Establishment of developmental gene silencing by ordered polycomb complex recruitment in early zebrafish embryos

Vertebrate embryos achieve developmental competency during zygotic genome activation (ZGA) by establishing chromatin states that silence yet poise developmental genes for subsequent lineage-specific activation. Here, we reveal the order of chromatin states in establishing developmental gene poising in preZGA zebrafish embryos. Poising is established at promoters and enhancers that initially contain open/permissive chromatin with 'Placeholder' nucleosomes (bearing H2A.Z, H3K4me1, and H3K27ac), and DNA hypomethylation. Silencing is initiated by the recruitment of Polycomb Repressive Complex 1 (PRC1), and H2Aub1 deposition by catalytic Rnf2 during preZGA and ZGA stages. During postZGA, H2Aub1 enables Aebp2-containing PRC2 recruitment and H3K27me3 deposition. Notably, preventing H2Aub1 (via Rnf2 inhibition) eliminates recruitment of Aebp2-PRC2 and H3K27me3, and elicits transcriptional upregulation of certain developmental genes during ZGA. However, upregulation is independent of H3K27me3 - establishing H2Aub1 as the critical silencing modification at ZGA. Taken together, we reveal the logic and mechanism for establishing poised/silent developmental genes in early vertebrate embryos.

Polycomb condensates can promote epigenetic marks but are not required for sustained chromatin compaction

Organization of the genome into transcriptionally active euchromatin and silenced heterochromatin is essential for eukaryotic cell function. Phase-separation has been implicated in heterochromatin formation, but it is unclear how phase-separated condensates can contribute to stable repression, particularly for heritable epigenetic changes. Polycomb complex PRC1 is key for heterochromatin formation, but the multitude of Polycomb proteins has hindered our understanding of their collective contribution to chromatin repression. Here, we show that PRC1 forms multicomponent condensates through hetero-oligomerization. They preferentially seed at H3K27me3 marks, and subsequently write H2AK119Ub marks. We show that inducing Polycomb phase-separation can cause chromatin compaction, but polycomb condensates are dispensable for maintenance of the compacted state. Our data and simulations are consistent with a model in which the time integral of Polycomb phase-separation is progressively recorded in repressive histone marks, which subsequently drive compaction. These findings link the equilibrium thermodynamics of phase-separation with the fundamentally non-equilibrium concept of epigenetic memory.

Loss of MGA repression mediated by an atypical polycomb complex promotes tumor progression and invasiveness

MGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated Mga in two mouse models of non-small cell lung cancer using a CRISPR-based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of non-canonical Polycomb ncPRC1.6 targets, including genes involved in metastasis and meiosis. Moreover, MGA deletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA-MAX, E2F6, and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes these ncPRC1.6 subunits. Lastly, we report that MGA loss also induces a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F target genes mediated by MGA-MAX associated with a non-canonical Polycomb complex.

DNA METHYLTRANSFERASE 3 (MET3) is regulated by Polycomb Group complex during Arabidopsis endosperm development

Complex epigenetic changes occur during plant reproduction. These regulations ensure the proper transmission of epigenetic information as well as allowing for zygotic totipotency. In Arabidopsis, the main DNA methyltransferase is called MET1 and is responsible for methylating cytosine in the CG context. The Arabidopsis genome encodes for three additional reproduction-specific homologs of MET1, namely MET2a, MET2b and MET3. In this paper, we show that the DNA methyltransferase MET3 is expressed in the seed endosperm and its expression is further restricted to the chalazal endosperm. MET3 is biallelically expressed in the endosperm but displays a paternal expression bias. We found that MET3 expression is regulated by the Polycomb complex proteins FIE and MSI1. Seed development is not impaired in met3 mutant, and we could not observe significant transcriptional changes in met3 mutant. Interestingly, we found that MET3 regulates gene expression in a Polycomb mutant background suggesting a further complexification of the interplay between H3K27me3 and DNA methylation in the seed endosperm.

Establishment of Developmental Gene Silencing by Ordered Polycomb Complex Recruitment in Early Zebrafish Embryos

Vertebrate embryos achieve developmental competency during zygotic genome activation (ZGA) by establishing chromatin states that silence yet poise developmental genes for subsequent lineage-specific activation. Here, we reveal how developmental gene poising is established de novo in preZGA zebrafish embryos. Poising is established at promoters and enhancers that initially contain open/permissive chromatin with 'Placeholder' nucleosomes (bearing H2A.Z, H3K4me1, and H3K27ac), and DNA hypomethylation. Silencing is initiated by the recruitment of Polycomb Repressive Complex 1 (PRC1), and H2Aub1 deposition by catalytic Rnf2 during preZGA and ZGA stages. During postZGA, H2Aub1 enables Aebp2-containing PRC2 recruitment and H3K27me3 deposition. Notably, preventing H2Aub1 (via Rnf2 inhibition) eliminates recruitment of Aebp2-PRC2 and H3K27me3, and elicits transcriptional upregulation of certain developmental genes during ZGA. However, upregulation is independent of H3K27me3 - establishing H2Aub1 as the critical silencing modification at ZGA. Taken together, we reveal the logic and mechanism for establishing poised/silent developmental genes in early vertebrate embryos.

Ancestry of the AUTS2 family–A novel group of polycomb-complex proteins involved in human neurological disease

Autism susceptibility candidate 2 (AUTS2) is a neurodevelopmental regulator associated with an autosomal dominant intellectual disability syndrome, AUTS2 syndrome, and is implicated as an important gene in human-specific evolution. AUTS2 exists as part of a tripartite gene family, the AUTS2 family, which includes two relatively undefined proteins, Fibrosin (FBRS) and Fibrosin-like protein 1 (FBRSL1). Evolutionary ancestors of AUTS2 have not been formally identified outside of the Animalia clade. A Drosophila melanogaster protein, Tay bridge, with a role in neurodevelopment, has been shown to display limited similarity to the C-terminal of AUTS2, suggesting that evolutionary ancestors of the AUTS2 family may exist within other Protostome lineages. Here we present an evolutionary analysis of the AUTS2 family, which highlights ancestral homologs of AUTS2 in multiple Protostome species, implicates AUTS2 as the closest human relative to the progenitor of the AUTS2 family, and demonstrates that Tay bridge is a divergent ortholog of the ancestral AUTS2 progenitor gene. We also define regions of high relative sequence identity, with potential functional significance, shared by the extended AUTS2 protein family. Using structural predictions coupled with sequence conservation and human variant data from 15,708 individuals, a putative domain structure for AUTS2 was produced that can be used to aid interpretation of the consequences of nucleotide variation on protein structure and function in human disease. To assess the role of AUTS2 in human-specific evolution, we recalculated allele frequencies at previously identified human derived sites using large population genome data, and show a high prevalence of ancestral alleles, suggesting that AUTS2 may not be a rapidly evolving gene, as previously thought.

Loss of MGA mediated Polycomb repression promotes tumor progression and invasiveness

ABSTRACTMGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated Mga in two mouse models of non-small cell lung cancer using a CRISPR based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of atypical Polycomb PRC1.6, E2F and MYC-MAX targets. Similarly, MGA depletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA, E2F6 and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes PRC1.6 subunits. Lastly, we report that MGA loss has also a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F targets mediated by an atypical Polycomb complex containing MGA-MAX dimers.