Mammalian Development and Evolution

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

KZFPs are rapidly evolving transcriptional repressors that first emerged in the common ancestor of coelacanth, birds, and tetrapods (1). They make up the single largest family of transcription factors in mammals (estimated to be more than 300-600 in humans and mice, respectively). Despite their abundance, little is known about their physiological functions. KZFPs consist of two conserved domains: 1) an N-terminal KRAB domain that binds the corepressor KAP1, which recruits the histone methyltransferase SETDB1 and heterochromatin protein 1 (HP1) to initiate heterochromatic silencing (2-4) and 2) a variable number of tandem C2-H2 type zinc finger domains (referred to as a zinc finger array) that mediates DNA binding. Each zinc finger within an array coordinates a zinc ion (using two cysteines and two histidines) and utilizes 3-4 amino acids at specific locations relative to a helical turn (referred to as “fingerprint” amino acids) that make base specific contacts with DNA (on three nucleotides on one strand and one nucleotide on the opposite strand). Arrays of zinc fingers “grip” DNA via consecutive interactions by individual fingers that coil around the major groove to achieve highly specific binding. The average KZFP in humans contains ~nine zinc fingers (5), and thus KZFPs can bind to long DNA motifs. Both the KRAB domain and zinc finger arrays found on KZFPs are being utilized in modern bioengineering approaches: the KRAB domain as a potent transcriptional silencer and the zinc finger array as a programmable DNA binding domain, often fused to nucleases for genome editing/engineering approaches. KZFPs are unique from other transcription factors in their sheer number and large DNA binding domains, but they are also remarkable from an evolutionary standpoint. Analysis of KZFP genes across the tree of life revealed that the gene family arose through repeated rounds of gene duplications followed by functional divergence (5). The KZFP gene family is thought to have originated with the meiotic recombination factor Prdm9 (6), the oldest gene that contains both a KRAB domain and zinc finger array (discussed further below). PRDM9’s KRAB domain, however, fails to interact with KAP1. Furthermore, PRDM9 contains an additional SET domain that methylates histone H3 on K4 and K36 (7). Other than PRDM9 (and the closely related PRDM7), and a handful of older KZFPs that also contain SCAN or DUF domains, the vast majority of KZFPs have only the KRAB and C-terminal zinc finger arrays (8, 9). Detailed phylogenetic analysis of the zinc fingerprint amino acids of all extant KZFPs makes it possible to determine orthologous relationships of KZFPs across species and thus the birthdate of most KZFP genes (1). This analysis has revealed a dramatic expansion of the KZFP gene family in the earliest mammals, with subsequent expansions along various evolutionary lineages, including a recent expansion in mice (10). These expansions of KZFP genes coincide with the accumulation of endogenous retroviruses and LTR retrotransposons in vertebrates (11). This observation first suggested that KZFP gene family expansion may be a response to retroviral invasion of the germline. Since these findings, we and others have provided several lines of direct evidence that KZFP family members play a direct role in ERV/TE silencing. First, the KZFP protein ZFP809 was isolated based on its ability to bind to the primer binding site for proline tRNA (PBSPro) of murine leukemia virus (MuLV) (12) and we found that Zfp809 knockout mice display loss of H3K9me3 at a set of ERVs containing the PBSpro motif (including VL30 elements), leading to activation of the “VL30Pro” elements in Zfp809 KO tissues (13). Second, deletion of the KZFP co-repressors Trim28 or Setdb1 leads to activation of many ERVs/TEs (14, 15). Third, recent ChIP-seq screens have revealed that the majority of human and mouse KZFPs bind primarily to specific TE families (1, 16-19). Finally, we demonstrated that deletion of entire clusters of KZFP genes caused ERV and TE activation phenotypes in mouse ESCs and tissues, resulting in increased rates of somatic retrotransposition in some cases (19). As mobile genetic elements, TEs are a major source of genetic diversity and mutagenic potential; the list of genetic diseases caused by TE activity is long and growing (20). It is thus essential to understand how TE activity is regulated. We have begun a systematic interrogation of KZFP function as a potential adaptive repression system against TEs (Project 1). The laboratory mouse has proven to be an exceptional model for understanding KZFP gene function because mice have recently undergone large expansions in KZFP gene number since their split with other rodents, while their genomes have also been infiltrated with numerous ERVs that remain active. Furthermore, mice also contain several dozen KZFP genes that emerged in the earliest mammals that have been maintained for over 100 million years by purifying selection. These genes open a window to the dawn of mammals and have been providing clues into the critical adaptations that coincided with viviparity. Our studies of KZFPs have also led us into a surprising but exciting new direction: the control of meiotic recombination by the ancestral member of the KZFP family, PRDM9 (Project 2). PRDM9 specifies meiotic recombination hotspots by binding DNA through its rapidly evolving zinc finger array and methylating histone H3 tails which results in SPO11 generated DSBs at hotspots (21-24). By exploring PRDM9’s unique histone