JB Special Review—Chromatin Structure and Function: Biological Implications in Epigenetics Histone variants: essential actors in male genome programming Received September 6, 2017; accepted October 13, 2017; published online November 20, 2017 Naghmeh Hoghoughi, Sophie Barral, Alexandra Vargas, Sophie Rousseaux and Saadi Khochbin* CNRS UMR 5309, Inserm, U1209, Universite´ Grenoble Alpes, Institut Albert Bonniot, Grenoble F-38700, France *Saadi Khochbin, Chromatin and Cell Signaling, Institute for Advanced Bioscience, Domaine de la Merci, La Tronche 38706, France. Tel: +33 4 76 54 95 83, Fax: 33 4 76 54 95 95, email: saadi.khochbin@univ-grenoble-alpes.fr Prior to its transmission to the offspring, the male genome has to be tightly compacted. A genome-scale histone eviction and the subsequent repackaging of DNA by protamines (Prms) direct this essential genome condensation step. The requirement for male germ cells to undergo such a dramatic and unique genome reorganization explains why these cells express the largest number of histone variants, including many testis-specific ones. Indeed, an open chromatin, nucleo- some instability and a facilitated process of histone dis- assembly are direct consequences of the presence of these histone variants in the chromatin of male germ cells. These histone-induced changes in chromatin first control a stage-specific gene expression program and then dir- ectly mediate the histone-to-Prm transition process. This review aims at summarizing and discussing a series of recent functional studies of male germ cell histone vari- ants with a focus on their impact on the process of his- tone eviction and male genome compaction. Keywords: H2A.L.2; H3.3; protamines; TH2B; tran- sition proteins. Spermatogenesis is a differentiation process which pro- duces mature spermatozoa, which will deliver the male genome to the oocytes in the female organism. Spermatogenic cells are continuously produced by an asymmetrical division of progenitor cells, named sper- matogonia. These cells on the one hand maintain the stem cell population and on the other hand generate meiotic cells, or spermatocytes, which themselves undergo two successive meiotic divisions. The second meiotic division produces haploid cells known as sperm- atids. The early post-meiotic spermatids are called round spermatids. These cells activate a specific gene expression program that will direct their transformation into spermatozoa, through a series of reorganizations of their various cellular constituents. These reorganiza- tions mostly take place in the subsequent stages of post- meiotic differentiation, in cells known as elongating and condensing spermatids. Large-scale histone post-trans- lational modifications (PTMs), essentially histone acetylation, signal and regulate the initiation of the process of histone-to-protamine (Prm) replacement in elongating spermatids (13). Although still largely unexplored, this genome-wide replacement of histones by Prms is the most dramatic of the known processes involved in chromatin remodelling, genome reorganization and reprogramming (4). In add- ition to Prms, early investigations also identified other basic non-histone proteins that appear at the time of histone replacement but, in contrast to Prms, totally disappear afterwards (5). These proteins have been named transition proteins (TPs). The early studies of sperm and testis histones in various species also revealed the existence of histones that are only expressed in male germ cells (6). Indeed testis-specific histones were among the first histone variants identified. Additionally, spermatogenic cells not only express a large number of various testis-specific histones, but are also characterized by the presence of some histone vari- ants, which almost entirely replace their canonical coun- terparts (Fig. 1). Therefore, prior to histone replacement, spermatogenic cell chromatin accumulates unique germline histones in replacement of their canon- ical counterparts. Histone variants, including the testis- specific ones, will eventually be replaced by Prms. An open question is whether and how this large number of histone variants, individually and collectively, are involved in directing the spermatogenic gene expres- sion program and in preparing chromatin for the final histone-to-Prm transition. Additionally, it is critical to decipher the individual contribution of each of the spe- cific or generalist histone variants to these unique events. Another related question is the interplay between his- tones, TPs and Prms in the process of histone eviction. Most of these questions have remained obscure until recently. It is only during the last few years, a long time after the discovery of the first testis-specific histone variants that several pilot studies have started to shed light on the role of histone variants in the stepwise pro- graming of the male genome, in the eviction of histones and in the final genome compaction. Here, we summarize these recent advances in our knowledge of the role of histone variants, specifically testis-specific histone variants, in spermatogenesis, with a specific focus on the process of genome-wide histone eviction and Prm assembly. TH2B One of the first histone variants discovered belongs to the H2B family and was named TH2B (TSH2B, according to the new nomenclature). Indeed, the protein was Featured Article J. Biochem. 2018;163(2):97–103 doi:10.1093/jb/mvx079 ß The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. 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