This is the accepted version of the journal article: Vara, Covadonga; Ruiz-Herrera, Aurora. «Unpacking chromatin remodelling in germ cells : implications for development and evolution». Trends in Genetics, Vol. 38, Issue 5 (May 2022), p. 422-425. DOI 10.1016/j.tig.2021.10.007 This version is available at https://ddd.uab.cat/record/264009 under the terms of the license 1 Unpacking chromatin remodeling in germ cells: 2 implications for development and evolution 3 4 Covadonga Vara1,2 and Aurora Ruiz-Herrera1,2,* 1 5 Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autònoma 6 de Barcelona (UAB), Cerdanyola del Vallès, 08193, Spain 2 7 Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina (IBB), 8 Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès, 08193, Spain 9 *Correspondence: aurora.ruizherrera@uab.cat (A. Ruiz-Herrera) 10 11 ORCID: 12 Covadonga Vara 0000-0002-8264-9262 13 Aurora Ruiz-Herrera 0000-0003-3868-6151 14 15 Abstract: Germ cells reflect the evolutionary history and the future potential of a 16 species. Understanding how the genome is organised in gametocytes is fundamental to 17 understanding fertility and its impact on genetic diversity and evolution of species. 18 Here, we explore principles of chromatin remodeling during the formation of germ cells 19 and how these are affected by genome reshuffling. 20 21 Key words: HiC; TADs; compartments; spermatocytes; oocytes; chromosomal fusions 22 1 23 The spatial conformation of genomes arises from a close interplay between gene 24 function and chromatin organisation. It is now well-stablished that the hierarchical 25 organisation of the genome is widely conserved across mammalian species comprising 26 as it does, chromosome territories (Glossary) in which chromatin is organised into 27 compartments (open/closed), topologically associated domains (TADs) and DNA 28 loops [1]. However, it was not until recently that the basis of chromatin remodeling 29 during the formation of germ cells and early development began to be understood [2–4]. 30 Here we provide an overview of the structural and functional plasticity characterising 31 higher-order genomic organisation and how this is transmitted to offspring, with a focus 32 on evolution. 33 34 Principles of genome organisation in mammalian germ cells 35 Highly differentiated haploid gametes (sperm in males and oocytes in females) are 36 generated during gametogenesis; a regulated cellular process in which the proliferation 37 and differentiation of gonium reproductive cells is followed by meiosis, which consists 38 of two rounds of cell divisions (meiosis I and II). 39 40 Male and female gametogenesis show fundamental differences in timing, cell 41 morphology and cell cycle regulation. During spermatogenesis, chromatin undergoes 42 dramatic structural changes, mainly correlated with transcriptional activity and cohesin 43 occupancy (Figure 1). Moreover, meiotic progression is accompanied by chromatin- 44 transcriptional relationships in DNA promoters, where promoters with high CG content 45 present active transcription marks (H3K4me3, H3K9ac and H3K27ac) and promoters 46 with low CG content appear to be methylated [5] (Figure 1). Interestingly, between 20- 47 45% of DNA methylated promoters also harbor active transcription marks and 5hmC 2 48 modifications (the so-called atypical promoters), that allow for wave-specific 49 transcription despite methylation, giving to poised transcriptional regulators during 50 gametogenesis [5]. 51 52 As for the dynamics of chromatin remodeling during spermatogenesis, spermatogonia 53 present a somatic-like genome organisation, which harbor clear A/B compartments and 54 TADs but exhibit changes in chromosome occupancy, DNA methylation, histone 55 modifications and transcription, highlighting their commitment to enter meiosis (Figure 56 1) [4,5]. It is during prophase I when major chromatin remodeling takes place in 57 primary spermatocytes; homologous chromosomes are organised into DNA loop 58 anchored to the chromosomal axes formed by the axial element of synaptonemal 59 complex and meiotic cohesins (i.e., REC8 or RAD21L). Evidence suggests a weak 60 compartmentalization [2,4] that would serve to accommodate the major events that take 61 place during prophase I, such as chromosomal movements, chromatin condensation and 62 the formation and repair of DNA double-strand breaks (DSBs). Moreover, meiotic 63 chromosomes accommodate transcriptional activity associated with cohesin occupancy 64 [4] that could result from transient chromatin contacts and be locally regulated. The 65 molecular mechanisms behind cohesin based organisation of active transcription in 66 primary spermatocytes remain to be elucidated. 