A, In, B); spermatocyte (Pl, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diakinesis; Mi, meiotic division); Ag, acrosomic granule; Ac, acrosomic cap. The diagram is drawn based on the illustration of Hess et al.71 2 NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 ARTICLE a II–III IX XII X IV VII–VIII II–III IX XII X IV VII–VIII II–III IX XII X IV VII–VIII ES ECS RS RS P CS Mi IV VII–VIII IX XII b H3K9me3 DAPI Chd5 H3K9me3 & Chd5 Merge VII–VIII VII–VIII VII–VIII VII–VIII VII–VIII IX–X IX–X IX–X IX–X IX–X c Mitosis Meiosis Spermiogenesis 13 14 15 15 15 15 16 16 Ag c 1 2 3 4 5 6 7 8 9 10 11 12 P P P P P P P P P P D Mi In In In B B Pl Pl L L Z Z A A A A A A I II III IV V VI VII VIII IX X XI XII Mouse spermatogenesis stages NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications 3 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 a b +/+ –/– +/+ –/– +/+ –/– KD 250 Chd5 150 50 β-Actin 37 c 4 DFI fold change 3 2 1 0 +/+ –/– d +/+ –/– e +/+ –/– +/+ –/– +/+ –/– I VI X VII II–III XI –VII IV–V IX XII f 1.2 1.00 Relative CHD5 1.0 expression 0.8 0.6 0.4 0.28 0.2 0.09 0.05 0.0 Normal Few late No Sertoli cell spermatids spermatids only Human testis pathology Figure 2 | Chd5 deficiency leads to defective spermatogenesis and chromatin condensation. (a) Western blot analyses indicates that Chd5 protein is not detectable in Chd5Aam1  /  (  /  ) testis. A validated antibody10 raised against amino acids 1,524–1,705 of mouse Chd5 (which is not disrupted by gene targeting), was used for western blotting. b-Actin serves as a loading control. (b) Representative abnormal head morphology of Chd5Aam1  /  sperm. Scale bar, 20 mm. (c) SCSA revealed impaired chromatin integrity of Chd5Aam1  /  sperm. DFI, DNA Fragmentation Index (see Methods). Data are presented as mean±s.d. from four independent experiments. (d) Transmission electron microscopy analyses of sperm from Chd5Aam1 þ / þ and Chd5Aam1  /  caudal epididymi. Chromatin is homogenously condensed in Chd5Aam1 þ / þ sperm, but appears loose and uneven with fibrillar texture and contains abnormal vacuoles in Chd5Aam1  /  sperm nuclei. Scale bar, 1 mm. (e) Staged comparison of periodic acid–Schiff (PAS)-stained Chd5Aam1 þ / þ and Chd5Aam1  /  testes. Roman numerals indicate the stages of the seminiferous tubules. A decrease in the number of elongated spermatids, especially at stages VII and VIII, is evident in Chd5Aam1  /  tubules. Arrows in stage IX and X mark abnormal retention of condensed spermatids. Scale bar, 10 mm. (f) Relative CHD5 expression in human testis with normal versus clinically defined abnormal spermatogenesis. Data are derived from published microarray data set (ArrayExpress: E-TABM-234) of 39 human testis biopsy samples from 29 men with highly defined testicular pathologies and 10 men with normal spermatogenesis. RNA was prepared from the testis biopsies and analysed for gene expression using Affymetrix GeneChip. Data were analysed through NextBio18. Arrow indicates increased severity of spermatogenic defect. 4 NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 ARTICLE Table 1 | Reduced sperm counts and motility in Fig. 7). The extent of histological abnormalities varied among Chd5Aam1  /  mice. individual Chd5Aam1  /  mice (Supplementary Fig. 7), in agreement with the variable severity of infertility observed in male Chd5-deficient mice. In contrast to post-meiotic defects, Genotype N Sperm/caudal All motile Progressive epididymis sperm (%) motile sperm (%) we did not observe differences in spermatogenic cells (spermato- (  106) gonia, spermatocytes, round spermatids) or in somatic cells Chd5Aam1 þ / þ 7 5.52±1.13 62.3±5.4 48.6±11.3 (Sertoli cells, Leydig cells) in Chd5Aam1  /  testes (Fig. 2e, Chd5Aam1 þ /  4 6.51±0.68 66.0±3.8 52.8±2.3 Supplementary Fig. 7). These findings indicate that Chd5 Chd5Aam1  /  11 2.74±0.68 42.4±10.3 21.0±5.7 deficiency disrupts the elongation and condensation steps of P( þ / þ versus þ /  ) 0.103 0.228 0.375 post-meiotic sperm maturation, consistent with Chd5’s peak of P( þ / þ versus  /  ) 0.0002* 0.0055* 0.0004* expression in step 7–8 round spermatids, the phase immediately preceeding extensive chromatin remodelling. N indicates the number of mice used for the indicated sperm analyses. P-value is two-tail Student’s t-test result between the indicated genotypes. To determine whether our findings from Chd5-deficient mice *indicates statistical significance. Data are presented as mean±s.d. þ / þ , Chd5Aam1 þ / þ ; might be relevant to human cases of male infertility, we analysed  /  , Chd5Aam1  /  ; þ /  , Chd5 þ /  . a previously established gene expression data set of testes biopsies from 39 men (29 men with highly defined testicular pathology and 10 men with normal spermatogenesis)18. This analysis the sperm that were produced had compromised motility and a revealed that men with spermatogenic defects had lower CHD5 higher proportion of morphological abnormalities (Table 1, Fig. 2b expression relative to controls and that the clinical grade of and Supplementary Table 2). Using in vitro fertilization (IVF), we spermatogenic defect correlated inversely with CHD5 expression found that Chd5Aam1  /  sperm failed to fertilize wild-type (Fig. 2f). While this is a correlation rather than evidence for oocytes (Supplementary Table 3). Although it might be expected causality, it suggests that future efforts should be made to that some functional sperm from sub-fertile Chd5Aam1  /  mice determine whether compromised CHD5 contributes to male should be able to fertilize oocytes, none of the sperm obtained infertility in humans. from three different Chd5Aam1  /  mice were able to generate blastocysts in vitro. This may be because each of the three males tested happened to be sterile rather than sub-fertile, or that IVF Chd5 deficiency disrupts histone-to-protamine replacement. conditions compromised the sperm that would have been able to During mammalian spermiogenesis, the majority of canonical fertilize oocytes under in vivo conditions. A possible explanation histones are removed and replaced by histone variants and for the range of severity of the fertility phenotype in different mice transition proteins, which are subsequently replaced by prota- was genetic background, as 129Sv embryonic stem cells were used mines6,7,19,20. This histone-to-protamine replacement process to generate the Chd5Aam1 allele, with mice being backcrossed for repackages the sperm genome at least sixfold more compact than over four generations onto the C57BL/6 background before its somatic counterpart8. To define the mechanism whereby Chd5 heterozygotes were intercrossed. To determine whether genetic deficiency compromises chromatin compaction during background affected fertility, we established a second Chd5- spermiogenesis, we used western blotting to assess expression of deficient mouse model (carrying