All ETDs from UAB

Advisory Committee Chair

Chengbei Chang

Advisory Committee Members

Kai Jiao

John Parant

Rosa Serra

Hengbin Wang

Document Type

Dissertation

Date of Award

2022

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The vertebrate nervous system comes from specific regions of the ectoderm that comprises of the neural plate and the neural crest. Although genetic mechanisms governing vertebrate neural development have been investigated in depth, there is a knowledge gap regarding the roles of epigenetic mechanisms in this process. As epigenetic modulators, the COMPASS (also known as SET1/MLL complex) and HP1 proteins are responsible for regulating chromatin accessibility to transcription factors. COMPASS is responsible for deposition of activating histone H3K4 methylation marks at promoters and enhancers and thus creates open chromatin domains. The critical structural and regulatory subunits of COMPASS, Dpy30 and Ash2l, are essential for the specificity of the complex. In contrast, HP1 proteins (HP1α, β, and γ) are associated with and plays a role in generating compact chromatin conformation. Similar to many other epigenetic factors, Dpy30, Ash2l, and HP1 isoforms show high conservation from yeast to human. In the first part of this dissertation, we investigated the role of HP1 isoforms in vertebrates early neural and neural crest development which was not well understood before. Using Xenopus laevis as a model system, we showed while different HP1 isoforms have similar expression pattern, only HP1β and HP1γ knockdown within the prospective neural tissues leads to head defects. By using gene expression analysis, we found solely late neural markers and neural crest specifier genes are downregulated as a iii result of HP1β and HP1γ knockdown whereas the expression of early neural and neural plate border genes did not show significant change. Furthermore, our work revealed that a pluripotency-associated gene, pou5f3.2/oct25, is expanded in the HP1β morphants embryos. Subsequently, we showed ectopic expression of pou5f3.2/oct25 mimics the effect of HP1β knockdown on marker expression, whereas simultaneous knockdown of HP1β and pou5f3.2/oct25 partially rescues expression of these genes. Collectively, our data suggest that HP1β regulates transition from precursor to more differentiated cell types during neural and neural crest development in Xenopus, and it does so at least partially via repression of the pluripotency-associated transcription regulator pou5f3.2/oct25. In the second study presented in this dissertation, we attempted to unravel the function of activating epigenetic factors Dpy30 and Ash2l in early vertebrates embryogenesis. Despite mounting in vitro and in vivo evidence indicating essential roles for dpy30 and ash2l in mesendodermal differentiation, their specific roles in neural development remain under-characterized. Using the amphibian Xenopus laevis, we demonstrate crucial roles for dpy30 and ash2l in vertebrate nervous system development. We show that targeted knockdown of dpy30 or ash2l within the developing neural tissues by antisense morpholino oligos (MOs) results in downregulation of the neural crest genes whereas the expression of the neural plate border (NPB) specifier genes remains largely intact. By using protein-protein interaction assays, we show that neural plate border transcription factors Msx1 and Tfap2a interact with Dpy30 and Ash2l. Further, via chromatin immunoprecipitation (ChIP) assay we showed enrichment of Ash2l and H3K4me3 marks upstream of the neural crest gene sox10. Such enrichment of Ash2l and H3K4me3 marks is significantly dwindled as a result of tfap2a knockdown. Taken iv together, our results demonstrate the importance of epigenetic factors, Dpy30 and Ash2l cooperating with specific transcription factors to regulate the expression of vertebrates neural and neural crest genes.

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