67 68 After male meiosis, haploid cells (round spermatids and sperm) adopt a distinctive 69 higher-order chromatin structure to accommodate histone-to-protamine transition and 70 cellular differentiation. Although A/B compartments are re-established in round 71 spermatids, TADs are not as defined as in other cell types, appearing more transient 72 (Figure 1). This phenomenon could represent an organisational preamble for the 3 73 spermiogenic chromatin remodeling, as flexibility and accessibility are needed for an 74 efficient protamine transition. Importantly, round spermatids harbor active transcription, 75 with cohesins present at the vicinity of promoters of genes relevant for fertilization and 76 embryogenesis [4]. Thus, the activation of transcription, which accompanies chromatin 77 remodeling during meiosis and spermiogenesis, might play a role in the development of 78 the future embryo by providing transcripts needed upon zygote genome activation. 79 80 In sperm, chromatin adopts a unique folding organisation in which highly condensed 81 DNA spatially constraints genome architecture. Chromosomes appear to be arranged 82 into chromosome territories, with increased pericentromeric interactions attributable 83 to centromere clustering into the chromocenter [4,6]. Moreover, sperm presents clear 84 A/B compartments and defined TADs with low variance scores defining their borders, 85 possibly linked to the sub-Mb scale toroidal organisation that characterises sperm 86 chromatin [7]. 87 88 In the case of female gametogenesis (oogenesis), late-stage oocytes present specific 89 chromatin configurations known as Polycomb-Associating Domains (PADs) (Figure 1). 90 PADs play a pivotal role during early embryogenesis, in which epigenetic 91 reprogramming of histone modifications and DNA methylation are essential for the 92 formation of the embryo [8] (see S1 in the supplemental information online). They are 93 compartment-like and cohesin-independent structures, which are marked by distinct 94 H3K27me3 profiles. PADs arise as A/B compartments that weaken in late-stage 95 oocytes, an organisational transition that might facilitate chromosomal segregation 96 [8,9]. Even though PADs have not been detected in MII oocytes, they are clearly 4 97 defined after fertilization at the 2-cell stage, in which maternal H3K27me3 might enable 98 the formation of PADs even before the zygote genome activation upon fertilization [10]. 99 100 Disruption of genome topology in germ cells by genome reshuffling 101 Exploring the implications of genome reshuffling on 3D genome folding in the germ 102 line can provide fertile grounds for investigating the evolutionary dynamics of genome 103 function and, ultimately, speciation. Large-scale chromosomal reorganisations can 104 potentially be fixed in a population eventually contributing to the formation of new 105 allelic variants on which natural selection can work (see S2 in the supplemental 106 information online). However, they can also trigger the development of inherited 107 diseases, genome instability and cancer by altering gene expression of the reorganised 108 genomic regions. 109 110 While it has been reported that disturbances of domain architecture due to inversions, 111 fusions or indels can lead to oncogene activation and novel gene functions [11], the role 112 of balanced chromosomal changes, such as Robertsonian (Rb) fusions, are just 113 beginning to be elucidated [12]. Rb fusions provide an example of genome plasticity, 114 representing the most common large-scale chromosomal structural change in nature. In 115 fact, there is evidence of Rb fusions having an impact on fertility as they are linked to 116 recurrent miscarriages and aneuploid offspring in humans [13]. However, recent data 117 suggest that the situation could be more complex and may also affect genome topology 118 in germ cells with associated functional and evolutionary implications [12]. 119 120 In fact, Rb fusions can drastically alter the 3D chromatin conformation in spermatocytes 121 and round spermatids [12] (Figure 2). This spatial chromosome reorganisation can 5 122 prompt novel interactions between domains, exposing them to new regulatory 123 environments that can potentially affect gene expression and/or regulation, as initially 124 proposed by the Integrative Breakage Model of genome architecture [14]. This can 125 have implications for both fertility and evolution that need further exploration. First, the 126 presence of new chromosomal interactions may rewire or attenuate gene networks, 127 providing new grounds for evolutionary novelty. Secondly, Rb fusions have a direct 128 impact on meiotic recombination, resulting in a detectable genomic footprint that has 129 implications for genetic diversity [12] (see S2 in the supplemental information online). 130 This represents the empirical demonstration of the interchromosomal effect [15], 131 showing that the presence of chromosomal fusions in the germ line can alter segregation 132 patterns. 