the Chd5Tm1b null allele15) that a panel of somatic histones, transition proteins and protamines in was in a 100% pure C57BL/6 genetic background, and assessed elutriation-purified spermatids at different steps of spermio- male fertility (Supplementary Fig. 5, Supplementary Table 4). We genesis (Supplementary Fig. 8). The core nucleosomal histones found that Chd5Tm1b  /  male mice were also either sterile or (H1, H2A, H2B, H3 and H4) were quickly depleted after the sub-fertile. Another Chd5-deficient mouse model in a pure 129E round spermatid steps in wild-type testes, with minimal retention background also showed that homozygous male mice exhibited in condensing and condensed spermatids (Fig. 3a). In contrast, variable pathology ranging from absence of sperm to near-normal these core histones were retained to a higher extent in sperm count, although six homozygous males tested did not Chd5Aam1  /  differentiated spermatids, which implied produce any progeny over the 2-month period analysed16. These ineffective histone removal. In addition, transition proteins data suggest that Chd5 deficiency compromises sperm production (Tnp1 and Tnp2) and protamines (Prm1 and Prm2) and male fertility and that this phenotype exhibits inherent had elevated expression in differentiated Chd5Aam1  /  variability in different individuals. spermatids (Fig. 3b,c), likely contributing to impaired fertility of To assess chromatin integrity in sperm from Chd5-deficient Chd5Aam1  /  mice, as precise control of levels of Prm1 and mice, we used the sperm chromatin structure assay (SCSA) Prm2 are critical for male fertility21,22. Prm2 is first translated as a (Fig. 2c). SCSA revealed that DNA fragmentation was enhanced precursor protein that is subsequently processed into mature in Chd5Aam1  /  sperm, reflecting a compromise in chromatin Prm2 through a multistep proteolytic cleavage. In Chd5Aam1  /  integrity17. Consistent with this finding, transmission electron spermatids, there was enhanced expression of the Prm2 precursor microscopy showed that whereas chromatin within nuclei of and partially processed Prm2 was present, indicating a defect in wild-type sperm is homogeneously condensed, less-condensed Prm2 processing, which may be because the overproduction of chromatin with a punctate texture and uneven density, as well as Prm2 in Chd5Aam1  /  spermatids exceeds the processing the presence of abnormal vacuoles, were observed in nuclei of capacity of the cells. In addition, both Tnp1 and Tnp2 Chd5Aam1  /  sperm (Fig. 2d). These findings show that Chd5 deficiency have been shown to increase levels of the Prm2 deficiency leads to defective chromatin compaction in sperm. precursor and partially processed forms of Prm2 (refs 23,24). Since compromised fertility can be caused by a deregulation of Thus, the abnormal elevation of Tnp1 and Tnp2 in sex hormones, we investigated this possibility (Supplementary Chd5Aam1  /  spermatids may also contribute to defective Fig. 6). However, we did not detect a significant alteration in sex Prm2 processing. Consistent with the observation in hormones in Chd5Aam1  /  male mice relative to controls. spermatids, western blotting of lysates from mature sperm also Histological analyses of testes revealed that seminiferous tubules revealed enhanced retention of core nucleosomal histones and of Chd5Aam1  /  mice contained fewer elongated spermatids elevated Prm1 and Prm2 expression in Chd5Aam1  /  sperm relative to controls, with an abnormal retention of condensed (Fig. 3d). Furthermore, immunofluorescent analyses of testes spermatids within stage IX and X tubules (Fig. 2e, Supplementary showed enhanced expression of Tnp1, Tnp2, Prm1 and Prm2 in NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications 5 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 a RS RES ECS CS c KD +/+ +/– –/– +/+ +/– –/– +/+ +/– –/– +/+ +/– –/– +/+ –/– 15 H4 - H1 20 H3 - H3/H2A/H2B - H4 15 - Tnp2 20 H2B - Prm2 precursor 15 - Intermediate Prm2 20 H2A 15 37 H1 - Prm2 50 β-Actin 37 +/+ –/– b RS RES ECS CS - Tnp1 KD +/+ +/– –/– +/+ +/– –/– +/+ +/– –/– +/+ +/– –/– - Tnp2 20 Tnp1 - Prm1 20 - Prm2 precursor Tnp2 - intermediate Prm2 15 Prm2 20 20 Prm1 - Prm2 50 β-Actin 37 d e Spermiogenesis +/+ –/– 15 Round Spermatids Elongating Condensing Condensed Spermatids 10 H4 20 15 H3 20 15 H2B 20 Step 1–6 7 8 9 10 11 12 13 14 15 16 Sperm 15 H2A Histones +/+ 37 –/– H1 Tnp1 +/+ Prm2 –/– 20 Tnp2 +/+ Prm1 –/– 20 Prm1 +/+ Tubulin –/– 37 Prm2 +/+ –/– Figure 3 | Chd5 modulates histone removal and homeostasis of transition proteins and protamines. (a,b) Western blot analyses of protein lysates from purified spermatids at different spermiogenic stages. RS, round spermatids; RES, round and early elongating spermatids; ECS, elongating and condensing spermatids; CS, condensed spermatids. Increased retention of histones (H4, H3, H2B, H2A and H1) as well as elevated transition proteins (Tnp1, Tnp2) and protamines (Prm1, Prm2) are observed in elongating, condensing and condensed spermatids, but not in round spermatids of Chd5Aam1  /  (  /  ) testis. b-Actin serves as a loading control. Levels of histones, transition proteins and protamines in Chd5Aam1 þ /  spermatids are in general similar to the levels in Chd5Aam1 þ / þ counterparts, with some variability among different Chd5Aam1 þ /  samples. (c) Top panel, Coomassie blue staining of basic protein extracts from Chd5Aam1 þ / þ and Chd5Aam1  /  sonication-resistant spermatids (SRS) separated using urea-acid gel electrophoresis. Equal amounts of proteins are loaded. Bottom panel, western blot analyses of the same basic protein extracts show an increase in Tnp1, Tnp2, Prm1 and Prm2 in Chd5Aam1  /  SRS. The increase in the Prm2 precursor and intermediate Prm2 indicate deficient processing of the Prm2 precursor in Chd5Aam1  /  SRS. (d) Western blot analyses of protein lysates of sperm prepared from caudal epididymi of Chd5Aam1 þ / þ ( þ / þ ) and Chd5Aam1  /  (  /  ) mice reveal increased retention of nucleosomal histones and elevated Prm1 and Prm2 in Chd5Aam1  /  sperm. H4, H2A and H1 are barely detectable in Chd5Aam1 þ / þ sperm, but are detected in Chd5Aam1  /  sperm. H3 and H2B are detectable in both Chd5Aam1 þ / þ and Chd5Aam1  /  sperm, but are higher in Chd5Aam1  /  sperm. Tubulin serves as a loading control. (e) Illustration of the dynamics of histones, transition proteins and protamines during spermiogenesis in Chd5Aam1 þ / þ ( þ / þ ) and Chd5Aam1  /  (  /  ) testes. For each nuclear protein, the darker colour of the bar in Chd5Aam1  /  testis indicates stronger expression of the protein at the indicated spermatogenic steps relative to Chd5Aam1 þ / þ counterparts. Chd5Aam1  /  spermatids (Fig. 3e, Supplementary Figs 9–12). In of nucleosomal histones and elevated levels of transition proteins addition, Tnp1 was precociously expressed in step 9–10 and