Genetics: Early Online, published on April 19, 2018 as 10.1534/genetics.118.300565 1 Interrogating the functions of PRDM9 domains in meiosis 2 Sarah Thibault-Sennett*†, Qi Yu*‡, Fatima Smagulova†,§, Jeff Cloutier‡, Kevin Brick‡, R. 3 Daniel Camerini-Otero‡^ and Galina V. Petukhova†^. 4 5 * These authors contributed equally to the manuscript. 6 † Department of Biochemistry and Molecular Biology, Uniformed Services University of 7 Health Sciences, Bethesda, Maryland 20814, USA 8 ‡ Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and 9 Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, Maryland 10 20892, USA 11 § Present address: 9 Avenue du Professeur Léon Bernard, 35000 Rennes, France 12 13 ^ Correspondence: [email protected] or [email protected]. 14 15 Data available at GEO (https://www.ncbi.nlm.nih.gov/geo/) with the accession number: 16 GSE104850. 1 Copyright 2018. 17 RUNNING TITLE: PRDM9 domains during meiosis 18 19 KEYWORDS: PRDM9, meiosis, homologous recombination and KRAB domain 20 21 CORRESPONDING AUTHORS: 22 Galina Petukhova, (301) 295-3564, [email protected], 23 Department of Biochemistry and Molecular Biology, USUHS, 4301 Jones Bridge Rd., 24 Bethesda, MD 20814, USA 25 R. Daniel Camerini-Otero, (301) 496-2710, [email protected] 26 National Institutes of Health, Building 5, Room 205A, 5 Memorial Dr., Bethesda, MD 27 20814 28 29 30 31 2 32 ABSTRACT 33 Homologous recombination is required for proper segregation of homologous 34 chromosomes during meiosis. It predominantly occurs at recombination hotspots that 35 are defined by the DNA binding specificity of the PRDM9 protein. PRDM9 contains 36 three conserved domains typically involved in regulation of transcription, yet, the role of 37 PRDM9 in gene expression control is not clear. Here we analyze the germline 38 transcriptome of Prdm9-/- male mice in comparison to Prdm9+/+ males and find no 39 apparent differences in the mRNA and miRNA profiles. We further explore the role of 40 PRDM9 in meiosis by analyzing the effect of the KRAB, SSXRD and post-SET zinc 41 finger deletions in a cell culture expression system and the KRAB domain deletion in 42 mice. We found that although the post-SET zinc finger and the KRAB domains are not 43 essential for the methyltransferase activity of PRDM9 in cell culture, the KRAB domain 44 mutant mice show only residual PRDM9 methyltransferase activity and undergo meiotic 45 arrest. In aggregate, our data indicate that domains typically involved in regulation of 46 gene expression do not serve that role in PRDM9, but are likely involved in setting the 47 proper chromatin environment for initiation and completion of homologous 48 recombination. 49 50 INTRODUCTION 51 Homologous recombination is required for the faithful segregation of homologous 52 chromosomes during meiosis. It assures that homologous chromosomes find each 53 other and stay connected until the first meiotic division, when they segregate to different 54 daughter cells. Failure to segregate properly results in aneuploidy in gametes and 3 55 offspring which accounts for 35% of miscarriages and developmental disabilities in 0.3% 56 of live births (Hassold et al. 2007). The most severe recombination defects lead to 57 meiotic arrest and consequent infertility (reviewed in (Handel and Schimenti 2010)). 58 59 During meiotic recombination the chromosomes undergo programmed DNA double- 60 stranded breaks (DSBs) (reviewed in (Keeney and Neale 2006)). This triggers a 61 genome-wide search for the intact homologous chromosome to be used for repair. 62 RAD51 and DMC1 recombinases bind to single-stranded tails of the resected DSB ends 63 and initiate recombinational repair of DSBs that results in either crossover or non- 64 crossover products (Keeney and Neale 2006). Meiotic DSBs preferentially occur in 65 discrete sites of the genome called recombination hotspots (reviewed in (Arnheim et al. 66 2007; Buard and de Massy 2007; Lichten 2008; Paigen and Petkov 2010; Clark et al. 67 2010)). In mice and humans, hotspot locations are defined by the DNA binding 68 specificity of the PR/SET domain containing 9 (PRDM9) protein that trimethylates 69 Lysine 4 of the histone H3 (H3K4me3) at the sites where recombination may 70 subsequently occur (Baudat et al. 2010; Parvanov et al. 2010; Myers et al. 2010; Grey 71 et al. 2011; Brick et al. 2012). The DNA binding domain of PRDM9 consists of an array 72 of zinc finger domains (ZFs) that vary primarily at three amino acids that define the 73 sequence specificity of DNA binding (reviewed in Wolfe et al. 2000; Persikov et al. 74 2009). Remarkably, the ZF array is extremely polymorphic and may consist of different 75 ZFs in different individuals, leading to a completely different distribution of 76 recombination hotspots. When PRDM9 is absent, DSBs are formed at so-called default 77 hotspots, the other available H3K4me3 sites within