GATA-1-Dependent and -Independent Modes of Establishing the Erythroid Cell Genetic Network.

Abstract 2083

The constant physiological demand to generate large numbers of red blood cells requires a complex genetic network established by the master regulatory transcription factor GATA-1, which orchestrates erythroblast survival, proliferation, and differentiation. Many questions remain regarding how GATA-1 instigates genetic networks and to what extent GATA-1-independent mechanisms regulate erythropoiesis. Med1, a component of the broadly expressed Mediator complex (Mediator), facilitates GATA-1-dependent transcriptional activation at select target genes, although its contribution to GATA-1 function in cell-based assays is considerably less than that of the cell type-specific coregulator Friend of GATA-1. Med1-nullizygous mice have hematopoietic, cardiac, and vascular defects, though the underlying mechanisms are not defined. Furthermore, whether Med1 coactivator activity is dedicated to GATA-1 in erythroid cells and whether it controls numerous or a restricted cohort of genes is also unclear. Using a genetic complementation assay in GATA-1-null erythroid cells and a functional genomics approach, we demonstrate that Med1 regulates a restricted gene ensemble in erythroid cells, consisting predominantly of genes not controlled by GATA-1. Of the 265 Med1-regulated genes and 1054 GATA-1-regulated genes, only 35 genes were regulated by Med1 and GATA-1. Given the preponderance of GATA-1-independent Med1 targets, it is attractive to propose that Med1 has important GATA-1-independent functions required to exert its crucial hematopoietic activities. Since Med1 is a Mediator subunit, it is presumed to function through Mediator to regulate target genes. However, Med1 interacts with various trans-acting factors, and therefore its gene regulatory activity may not invariably rely on Mediator or a Mediator subcomplex. As Mediator is largely unstudied in erythroid cells, we asked whether Mediator subunit expression is regulated upon primary human erythroid cell maturation ex vivo. Mining the Human Erythroblast Maturation Database revealed that Med25 is strongly up-regulated during maturation. Knockdown of Med25 significantly dysregulated all ten of the highest responding Med1 target genes. Simultaneous knockdowns of Med1 and Med25 altered expression of 9 of the 10 top Med1 target genes, resembling the individual factor knockdowns. These results support the hypothesis that Med1 and Med25 function in the erythroid Mediator complex to regulate these genes. Med1 regulated these genes in a cell type-specific manner, as 8 of the 10 top Med1 targets in G1E-ER-GATA-1 proerythroblast-like cells and Mouse Erythroleukemia Cells were not dysregulated upon Med1 knockdown in Mouse Embryonic Fibroblasts. As Med1 modulated, but was not essential for, GATA-1-dependent transcription, we reasoned that certain Med1 target genes may exert GATA-1-independent activities to control erythroid cell development and/or function. The Med1 target gene Rrad encodes a small GTPase induced during primary human erythroid cell maturation, but its regulation/function has not been described in the hematopoietic system. Loss-of-function analysis in G1E-ER-GATA-1 cells indicated that Rrad confers survival. Knocking-down Rrad increased early apoptosis 2.5 fold (p < 0.05). The Rrad requirement for survival was more pronounced when cells were deprived of Erythropoietin (Epo) and Stem Cell Factor (SCF). In cells cultured without Epo, early apoptosis increased 7.0 fold upon Rrad knockdown [from 1.0% ± 0.1% to 7.2% ± 0.5% (p < 0.05)]. Removing SCF from the media significantly increased apoptotic cells, and Rrad knockdown elevated this further from 28% ± 2.4% to 46% ± 2.8% (p < 0.01), while the number of live cells decreased 4.7 fold (p < 0.01). These studies established a dual role for Mediator in erythroid cell regulation as a context-dependent GATA-1 coregulator and a GATA-1-independent regulator of cell type-specific genes, including potentially critical regulators of erythroid cell development, survival, and function. Mechanistically, given the greater than twenty components of the canonical Mediator, it will be particularly instructive to compare our findings to that of other key Mediator components, which shall yield a comprehensive understanding of their regulation and function during the progressive transitions from erythroid precursors to the erythrocyte.