Mammalian Development and Evolution

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Kruppel associated box zinc finger proteins (KRAB-ZFPs) have emerged as candidates that recognize ERVs. KRAB-ZFPs are rapidly evolving transcriptional repressors that emerged in in a common ancestor of coelacanth, birds, and tetrapods. They make up the largest family of transcription factors in mammals (estimated to be several hundred in mice and humans). Each species has its own unique repertoire of KRAB-ZFPs, with a small number shared with closely related species and a larger fraction specific to each species. Despite their abundance, little is known about their physiological functions. KRAB-ZFPs consist of an N-terminal KRAB domain that binds the co-repressor KAP1 and a variable number of C-terminal C2H2 zinc finger domains that mediate sequence-specific DNA binding. KAP1 directly interacts with the KRAB domain, which recruits the histone methyltransferase (HMT) SETDB1 and heterochromatin protein 1 (HP1) to initiate heterochromatic silencing. Several lines of evidence point to a role for the KRAB-ZFP family in ERV silencing. First, the number of C2H2 zinc finger genes in mammals correlates with the number of ERVs. Second, the KRAB-ZFP protein ZFP809 was isolated based on its ability to bind to the primer binding site for proline tRNA (PBSPro) of murine leukemia virus (MuLV). Third, deletion of the KRAB-ZFP co-repressors Trim28 or Setdb1 leads to activation of many ERVs. Thus we have begun a systematic interrogation of KRAB-ZFP function as a potential adaptive repression system against ERVs. We initially focused on ZFP809 as a likely ERV-suppressing KRAB-ZFP since it was originally identified as part of a repression complex that recognizes infectious MuLV via direct binding to the 18 nt Primer Binding Site for Proline (PBSpro) sequence. We hypothesized that ZFP809 might function in vivo to repress other ERVs that utilized the PBSpro. Using ChIP-seq of epitope tagged ZFP809 in ESCs and embryonic carcinoma (EC) cells, we determined that ZFP809 bound to several sub-classes of ERV elements via the PBSpro. We generated Zfp809 knockout mice to determine whether ZFP809 was required for VL30pro silencing. We found that Zfp809 knockout tissues displayed high levels of VL30pro elements and that the targeted elements display an epigenetic shift from repressive epigenetic marks (H3K9me3 and CpG methylation) to active marks (H3K9Ac and CpG hypo-methylation). ZFP809-mediated repression extended to a handful of genes that contained adjacent VL30pro integrations. Furthermore, using a combination of conditional alleles and rescue experiments, we determined that ZFP809 activity was required in development to initiate silencing, but not in somatic cells to maintain silencing. These studies provided the first demonstration for the in vivo requirement of a KRAB-ZFP in the recognition and silencing of ERVs. As a follow-up to our studies on ZFP809, we began a systematic analysis of KRAB-ZFPs using a medium throughput ChIP-seq screen and functional genomics of KRAB-ZFP clusters and individual KRAB-ZFP genes. Our ChIP-seq data demonstrates that the majority of recently evolved KRAB-ZFP genes interact with and repress distinct and partially overlapping ERV targets. This hypotheses is strongly supported by the distinct ERV reactivation phenotypes we observed in mouse ESC lines lacking one of five of the largest KRAB-ZFP gene clusters. Furthermore our preliminary evidence suggests that KRAB-ZFP cluster KO mice are viable, but have elevated rates of somatic retrotransposition of specific retrotransposon families, providing the first direct genetic link between KRAB-ZFP gene diversification and retrotranspsoson mobility. Although our data shows that many KRAB-ZFPs repress ERVs, we also found that more ancient KRAB-ZFPs that emerged in a human/mouse common ancestor do not bind and repress ERVs. One of these KRAB-ZFPs, ZFP568 plays an important role in silencing a key developmental gene that may have played a critical role in the onset of viviparity in mammals. Using ChIP-seq and biochemical assays, we determined that ZFP568 is a direct repressor of a placental specific isoform of the Igf2 gene called Igf2-P0. Insulin-like growth factor 2 (Igf2) is the major fetal growth hormone in mammals. We demonstrated that loss of Zfp568, which causes gastrulation failure, or mutation of the ZFP568 binding site at the Igf2-P0 promoter causes inappropriate Igf2-P0 activation. We also showed that this lethality could be rescued by deletion of Igf2. These data highlight the exquisite selectivity by which members of the KRAB-ZFP family repress their targets and identifies an additional layer of transcriptional control of a key growth factor regulating fetal and placental development. In an exciting follow-up to these studies, we determined that ZFP568 is highly conserved and under purifying selection in eutheria with the exception of human. Human ZNF568 allele variants have lost the ability to bind and repress Igf2-P0, which may have been driven by the loss of the Igf2-p0 transcript in human placenta. We solve the crystal structure of mouse ZFP568 zinc fingers bound to the Igf2-P0 binding site that reveals several non-canonical ZF-DNA contacts, highlighting the ability of individual ZFs to change confirmation depending upon ZF context and DNA structure. These structures also explain how mutations in human ZNF568 alleles disrupt Igf2-P0 interactions, which contain either deleted ZFs or mutations to key ZF-DNA contact residues. Since these studies on ZFP568, we have also made important progress studying two additional mammalian conserved KRAB-ZFPs, ZFP110 (called ZNF274 in humans) and Zfp661(called ZNF2 in humans). We have found that Zfp110 is essential for development in mice, and like its human ortholog, binds to the 3' end of KRAB-ZFP genes. In contrast, Zfp661 is not necessary for survival, but Zfp661 mutants have dendrite arborization defects and display autism-like behaviors. Mechanistically, ZFP661 balances the expression of clustered protocadherin genes, increasing their diversity in neurons, by binding antagonistically near CTCF sites, preventing CTCF from trapping cohesin, allowing cohesin to pass through CTCF barriers. These studies highlight how the deep exploration of KZFP gene function is revealing critical adaptations that occurred in the earliest mammals We have also begun to explore the ancestral gene of the KRAB-ZFP family, PRDM9. PRDM9 contains a DNA-bind zinc finger array and a KRAB domain, like other KZFPs, but it is unique in several respects. First, PRDM9 does not interact with KAP1. Second, PRDM9 also contains a histone methyltransferase domain that methylates histone H3 on both K4 and K36 to generate the K4me3 and K36me3 dual mark. Third, PRDM9 is exclusively expressed at a brief window of time during meiotic prophase, where its activity directs the programmed DNA double strand break machinery to initiate meiotic recombination. We began an in silico search for factors that may function downstream of PRDM9. We identified two factors, Zcwpw1 and Zcwpw2, that binds to the double marked H3K4me3 and H3K36me3 mark in vitro and at hotpots in vivo. We show that loss of Zcwpw1 in mice leads to complete male sterility, meiotic arrest and failed synapsis. Strikingly we determine that the positioning of DSBs are not altered in Zcwpw1 KOs, demonstrating that Zcwpw1 is required not for the initiation but for the repair of PRDM9-induced DSBs. However in Zcwpw2 KOs, DSBs partially relocated towards promoters. This suggests that Zcwpw2 plays a role linking PRDM9-induced histone methylation marks to efficient production of DNA double strand breaks at hotspots, while Zcwpw2 plays a critical role in ensuring efficient homologous DNA repair.