protamines. spermatids and had extended expression until step 15 spermatids in Chd5Aam1  /  testes. These findings indicate that Chd5 deficiency perturbs the histone-to-protamine transition Chd5 mediates histone removal and DNA repair. To assess the that occurs during spermiogenesis, leading to aberrant retention mechanism of how Chd5 deficiency leads to aberrant histone 6 NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 ARTICLE removal we focused on H4 hyperacetylation, a molecular event spermiogenesis (Fig. 4a). Consistent with this hypothesis, occurring in early elongating spermatids that is essential for immunofluorescent analyses revealed that H4 acetylation was histone-to-protamine replacement in Drosophila25, and compromised in Chd5Aam1  /  spermatids (Fig. 4b). In considered the same for spermiogenesis in mammals6,25,26. Chd5Aam1 þ / þ testis, H4 hyperacetylation was detected in step Using immunofluorescence, we determined that Chd5 had 9 spermatids and showed peak expression in step 10–12. H4 similar expression dynamics as H4 hyperacetylation during acetylation decreased in step 13 spermatids, and was not spermiogenesis, but immediately preceded it (Fig. 4a). In step detectable at later steps. Whereas H4 acetylation was detected 9–10 spermatids, Chd5 colocalized with H4 acetylation, within step 9–13 spermatids of Chd5Aam1  /  testis, its suggesting that Chd5 modulates H4 hyperacetylation during expression was substantially reduced in Chd5Aam1 þ / þ a Chd5 H4Ac DAPI Merge XI XI XI XI X X X X VIII VIII VIII VIII IX IX IX IX VII VII VII VII b H4Ac DAPI Merge H4Ac DAPI Merge XI XI XI +/+ IX IX IX –/– IX IX IX XI XI XI I I I +/+ X X X XII XII XII –/– X X X XII XII XII I I I c RS RES ECS CS KD +/+ +/– –/– +/+ +/– –/– +/+ +/– –/– +/+ +/– –/– 15 H4Ac 10 15 H4 50 β-Actin 37 Figure 4 | Chd5 deficiency leads to compromised H4 acetylation during spermiogenesis. (a) Co-staining of Chd5 and H4 acetylation (H4Ac) in wild type testes. Roman numerals indicate spermatogenic stages of the marked tubular areas. Top and bottom inserts show higher magnification view of step 10 and step 9 spermatids, respectively. Both Chd5 and H4Ac are enriched in DAPI-intense regions within nuclei of step 9 and 10 spermatids in wild-type testes. H4Ac is detected by a mouse monoclonal antibody against pan-H4K5/8/12/16Ac. Scale bars, 20 mm in main panels and 1 mm for inserts. (b) Immunofluorescence analyses of H4 acetylation in Chd5Aam1 þ / þ ( þ / þ ) and Chd5Aam1  /  (  /  ) seminiferous tubules. Roman numerals indicate spermatogenic stages of the marked tubules. H4 hyperacetylation starts at step 9 spermatids of stage IX tubules, exhibits peak expression from step 10 of stage X tubules to step 12 spermatids of stage XII tubules and decreases in step 13 spermatids in stage I tubules in Chd5Aam1 þ / þ ( þ / þ ) testis. H4 acetylation is weaker from step 9 to 13 spermatids in Chd5Aam1  /  (  /  ) testis than in the Chd5Aam1 þ / þ counterparts. H4Ac was detected by a rabbit polyclonal antibody against pan-acetylation of H4. Scale bar, 20 mm. (c) Western blot analyses of purified spermatids at different spermiogenic stages. Histone H4 becomes transiently hyperacetylated in ECS of Chd5Aam1 þ / þ ( þ / þ ) testes, but not in ECS of Chd5Aam1  /  (  /  ) testes. H4Ac was detected by a rabbit polyclonal antibody against pan-acetylation of H4. b-Actin serves as a loading control. RS, round spermatids; RES, round and early elongating spermatids; ECS, elongating and condensing spermatids; CS, condensed spermatids. NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications 7 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 spermatids. Furthermore, western blotting of lysates from Nucleosome eviction generates DNA supercoiling tension that elutriation-purified spermatid fractions showed that whereas needs to be relieved. Previous studies indicate that topoisomerase more total H4 was retained, H4Ac was severely compromised II beta (Top2b) catalyses resolution of such supercoils in in differentiated Chd5Aam1  /  spermatids (Fig. 4c). Consistent elongating spermatids, during which double-strand breaks with the findings in Chd5Aam1  /  mice, Chd5Tm1b  /  (DSBs) are generated. A DNA damage response is then triggered testes also showed compromised histone H4 acetylation to repair the DSBs in order to maintain genome integrity30–33. (Supplementary Fig. 13). These findings indicate that acetylated We found that Chd5 expression is induced by DNA damage H4 is compromised in Chd5-deficient testes. (Fig. 6a), suggesting that Chd5 plays a role in the DNA damage Following H4 hyperacetylation, acetylated histone tails are response as well. Indeed, TUNEL assays revealed an increase of recognized by Brdt, a testis-specific member of the BRD DNA breaks in differentiated Chd5Aam1  /  spermatids, most bromodomain-containing protein family that has been shown notably at steps 13–14 (Fig. 6b), a time point when most DNA to induce eviction of nucleosomes6,27,28. However, how Brdt breaks are repaired in wild-type testes. In agreement with this mediates nucleosome eviction is not clear. Likely, Brdt recruits finding, immunofluorescent analyses showed that whereas chromatin remodelers to remodel and evict hyperacetylated g-H2A.X (a marker for the DSB-activated DNA damage nucleosomes. We therefore asked whether the compromised H4 response) is cleared in wild-type spermatids after step 12, it is acetylation and Chd5 deficiency perturbed nucleosome eviction detected in Chd5Aam1  /  spermatids as late as step 14 (Fig. 6c). in Chd5Aam1  /  spermatids. Using an antibody specific for Western blot analyses further confirmed an increase of g-H2A.X intact nucleosomes29, we found that nucleosomes were detectable in differentiated Chd5Aam1  /  spermatids (Fig. 6d). Together, through step 11 of spermatid maturation in wild-type testes, but these findings indicate that Chd5 deficiency impairs H4 were depleted at later steps (Fig. 5a). However, we found that hyperacetylation, nucleosome eviction, and DNA damage repair nucleosomes were aberrantly retained in Chd5Aam1  /  during spermiogenesis. spermatids as late as step 14 (Fig. 5b), indicating that nucleosomes were ineffectively evicted. Thus, both H4 acetylation and nucleosome eviction are compromised by Chd5 Chd5 loss alters gene expression in spermatids. Tnp1, Tnp2, deficiency. Prm1 and Prm2 are transcribed in round spermatids, but their a Mi Mi XII XII XI XI * * Mi Mi XII XII XI XI * * b Nucleosome DAPI Lectin Merge II II II II +/+ II II II II * * * * –/– * * * * Figure 5 | Chd5 deficiency leads to inefficient nucleosome eviction during spermiogenesis. Roman numerals indicate the spermatogenic stages of tubular areas. (a) Nucleosomes are detected in step 11 spermatids (indicated by arrow head) of stage XI seminiferous tubules, but are depleted afterwards and not detectable in step 12 