The organization of the nucleus and the regulated folding of the genome plays essential roles in regulating gene expression, chromosome segregation and chromosome structure. Long range interactions in chromatin are required for activation of gene transcription and repression of genes during the differentiation of eukaryotic cells. Long-range contacts between different chromosomal loci also regulate processes such as antibody diversity and mitotic chromosome condensation. Despite the wide range of processes that involve chromatin loops we know very little about the mechanisms that drive chromatin folding and stabilize long range interactions in the genome. This proposal is focused on developing new methods to measure the formation of looped domains dependent upon the activity of the chromatin protein CTCF. CTCF is known to be required for the stabilization of looped regions in the genome but how it generates or stabilizes looped domains is unknown. We propose to first characterize the dynamics of CTCF dependent looping using defined chromatin substrates in vitro and on chromatinized plasmids in cell extracts. Using mutagenesis and depletion we will alter the binding affinity and dimerization properties of CTCF and its interaction with the loop stabilizing protein cohesin to determine how these activities regulate the frequency of loop generation. Using the insight we gain from these in vitro experiments we will compare the dynamics of loop formation to the statistics of long range interactions at the human globin locus. By depleting CTCF and cohesin we will relate the cellular statistics of loop formation to the in vitro mechanics of loop stabilization. Our studies should provide unique and novel insight into the processes that regulate the formation of long range chromatin interactions and how they relate to essential developmental and cell biological processes.
The organization of chromosomes in the eukaryotic nucleus plays essential roles in controlling the development and proliferation of all eukaryotic cells. Despite the importance of chromatin folding in the nucleus we have very little mechanistic understanding of how chromatin folding is accomplished. This proposal is aimed at understanding how looped domains in chromatin are generated so that they can properly control cell type specific gene expression that is essential for normal human development.
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| Bell, Jason C; Jukam, David; Teran, Nicole A et al. (2018) Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts. Elife 7: |
| Risca, Viviana I; Denny, Sarah K; Straight, Aaron F et al. (2017) Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping. Nature 541:237-241 |
