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Mouse BRWD1 Is Critical for Spermatid Postmeiotic Transcription and Female Meiotic Chromosome Stability

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Mouse BRWD1 Is Critical for Spermatid Postmeiotic Transcription and Female Meiotic Chromosome Stability JCB: Article Mouse BRWD1 is critical for spermatid postmeiotic transcription and female meiotic chromosome stability Shrivatsav Pattabiraman,1,2 Claudia Baumann,3 Daniela Guisado,1,2 John J. Eppig,4 John C. Schimenti,1,2 and Rabindranath De La Fuente3 1Department of Biomedical Sciences and 2Center for Vertebrate Genomics, Cornell University, College of Veterinary Medicine, Ithaca, NY 14853 3Department of Physiology and Pharmacology, University of Georgia College of Veterinary Medicine, Athens, GA 30602 4The Jackson Laboratory, Bar Harbor, ME 04609 ostmeiotic gene expression is essential for devel- Brwd1 ablation caused severe chromosome condensa- opment and maturation of sperm and eggs. We re- tion and structural defects associated with abnormal P port that the dual bromodomain-containing protein telomere structure but only minor changes in gene ex- BRWD1, which is essential for both male and female pression at the germinal vesicle stage, including more fertility, promotes haploid spermatid–specific transcrip- than twofold overexpression of the histone methyltrans- tion but has distinct roles in oocyte meiotic progression. ferase MLL5 and LINE-1 elements transposons. Thus, Brwd1 deficiency caused down-regulation of 300 loss of BRWD1 function interferes with the completion of mostly spermatid-specific transcripts in testis, includ- oogenesis and spermatogenesis through sexually di- ing nearly complete elimination of those encoding the morphic mechanisms: it is essential in females for epi- protamines and transition proteins, but was not asso- genetic control of meiotic chromosome stability and in ciated with global epigenetic changes in chromatin, males for haploid gene transcription during postmeiotic which suggests that BRWD1 acts selectively. In females, sperm differentiation. Introduction During spermiogenesis, round spermatids undergo complex one of the activator isoforms of CREM that is specific to testis and biochemical and morphological changes as they differentiate is transcribed exclusively in mid-late pachytene spermatocytes into sperm. The spermatids elongate, acquire an acrosomal cap, and translated in round spermatids (Foulkes, 1992; Weinbauer and develop a tail. Most of the cytoplasm is extruded in the form et al., 1998). Before meiosis, a repressor isoform of CREM is of residual bodies, and the nucleus undergoes extensive conden- expressed. Therefore, CREM-–activated genes are expressed sation as the histones are replaced by protamines. This differen- exclusively after meiosis. Crem knockout mice are sterile and THE JOURNAL OF CELL BIOLOGY tiation is orchestrated by a wave of gene expression that occurs arrest at the round spermatid stage (Blendy et al. 1996; Nantel exclusively after meiosis. Examples of genes transcribed post- et al., 1996). Chromatin immunoprecipitation (ChIP)-Seq ex- meiotically include protamines, transition proteins, and outer periments revealed that CREM- binds the promoters of >6,000 dense fiber and fibrous sheath proteins of the tail. Transcrip- genes in male germ cells, including those of the protamines, tion of haploid-expressed genes eventually ceases when round transition proteins, and other postmeiotic genes (Martianov et al., spermatids begin to elongate and undergo nuclear compaction 2010). CREM- transcriptional activity is regulated by its co- (Sassone-Corsi, 2002). activator activator of CREM in testis (ACT; Kotaja et al., 2004). One of the key proteins regulating haploid gene expression Apart from CREM-, there are many testis-specific transcrip- is cAMP response element modulator (CREM)-. CREM- is tion factors that are either paralogues of the TFIID compo- nents (such as TAF4B and TAF7L) or are testis-specific isoforms of general transcription factors such as TATA-binding protein Correspondence to Rabindranath De La Fuente: [email protected]; or John C. Schimenti: [email protected] © 2015 Pattabiraman et al. This article is distributed under the terms of an Attribution– Abbreviations used in this paper: ACT, activator of CREM in testis; CREM, cAMP Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication response element modulator; GV, germinal vesicle; IAP, intracisternal A particle; date (see http://www.rupress.org/terms). After six months it is available under a Creative MGI, Mouse Genome Informatics; SN, surrounded nucleolus; TSA, trichostatin A; Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described WT, wild type. at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 208 No. 1 53–69 www.jcb.org/cgi/doi/10.1083/jcb.201404109 JCB 53 Figure 1. Gene expression