spermatids (indicated by *) of stage XII seminiferous tubule in wild-type testes. Green, nucleosomes; Red, lectin (visualizing acrosome for staging seminiferous tubules); Blue, DAPI; Mi, meiotic figure, a hallmark of stage XII tubules. (b) Immunostaining for nucleosomes shows that nucleosomes are retained in as late as step 14 spermatids of stage II seminiferous tubules in Chd5Aam1  /  (  /  ) testes, while being absent in the Chd5Aam1 þ / þ ( þ / þ ) counterpart. Asterisks (*) mark nucleosome-positive condensing spermatids (step 14) in Chd5Aam1  /  tubules. Scale bars, 10 mm. 8 NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 ARTICLE a 8.0 b 100 Relative Chd5 expression +/+ –/– TUNEL positive (%) 6.0 80 60 4.0 40 2.0 20 0.0 0 Adr 0 h Adr 24 h Step 13 14 15 c γ-H2A.X DAPI Lectin Merge I I I I +/+ I I I I * * * * –/– * * * * II II II II d RS RES ECS CS KD +/+ +/– –/– +/+ +/– –/– +/+ +/– –/– +/+ +/– –/– 20 γ-H2A.X 15 50 β-Actin 37 Figure 6 | Increased DNA damage in Chd5Aam1  /  spermatids. (a) qRT–PCR analyses shows that Chd5 expression is induced approximately fivefold when wild-type mouse embryonic fibroblasts are treated with the DNA-damaging agent adriamycin (0.4 mg ml  1) for 24 h. Results are normalized to Actb and the expression level at 0 h is defined as 1. Data are presented as mean±s.d. from three independent experiments. Adr, adriamycin. (b) Increased TUNEL-positive condensing spermatids (steps 13–15) in Chd5Aam1  /  testis. Data are presented as mean±s.d. from three independent experiments. (c) Immunofluorescence analyses of g-H2A.X in Chd5Aam1 þ / þ ( þ / þ ) and Chd5Aam1  /  (  /  ) seminiferous tubules. Asterisks (*) mark g-H2A.X-positive spermatids (step 13 in stage I and step 14 in stage II tubules) in Chd5Aam1  /  testis. Scale bar, 20 mm. (d) Western blot analyses of purified spermatids at different spermiogenic stages show a transient upregulation of g-H2A.X in wild-type ECS, and increased accumulation of g-H2A.X in Chd5Aam1  /  ECS and CS compared with wild-type counterparts. b-Actin serves as a loading control. RS, round spermatids; RES, round and early elongating spermatids; ECS, elongating and condensing spermatids; CS, condensed spermatids. transcripts are stored in translationally repressed ribonucleo- As described above, a Chd5-intense focus near the edge of protein particles before being later translated into protein within chromocentre in round spermatids showed proximity to the elongating spermatids34–36. To determine whether the increase in nucleolar marker fibrillarin (see Supplementary Fig. 3b), suggest- transition proteins and protamines within Chd5-deficient ing that Chd5 may play a role in rRNA biogenesis and spermatids was due to enhanced expression at the transcript translational control. qRT–PCR analyses of 45S and 28S rRNA level, we used qRT–PCR to assess Tnp1, Tnp2, Prm1 and Prm2 expression revealed that 45S rRNA was reduced by B30%, and expression in Chd5Aam1  /  and Chd5Aam1 þ / þ round 28S rRNA expression was reduced by 450%, in Chd5Aam1  /  spermatids. Whereas only a slight increase of Tnp1, Tnp2 and round spermatids (Fig. 7c), indicating compromised rRNA Prm2 transcripts were detected, Prm1 transcript was increased biogenesis in Chd5Aam1  /  spermatids. These results suggest a B2.5-fold in Chd5Aam1  /  round spermatids (Fig. 7a). role of Chd5 in rRNA biogenesis during spermiogenesis, which is Using chromatin immunoprecipitation-qPCR (ChIP-qPCR) consistent with the known function of other CHD proteins analyses of nuclear lysates from testicular cells, we found an such as CHD4 and CHD7 in positively regulating rRNA enrichment of Chd5 binding at the Prm1 promoter (region P: biogenesis37,38.  77 bp to þ 135 bp) (Fig. 7b). Less-pronounced Chd5 binding In addition to H4 hyperacetylation, replacement of canonical was observed at region A (  860 to  672 bp), B (  444 bp to core nucleosomal histones with histone variants including those  239 bp) and C ( þ 397 bp to þ 585 bp), but not at region that are testis-specific is another key mechanism facilitating 50 (  1,319 bp to  1,164 bp). Collectively, these data suggest nucleosome destabilization and histone removal during sperma- that Chd5 represses Prm1 transcription, whereas it negatively togenesis39–41. We examined expression of a panel of general and modulates expression of Tnp1, Tnp2 and Prm2 mainly testis-specific histone variants in Chd5Aam1 þ / þ and post-transcriptionally. Chd5Aam1  /  round spermatids via qRT–PCR, and discovered NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications 9 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 +1 100 bp a b Prm1 3.0 +/+ 1,319 –860 –444 –77 585 bp –/– 5′ A B P C 2.5 Relative mRNA expression 5.0 IgG 2.0 a-Chd5 Fold of enrichment 4.0 1.5 3.0 1.0 2.0 0.5 1.0 0.0 0.0 Tnp1 Tnp2 Prm1 Prm2 5′ A B P C c 1.2 +/+ d +/+ –/– –/– 5.5 Relative mRNA expression 1.0 Relative mRNA expression 0.8 3.5 1.2 0.6 1.0 0.8 0.4 0.6 0.2 0.4 0.2 0.0 0.0 45S rRNA 28S rRNA His1h2bc Hist1h1e Hist2h3c1 H1t Figure 7 | Altered transcription of Prm1 and histone variants in Chd5-deficient spermatids. (a) qRT–PCR analyses of transition protein and protamine genes in Chd5Aam1 þ / þ and Chd5Aam1  /  round spermatids. Results are normalized to Actb expression. Prm1 expression is increased by B2.5-fold in Chd5Aam1  /  round spermatids. Data are presented as mean±s.d. from four independent experiments. (b) Top panel, diagram of mouse Prm1 gene and location of primer sets used for ChIP-qPCR. Bottom panel, ChIP-qPCR analyses reveals enrichment of Chd5 at promoter region P (  77 bp to þ 135 bp) of Prm1 gene. The results are normalized to IgG control and are shown as fold of enrichment. Data are presented as mean±s.d. from four to five independent experiments. (c) qRT–PCR analyses of rRNAs shows compromised expression of 28S and 45S ribosomal RNAs in Chd5Aam1  /  (  /  ) round spermatids. Results are normalized to Actb expression. Data are presented as mean±s.d. from four to five independent experiments. (d) qRT–PCR analyses of histone variants revealed an approximately fivefold increase in expression of histone H2B variant Hist1h2bc and modest decreases in expression of histone variants Hist1h1e, Hist2h3c1 and H1t in Chd5Aam1  /  (  /  ) round spermatids. Results are normalized to Actb expression. Data are presented as mean±s.d. from three to five independent experiments. that expression of the H2B variant Hist1h2bc was elevated over transcripts, or 40.2%, were upregulated in Chd5Aam1  /  round fivefold in Chd5Aam1  /  round spermatids (Fig. 7d). Although spermatids. Together, these results suggest that Chd5 deficiency little is known about the functions of Hist1h2bc, its substantial leads to a rather limited alteration in global gene expression in increase in Chd5Aam1  /  round spermatids suggests that it round spermatids, and that Chd5 both activates and suppresses