Medical Biology - Theses

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Author: Benetti, Natalia Jane × End date: 2023 × Start date: 2020 × Subject: Epigenetics ×

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    Developmental Control of Hox Genes by the Epigenetic Regulator SMCHD1
    Benetti, Natalia Jane ( 2023-05)
    Epigenetic processes govern transcription to ensure precise timing and location of gene expression during development. Structural Maintenance of Chromosomes Hinge Domain containing 1 (SMCHD1) plays a key role in the epigenetic silencing of the inactive X chromosome (Xi) and several autosomal clustered gene families, including the Hox clus- ters. SMCHD1 also facilitates ultra-long-range chro- matin interactions on the autosomes and the Xi, and CTCF binding is increased in its absence, suggesting that SMCHD1 functionally opposes CTCF. To elucidate SMCHD1’s mechanism of Hox gene silencing, I used a range of genomic and transcriptomic techniques to profile gene expression and chromatin architecture in vivo and in vitro from several Smchd1 mutant mouse lines. Recent work has shown that maternal SMCHD1 contributes to the silencing of H3K27me3- controlled non-canonical imprinted genes. As Hox genes are also marked by H3K27me3, I asked whether maternal SMCHD1 plays a role in their regulation. I found that knocking out Smchd1 in the oocyte caused an overexpression of Hox genes and homeotic transformations in the axial skeleton of the post-implantation embryo. This is the first case of a maternal effect on Hox genes in mammals, as genetically identical offspring with SMCHD1 in the oocyte exhibited wildtype axial patterning and Hox expression. I also demonstrated that Hox gene upregulation in the absence of maternal SMCHD1 did not coincide with detectable changes in Polycomb marks H3K27me3 and H2AK119ub, DNA methylation or the chromatin architecture of the Hox clusters. I therefore propose that SMCHD1 is required to set up an as yet undefined chromatin state preimplantation that persists through development to ensure correct timing of Hox expression post-implantation. Evidence from the Blewitt Lab and others has shown that SMCHD1 is reliant upon the H2AK119 ubiquitination capacity of PRC1 to bind to the Xi. To test whether this is also the case for SMCHD1’s autosomal targets, including the Hox genes, I carried out H2AK119ub ChIP-seq in Smchd1 wildtype and null cells and found no difference in H2AK119ub between the genotypes, suggesting that SMCHD1 does not act upstream of H2AK119ub. I also optimised SMCHD1 ChIP-seq and identified 14,923 SMCHD1-bound loci, most of which are DNA-binding transcription factors with important roles in development. Three-quarters of these SMCHD1 targets were also enriched for H2AK119ub, suggesting that that SMCHD1 may act downstream of PRC1 to regulate their shared targets. Taken together, I propose that both maternal and zygotic SMCHD1 regulate Hox gene expression by acting downstream of Polycomb to form ultra-long-range chromatin in- teractions that functionally oppose CTCF to prevent precocious Hox gene activation. Maternal SMCHD1 has an additional unique role of setting up a mitotically heritable chromatin state preimplantation that ensures correct timing of Hox gene expression post-implantation.