Regulation of X-Linked Gene Expression During Early 3 Mouse Development by Rlim 4 5 6 Feng Wang1‡, Jongdae Shin1,2‡, Jeremy M

1 2 Regulation of X-linked gene expression during early 3 mouse development by Rlim 4 5 6 Feng Wang1‡, JongDae Shin1,2‡, Jeremy M. Shea3, Jun Yu1, Ana Bošković3, Meg Byron4, 7 Xiaochun Zhu1, Alex K. Shalek5,6,7, Aviv Regev6,8, Jeanne B. Lawrence4, Eduardo M. 8 Torres1 , Lihua J. Zhu1,9, Oliver J. Rando3, Ingolf Bach1* 9 10 1Department of Molecular, Cell and Cancer Biology, University of Massachusetts 11 Medical School, Worcester, MA 01605 12 2Department of Cell Biology, College of Medicine, Konyang University, Daejeon, 13 302-832, Korea 14 3Department of Biochemistry and Molecular Pharmacology, University of Massachusetts 15 Medical School, Worcester, MA 01605 16 4Department of Cell and Developmental Biology, University of Massachusetts Medical 17 School, Worcester, MA 01605 18 5Department of Chemistry and Institute for Medical Engineering & Science, 19 Massachusetts Institute of Technology, Cambridge, MA, 02139 20 6Broad Institute of MIT and Harvard, Cambridge, MA 02142 21 7Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, 02139 22 8Howard Hughes Medical Institute, Department of Biology, MIT, Cambridge, MA 02140 23 9Program in Bioinformatics and Integrative Biology, University of Massachusetts 24 Medical School, Worcester, MA 01605 25 26 * Corresponding author 27 Tel: 508 856 5627 28 Fax: 508 856 4650 29 [email protected] 30 31 ‡ equal contribution 32 33 34 35 key words: preimplantation development, whole embryo RNA-seq, mouse genetics, X 36 chromosome, epigenetic gene silencing, X chromosome inactivation, X upregulation, 37 Rlim, Rnf12, Xist 38 39 1 40 Abstract 41 Mammalian X-linked gene expression is highly regulated as female cells contain two 42 and male one X chromosome (X). To adjust the X gene dosage between genders, 43 female mouse preimplantation embryos undergo an imprinted form of X 44 chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and 45 the long non-coding RNA Xist. Moreover, it is thought that gene expression from the 46 single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. 47 We have combined mouse genetics with RNA-seq on single mouse embryos to 48 investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our 49 results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist 50 clouds and X-silencing in female embryos at blastocyst stages, while initial Xist 51 expression appears Rlim-independent. We find further that X/A upregulation is 52 initiated in early male and female preimplantation embryos. 53 54 55 56 57 58 59 60 61 2 62 Introduction 63 Most mammalian cells contain two transcriptionally active copies of autosomal 64 chromosomes but only one active X chromosome (X). This is due to the fact that female 65 cells inactivate one X in a process known as X chromosome inactivation (XCI), to correct 66 for male/female (F/M) gene dosage imbalances caused by the presence of two X 67 chromosomes. In addition, to adjust for X/autosomal (X/A) imbalances arising from 68 transcription of both autosomal copies of most genes, it is thought that male and female 69 cells upregulate gene expression from the single active X two-fold. 70 Beginning at the 4-cell stage in female mouse embryos, imprinted XCI (iXCI) 71 exclusively silences the paternally inherited X (Xp), and this pattern of XCI is maintained 72 in extraembryonic trophoblast cells. In contrast, epiblast cells in the inner cell mass 73 (ICM) of blastocysts that will give rise to the embryo reactivate the Xp and undergo a 74 random form of XCI (rXCI) around implantation (E5-E5.5) (Disteche, 2012, Payer and 75 Lee, 2014, Galupa and Heard, 2015). During XCI the long non-coding RNA Xist 76 progressively paints the X chromosome from which it is synthesized, thereby triggering 77 repressive histone modifications including H3K27me3 (Plath et al., 2003) and 78 transcriptional silencing of X-linked genes (Disteche, 2012, Payer and Lee, 2014, Galupa 79 and Heard, 2015). Xist is required both for iXCI and rXCI (Marahrens et al., 1997, Penny 80 et al., 1996). The X-linked gene Rlim encodes a ubiquitin ligase (Ostendorff et al., 2002) 81 that shuttles between the nucleus and cytoplasm (Jiao et al., 2013) and modulates 82 transcription via regulating nuclear multiprotein complexes (Bach et al., 1999, Gungor et 83 al., 2007). Rlim promotes the formation of Xist clouds both during iXCI in female mice 3 84 (Shin et al., 2010) and in female embryonic stem cells (ESCs) undergoing rXCI in culture 85 (van Bemmel et al., 2016). In mice, however, Rlim is dispensable for rXCI in epiblast 86 cells (Shin et al., 2014). While both Rlim and Xist play essential roles during iXCI in 87 mice (Marahrens et al., 1997, Shin et al., 2010), questions remain with regards to their 88 functions on the general kinetics of X-linked gene expression, their functional 89 interconnection and the contribution of maternally vs embryonically expressed RLIM for 90 iXCI. Moreover, while evidence for X/A upregulation was observed not only in adult 91 mouse tissues but also in mouse ES cells and epiblast cells at blastocyst stages (Deng et 92 al., 2013, Deng et al., 2011, Lin et al., 2007), details on the developmental X upregulation 93 are lacking. 