might impact histone variant exchanges during spermiogenesis. gene expression in round spermatids with a slight preference for In addition, an B30% decrease in expression of histone H1 activation. Gene ontology (GO) analysis of the gene expression variant Hist1h1e, histone H3 variant Hist2h3c1 and testis-specific changes showed clustering of GO terms including chromosome H1 variant H1t were consistently observed in Chd5Aam1  /  organization, response to DNA damage, acetylation, alternative spermatids (Fig. 7d). These findings indicate that Chd5 deficiency splicing, nuclear export, protein transport, ubl (ubiquitin) alters transcript levels of specific histone variants during conjugation, intracellular transport, endocytosis, cell cycle and spermiogenesis. MAPK pathway (Supplementary Fig. 14). Since Chd5Aam1 þ /  In order to gain insight into the global gene expression changes male mice were fertile, we reasoned that genes that had resulting from Chd5 deficiency and to identify candidate Chd5 expression changes only in Chd5Aam1  /  spermatids but not targets, we performed RNA sequencing (RNA-Seq) (Fig. 8). Chd5 in Chd5Aam1 þ /  spermatids, or genes that had gradual is mainly expressed in round spermatids, where transcription is expression changes dependent on Chd5 dosage, would be most active during spermiogenesis, with transcription globally candidates most likely to contribute to the infertility of being ceased afterwards. Using RNA-Seq, we compared mRNA Chd5Aam1  /  male mice. We thus further filtered the 261 expression profiles of round spermatids that had been elutriation- transcripts through clustering with manual examination and purified from five sets of Chd5Aam1 þ / þ , Chd5Aam1 þ /  and identified a list of 155 transcripts that had expression changes Chd5Aam1  /  littermates. Expression of 14,206 transcripts was only in Chd5Aam1  /  spermatids, or that had gradual expression detected in at least one of the three genotypes. Two-hundred and changes that correlated with Chd5 dosage (Fig. 8a, Supplementary sixty-one transcripts, or 1.8% of all the transcripts detected, Fig. 15). Among the list, 90 transcripts were downregulated and showed a twofold or greater expression change in Chd5Aam1  /  65 transcripts, including Hist1h2bc, were upregulated in round spermatids compared with Chd5Aam1 þ / þ counterparts Chd5Aam1  /  spermatids (Fig. 8a). qRT–PCR analyses for (false discovery rate q ¼ 0.05). Among the 261 transcripts, 156 expression of 11 genes from this list, which included the positive transcripts or 59.8% were downregulated, whereas 105 control Hist1h2bc and represented a range of expression changes, 10 NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 ARTICLE a b 0.0 1.0 6 +/+ –/– +\+ +\– –\– Relative mRNA expression 5 4 65 3 2 1 90 0 lt 1 k1 1 8 18 b9 bc p2 2 a5 5 m pm ar sl xy Si h2 p6 k1 Pn ar Ac Pr C Tr G Sc Kl t1 Zf is H c GO terms Number of genes classified P value Regulation of DNA metabolic process 3 4.70E–02 MAPK signalling pathway 5 4.30E–02 Response to DNA damage stimulus 6 2.80E–02 Chromosome organization 7 3.30E–02 Cellular response to stress 8 1.00E–02 Cell cycle 9 2.90E–02 ubl conjugation 9 1.70E–02 Alternative splicing 47 1.10E–04 0 10 20 30 40 50 d 1.2 +/+ –/– 1 0.8 0.6 0.4 0.2 0 1 3 t ik f2 ip ne R st rn 17 Sy C W 9G 01 00 17 Figure 8 | RNA-Seq reveals global gene expression changes in Chd5Aam1  /  spermatids. (a) Heat map presentation of gene expression in Chd5Aam1 þ / þ , Chd5Aam1 þ /  and Chd5Aam1  /  round spermatids revealed by RNA-Seq. Green to red (0 to 1) represents the gradient increase of expression levels. Sixty-five transcripts show upregulation, and 90 transcripts show downregulation, in Chd5Aam1  /  round spermatids. (b) qRT–PCR validation of RNA-Seq revealed gene expression changes in round spermatids. Results are normalized to Actb expression and data are presented as mean±s.d. from four to five independent experiments. (c) GO analysis of the 155 transcripts that either show expression changes only in Chd5Aam1  /  spermatids but not in Chd5Aam1 þ /  spermatids, or that show gradual expression change from Chd5Aam1 þ / þ spermatids to Chd5Aam1 þ /  spermatids to Chd5Aam1  /  spermatids. Numbers next to bars indicate the number of genes classified to the corresponding GO term. P-value is calculated using modified Fisher’s exact test by DAVID (v6.7)70. (d) qRT–PCR verification of compromised expression of genes implicated in histone acetylation, DNA damage response, RNA processing and nuclear integrity in Chd5Aam1  /  spermatids. Results are normalized to Actb expression and Data are presented as mean±s.d. from four to five independent experiments. was used to validate the expression changes revealed by RNA-Seq, impacts of Chd5 deficiency on chromatin compaction, attesting to the reliability of the RNA-Seq data and analyses DNA damage response and post-transcriptional modulation of (Fig. 8b). GO analysis of this list showed clustering of a subset of transition proteins and protamines during spermiogenesis. GO terms revealed from the original list, which include Alterations in gene expression within these GO terms are likely chromosome organization, response to DNA damage stimulus, contributing, at least partially, to the spermatogenic abnormalities alternative splicing, ubl conjugation, regulation of DNA meta- of Chd5Aam1  /  male mice. bolic process, cell cycle and MAPK signalling (Fig. 8c). A number of candidates implicated in acetylation (1700019G17Rik), DNA damage response (Wrnip1), RNA processing and translational Discussion control (Cstf2t) and nuclear structure maintenance (Syne3) were In this study, we show that Chd5 mediates a cascade of molecular further verified using qRT–PCR (Fig. 8d, Supplementary Fig. 16). events underlying histone removal during spermiogenesis Altogether, these GO terms are consistent with the phenotypic including H4 hyperacetylation, histone variant expression, NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications 11 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 nucleosome eviction and DNA damage repair. Chd5 deficiency spermiogenesis. Lu et al.50 revealed that Rnf8 mediates H2A leads to disruption of these biological processes and increases and H2B ubiquitination in elongating spermatids, and is critical histone retention in both spermatids and sperm. We reveal that for H4K16 acetylation and histone-to-protamine replacement. Chd5 also modulates the homeostasis of transition proteins and However, Sin et al.51 later reported that Rnf8 deficiency does not protamines by suppressing expression of Prm1 transcriptionally affect H4K16 acetylation and histone-to-protamine exchange in and expression of Tnp1, Tnp2 and Prm2 post-transcriptionally. spermatids, but instead compromises gene activation from Chd5 deficiency results in elevated levels of both transition inactive sex chromosomes