Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3)

Background: EKLF is a transcription factor that is critical for red cell proliferation and differentiation. Results: EKLF interactions with the PIAS family of proteins repress or superactivate expression of its target genes. Conclusion: PIAS3 interaction modulates EKLF activity in a promoter-dependent and SUMO-independent manner. Significance: Our studies reveal a novel group of coregulators that affect differentiation of red blood cells. Erythroid Kruppel-like factor (EKLF or KLF1) is a transcription factor crucial for red cell development that is directly involved in regulation of a large number of erythroid genes. EKLF serves mostly as an activator of expression of these genes; however, it can act also as a repressor. Here, we present evidence that EKLF interacts with proteins from the PIAS (protein inhibitor of activated STAT) family that convey repressive activity to EKLF in the absence of sumoylation. Our studies identify PIAS3 as a transcriptional corepressor of EKLF for at least a subset of its target genes during erythropoiesis ( e.g. β-globin, α-hemoglobin stabilizing protein). We demonstrate an interaction between EKLF and PIAS proteins confirmed by in vivo coimmunoprecipitation assays with both exogenous and endogenous proteins. We identified an L XX LL signature motif located near the N terminus of PIAS proteins that, although not involved in the EKLF-PIAS3 interaction, is required for the transrepression activity. Knockdown of endogenous PIAS3 accelerates differentiation of both murine erythroleukemia cells, as well as fetal liver cells, whereas an increase in PIAS3 levels inhibits this increase. Using chromatin immunoprecipitation assays, we show that PIAS3 preferentially occupies the β-globin promoter in undifferentiated murine erythroleukemia cells. Together these results demonstrate that an interaction between EKLF and PIAS3 provides a novel mode of regulation of EKLF activity in the absence of sumolylation and furthermore shows an important involvement of PIAS proteins in erythropoiesis.