CORDIS - EU research results

B-cell development and gene regulation in three dimensions

Final Report Summary - GENOMIC INTERACTIONS (B-cell development and gene regulation in three dimensions)

The blood system consists of a highly specialized cell-types that all originate from hematopoietic stem cells in the bone marrow. Proper development (differentiation) of mature blood cells depend on cell-type and developmental stage specific gene expression.

This spatio-temporal control is achieved through the integrated action of transcription factors binding to promoters and enhancers as well as other elements. While promoters are located in direct proximity of their regulatory targets, enhancers are located in a much more sporadic manner often being located long genomic distances from prospective target genes.

The contact between distal enhancers and promoters is mediated by chromatin looping which puts regions that are far apart in terms of genomic position into close vicinity. This allows for transcription factors bound to enhancer elements to influence transcription from the target promoter. Thus, to accurately describe a stage and lineage specific transcriptional network, these promoter-enhancer interactions need to be experimentally determined.

The main goal of the project is to map promoter-enhancers interactions throughout hematopoietic development. This will greatly contribute to our understanding of transcription factor function and the regulatory code that links distally located elements together. In a larger perspective, these experiments will be key to our understanding of the functional consequences of genetic variants in control regions, be these mutations identified in blood cancer or natural human variation associated with complex genetic traits or hereditary diseases.

To be able to conduct these studies on often rare cells subsets we established ChIPseq, RNAseq and ATACseq platforms compatible with the use of limited numbers enough cell numbers to allow for analysis of hematopoietic stem cells as well as later progeny. Based on this methodological platform we have established enhancer maps of B-cell development as well the myeloid developmental pathway. This is complemented with maps of open chromatin as well as RNA expression profiles. Our initial analysis of these studies show clear pre-establishment of B-cell related enhancers in early progenitor cells. These elements however mainly remain functionally inactive. This pre-establishment is presently being pursued in transcription factor knock-out models know to be important for early lymphoid development.

To map interactions between promoters and enhancers on a genome-wide level we further established a HiC protocol adapted for the use of limited cell-numbers while maintaining a high library complexity. This protocol has subsequently coupled to a custom-promoter capture (including all promoters active in mouse progenitor cells as determined by CAGE as well as all previously known promoters) to allow for specific and deep re-sequencing of interactions involving promoters. This data is presently being generated following the validation of the methodology.

Project will upon completion greatly contribute to our understanding of transcription factor mediated control of differentiation within the context of long-range genomic interactions mediating promoter-enhancers contacts.