Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)
The overall aim of this project is to better understand gene regulation and chromatin folding in the spatial context of the nucleus. Most of our knowledge on the organisation of chromatin in the nucleus comes from light- and fluorescent microscopy studies, and although spectacular advances have been made in this field, optical constraints set limits to what can be resolved. Thus, it is not possible as yet to visualize the structural organization of a single gene locus that spans, for example, 200 kilobases of genomic DNA. Intricate structural organisations are to be expected at this level of resolution though, for example in cases where enhancers or other transcriptional regulatory elements communicate with distant promoters located in cis. Novel 3C technology allows the structural analysis of genomic regions at a level of resolution not studied before. Originally developed to be applied in yeast, we adapted 3C technology for (much more complex) mammalian cells and addressed how genes communicate with distant regulatory DNA elements for their activation. We found that active b-globin genes and multiple cis-regulatory elements form a spatial unit called the Active Chromatin Hub (ACH). A sub-structure precedes ACH formation during erythroid differentiation and globin genes switch interaction with this compartment during development, correlating with their switch in expression. In this project, we want to exploit our (temporal) technical advantage and extensive resource of mutated loci and transcription factors to test predictions from our work and we propose to further explore the relationships between the structural organisation of genomic loci and gene expression. Also, we aim to increase the resolution of 3C. Key objectives of this project are to (1) provide independent evidence for b-globin compartmentalization, (2) identify proteins involved in b-globin ACH formation (3) further investigate when and how the b-globin ACH is formed, (4) analyse the spatial organisation at other genomic loci, and (5) identify genes and/or other DNA elements that co-localize with the b-globin ACH in the nuclear space. To address these issues, we plan to further develop 3C technology and apply/set up independent assays (DNA-TRAP, RNA-protein immuno-FISH) that can corroborate and extend our findings by 3C.