methyltransferase domain that deposits both histone H3K4me3 and H3K36me3 marks at hotspots, we have identified two members of a family of conserved dual histone methylation readers, ZCWPW1 and ZCWPW2. These factors mediate key steps in DNA double strand break formation and repair during meiotic recombination (25-27). Our ongoing studies explore the mechanisms of action of this novel histone writer/reader system in hotspot specification, that includes studies in the genetically tractable mouse model. We will further expand our efforts to include translational studies in humans with infertility, in a new partnership with the NICHD Reproductive Endocrinology and Infertility training program and Shady Grove Fertility Center, which operates in Maryland. These studies will shed important light on the role that hotspot specification machinery plays in human germ cell development and infertility. Progress Report Project 1: KZFP and TE function in mammalian development and evolution KZFP diversification in response to TEs KZFPs comprise the largest family of mammalian transcription factors, rapidly evolving within and between species. Most KZFPs repress endogenous retroviruses (ERVs) and other retrotransposons, with KZFP gene numbers correlating with the ERV load across species, suggesting co-evolution (28, 29). How new KZFPs emerge in response to ERV invasions is currently not known. Using a combination of long read sequencing technologies (PacBio HiFi and ONT Ultra long reads) and de novo genome assembly, we presented a first detailed comparative analysis of young KZFP gene clusters in the mouse lineage, which has undergone recent KZFP gene expansion and ERV infiltration. Detailed annotation of KZFP genes in a cluster on Mus musculus Chr4 revealed parallel expansion and diversification of this locus in different mouse strains (C57BL6J, 129S1/SvImJ and CAST/EiJ) and species (Mus spretus and Mus pahari). Our data supports a model by which new ERV integrations within young KZFP gene clusters likely promoted recombination events leading to the emergence of new KZFPs that repress them. At the same time, ERVs also increased their numbers by duplication instead of retrotransposition alone, unraveling a new mechanism for ERV enrichment at these loci. ZFP661 regulates clustered PCDH diversity in the mammalian brain Through analysis of our own and published ChIP-seq data for KZFPs in mice and humans, we identified ZFP661 (ZNF2 in humans) as a KZFP that is strongly associated with CTCF binding (1, 30). CTCF plays a crucial role in organizing the genome into large Topologically Associated Domains, or TADs, as well as other smaller scale genomic loops, by serving as a barrier to the cohesin complex, which utilizes ATP to extrude chromatin (31-33). We found that ZFP661 binding was always located within CTCF barriers, adjacent to CTCF motifs, in a position overlapping the typical position occupied by “trapped” cohesin. There was also a strong “head-to-head” bias of ZFP661 motifs relative to CTCF motifs. Using Zfp661 gain- and loss-of-function models and capture Hi-C analyses in mouse ESCs, we demonstrated that ZFP661 binding adjacent to CTCF promoted cohesin bypassing CTCF barriers. Using in vitro protein:protein interaction assays and CoIP experiments, we demonstrated that this bypassing function is achieved via ZFP661 direct binding to CTCF, which directly competes for cohesin binding. Our results unveiled a novel mechanism that regulates the permeability of CTCF barriers to cohesin. Despite the strong effect of ZFP661 on a relatively small number of CTCF/cohesin barriers, we found that Zfp661 KO mice were viable with no overt phenotypes. We reasoned it may play a more subtle role in development, and focused on the developing nervous system, where Zfp661 displayed somewhat elevated expression. Using gene ontology analysis of the small number of ZFP661 binding sites we were led to a fascinating gene cluster, the aptly named “clustered protocadherin” or cPcdh genes. cPcdh genes plays a crucial role in neuronal self-avoidance and in neural dendritic projection during development (34-39). A small number amongst dozens of alternative cPcdh isoforms are expressed in each neuron via a tightly controlled alternative promoter selection mechanism mediated by CTCF and cohesin, with CTCF binding sites found at each of the dozens of alternative cPcdh promoters (40-42). The expressed cPcdh isoforms in each cell create a unique cell surface protocadherin “barcode” that allows each neuron to identify “self” from “other” via a phenomenon called contact dependent repulsion. This is mediated by homophilic interactions of protocadherins across neurons (43, 44). We demonstrated that ZFP661 binds to only a few of the cPcdh promoters/CTCF barriers at the cPcdh locus that are most proximal to an upstream enhancer. Using single cell RNA-seq and capture Hi-C analysis, we demonstrated that the loss of Zfp661 caused cohesin to be trapped at these proximal barriers, resulting in greater association of the upstream enhancer with proximal promoters, and more frequent usage of these proximal isoforms in individual neurons. These results demonstrate that ZFP661 delicately balances cPcdh isoform usage by enhancing their overall expression diversity, by allowing more frequent use of the many enhancer distal isoforms. As a consequence, loss of Zfp661 caused reduced cortical dendritic projection and autism-like social defects in mice, phenocopying mice harboring deletion of several cPcdh genes (45-47). Both genetic variation at the cPcdh locus and copy number variation of the human ortholog of Zfp661, ZNF2, are