in round spermatids. Thus, while a proteins and protamines. These findings unravel pleiotropic number of nuclear proteins have been implicated in spermato- functions of Chd5 and highlight its multi-faceted role in genesis, Chd5, to our knowledge, is the first chromatin remodeller orchestrating the extensive histone-to-protamine remodelling identified to play an orchestrating role in chromatin remodelling that occurs during male germ cell development. during post-meiotic spermiogenesis. Chd5 contains multiple domains (PHD domains, chromodo- Consistent with Chd5 deficiency disrupting histone acetylation, mains, SNF2-like ATPase domain, DEAD/DEAH-box Helicase DNA damage response and homeostasis of transition proteins domain, SANT domain and DNA-binding motifs), which may and protamines during spermiogenesis, RNA-Seq reveals that enable its diverse functions during spermiogenesis. The SNF2-like Chd5 deficiency alters expression of genes encoding proteins ATPase domain defines the nucleosome remodelling function of underlying these processes, but does not cause a major change in CHD proteins42,43. Chd5 may facilitate nucleosome eviction global gene expression in round spermatids. We validate a during spermiogenesis through its ATPase domain, whose number of candidate Chd5 target genes (for example, Cstf2t, absence in Chd5-deficient spermatids would thus lead to 1700019G17Rik, Wrnip1 and Syne3) implicated in these pro- inefficient nucleosome eviction. The ATPase domain is also cesses. Notably, Cstf2t encodes the RNA polyadenylation protein implicated in DNA damage repair in somatic cells44 and may play tauCstF-64, which is expressed during haploid spermatid a direct role in the DNA damage response during spermiogenesis differentiation52. Deletion of Cstf2t in mice disrupts post- and contribute to the increased DNA damage in Chd5-deficient meiotic development and leads to male infertility52. Similar to spermatids. The DEAD/DEAH-box helicase domain may the heterogeneity of histopathology among Chd5Aam1  /  testes, implicate Chd5 in RNA processing and the characteristic Cstf2t  /  male mice also display variable expressivity of sperm repression of mRNA translation in round spermatids, which defects52. These intriguing parallels suggest that deficient could contribute to the abnormal post-transcriptional elevation of expression of Cstf2t in Chd5Aam1  /  spermatids may transition proteins and protamines in Chd5-deficient spermatids. contribute to the infertile phenotypes of Chd5Aam1  /  mice. It is known that the dual PHD domains of Chd5 preferentially In addition, the compromised expression of Cstf2t in bind H3 tails lacking H3K4me3 in mouse embryonic Chd5Aam1  /  spermatids may compromise polyadenylation fibroblasts10, whereas the chromodomains bind to H3K27me3 and translational repression of transition protein and protamine in neurons, both of which are important for Chd5 to mediate mRNAs, thereby leading to an elevation in protein production, gene expression in somatic cells10,45. We also observe such since translational repression of mRNAs in spermatids involves patterns in mouse spermatids, as Chd5 is enriched in the binding of protein repressors to poly (A) tails, and shortening of chromocentre of spermatids, a heterochromatic region marked poly (A) tails of Tnp1, Tnp2, Prm1 and Prm2 mRNAs with H3K9me3 and H3K27me3, but lacking H3K4me3 (Fig. 1, accompanies their translation activation53,54. 1700019G17Rik Supplementary Fig. 2). This suggests that the PHD and/or encodes a putative N-acetyltransferase that is highly enriched in chromodomains of Chd5 may also play important roles in mouse testis but is absent in most other tissues (Supplementary regulating gene expression in spermatids, as RNA-Seq revealed Fig. 16a). We also found that 1700019G17Rik expression increases that Chd5 deficiency leads to expression changes of specific gene as round spermatids differentiate into elongating spermatids, the sets. These multiple functional domains of Chd5 may work time point at which H4 hyperacetylation occurs (Supplementary independently or in concert, enabling the diverse functions of Fig. 16b). These data suggest 1700019G17Rik as a potential Chd5 observed during spermiogenesis. acetyltransferase affecting H4 acetylation during spermiogenesis, It is possible that there is a functional connection between the and the 480% decrease in expression may compromise H4 histone removal process and homeostasis of transition proteins acetylation in Chd5Aam1  /  spermatids. Wrnip1 stimulates the and protamines. For example, transition proteins have been activity of DNA polymerase delta, rapidly accumulates at laser- implicated in DNA repair during spermiogenesis46,47, thus irradiated sites and is required for maintaining genome the abnormal levels of transition proteins may contribute to the integrity55–57. Syne3 is a component of the nuclear envelope increased DNA damage in Chd5Aam1  /  spermatids. On the that tethers the nucleus to the cytoskeleton, and is critical for other hand, deficient H4 acetylation may alter the chromatin maintaining nuclear organization and structural integrity, as well structure of the Prm1 locus, thereby contributing to its increased as for development of the sperm head58–62. Compromised transcription, although it is counterintuitive that a decrease in H4 expression of Wrnip1 and Syne3 may contribute to the acetylation would result in transcriptional activation. enhanced DNA damage and abnormal sperm head Chromatin remodellers are thought to be critical for the morphology, respectively, in Chd5Aam1  /  testis. However, it extensive chromatin remodelling taking place during the post- is not yet clear whether these genes and other candidates revealed meiotic phase of spermiogenesis; however, little is known about by RNA-Seq are direct Chd5 target genes, a question that could their roles in this process. The chromatin remodeller Brg1 is be addressed by defining the global pattern of Chd5-bound loci in essential for meiosis, and its deficiency leads to meiotic arrest spermatids. A small proportion of histones and nucleosomes are with global alterations in histone modifications and chromatin retained in chromatin of wild-type sperm, preferentially at loci structure in mice48. Acf1, which binds to chromatin remodeller encoding proteins of developmental importance63. It would be Snf2h within the ACF complex, plays an essential role during interesting to know whether the aberrant histone retention in post-meiotic spermiogenesis49. Deletion of Acf1 results in male Chd5Aam1  /  sperm disrupts such a pattern, where the infertility with increased DNA damage and spermiation defects, aberrantly retained histones locate in the genome of but without any detectable alterations in chromatin Chd5Aam1  /  sperm, and to understand these implications for composition49. Previous studies