the genome, mostly at gene 4 78 promoters and enhancer elements (Brick et al. 2012). These default sites are almost 79 never used in wild type mice, suggesting a dominant direct interaction between PRDM9 80 and the DSB formation machinery. Indeed, recent studies implicated the KRAB domain 81 of PRDM9 in tethering the hotspot DNA to the chromosome cores, where the DSB 82 machinery resides, through an interaction with CXXC1 (Imai et al. 2017; Parvanov et al. 83 2017). Prdm9-/- mice undergo meiotic arrest due to recombination failure, leading to 84 sterility in both sexes (Hayashi et al. 2005). Interestingly, the loss of Prdm9 does not 85 universally result in sterility as Prdm9 knockouts in some species remain fertile 86 (Narasimhan et al. 2016), and the functional Prdm9 gene has been lost in several 87 lineages (Ponting 2011; Baker et al. 2017). 88 89 PRDM9 belongs to the PRDM protein family, a group that is defined by the presence of 90 a ZF array and the PR/SET domain (reviewed in (Hohenauer and Moore 2012)). The 91 PR/SET domain of PRDM9 is responsible for histone methyltransferase activity, 92 including mono-, di- and trimethylation of the H3K4 (Hayashi et al. 2005; Wu et al. 93 2013), as well as H3K36 trimethylation (Eram et al. 2014, Powers et al. 2016). This 94 domain is distantly related to the classic SET domain found in the histone lysine 95 methyltransferases, a protein family involved in regulation of gene expression. The 96 crystal structure of the PRDM9 PR/SET domain suggests that the methyltransferase 97 activity of PRDM9 is autoregulated and the lone ZF located in the post-SET region is 98 involved in this regulation (Wu et al. 2013). 99 100 PRDM family members either directly methylate their histone targets or recruit other 5 101 histone modifiers to chromatin (Hohenauer and Moore 2012). Owing to these activities 102 the PRDM family is involved in a diverse array of developmental processes through the 103 regulation of gene expression (Hohenauer and Moore 2012). PRDM9, however, is 104 unique in that it also has the Kruppel Associated Box (KRAB) domain (Liu et al. 2014). 105 This leads to inclusion of PRDM9 in a second family of transcription factors, the KRAB- 106 ZFPs (KRAB Zinc Finger Proteins). Members of this family also contain a ZF DNA- 107 binding array, but have a KRAB domain instead of PR/SET (Lupo et al. 2013). 108 109 The KRAB domain is typically involved in gene silencing through interaction with the 110 TRIM28 protein, also known as KAP-1 (reviewed in (Iyengar and Farnham 2011)). 111 TRIM28 interacts with Heterochromatin Protein 1 (HP-1) and serves as a scaffold for 112 recruiting chromatin modifying enzymes and remodeling complexes to promoters of 113 target genes bound by the KRAB-ZFPs (Iyengar and Farnham 2011). Interestingly, the 114 KRAB domain of PRDM9 is missing the amino acids required for interaction with 115 TRIM28 (Liu et al. 2014), and recent studies found no direct interaction between these 116 two proteins (Patel et al. 2016; Imai et al. 2017). In fact, the KRAB domain of PRDM9 117 has higher similarity to the atypical KRAB domain found in the Synovial Sarcoma X 118 chromosome breakpoint (SSX) protein family (Lim et al. 1998). PRDM9 and the SSX 119 family members have another similarity: they both contain the SSX Repressive Domain 120 (SSXRD) following the atypical KRAB domain. Although the SSXRD was shown to be a 121 potent transcription repressor (Lim et al. 1998) the molecular mechanism behind this 122 function has not been described. 123 6 124 Considering that PRDM9 has three domains implicated in regulation of transcription it 125 was originally thought of as a transcription factor (Hayashi et al. 2005; Matsui and 126 Hayashi 2007; Mihola et al. 2009; reviewed in Nowick et al. 2013; Capilla et al. 2016). 127 This role was attractive, because Prdm9 is the only known speciation gene in mammals, 128 and the conflict between regulatory mechanisms is perceived as the most likely cause 129 of hybrid incompatibilities (Nowick et al. 2013; Mack and Nachman 2017). Although later 130 studies suggested a different role of Prdm9 in speciation (Smagulova et al. 2016; 131 Davies et al. 2016) they did not rule out the possibility of additional mechanisms that 132 may involve misregulation of transcription. Furthermore, it was recently reported that 133 during meiosis, PRDM9 binds to some active promoters (Grey et al. 2017), bringing 134 back the question of the possible regulatory function of PRDM9 in gene expression. In 135 this study we evaluate the potential role of PRDM9 in transcription and the role of 136 transcription-related domains within PRDM9 in PRDM9 function. 137 138 We first used mRNA sequencing to demonstrate that inactivation of Prdm9 does not 139 lead to changes in gene expression. We then showed that PRDM9 does not appear to 140 regulate miRNA expression as well.