Transcriptome Profiling of Histone Acetylation and Methylation Writers and Erasers Families Across Male Germ Cell Development and Pre-Cleavage Zygote

20 Accumulating evidence put on the focus that male spermatogenesis presents windows of 21 vulnerability for epigenetic reprogramming by environmental stressors that not only can 22 affect fertility but even transmit developmental, metabolic, and behavioral traits to 23 offspring. This work analyzed the transcriptomic programs of epigenetic enzymes involved 24 in histone K acetylation and methylation from undifferentiated spermatogonia to mature 25 sperm and pre-cleavage zygote in the mouse. We dissected the peak expression at each 26 stage for families and individual enzymes, their target histone PTMs functions and reported 27 dynamics during spermatogenesis and early embryogenesis. This approach allowed us to 28 identify epigenetic enzymes with known functions during spermatogenesis and, by the rule 29 of “guilty by association”, many others not even described yet in male germ cell 30 development. Moreover, some of these enzymes’ mRNA were detected higher in the pre31 cleavage zygote than in the transcriptionally silent MII oocyte, suggesting a paternal 32 contribution. Our study contributes to the mechanistic aspects behind transgenerational 33 epigenetics, described for the first time in such a comprehensive approach. 34 35 36 INTRODUCTION 37 In the last years, accumulating research has documented that male germ cell 38 maturation presents windows of vulnerability for epigenetic reprogramming by 39 environmental stressors that not only can affect fertility but even transmit developmental, 40 metabolic and behavioral traits to offspring (Bale, 2015). This type of non-genetic 41 “Lamarckian” transmission has been established for paternal lifestyle and different forms of 42 chronic stress, drug abuse, dietary change, and social defeat, involving changes in non43 coding RNAs cargo, DNA methylation, and histone PTMs (Bale, 2105). However, most of 44 the research in epigenetic inheritance has documented the role of non-coding RNAs and 45 DNA methylation patterns, whereas the mechanisms by which the environment can trigger 46 specific changes on histone PTMs are still largely unexplored (Skvortsova et al., 2018). 47 Spermatogenesis is a finely regulated process where unipotent spermatogonia 48 undergo meiosis and, subsequently, spermiogenesis, to become sperm cells. During 49 developmental stages of spermatogenesis, male germ cells experience dramatic chromatin 50 reorganization, where approximately 90% (human) to 95% (mouse) of histones are evicted 51 and replaced by protamines to compact the paternal genome (Rajender et al., 2011, Carrel 52 2012). Importantly, a small fraction of histones in the sperm genome is retained on specific 53 locations and carries several post-translational modifications (PTMs) that play critical roles 54 in epigenetic regulation of spermatogenesis and early embryonic development (Luense et 55 al., 2016). The epigenetic program of histone PTMs that controls spermatogenesis is driven 56 by enzymatic families known as writers and erasers of the so-called “histone code”, that 57 modulate chromatin structure and transcription (Kouzarides, 2007). These epigenetic 58 enzymes catalyze the deposition or removal of specific PTMs such as acetylation, 59 methylation, phosphorylation, crotonylation, etc, at specific amino acid residues on the 60 nucleosome’s core histones H2A, H2B, H3, and H4, and also on the linker H1/H5 (Luense 61 et al., 2016). In male germ cells, histone PTMs not only control active vs repressed 62 chromatin states but also a range of processes including DNA replication and repair, 63 chromosome maintenance and histone eviction (Luense et al., 2016). So far, the most 64 studied histone PTMs related to the control of germ cells mitosis, meiosis, spermiogenesis 65 and early embryo development are methylated and acetylated lysines (K) on H2A/B, H3 66 and H4. Histone K acetylation can be dynamically regulated by the opposing action of 67 acetyltransferases (HATs) and deacetylases (HDACs). Acetylation of K residues 68 neutralizes the positive charge on histones, allowing DNA-binding proteins better access to 69 the DNA and resulting in activation of gene expression as well as histone eviction 70 (Rajender et al., 2011). Unlike acetylation, methylation does not affect histone charge but 71 regulates recognition and interaction with chromatin-binding proteins that control the 72 transcription or respond to DNA damage (Rajender et al., 2011). Histone K methylation is 73 finely regulated by methyltransferases (KMTs) and demethylases (KDMs) that control the 74 mono-, di-, and/or tri-methylation of specific residues, and this can either activate or repress 75 transcription depending on the residue position, the number of methylations and the 76 presence of other methyl or acetyl groups in the vicinity (Black et al., 2012). 77 The role of epigenetic enzymes comes to focus, as they are responsible for the 78 histone’s PTMs writing and erasing balance. Moreover, histone-modifying enzymes have 79 been shown to respond to the organism physiology and metabolism, several disease 80 conditions, and environmental stressors (Fan et al., 2016; Nebbioso et al., 2018; Gonzalez 81 et al., 2020). Even though epigenetic enzymes are the bridge linking the environment with 82 epigenetic inheritance through retained histones PTMs, an in-depth analysis of their 83 expression patterns throughout the spermatogenic process is still lacking. Moreover, these 84 enzymes provide new targets for therapies of numerous diseases (Prachayasittikul et al., 85 2017) but the potential effect of these compounds on epigenetic inheritance is generally 86 overlooked. Here, we curated transcriptomic data from spermatogonia to mature sperm 87 populations and pre-cleavage zygote and analyzed the expression profile and dynamic 88 changes of all the known families of histone K acetylation and methylation writers and 89 erasers. We provide a complete picture of the epigenetic enzymes across the 90 spermatogenesis in mice and the developmental windows when the transcription of these 91 enzymes may be more susceptible to environmental disruption. Not only we expand the 92 knowledge on enzymes with recognized roles in spermatogenesis, but provide evidence of 93 many more whose role in male germ cells development and zygote have not been described 94