Nodal signaling is one of the principal players in the process of gastrulation, during which the primary germ layers (endoderm, mesoderm, and ectoderm) are formed and organized in their proper locations. During my PhD I studied how Smad2 and FoxH1 transcription factors regulate the expression of Nodal target genes, and the relationship between the chromatin state of target genes and their expression.
In order to carry out in depth analysis of FoxH1 function I generated a complete mutant of this transcription factor and used deep sequencing to identify which genes FoxH1 regulates in zebrafish development. Using ChIP-Seq experiments, I also found the binding sites of FoxH1 and found that FoxH1 is capable of binding to genomic DNA in the absence of Nodal signaling. This finding suggests that it may act as a pioneer factor, preparing target genes for rapid activation when gastrulation starts. I also identified 54 direct FoxH1 target genes that are not Nodal-dependent, as well as 13 genes that are repressed, rather than activated, by FoxH1.
To identify Smad2 binding sites, I carried out ChIP-Seq in embryos overexpressing Nodal signal Squint, thus detecting loci that bind Smad2 after exposure to high Nodal levels. I tested the identified Smad2-bound DNA elements for their gene regulatory potential, and discovered that they are sufficient to drive gene expression in a Nodal-dependent manner. I also identified 26 previously unpublished Nodal target genes, and 55 genes that are bound by Smad2 upon exposure to high, but not low levels of Nodal signaling.
In the last chapter I describe the study of the interaction between Nodal signaling in early zebrafish development and chromatin marks. I found that exposure of embryonic cells to high levels of Nodal is associated with low levels of H3K27me3 and high levels of H3K4me3 marks on Nodal target genes, compared to unexposed cells. I also describe a Cas9-based system that we used to change H3K27me3 levels in a targeted manner, and tested it in a developing embryo on a Nodal-responsive fgf8a gene. Our results suggest that reduction of H3K27me3 mark on its own is not sufficient to affect the expression of this gene, and additional mechanisms are involved in target gene activation by Smad2. / Biology, Molecular and Cellular
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/33493493 |
| Date | 26 July 2017 |
| Creators | Akhmetova, Laila |
| Contributors | Mango, Susan, Schier, Alexander |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation, text |
| Format | application/pdf |
| Rights | open |
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