133 134 Concluding remarks 135 The spatial folding of chromosomes, and their organisation in the nucleus, has a 136 significant regulatory impact on gene expression and, as a result, potentially important 137 evolutionary consequences. Understanding the structural and functional implications of 138 genome reshuffling in the germ line will be fundamental for elucidating the 139 evolutionary forces that drive genome plasticity. Here we have summarized how large 140 scale chromosome reorganisations (such as Rb fusions) can alter the 3D topology in 141 germ cells. Further functional studies on the effect of CTCF/cohesins mutations in 142 disrupting genome topology in germ cells will certainly provide new insights into our 143 understanding of the mechanism responsible of the heritability of 3D genome folding. 144 In this context, the advent of multidisciplinary approaches that combine computational 145 and experimental methods, underpinned by high-throughput technologies, will provide 146 impetus for the broader exploration of the functional and structural basis of genomes 6 147 reinforcing the link between the 3D genome architecture, developmental biology, 148 fertility and evolution. 149 150 Acknowledgments 151 We apologize to our colleagues whose work we were unable to cite due to space 152 restrictions. Research at A.R.H.’s laboratory is supported by the Spanish Ministry of 153 Economy, Industry and Competitiveness (CGL2014-54317-P, CGL2017-83802-P), the 154 Spanish Ministry of Science and Innovation (PID2020-112557GGB-I00) and the 155 Agència de Gestió d’Ajuts Universitaris i de Recerca, AGAUR (DI2015 and 156 SGR1215). C.V. was supported by a FPI predoctoral fellowship from the Ministry of 157 Economy, Industry and Competitiveness (BES-2015-072924). 158 159 Declaration of interests 160 None declared by authors. 161 162 References 163 1 Rao, S.S.P. et al. (2014) A 3D map of the human genome at kilobase resolution 164 reveals principles of chromatin looping. Cell 159, 1665–1680 165 2 Alavattam, K.G. et al. (2019) Attenuated chromatin compartmentalization in 166 meiosis and its maturation in sperm development. Nat. Struct. Mol. Biol. 26, 167 175–184 168 3 Patel, L. et al. (2019) Dynamic reorganization of the genome shapes the 169 recombination landscape in meiotic prophase. Nat. 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(2015) An Integrative Breakage Model of genome architecture, 197 reshuffling and evolution. BioEssays 37, 479–488 198 15 Lucchesi, J.C. and Suzuki, D.T. (1968) The interchromosomal control of 199 recombination. Annu. Rev. Genet. 2, 53–86 200 201 9 202 Figure Legends 203 Figure 1: Genome organisation in the mammalian germline and early stages of 204 embryonic development. (A) Representation of chromatin organisation during 205 spermatogenesis. Spermatogonia present a somatic-like organisation with the genome 206 folded into compartments (A and B) and subsequent TADs. Active transcription (RNA- 207 seq) correlates with A (open) compartments. Subsequently, there is an attenuation of 208 compartments and TADs in primary spermatocytes (here exemplified as leptonema, 209 zygonema, pachynema and diplonema stages). In pachynema and diplonema active 210 transcription correlates to meiotic cohesin occupancy in promoters of transcriptionally 211 active genes. Post-meiotic cells (round spermatids and spermatozoa) recover a somatic- 212 like configuration, although with particularities such as that TAD borders are not clearly 213 defined, but active transcription correlates with A compartments. Adapted from [4]. (B) 214 Representation of chromatin organisation in the female germline. Late oocyte stages 215 (growing oocytes (GO) I and II) show blurrier TAD organization than in somatic cells, 216 as transitioning to specific local chromatin-interacting regions known as Polycomb- 217 Associating Domains (PADs), which are associated to H3K27me3 marks. Chromatin 218 organisation dramatically changes in MII oocytes, where neither PADs nor other 219 folding configurations are detected. Adapted from [8]. (C) After fertilization, the PAD 220 organisation detected in late oocytes re-appears, being the most evident at the two-cell 221 stage of the embryo. Adapted from [8]. (D) Schematic representation of promoter 222 activity during gametogenesis (from spermatogonia to round spermatids). Active 223 promoters (green arrows) can be either the typical high CG-content promoters with 224 active transcription marks, or atypical promoters that present both methylation (black 225 lollipops) and 5hmC modifications (green lollipops) accompanied by active 10 226 transcription marks. Low-CG-containing promoters present DNA methylation and are 227 transcriptionally silent (red arrow). Adapted from [5]. 11 228 Figure 2: Impact of Rb fusions in genome folding. Schematic representation of 229 chromatin organisation described in standard (no chromosomal fusions) and Rb mice 230 (Robertsonian fusions) depicting chromosomes non-involved in Rb fusions 231 (chromosomes 1 and 2) and chromosomes involved in Rb fusions (chromosomes 3 and 232 8) as examples in (A) somatic interphase (fibroblasts), (B) primary spermatocytes 233 (meiotic cells) and (C) round spermatids (post-meiotic cells). Adapted from [12]. 