TF-bind Determinants · Genomic binding of transcription factors as a function of DNA affinity and chromatin.
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2017-04-01 → 2019-03-31
- EU contribution
- €187,420
- Participants
- 1
- Scheme
- MSCA-IF
Lines connect the coordinator with its partners.
Results in brief
Genomic binding of transcription factors as a function of DNA affinity and chromatin.
The development of complex multicellular organisms requires varied expression patters (i.e. gene activation and silencing) in different cell types. Transcription factors (TFs) are proteins able to interpret DNA sequence by binding to specific motifs and ultimately enable the correct recruitment levels of transcriptional machinery at gene promoters. However, chromatin proteins such as nucleosomes represent a barrier for TF binding to genomic DNA in vivo. Here, we used sophisticated biochemical and genomic approaches to investigate to what extent TF and chromatin properties affect genomic binding in mouse embryonic stem (mES) cells. As transcription factors are drivers of gene expression changes during differentiation and in disease settings, increased understanding of TF-sensitivities to chromatin will be of major importance to the development of targeted genomic therapeutics. These are potentially useful to optimise design parameters for synthetic transcriptional activators and repressors and are especially relevant to addressing the risk of off-target effects, which limit the applicability of these genomic therapeutics. The overall objectives of the project were to gain a comprehensive understanding of the binding determinants of a model TF, as a function of the properties of the TF (i.e. affinity for DNA, expression level etc..) and chromatin marks enriched over potential binding motifs across the mammalian genome. By utilizing high throughput in vivo and in vitro approaches, we were able to assess binding of TFs as a function of chromatin, with the inclusive aim of establishing generalizable principle of TF-chromatin interactions.
Data: CORDIS, © European Union
Project objective
Transcription factors (TFs) read cis-regulatory information encoded in the genome and by doing so, they establish gene expression patterns. However, TFs must compete with structural chromatin proteins such as nucleosomes for access to DNA, and this may partially explain why only a fraction of their sequence motifs in the genome are actually bound. It is unclear what features discriminate used from unused sites with similar motifs, in vivo, though it is likely that sensitivity of TFs to chromatin is critical for normal gene expression. For example, some TFs have reduced chromatin sensitivity, termed pioneer-factors, and are able to drive differentiation; even these bind a minority of cognate motifs in the genome. Such factors may nevertheless modify chromatin and expose motifs for TFs with higher sensitivity. Alternatively, chromatin proteins and modifications may be intrinsically directed by unidentified DNA sequence features. Though little is known about determines of TF-chromatin sensitivity, it is likely a function of DNA affinity and the ability to recruit chromatin-modifying activity.The complexity of chromatin/TF interactions necessitates a reductionist approach. The objectives of this project is to gain mechanistic understanding of chromatin-sensitivity and will use established methods to measure binding of ectopic TFs as a function TF properties and chromatin components. I will express exogenous TFs in mammalian cells and compare in vivo binding to in vitro DNA affinity and analyse their chromatin sensitivity (WP1). This setup enables me to modify and profile chromatin before and after expression, which will test the contribution these factors have on TF binding (WP2). Finally, I will test if manipulating the chromatin-modifying activity of an ectopic TF affects binding (WP3). These results will provide mechanistic insight into TF sensitivity to chromatin and should reveal general principles of binding hierarchies and the logic of cis-regulatory regions.
Original text from CORDIS.
Participants
- FRIEDRICH MIESCHER INSTITUTE FOR BIOMEDICAL RESEARCH FONDATION · BASELCoordinatorSwitzerland
Links
Data: CORDIS, © European Union