have also suggested roles of the developmental potency of the sperm. Future studies to chromatin modifier Rnf8, a E3 ubiquitin ligase, in characterize genome-wide distribution of Chd5, nucleosomal 12 NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 ARTICLE histones and specific histone modifications in Chd5-deficient Chd5Tm1a(EUCOMM)Wtsi allele, which has exon 2 of Chd5 locus flanked by LoxP spermatids and sperm should shed additional light on these sites, were obtained from EUCOMM (European Conditional Mouse Mutagenesis)65. ES cells were from the C57BL/6N-A/a background and were questions. injected into albino B6 (C57BL/6J-Tyr c-2J) blastocysts through standard We observed variation ranging from sterility to sub-fertility procedures. Progeny resulting from germline transmission, designated as among individual Chd5Aam1  /  male mice in a mixed C57BL/6 Chd5Tm1a þ /  mice harboured a Chd5 allele with exon 2 flanked by LoxP sites. and 129S background, as well as among individual Chd5Tm1b  /  Chd5Tm1a þ /  mice were mated to CMV-Cre mice in the C57BL/6 background to male mice in a pure C57BL/6 background. Such variability in obtain Chd5Tm1b þ /  mice (which had a Chd5 allele with exon 2 excised), and Chd5Tm1b þ /  mice were intercrossed to obtain Chd5Tm1b  /  progeny. infertility is similarly observed in Tnp1  /  and Tnp2  /  male mice, as B40 and 89% of Tnp1  /  and Tnp2  /  male mice, Genotyping. For Southern blot genotyping of the Chd5Aam1 model, the respectively, are sub-fertile23,24. The aberrant Tnp1 and Tnp2 MHPN20h05 MICER vector was cut with AflII and the 2.6 kb excised fragment was levels in Chd5-deficient testes may contribute to the variable gel-purified and used as a probe. Southern blotting of genomic DNA digested with infertility among individual Chd5-deficient male mice. In BglII yielded the expected 7.8 and 10.4 kb endogenous and targeted alleles, addition, we observed increased DNA damage in Chd5- respectively. Genotypes were differentiated based on dosage of the targeted allele (Chd5Aam1 þ / þ , 0 copies; Chd5Aam1 þ /  , 1 copy; Chd5Aam1  /  , 2 copies). All deficient spermatids and sperm. The intrinsically variable extent genotypes had two copies of the endogenous band, which serve as an internal of DNA damage may be more severe in testes of some Chd5- loading control and reference for dosage. For PCR-based genotyping, relative deficient mice than in others, thus contributing to the variability dosage of the neo-cassette in different genotypes (Chd5Aam1 þ / þ , 0 copies; of infertility. Chd5Aam1 þ /  , 1 copy; Chd5Aam1  /  , 2 copies) was quantified using qPCR, with H4 hyperacetylation in early elongating spermatids is shown to Neo-cassette dosage being normalized to dosage of Actb. Genotyping of Chd5Tm1b mice was performed by PCR using primers (Supplementary Fig. 16) that amplify a be essential for histone-to-protamine replacement in Drosophila25 674-bp endogenous band specific for the wild-type Chd5 allele and a 456-bp and has been considered the same for mammalian targeted band specific for the targeted Chd5Tm1b allele. spermiogenesis6,25,26. Our study shows that Chd5 deficiency leads to substantial deficiency in H4 acetylation in elongating Antibodies. Antibodies used for western blot, immunoflurorescence and ChIP spermatids and subsequent defects in nucleosome eviction and are as follows: anti-H3K27me3 (Cell Signaling no. 9756, 1:100 for IF, that is, histone removal, supporting the notion that H4 hyperacetylation immunofluorescence), anti-H2A (Cell Signaling no. 2578, 1:300 for WB, that is, is indeed critical for efficient nucleosome eviction and histone Western blotting; for uncropped images, see Supplementary Fig. 17), anti-H4 (Cell Signaling no. 2935, 1:800 for WB), anti-g-H2A.X (Cell Signaling no. 9718, 1:200 for removal during mouse spermiogenesis. However, most IF; 1:1,500 for WB), anti-H3K9me3 (Active Motif no. 39385, 1:100 for IF), anti-H1 nucleosomes and histones eventually get removed in late (Active Motif no. 39707, 1:800 for WB), anti-H2B (Active Motif no. 39125, 1:800 Chd5Aam1  /  spermatids. This suggests that whereas H4 for WB), anti-H4Ac-pan (Active Motif no. 39243, 1:1,000 for WB; 1:200 for IF), hyperacetylation is important for efficient nucleosome eviction anti-H3 (Abcam no. ab1791, 1:15,000 for WB), anti-Chd5 (Santa Cruz Bio- technology no. sc-68389, 1:1,500 for WB; 1:200 for IF), anti b-Actin (Sigma no. and histone removal during mammalian spermiogenesis, it seems A2228, 1:2,000 for WB), anti-H4K5/8/12/14Ac (Millipore no. 05-1335, 1:100 for IF non-essential. Thus, whether H4 hyperacetylation is required ), anti-lectin PNA Alex Fluor 568 (Invitrogen no. L32458, 1:4,000 for IF), anti- for histone-to-protamine replacement during mammalian Prm1 (Briar Patch Biosciences, Hup1N, 1:300 for WB; 1:150 for IF), antiPrm2 spermiogenesis warrants further investigation. In addition, the (Briar Patch Biosciences, Hup2B, 1:800 for WB; 1: 200 for IF), anti-Tnp1 (gift from Dr Stephen Kistler, University of South Carolina, 1:500 for WB; 1: 100 for IF ), enzymes responsible for H4 hyperacetylation during anti-Tnp2 (gift from Dr Stephen Kistler, University of South Carolina, 1:1,000 for spermiogenesis remain unidentified and are of great interest in WB; 1: 200 for IF) and anti-nucleosome (mab no. 32, gift from Dr Jo H.M. Berden, the field. Our study identifies 1700019G17Rik as a candidate Radboud University Nijmegen Medical Center, 1:300 for IF). acetyltransferase for H4 hyperacetylation, providing a new promising target for future study. Histology and immunostaining. Testes were fixed in Bouin’s fixative or 4% The cascade of defects in H4 hyperacetylation, nucleosome paraformaldehyde, embedded in paraffin and sectioned at 5 mm. Sections were eviction and DNA break repair during Chd5Aam1  /  spermatid deparaffinized in xylene and subjected to either PAS staining for histological analyses or immunofluorescent staining using the indicated antibodies. maturation provide functional evidence demonstrating the sequential order of these events, and suggest a model for the molecular events underlying the histone-to-protamine replace- Sperm counts and motility analysis. Individual caudal epididymi were minced in 200 ml HTF medium (Irvine Scientific). After 30 min incubation at 37 °C, the tissue ment process during spermiogenesis: H4 is first hyperacetylated, pieces were separated from sperm by pipetting and passaging through a 70-mm which along with other epigenetic modifications, leads to filter. Sperm counts and motility assessment were performed using the DRM-600 chromatin loosening and nucleosome eviction to facilitate histone CELL-VU Sperm Counting Cytometer. removal and exposure of the DNA to allow for deposition of transition proteins and eventually protamines. DSBs are gener- Sperm morphology. Air-dried smears were prepared from sperm suspended