Analysis of chromatin dynamics during glucocorticoid receptor activation.

Steroid hormone receptors initiate a genetic program tightly regulated by the chromatin environment of the responsive regions. Using the glucocorticoid receptor (GR) as a model factor for transcriptional initiation, we classified chromatin structure through formaldehyde-assisted isolation of regulatory elements (FAIRE). We looked at dynamic changes in FAIRE signals during GR activation specifically at regions of receptor interaction. We found a distribution of GR-responsive regions with diverse responses to activation and chromatin modulation. The majority of GR binding regions demonstrate increases in FAIRE signal in response to ligand. However, the majority GR-responsive regions shared a similar FAIRE signal in the basal chromatin state, suggesting a common chromatin structure for GR recruitment. Supporting this notion, global FAIRE sequencing (seq) data indicated an enrichment of signal surrounding the GR binding site prior to activation. Brg-1 knockdown showed response element-specific effects of ATPase-dependent chromatin remodeling. FAIRE induction was universally decreased by Brg-1 depletion, but to varying degrees in a target specific manner. Taken together, these data suggest classes of nuclear receptor response regions that react to activation through different chromatin regulatory events and identify a chromatin structure that classifies the majority of response elements tested.

Defective erythropoiesis in transgenic mice expressing dominant-negative upstream stimulatory factor.

Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of proteins. It binds with high affinity to E-box elements and, through interaction with coactivators, aids in the formation of transcription complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and beta-globin. Here, we show that the erythroid cell-specific expression of a dominant-negative mutant of USF, A-USF, in transgenic mice reduces the expression of all beta-type globin genes and leads to the diminished association of RNA polymerase II with locus control region element HS2 and with the beta-globin gene promoter. We further show that the expression of A-USF reduces the expression of several key erythroid cell-specific transcription factors, including EKLF and Tal-1. We provide evidence demonstrating that USF interacts with known regulatory DNA elements in the EKLF and Tal-1 gene loci in erythroid cells. Furthermore, A-USF-expressing transgenic mice exhibit a defect in the formation of CD71(+) progenitor and Ter-119(+) mature erythroid cells. In summary, the data demonstrate that USF regulates globin gene expression indirectly by enhancing the expression of erythroid transcription factors and directly by mediating the recruitment of transcription complexes to the globin gene locus.

Calpeptin increases the activity of upstream stimulatory factor and induces high level globin gene expression in erythroid cells.

Differentiation of erythroid cells is regulated by cell signaling pathways including those that change the intracellular concentration of calcium. Calcium-dependent proteases have been shown previously to process and regulate the activity of specific transcription factors. We show here that the protein levels of upstream stimulatory factor (USF) increase during differentiation of murine erythroleukemia (MEL) cells. USF was subject to degradation by the Ca(2+)-dependent protease m-calpain in undifferentiated but not in differentiated MEL cells. Treatment of MEL cells with the specific calpain inhibitor calpeptin increased the levels of USF and strongly induced expression of the adult alpha- and beta-globin genes. The induction of globin gene expression was associated with an increase in the association of USF and RNA po ly mer ase II with regulatory elements of the beta-globin gene locus. Calpeptin also induced high level alpha- and beta-globin gene expression in primary CD71-positive erythroid progenitor cells. The combined data suggest that inhibition of calpain activity is required for erythroid differentiation-associated increase in globin gene expression.

Recruitment of coregulator complexes to the beta-globin gene locus by TFII-I and upstream stimulatory factor.

Upstream stimulatory factor and TFII-I are ubiquitously expressed helix-loop-helix transcription factors that interact with E-box sequences and or initiator elements. We previously demonstrated that upstream stimulatory factor is an activator of beta-globin gene expression whereas TFII-I is a repressor. In the present study, we demonstrate that upstream stimulatory factor interacts with the coactivator p300 and that this interaction is restricted to erythroid cells expressing the adult beta-globin gene. Furthermore, we demonstrate that Suz12, a component of the polycomb repressor complex 2, is recruited to the beta-globin gene. Reducing expression of Suz12 significantly activates beta-globin gene expression in an erythroid cell line with an embryonic phenotype. Suz12 also interacts with the adult beta-globin gene during early stages of erythroid differentiation of mouse embryonic stem cells. Our data suggest that TFII-I contributes to the recruitment of the polycomb repressor complex 2 complex to the beta-globin gene. Together, these data demonstrate that the antagonistic activities of upstream stimulatory factor and TFII-I on beta-globin gene expression are mediated at least in part by protein complexes that render the promoter associated chromatin accessible or inaccessible for the transcription complex.

Antagonistic regulation of beta-globin gene expression by helix-loop-helix proteins USF and TFII-I.

The human beta-globin genes are expressed in a developmental stage-specific manner in erythroid cells. Gene-proximal cis-regulatory DNA elements and interacting proteins restrict the expression of the genes to the embryonic, fetal, or adult stage of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region contributes to the temporal regulation of the genes. We have previously shown that transcription factors TFII-I and USF interact with the beta-globin promoter in erythroid cells. Herein we demonstrate that reducing the activity of USF decreased beta-globin gene expression, while diminishing TFII-I activity increased beta-globin gene expression in erythroid cell lines. Furthermore, a reduction of USF activity resulted in a significant decrease in acetylated H3, RNA polymerase II, and cofactor recruitment to the locus control region and to the adult beta-globin gene. The data suggest that TFII-I and USF regulate chromatin structure accessibility and recruitment of transcription complexes in the beta-globin gene locus and play important roles in restricting beta-globin gene expression to the adult stage of erythropoiesis.

Recruitment of transcription complexes to the beta-globin locus control region and transcription of hypersensitive site 3 prior to erythroid differentiation of murine embryonic stem cells.

Eukaryotic chromosomal DNA is densely packaged in the nucleus and organized into discrete domains of active and inactive chromatin. Gene loci that are activated during the process of cell differentiation undergo changes that result in modifications of specific histone tail residues and in loosening of chromatin structure. The beta-globin genes are expressed exclusively in erythroid cells. High-level expression of these genes is mediated by a locus control region (LCR), a powerful DNA regulatory element composed of several DNase I hypersensitive (HS) sites and located far upstream of the beta-globin genes. Here we show that RNA polymerase II and specific histone modifications that mark transcriptionally active chromatin domains are associated with the LCR core elements HS2 and HS3 in murine embryonic stem cells prior to differentiation along the erythroid lineage. At this stage HS3 is abundantly transcribed. After in vitro differentiation, RNA Polymerase II can also be detected at the embryonic epsilon- and adult beta-globin genes. These results are consistent with the hypothesis that activation of the beta-globin gene locus is initiated by protein complexes recruited to the LCR.