profiling of Brwd1/ spermatids. (A) Heat map visualiza- tion of 286 down-regulated transcripts in mu- tant testes from 27-d-old animals as detected by gene expression microarray analyses (H1–H4 are heterozygotes, M1–M4 are mutants). (B) Real-time PCR validation of the most down- regulated genes in 27-d-old mice. The hatched boxes represent mutant levels. (C) Flow cytom- etry count of testicular cells based on DNA con- tent (C) shows that the percentage of haploid spermatids in both WT and mutant testes are the same. The 1C cells are haploid sperma- tids, the 2C cells correspond to somatic and spermatogonial cells that have not undergone DNA replication, and the 4C cells correspond to spermatocytes (predominantly), spermato- gonia, and somatic cells that have completed S phase but not cell division. (D) Real-time PCR shows significant differences in Tnp1, Tnp2, and Prm1 between WT and mutant transcript levels even in 21-d-old mice. Error bars indi- cate ±SEM. The data are the mean of three independent replicates. (Martianov, et al., 2001). The usage of testis-specific transcrip- grown oocyte (De La Fuente, 2006). For example, analysis of tional factors and regulators indicates that unique pathways nucleoplasmin (Npm2/) oocytes indicates that large-scale may be used in the regulation of haploid genome expression. chromatin remodeling in the germinal vesicle and redistribution Epigenetic modifications also play an important role in of major satellite sequences around the nucleolus induce the regulating transcription during both male and female germ cell formation of a prominent heterochromatin rim or karyosphere development (De La Fuente, 2006; Sasaki and Matsui, 2008). that coincides with global transcriptional repression in preovu- After meiosis, histone modifications in the spermatid nucleus latory oocytes (De La Fuente et al., 2004). Although chromatin are necessary for transcriptional activation of the haploid ge- remodeling into this surrounded nucleolus (SN) configuration nome. For instance, the promoter of the “protamine domain” and global transcriptional repression are regulated through dif- containing the protamine 1 (Prm1), protamine 2 (Prm2), and ferent pathways, the specialized nuclear architecture and tran- transition protein 1 (Tnp1) genes undergoes extensive epigen- scriptional quiescence of the SN configuration is essential for etic changes as the germ cells undergo meiosis and become the acquisition of both meiotic and developmental potential round spermatids. Coincident with the transcriptional activa- (De La Fuente, 2006; Abe et al., 2010). tion, acetylation of histones H3 and H4 increases in the promot- Mice of both sexes lacking BRWD1 (bromo- and WD- ers and coding sequences within the protamine domain (Martins containing protein-1) are infertile (Philipps et al., 2008). Sperm and Krawetz, 2007). H3K9 demethylation by KDM3A is also of mutant males exhibit aberrant morphologies including mis- important for the transcriptional activation of the protamine shapen heads, a ragged mid-piece, and impaired motility. Only domain (Okada et al., 2007). However, the mechanisms under- 44% of mutant oocytes developed to metaphase II when sub- lying epigenetic alterations specific to haploid gene expression jected to in vitro maturation, and those that reached metaphase in spermatids are not well understood, largely due to the lack of II did not cleave to the two-cell stage after in vitro fertilization suitable in vitro model systems. Consequently, mouse models with wild-type (WT) sperm. Importantly, meiosis in males ap- provide the most robust system for addressing the epigenetic peared completely normal, indicating that BRWD1 deficiency control of gene expression in the male germline. impacts spermiogenesis exclusively. Functional differentiation of chromatin structure is also Whereas most infertility mutations affecting both sexes essential for meiotic and developmental potential of the fully occur either in genes essential for primordial germ cells, meiosis, 54 JCB • volume 208 • numBer 1 • 2015 or the endocrine system (Matzuk and Lamb, 2008), Brwd1 is unique in that it appears to affect entirely different processes in sperm and oocytes (Philipps et al., 2008). BRWD1 contains two tandem bromodomains and eight WD repeats. Bromodomains are highly conserved 110–amino acid motifs that recognize acetyl-lysine residues (Hudson et al., 2000; Bottomley, 2004). This interaction is pivotal for many cellular processes, in partic- ular chromatin remodeling and transcriptional activation (Zeng and Zhou, 2002). Here, we provide evidence indicating that loss of BRWD1 exhibits a sexually dimorphic phenotype in male and female germ cells due to drastically different underlying mechanisms. Our results demonstrate that Brwd1 is essential for haploid gene expression during postmeiotic germ cell
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