in ated during nucleosome eviction to resolve supercoiling tension, PBS, stained with haematoxylin, and examined using light microscopy at  100 magnification. Head, neck and tail morphology was determined independently for and DNA damage response is activated to repair the DSBs, each mouse, with separate counts of at least 100 cells per sample. ensuring integrity of the sperm genome. This sequence of molecular events is in agreement with the model proposed by Sperm chromatin structure assay. SCSA was carried out as previously descri- Leduc et al.30. Our findings establish functional evidence bed24 using a LSR II flow cytometer (Becton Dickinson). Briefly, a 0.2 ml aliquot of revealing the cascade of major molecular events underlying the sperm nuclei in TNE buffer (0.1 M Tris, 0.15 M NaCl and 1 mM EDTA (pH 7.4)) histone-to-protamine replacement process, and provide a was mixed with 0.4 ml acid detergent solution (0.15 M NaCl, 0.08 N HCl and 0.1% foundation to further elucidate this critical but elusive process. Triton X-100, pH 1.4). After 30 s, 1.2 ml acridine orange staining solution (6 mg ml  1 acridine orange, 0.1 M citric acid, 0.2 M Na2HPO4, 1 mM EDTA and 0.15 M NaCl, pH 6.0) were added to the denatured sperm nuclei. After staining for 3 min, samples were measured for green and red fluorescence using LSR II with a Methods 488-nm excitation wavelength. For the SCSA assay, acid-treated sperm were Generation of Chd5-deficient mouse models. To generate the Chd5Aam1 mouse stained with acridine orange, which emits red or green fluorescence when binding model, Chd5 locus was disrupted in AB2.2 ES cells using the MHPN20h05 MICER to single-stranded or double-stranded DNA, respectively. Sperm with impaired vector64, and targeted ES cells were injected into C57BL/6 blastocysts through chromatin generate more single-stranded DNA after denaturation with acid standard procedures. All animals were housed and utilized according to the Cold treatment, and therefore emit more red fluorescence. DNA Fragmentation Index Spring Harbor Institutional Animal Care and Use Committee (IACUC) and the (DFI) is defined as the percentage of red/green þ red fluorescence. Association for Accreditation of Laboratory Animal Care International (AAALAC) policies. Progeny resulting from germline transmission were backcrossed to wild- type C57BL/6 mice, and Chd5Aam1 þ /  mice were intercrossed to obtain Transmission electron microscopy. Testes were fixed with 2% paraformaldehyde homozygotes. To generate the Chd5Tm1b mouse model, ES clones with and 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4), dehydrated and NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications 13 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4812 embedded in Epon. Sections were contrasted and imaged in a Hitachi H7000T 4. Oakberg, E. F. A description of spermiogenesis in the mouse and its use in transmission electron microscope. analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat. 99, 391–413 (1956). Basic protein extraction and acid-urea gel electrophoresis. Testes were surgi- 5. Ahmed, E. A. & de Rooij, D. G. Staging of mouse seminiferous tubule cross- cally decapsulated and homogenized in buffer (10 mM Tris–HCl (pH 7.2), 0.32 M sections. Methods Mol. 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This project was sup- spermatogenesis. PLoS Genet. 9, e1003945 (2013). ported by the Office of the Director, National Institutes of Health under award numbers 50. Lu, L. Y. et al. RNF8-dependent histone modifications regulate nucleosome R01CA127383 (to A.A.M.) and R21OD018332 (to A.A.M.), NIH HG001696 (to M.Q.Z.), removal during spermatogenesis. Dev. Cell 18, 371–384 (2010). and NSFC 91019016 (to M.Q.Z.); the content is the sole responsibility of the authors. 51. Sin, H. S. et al. RNF8 regulates active epigenetic modifications and escape gene This work was performed with assistance from Cold Spring Harbour Laboratory Shared activation from inactive sex chromosomes in post-meiotic spermatids. Genes Resources, which are funded, in part, by the Cancer Center Support Grant Dev. 26, 2737–2748 (2012). 5P30CA045508. 52. Dass, B. et al. Loss of polyadenylation protein tauCstF-64 causes spermatogenic defects and male infertility. Proc. Natl Acad. Sci. USA 104, 20374–20379 (2007). 53. Steger, K. Haploid spermatids exhibit translationally repressed mRNAs. Anat. Author contributions Embryol. (Berl.). 203, 323–334 (2001). W.L. and A.A.M. designed the experiments and prepared the manuscript with input 54. Kleene, K. C. Poly(A) shortening accompanies the activation of translation of from co-authors. W.L. and A.A.M. generated Chd5-deficient mouse models. W.L. five mRNAs during spermiogenesis in the mouse. Development 106, 367–373 performed experiments with assistance from S.-Y.K. for sperm analyses and IVF, with (1989). assistance from S.A.H. for electron microscopy, and with assistance from M.Z. and 55. Nomura, H. et al. WRNIP1 accumulates at laser light irradiated sites rapidly via M.L.M. for centrifugal elutriation. W.L. and A.A.M. interpreted the data, with RNA-Seq its ubiquitin-binding zinc finger domain and independently from its ATPase data being analysed by J.W., W.L. and M.Q.Z. domain. Biochem. Biophys. Res. Commun. 417, 1145–1150 (2012). 56. Hayashi, T. et al. Vertebrate WRNIP1 and BLM are required for efficient maintenance of genome stability. Genes Genet. Syst. 83, 95–100 (2008). Additional information 57. Tsurimoto, T., Shinozaki, A., Yano, M., Seki, M. & Enomoto, T. Human Accession codes: RNA-seq data are deposited in the NCBI Short Read Archive under Werner helicase interacting protein 1 (WRNIP1) functions as a novel accession number SRPO40711. modulator for DNA polymerase delta. Genes Cells 10, 13–22 (2005). 58. Morimoto, A. et al. A conserved KASH domain protein associates with Supplementary Information accompanies this paper at http://www.nature.com/ telomeres, SUN1, and dynactin during mammalian meiosis. J. Cell Biol. 198, naturecommunications 165–172 (2012). 59. Gob, E., Schmitt, J., Benavente, R. & Alsheimer, M. Mammalian sperm head Competing financial interests: The authors declare no competing financial interests. formation involves different polarization of two novel LINC complexes. PLoS Reprints and permission information is available online at http://npg.nature.com/ ONE 5, e12072 (2010). reprintsandpermissions/ 60. Nery, F. C. et al. TorsinA binds the KASH domain of nesprins and participates in linkage between nuclear envelope and cytoskeleton. J. Cell Sci. 121, How to cite this article: Li, W. et al. Chd5 orchestrates chromatin remodelling during 3476–3486 (2008). sperm development. Nat. Commun. 5:3812 doi: 10.1038/ncomms4812 (2014). NATURE COMMUNICATIONS | 5:3812 | DOI: 10.1038/ncomms4812 | www.nature.com/naturecommunications 15 & 2014 Macmillan Publishers Limited. All rights reserved.