234 Centromeres are presented as black circles whereas telomeres are shown as red circles 235 at one tip of the chromosomes. Two types of interactions are represented for each cell 236 type: genome-wide interactions and heterologous chromosomal interactions. Grey 237 represents no interactions, while the scale color towards red represents increasing 238 interactions, where orange represents detectable interactions and red highlights an 239 increase of interactions. (A) In somatic interphase, genome-wide interactions HiC maps 240 depict cis (diagonal for each chromosome) and trans interactions (shadowed squared 241 outside the diagonal). In the case of Rb mice, genome-wide HiC maps represent high 242 interactions between fused chromosomes (3 and 8). Representation of heterologous 243 interactions between pairs of chromosomes (1 vs. 2 and 3 vs. 8) shows high interaction 244 at the centromeric regions as the result of Rb fusions. (B) Genome-wide HiC maps in 245 primary spermatocytes present attenuated compartments in both standard and Rb mice 246 and centromeric interactions in fused chromosomes. Heterologous interactions maps 247 show how the presence of Rb fusions disrupts interaction patterns in both fused (3 vs. 8) 248 and non-fused (1 vs. 2) chromosomes. (C) Post-meiotic cells present a somatic-like 249 chromatin configuration genome-wide. The presence of the chromocenters (centromeric 250 associations present in mouse round spermatids) results in a reduction of heterologous 251 contacts genome-wide. As such, the presence of Rb fusions restricts interactions 252 between non-fused chromosomes in round spermatids. 12 253 13 254 Glossary: 255 Cohesins: Ring-shaped protein complexes that are essential for sister chromatid 256 cohesion. Additionally, they have a determinant role in the assembly of DNA 257 replication factories, chromosome condensation and mitotic spindle assembly, among 258 others. 259 Chromosome territories: Regions within the nucleus that are preferentially occupied 260 by specific chromosomes. Chromosome territoriality can be influencing by many 261 factors, such as chromosomal size, gene density, and gene expression. 262 Chromosome reorganisation: The reshuffling of chromosomal regions and can be 263 classified in unbalanced or balanced depending on whether they alter gene dosage. 264 Balanced reorganisations include inversions, reciprocal translocations, fissions, and 265 fusions, while unbalanced reorganisations include duplications and deletions. 266 Compartments: A hierarchical level of the 3D genome organisation provided by the 267 first principal component of Hi-C interaction matrices and captured by the 268 correspondent eigenvector, which discriminates between interaction frequencies. 269 Chromosomes are organised in A/B compartments that can vary between 1 to 10 Mb in 270 mammals. 271 DNA Double-Strand Breaks: Programmed DNA breaks catalyzed by the Spo11 272 endonuclease during early stages of the first meiotic division. 273 Gametogenesis: The biological process by which haploid gametes are generated. This 274 process is divided into two main stages: (i) proliferation and differentiation of gonium 275 and (ii) meiosis, which consists of two rounds of cell divisions (meiosis I and II). 276 Integrative Breakage Model: A multidisciplinary hypothesis for the study of genome 277 plasticity that considers that genomic regions involved in evolutionary reshuffling: (i) 278 interact physically inside the nucleus during the formation of the germ line, (ii) present 14 279 accessible epigenetic features providing structural and functional accessibility, and (iii) 280 only those reorganisations that do not disturb essential genes and/or gene expression 281 will likely be fixed within populations, thus providing grounds for evolution. 282 Interchromosomal effect: The mechanism by which chromosomes involved in 283 chromosome reorganisations are proposed to interfere with correct segregation during 284 meiosis of other chromosomes not involved in reorganisations. 285 Meiosis: Specialized cell division in sexually reproducing organisms that produce 286 haploid gametes. 287 Recombination: The exchange of genetic material between homologous chromosomes 288 during the first meiotic division. 289 Robertsonian fusion: A chromosome rearrangement involving centric fusion of two 290 non-homologous acrocentric chromosomes to form a single metacentric chromosome. 291 Synaptonemal complex: A proteinaceous scaffold, which consists of two lateral 292 elements and a central region that includes the central element and the transverse 293 filaments. The synaptonemal complex maintains the physical connection of homologous 294 chromosomes during the prophase of the first meiotic division and plays a role in 295 meiotic recombination. 296 Topologically associated domains: Represent genomic loci that preferentially interact 297 with the neighboring cis chromatin domains rather than with other regions, conforming 298 functional chromatin domains. 15