EKLF directly activates the p21WAF1/CIP1 gene by proximal promoter and novel intronic regulatory regions during erythroid differentiation.

Central to the homeostasis of the hematopoietic system is the correct balance of progenitor cell proliferation versus lineage-committed differentiation. Accordingly, impaired differentiation and unrestricted proliferation due to somatic mutations and chromosomal translocations of hematopoietic transcription factors are directly associated with the development of hematopoietic malignancies (37). One of the defining features of the switch from the proliferative to the differentiating state is the exit from the cell cycle. Upon establishment of lineage commitment, cell cycle arrest occurs in the G1 phase as differentiation begins. Cellular proliferation is controlled through the interactions of cyclin-dependent kinases (CDKs) and their inhibitors. In contrast to Ink4 family members such as p16Ink4A and p18Ink4C that inhibit cyclin D-CDK4 complexes and act early in G1, the CIP/KIP family members p21WAF1/CIP1 and p27Kip1 regulate cell cycle transition through inhibition of cyclin E-CDK2 complexes at later stages in G1 (65) that can be p53 independent (40, 43). While p21WAF1/CIP1 expression is required in hematopoietic stem cells to maintain quiescence, its transcriptional upregulation in progenitor cells promotes differentiation (52). Erythropoiesis has served as a useful model to elucidate the mechanisms by which hematopoietic transcription factors regulate the induction of cell cycle inhibition to promote differentiation upon lineage commitment (52, 65). Gata1 (41) and erythroid Kruppel-like factor (EKLF [KLF1]) (31) are highly expressed in the megakaryocytic erythroid progenitor and play directive roles in erythroid versus megakaryocytic commitment and differentiation (reviewed in references 5 and 16). Both are required for terminal erythroid differentiation, as the ablation of either gene leads to an arrest in maturation (10, 58). Analyses of global gene expression profiles in response to either Gata1 or EKLF activity have revealed a plethora of target genes (14, 19, 59), within which a subset is of particular relevance to the balance of proliferation versus differentiation. Gata1-dependent erythroid differentiation correlates with an inhibition of mitogenic signals (32, 47) and cell cycle progression as p18Ink4C and p27Kip1 are upregulated (47). A substantive role for EKLF in this process is suggested by its direct activation of p18Ink4C (54) and E2F2 (42, 53) and by studies showing that EKLF gain of function promotes precocious terminal differentiation (16). Consistent with these molecular effects, EKLF-null fetal livers accumulate progenitor but not Ter119-expressing erythroid cells, showing that erythroid commitment and differentiation cannot proceed in the absence of EKLF expression (19, 42). In the present study, we have used inducible cellular differentiation systems (murine erythroleukemia [MEL] and G1E-ER-Gata1 cells) to further illuminate the role of EKLF in these later stages of the erythroid terminal differentiation process. Using a combination of EKLF gain of function, RNA interference (RNAi)-mediated knockdown, gel shift analyses, cell cycle profiling, luciferase reporter assays, and chromatin immunoprecipitation, we show that p21WAF1/CIP1 activation is highly dependent on cellular EKLF levels and provides another avenue by which EKLF affects terminal differentiation; intriguingly, it performs this in part by use of a novel conserved p21WAF1/CIP1 intronic regulatory region.