Normal gene expression is controlled, in part, through careful coordination of gene regulatory elements, including transcription factors, promoter/enhancer interactions, and 3-dimensional chromatin structure. The cohesin complex is essential in maintaining the integrity of local interactions within structurally defined topologically associating domains (TADs) and in influencing dynamic cell-type specific transcriptional programs. Genes which contribute to transcriptional regulation, including members of the cohesin complex, are frequently mutated in human cancers, including leukemias, glioblastoma multiforme, bladder cancer and Ewing sarcoma, yet the specific role of STAG2 in gene regulation, hematopoietic function, and tumor suppression has not been delineated. I show that somatic Stag2 deletion in hematopoietic stem/progenitor cells (HSPC) results in altered hematopoietic function, increased self-renewal and impaired differentiation consistent with myelodysplasia. Chromatin immunoprecipitation sequencing of Stag2-deficient HSPCs revealed that Stag2 and Stag1 have both common and non-redundant cistromes. Hi-C analyses identified a role for Stag2 in maintaining short-range chromatin interactions. While co-deletion of Stag2 and Stag1 resulted in synthetic lethality, Stag2-loss alone resulted in decreased chromatin accessibility and transcriptional output at key loci involved in lineage-specification, including reduced Ebf1 and Pax5 resulting in impaired B-lineage differentiation. My data illustrate a key role for Stag2 loss in transformation and transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation. Moreover, low cell input Hi-C technology has enabled us to perform high order chromatin assays on low numbers of sorted cell populations and identify DNA-DNA loop interactions that are cell type and developmental stage specific. We have found that the promoter of the hematopoietic stem cell gene Evi1 remains open and hypomethylated in normal LSK but during myeloid fate commitment the upstream enhancer has dynamic loss of accessibility and becomes methylated consistent with the loss of enhancer-promoter interaction measured by Hi-C. We are now applying these technologies to Stag2- and Smc3-mutant leukemia models that represent opposite ends of this stem-to-progenitor transition point. Cooperativity in leukemogenesis has been observed for both cohesin alleles in combination with Npm1c (myeloid progenitor-predominant blasts) and Flt3ITD (stem-predominant blasts). We observe the stem-to-progenitor dynamic change at the Evi1 locus to be persistent with Stag2 loss of function leading to higher Evi1 expression in myeloid progenitors and may explain the aberrant self-renewal phenotype. Taken together, we illustrate a critical link between cohesin, chromosomal contacts, and gene regulation that contributes to hematopoietic transformation.
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