PAF1 complex interactions with SETDB1 mediate promoter H3K9 methylation and transcriptional repression ofHoxa9andMeis1in acute myeloid leukemia

Abstract

The Polymerase Associated Factor 1 complex (PAF1c) is an epigenetic co-modifying complex that directly contacts RNA polymerase II (RNAPII) and several epigenetic regulating proteins. Mutations, overexpression and loss of expression of subunits of the PAF1c are observed in various forms of cancer suggesting proper regulation is needed for cellular development. However, the biochemical interactions with the PAF1c that allow dynamic gene regulation are unclear. We and others have shown that the PAF1c makes a direct interaction with MLL fusion proteins, which are potent oncogenic drivers of acute myeloid leukemia (AML). This interaction is critical for the maintenance of MLL translocation driven AML by targeting MLL fusion proteins to the target genes Meis1 and Hoxa9. Here, we use a proteomics approach to identify protein-protein interactions with the PAF1c subunit CDC73 that regulate the function of the PAF1c. We identified a novel interaction with a histone H3 lysine 9 (H3K9) methyltransferase protein, SETDB1. This interaction is stabilized with a mutant CDC73 that is incapable of supporting AML cell growth. Importantly, transcription of Meis1 and Hoxa9 is reduced and promoter H3K9 trimethylation (H3K9me3) increased by overexpression of SETDB1 or stabilization of the PAF1c-SETDB1 interaction in AML cells. These findings were corroborated in human AML patients where increased SETDB1 expression was associated with reduced HOXA9 and MEIS1. To our knowledge, this is the first proteomics approach to search for CDC73 protein-protein interactions in AML, and demonstrates that the PAF1c may play a role in H3K9me3-mediated transcriptional repression in AML.

SETDB1 mediated histone H3 lysine 9 methylation suppresses MLL-fusion target expression and leukemic transformation

“…Lin – mouse bone marrow cells treated with UNC0638 exhibit a reduction in both H3K9me2 and H3K9me3 ( Figure 3A ). 28 , 29 Given our data demonstrating Hoxa9 and Meis1 are repressed by H3K9 methylation, 5 we tested the effect of UNC0638 on HSPC self-renewal. Previous reports have demonstrated that mouse Lin – Ska1 + cKit + cells (LSK) are preserved in culture following treatment with UNC0638.…”

“…The PAF1c-SETDB1 interaction mediates promoter H3K9me3 and repression of Hoxa9 and Meis1 expression. 5 Further, SETDB1 expression is inversely correlated with HOXA9 and MEIS1 expression in AML patient samples. 5 These data imply a more complex role for H3K9 methylation in AML similar to skin tumors whereby H3K9 methyltransferases display both oncogenic and suppressive roles.…”

“… 5 Further, SETDB1 expression is inversely correlated with HOXA9 and MEIS1 expression in AML patient samples. 5 These data imply a more complex role for H3K9 methylation in AML similar to skin tumors whereby H3K9 methyltransferases display both oncogenic and suppressive roles. 20 , 21 Thus, further investigation into the role of H3K9 methyltransferases in AML is required.…”

“… 4 Our lab and others have previously demonstrated that members of the SUV39 family of H3K9 methyltransferases bind to the polymerase associated factor complex (PAF1c). 5 , 6 SETDB1, G9a ( EHMT2 ), and GLP ( EHMT1 ), were identified in a proteomics study exploring the interactome of the PAF1c in AML. 5 The PAF1c is an epigenetic regulator complex that physically associates with RNA polymerase II (RNAPII) and both positively and negatively regulates gene transcription.…”

“… 5 , 6 SETDB1, G9a ( EHMT2 ), and GLP ( EHMT1 ), were identified in a proteomics study exploring the interactome of the PAF1c in AML. 5 The PAF1c is an epigenetic regulator complex that physically associates with RNA polymerase II (RNAPII) and both positively and negatively regulates gene transcription. 7–11 In AML, the PAF1c is critical for the regulation of a proleukemic HOXA gene program in AML cells through the recruitment of MLL and MLL-fusion proteins to the HOXA locus via direct physical interactions.…”