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Title: Mechanism of SMAD-mediated transcription on chromatin in response to Activin/Nodal signalling
Author: Gaarenstroom, T. Y.
ISNI:       0000 0004 8503 7397
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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Ligands of the TGF-β/Activin/Nodal family signal through SMAD2/3 transcription factors, which recognize a very short DNA consensus sequence. Their genomic target sites therefore are largely determined by other transcription factors, enabling the pathway to induce a variety of cellular responses depending on signalling and co-factor context. In addition, SMADs require a chromatin template to activate transcription and do so via recruitment of chromatin remodelling enzymes, although the exact sequence of events initiated by SMAD binding is unclear. Using a combination of RNA-seq, ChIP-seq and in silico data analysis, I investigated how SMAD proteins regulate transcription of target genes displaying different expression kinetics in response to Activin/Nodal stimuli. P19 embryonic teratoma cells represent a useful system to study the signalling pathway over time as we can inhibit all SMAD2-mediated signalling, followed by an acute Activin induction, while the cells secrete their own Nodal ligand when left untreated. I have identified SMAD2 transcriptional targets and genomic binding sites in response to acute or sustained signal activation or inhibition. SMAD2 displays a dynamic binding pattern, mainly binding to enhancer elements. SMAD2 binding increases in response to prolonged signalling, whereas target genes are up-or downregulated with varying kinetics. I examined changes in the chromatin landscape that allow the SMADs to regulate gene expression over time, and find that SMAD binding correlates with nucleosome displacement and increased histone acetylation. A correct expression pattern for a subset of genes requires de novo protein synthesis, highlighting a feedback response where SMADs regulate co-factor expression required for secondary induction or repression. DNA motif analysis identified several sequence-specific candidate transcription factors, including FOXH1 and POU5F1, as well as a potential novel transcriptional cofactor ZIC3. Knockdown of these different candidates allowed me to define subsets of target genes that rely on specific cofactors, indicating that SMAD2 target genes are regulated in different manners, both based on required transcription factors and the changes in chromatin landscape.
Supervisor: Hill, C. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available