The Cohesin Subunit Rad21 Is a Negative Regulator of Hematopoietic Self-Renewal through Epigenetic Repression of HoxA7 and HoxA9

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy with a poor prognosis. Developing novel treatments for AML has been difficult due to the heterogeneity of genetic mutations observed across patients. Recent genomic sequencing of AML patients have identified recurrent somatic mutations in cohesin complex genes in a subset of patients (10-20%). The canonical function of the cohesin complex is to maintain sister chromatid cohesion permitting proper segregation of genomic information to each daughter cell in mitosis. Interestingly, somatic cohesin mutations in AML are uniformly heterozygous and not associated with dramatic changes in chromosomal number, implying that cohesin mutations promote AML through a function independent of their role during mitosis. Recent studies have demonstrated that haploinsufficiency of the cohesin complex leads to enhanced self-renewal in Hematopoeitic Stem and Progenitor Cells (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal since this represents an early step of oncogenic transformation.

In the present study we confirmed previous studies showing that reduced cohesin levels elicit enhanced self-renewal of murine HPSCs in vitro in serial-replating assays. Gene arrays and RT-qPCR identified significant increases in the HSPC self-renewal factors Hoxa7 and Hoxa9. We confirmed the gene array data by RT-PCR and observed that Hoxa9 levels increase within 24 hours following Rad21 depletion. Consistent with these data, multiple approaches demonstrated a high correlation between Rad21 depletion and Hoxa9 target gene expression programs. We next sought to identify the mechanism that leads to this abberant expression of Hoxa9. GSEA identified derepression of Polycomb Repressive Complex 2 (PRC2) target genes in Rad21-depleted HSPCs. Given the well-described silencing of Hox clusters by the PRC2 complex, we examined if Rad21-depletion-induced HoxA9 gene activation could be mediated by epigenetic changes. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, indicating that Rad21 plays a role in proper PRC2 mediated silencing of these genes. Using immunoprecipitation experiments we further demonstrate that cohesin and PRC2 interact and are bound in close proximity to Hoxa7 and Hoxa9, arguing that Hoxa7 and Hoxa9 derepression is directly mediated by Rad21-targeting of PRC2. Consistent with this finding we observed reduced global levels of PRC2's repressive histone mark H3K27me3 arguing that PRC2 requires the cohesin complex for proper targeting. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying both are critical downstream effectors of reduced cohesin levels. Interestingly, inhibition of Dot1L function also reduced the enhanced self-renewal capacity elicited by cohesin depletion, consistent with the Hoxa locus being regulated by H3K79 methylation. Our data demonstrate that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis and provide a possible therapeutic treatment for AML patients harboring cohesin mutations.