Runx1 Regulates Pu.1 Coactivator/Corepressor Exchange

Abstract 2372

RUNX1 cooperates with lineage-specifying transcription factors (TF) (eg., PU.1) to activate differentiation genes. One mechanism for this cooperation is RUNX1 regulation of SIN3A/ETO2/HDAC2 corepressor recruitment by PU.1, (Hu et al, Blood 2011). However, hematopoietic precursors express many other corepressors, as well as coactivators, at high levels. To develop a more complete picture of the corepressors and coactivators that interact with Pu.1, and to determine if Runx1 differentially regulates these interactions, affinity purification and tandem mass spectrometry (AP-MS), complemented by Western blot, was used to analyze the effects of Runx1 deficiency on the Pu.1 interactome in a model of Pu.1 driven macrophage differentiation (PUER cells) and in primary murine bone marrow cells. The functional implications with regards to acute myeloid leukemia (AML) pathophysiology and therapy were also examined.

In both models of hematopoiesis, and by AP-MS and Western blot, multiple corepressors (CHD4, LSD1, MBD2, RBBP4, REST) demonstrated major interactions with Pu.1 in Runx1 deficient cells (PUER shRunx1 and Runx1+/− primary cells) compared to controls (Table 1 ). The converse was noted for coactivators: in both models of hematopoiesis, and by both AP-MS and Western blot, multiple coactivators (Brg1, Ncoa5, Ruvb1 and Ruvb2) had major interactions with Pu.1 in control cells compared to Runx1 deficient cells (Table 1 ). One of the experimental hypotheses was that Runx1 regulates some Pu.1 corepressor/coactivator interactions but not others. However, the pattern of impaired corepressor/coactivator exchange was similar for all identified interactions (Table 1 ). Consistent with a broad effect of Runx1 on Pu.1 corepressor/coactivator exchange, in both LCMSMS and Western blot analysis, compared to PUER Empty Vector, PUER shRunx1 demonstrated increased global expression of the histone repression marks H3K27me3 (3-fold) and H3K9me3 methylation (2- fold), and decreased global expression of the histone activation marks H3K27Ac (2.5-fold) and H4K16Ac (4 –fold) (Western blot fold-change measured by densitometry).

A functional consequence of selective disruption to Pu.1-mediated activation, but preserved Pu.1-mediated repression, was repression of stem cell genes (eg., Hoxb4, c-Kit) upon nuclear introduction of Pu.1 in PUER shRunx1, but epigenetic repression of key late-differentiation genes (eg., Mcsfr, Gmcsfr) (locus specific decrease in histone acetylation was measured by chromatin-immunoprecipitation). This unbalanced recruitment of corepressors over coactivators at Pu.1 target differentiation genes was reversible by treatment of Runx1 deficient cells with histone deacetylase inhibitor drugs.

In conclusion, the mechanism of Runx1 cooperation with a key lineage-specifying TF could explain the partial differentiation but aberrant epigenetic repression of key late-differentiation genes that is characteristic of AML cells (Hu et al, Blood 2011; Negrotto et al, Leukemia 2011; Ng et al, Leukemia 2011). This unbalanced recruitment of corepressors over coactivators at lineage-specifying TF target genes provides a mechanistic rationale for use of corepressor antagonists to redress the imbalance, restore differentiation gene expression and induce terminal maturation in AML cells containing Runx1 abnormalities.