associated with autism, and ZNF2 binds adjacent to CTCF sites at the human cPCDH locus, suggesting this mechanism is conserved across mammals (48-50). Our study unveils a mechanism that finely tunes CTCF barrier permeability by modulating cohesin trapping and demonstrates the adaptation of this mechanism to enhance cPcdh diversity for increased cortical dendritic projection in more densely packed mammalian brains. TEs and the evolution of the regulatory landscape in the placenta In mammals, the placenta mediates maternal-fetal nutrient and waste exchange and provides immunomodulatory actions that facilitate maternal-fetal tolerance (51). The placenta is highly diversified among mammalian species, yet the molecular mechanisms that distinguish the placenta of human from other mammals are not fully understood. Using an interspecies transcriptomic comparison of human, macaque, and mouse placentae, we identified hundreds of genes with lineage-specific expression – including dozens that are placentally-enriched and potentially related to pregnancy (52). We further annotated the enhancers for different human tissues using epigenomic data and demonstrate that the placenta and chorion are unique in that their enhancers display the least conservation relative to other organs. We identified numerous lineage-specific human placental enhancers, and found they are over-represented for specific families of endogenous retroviruses (ERVs), including MER21A, MER41A/B and MER39B. Among these ERV families, we further demonstrate that MER41 insertions create dozens of lineage-specific Serum Response Factor (SRF) binding loci in human. These binding sites include one adjacent to FBN2, a placenta-specific gene with increased expression in humans that produces the peptide hormone placensin to stimulate glucose secretion and trophoblast invasion (53). Our results demonstrate that lineage-specific human placental enhancers are frequently derived from ERV insertions, likely facilitating the lineage-specific evolution of the placenta. Project 2: The control of meiotic recombination hotspots by PRDM9 ZCWPW2 is essential for efficient DSB formation and synapsis In mice and humans, meiotic recombination occurs across thousands of 1-2kb regions called “hotspots” determined by the rapidly evolving DNA binding domain of the oldest KZFP, PRDM9. PRDM9 is unique from other KZFPs in that it possesses an accessory PR/SET domain that methylates histone H3 on K4 and K36 at meiotic hotspots, and this activity is essential for the formation of DNA double strand breaks (DSBs) at PRDM9 directed hotspots that initiate crossovers (7). In Prdm9 null mice, DSBs are still produced in spermatocytes, but are instead located at default positions near gene transcription start sites (TSSs) (54). We have identified the dual histone methylation reader proteins ZCWPW1 and ZCWPW2, that contain two highly conserved H3K4me3 and H3K36me3 reader domains called Zf-CW and PWWP. We demonstrate that in vitro, ZCWPW1 and ZCWPW2 interact with histone peptides containing the dual H3K4me3 and H3K36me3 modifications with high affinity (27, 55). Importantly ZCWPW2 is tightly co-expressed with PRDM9 during meiosis, and like its paralog ZCWPW1, binds to the genome in a PRDM9-dependent fashion. We demonstrate that ZCWPW2 is a critical meiotic factor that plays essential roles in synapsis and DSB positioning. Zcwpw2 mutants are sterile due to meiotic arrest caused by synapsis defects that are more severe than both Prpm9 or Zcwpw1 mutants, with fewer chromosomes synapsing on average and a larger number of “partner switching” events, suggesting Zcwpw2 plays at least a partial role independent of PRDM9 activity. DNA DSBs are also partially, but not completely, relocated towards default sites in Zcwpw2 mutants reminiscent of Ankrd31 mutants (56). We propose that ZCWPW2 plays a critical role in linking PRDM9-dependent histone marks to the DSB machinery, outcompeting the ancestral machinery that posits DSBs at TSSs, as well as a separate role in facilitating homology search and synapsis. Uncovering principles and consequences of hotspot evolution by mapping PRDM9 binding Prdm9 KO mice are sterile and PRDM9 variants/mutations have been associated with human infertility (57-59). PRDM9’s ZNF array is rapidly evolving, leading to differential hotspot usage between individuals. Although humans and mice have >100 distinct PRDM9 alleles, the binding sites have only been mapped for a few variants (human A & C, mouse Dom2 & Cst). Thus, the drivers and biological consequences of PRDM9 allelic heterogeneity are unclear. We developed a cell-based high-throughput CUT&RUN assay to map where PRDM9 variants bind genome-wide using PRDM9-dependent H3K4me3 deposition as a proxy. We determined the binding sites and motifs of 89 previously uncharacterized human PRDM9 variants, including two novel/low-frequency variants we identified in a cohort of men with azoospermia (A7, C11). We found that, despite extensive ZNF composition differences, most human variants bound to known A or C hotspots, suggesting many extant variants are functionally redundant. Some variants (n=8), including A7, bound few locations (< 5000) and had poorly defined motifs, suggesting they are nonfunctional. Other alleles (n=8), such as C11, bound many more sites (>30,000), most of which were distinct from the A/C variants. We mimicked the genotypes of the PRDM9-A/A7 and PRDM9-A/C11 in men with azoospermia by co-expressing both variants and found that A7 contributed minimally to PRDM9-A binding whereas C11 largely overrode it. 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