94 We have carried out RNA-seq on single mouse embryos to investigate the regulation 95 of X-linked gene expression during pre- and peri-implantation development by Rlim. 96 Analyses of WT, Rlim-knockout (KO) or Xist-KO embryos reveal that Rlim is required for 97 maintenance of Xist clouds and iXCI in female embryos at blastocyst stages. In addition, our 98 data uncover that X dosage compensation via iXCI in female mice occurs concurrently with a 99 general X/A upregulation in both male and female preimplantation embryos. These results 100 represent a comprehensive view on the regulation of X-linked gene expression during 101 early mouse embryogenesis by Rlim and Xist. 102 103 Results 104 Elucidation of the mouse preimplantation transcriptome by single embryo RNA-seq 105 Several studies have investigated the dynamics of XCI and X-silencing in female ES cells 4 106 (Lin et al., 2007, Marks et al., 2015) or trophoblasts (Calabrese et al., 2012) using RNA- 107 seq. To assess the general dynamics of X-linked gene expression and iXCI in vivo, we 108 have adapted single cell RNA-seq technology (Shalek et al., 2013, Jaitin et al., 2014) to 109 elucidate the pre- and peri-implantation transcriptome using single mouse embryos 110 (Sharma et al., 2016). This is a valid strategy because 1) early embryos consist of a 111 limited number of cell types: essentially one totipotent cell type up to E3, and three cell 112 types at blastocyst stages - epiblast, trophoblast and primitive endoderm cells - that 113 express known marker genes 2) cells of preimplantation embryos undergo iXCI that 114 exclusively silences the Xp and 3) in mice there is only a relatively small number of 115 genes that escape XCI (Yang et al., 2010, Finn et al., 2014). Thus, using RNA-seq we 116 examined embryos at the 4- and 8-cell stages, early and late morula (E2.5 and E3.0), and 117 blastocyst stages (E3.5, E4.0 and E4.5), comparing global changes in gene expression. 118 We also included trophoblasts isolated from blastocyst outgrowths of cultured E4.0 119 embryos in our analyses. In contrast to previous studies on single cells of preimplantation 120 embryos that were performed in a mixed genetic background (Deng et al., 2014), 121 embryos were generated in a C57BL/6 background to exclude potential background 122 influences on the general kinetics of iXCI and/or X upregulation. Moreover, because the 123 known functions of Rlim during mouse embryogenesis are restricted to XCI in females 124 (Shin et al., 2010, Shin et al., 2014), RNA-seq experiments were performed on WT and 125 RlimKO embryos. Based on a mouse model allowing a conditional KO (cKO) of the Rlim 126 gene, we have previously generated females carrying a maternally transmitted Sox2-Cre- Δ 127 mediated cKO allele and a paternally transmitted germline KO allele (RlimcKOm/ p-SC). 5 128 These females appear healthy, fertile and lack RLIM in all somatic tissues as well as their 129 germline (Figures 1A, B) (Shin et al., 2014). Thus, to efficiently generate male and 130 female germline Rlim KO embryos (Δ/Y, Δ/Δ) and to eliminate potential influences of 131 maternal RLIM in oocytes on the iXCI process, all Rlim KO embryos were generated by Δ Δ 132 crossing RlimcKOm/ p-SC females with germline KO males (Rlim /Y) (Figure 1 – figure 133 supplement 1A). RNA was prepared and barcoded RNA-seq libraries were constructed 134 from 187 samples that were distributed between two 96-well plates, each with similar 135 numbers of embryos from each stage and mating. Libraries were pooled and sequenced to 136 an average read depth of 2.95 million reads. This read depth lies within the range 137 typically obtained by using single-cell technology (Shalek et al., 2013). The gender of 138 each embryo was determined by assessing expression of Y-linked genes, which occurs 139 only in male embryos, and Xist, which is expressed only in females (Figures 1C, D). 12 140 samples for which gender could not be assigned or displayed less than 280,000 total reads 141 were removed, leaving 175 samples for further analysis (Figure 1 – figure supplement 142 1B; Supplementary file 1). Mapping reads from WT and KO embryos to the Rlim locus 143 showed that the deleted region in KO embryos was not represented (Figure 1E; data not 144 shown), validating both the mating strategy and the specificity of the data obtained via 145 whole embryo RNA-seq.

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