Transcriptional Regulation of Red Cell Specific Gene Expression
The molecular events that confer the ability to express lineage-specific genes upon an initially uncommitted, pluripotent hematopoietic stem cell remain a major question in cell differentiation. Use of an immortalized erythroid cell line as a means to isolate genes that may be important for erythroid function allowed us to identify a novel, erythroid-specific gene, which was named EKLF (erythroid Krüppel-like factor).
EKLF binds to and activates transcription from the CACCC element, one of a trio of localized promoter and enhancer DNA binding sites known to be crucial for transcription of globin and other erythroid cell-specific genes. Biological analyses reveal that murine EKLF is expressed in primitive erythroid cells by embryonic day 7.5, and in definitive erythroid cells within the hepatic primordia by embryonic day 9.5. However, its ability to preferentially activate an adult ß-globin promoter over a linked fetal γ-globin promoter led us to propose that EKLF may be an important factor for γ- to ß-globin gene switching. This was verified by studies showing that EKLF is absolutely required for normal red cell development, since its genetic disruption leads to death by embryonic day 14-16 (precisely the time of the switch in mice) due to a deficiency of mature, definitive red cells. EKLF-deficient mice exhibit drastically low ß-globin expression at the transcriptional level, i.e., a severe ß-thalassemia phenotype, and contain an altered chromatin structure at the ß-globin locus.
These molecular and biological studies have established that EKLF is an essential component required for globin switching and completion of the definitive erythroid program. Disorders of hemoglobin expression can lead to a variety of hemoglobinopathies, including sickle cell anemia and ß–thalassemia (Cooley’s anemia). As a result, our examination of EKLF’s mechanism of action has illuminated how it regulates the globin locus, and has provided us with a way to reconstruct EKLF so that it can potentially rectify one type of hemoglobin disorder.
Our discovery of EKLF has stimulated other investigators around the world to search for analogous genes that can work in a similar fashion to regulate unique targets in other tissues. EKLF is now the founding member (KLF1) of a family of 16 proteins, some of which have been directly implicated in suppression of a specific subset of cancers. We are vigorously continuing the study of EKLF (KLF1) using a number of approaches, including biochemical and structure/function analyses of the EKLF protein, identification of its protein partners, and monitoring how EKLF expression itself is so precisely regulated during development.
Our most recent studies show that EKLF becomes acetylated by virtue of its association with a subset of coactivators, leading to enhanced interaction with chromatin remodelers that leads to activated transcription. Surprisingly, EKLF can also associate with corepressors and decrease transcription at selected promoters, suggesting other activities beyond activation of the adult ß-globin gene. Finally, the BMP4/Smad pathway plays a critical role in transcriptional activation of EKLF in the erythroid cell, likely via a relatively small promoter region proximal to its initiation site.