(PROJECT 2) Project 2 seeks to elucidate the role of enhancer dysfunction in modulating the spatio-temporal organization of chromatin and information transfer in cancer. Multiple lines of genetic and biological evidence suggest that a subset of cases of acute myeloid leukemia (AML) and myelodysplasia (MDS) suffers from enhancer dysfunction. We hypothesize that enhancer dysfunction leads to an altered physical state of chromatin, including an abnormal state of 3-D looping, resulting in aberrant formation of transcriptional, initiating and elongating complexes and destroying the normal regulation of gene expression in the cell. The sequelae of these events are ineffective hematopoiesis and clonal expansion of MDS and AML precursors. Emerging CRISPR technology, sophisticated chromatin confirmation capture and genome wide chromatin surveys and the nanocytometry and animal cores will be used to elucidate the aberrant structure and function of chromatin. Initial studies will test the hypothesis that reduced expression of chromatin modifiers, cohesin and MLL3, which encodes a specific histone methyl transferase, leads to an imbalance between cell differentiation and proliferation and cytokine-guided differentiation in myeloid and erythroid cell lines. This will be accomplished by using loss-of-function mutants to probe the role of these chromatin modulators in cell proliferation, cell cycle regulation, apoptosis, and DNA repair and gene expression. This model system will then be used to relate these biological outcomes to changes in chromatin conformation by measuring their impact on enhancer/promoter looping, chromosomal compartmentalization and folding. Large data sets of RNA-Seq, ChIP seq and ?5C? chromosome conformation capture data will be integrated and 3D models of the normal and malignant chromatin will be constructed to explain how the loss of a single allele of these chromatin regulators can reprogram the cell and push it on the pathway to over malignancy. These in vitro observations will be further tested using an in vivo model of normal myelopoiesis, an animal model of cohesin and MLL3 loss, and analysis of primary human specimens, the latter in collaboration with the PDX Tumor Model Core. Finally we will determine if aberrant chromatin configurations lead to enhanced susceptibility to epigenetically targeted drugs. The insights gained from these studies of the functional consequences of changes in chromatin structure at the kilobase-scale will interlock with those generated by studying the impact of ion concentrations in changing chromatin structure in malignant cells (Project 1) and the role of condensin-mediated folding in chromatin structure and function at the megabase and chromosomal scale (Project 3).
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| Gibcus, Johan H; Samejima, Kumiko; Goloborodko, Anton et al. (2018) A pathway for mitotic chromosome formation. Science 359: |
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