Christine M. Dorman

A GATA-1-regulated microRNA locus essential for erythropoiesis

MicroRNAs (miRNAs) control tissue development, but their mechanism of regulation is not well understood. We used a gene complementation strategy combined with microarray screening to identify miRNAs involved in the formation of erythroid (red blood) cells. Two conserved miRNAs, miR 144 and miR 451, emerged as direct targets of the critical hematopoietic transcription factor GATA-1. In vivo, GATA-1 binds a distal upstream regulatory element to activate RNA polymerase II-mediated transcription of a single common precursor RNA (pri-miRNA) encoding both mature miRNAs. Zebrafish embryos depleted of miR 451 by using antisense morpholinos form erythroid precursors, but their development into mature circulating red blood cells is strongly and specifically impaired. These results reveal a miRNA locus that is required for erythropoiesis and uncover a new regulatory axis through which GATA-1 controls this process.

Dynamics of the epigenetic landscape during erythroid differentiation after GATA1 restoration

Interplays among lineage-specific nuclear proteins, chromatin modifying enzymes, and the basal transcription machinery govern cellular differentiation, but their dynamics of action and coordination with transcriptional control are not fully understood. Alterations in chromatin structure appear to establish a permissive state for gene activation at some loci, but they play an integral role in activation at other loci. To determine the predominant roles of chromatin states and factor occupancy in directing gene regulation during differentiation, we mapped chromatin accessibility, histone modifications, and nuclear factor occupancy genome-wide during mouse erythroid differentiation dependent on the master regulatory transcription factor GATA1. Notably, despite extensive changes in gene expression, the chromatin state profiles (proportions of a gene in a chromatin state dominated by activating or repressive histone modifications) and accessibility remain largely unchanged during GATA1-induced erythroid differentiation. In contrast, gene induction and repression are strongly associated with changes in patterns of transcription factor occupancy. Our results indicate that during erythroid differentiation, the broad features of chromatin states are established at the stage of lineage commitment, largely independently of GATA1. These determine permissiveness for expression, with subsequent induction or repression mediated by distinctive combinations of transcription factors.

Activated Raf inhibits avian myogenesis through a MAPK-dependent mechanism.

Chronic overexpression of the oncogenic form of Ras is a potent inhibitor of skeletal myogenesis. However, the intracellular signaling pathways that mediate the repressive actions of Ras on myogenic differentiation have yet to be identified. We examined the role of Raf-mediated signaling as a modulator of avian myogenesis. Raf overexpression elicited pronounced effects on both myoblasts and mature myocytes. Most notably, the embryonic chick myoblasts overexpressing a constitutively active form of Raf (RCAS-Raf CAAX or RCAS-Raf BXB) fail to form the large multinucleated myofibers characteristic of myogenic cultures. While residual myofibers were apparent in the RCAS-Raf BXB and RCAS-Raf CAAX infected cultures, these fibers had an atrophic phenotype. The altered morphology is not a result of reinitiation of the myonuclei cell cycle nor is it due to apoptosis. Furthermore, the mononucleated myoblasts misexpressing Raf BXB are differentiation-defective due to overt MAPK activity. Supplementation of the culture media with the MAPK kinase (MEK) inhibitor, PD98059, caused a reversal of the phenotype and allowed the formation of multinucleated myofibers at levels comparable to controls. Our results indicate that the Raf/MEK/MAPK axis is intact in chick myoblasts and that persistent activation of this signaling cascade is inhibitory to myogenesis.

Transcriptional enhancement by GATA1-occupied DNA segments is strongly associated with evolutionary constraint on the binding site motif

Tissue development and function are exquisitely dependent on proper regulation of gene expression, but it remains controversial whether the genomic signals controlling this process are subject to strong selective constraint. While some studies show that highly constrained noncoding regions act to enhance transcription, other studies show that DNA segments with biochemical signatures of regulatory regions, such as occupancy by a transcription factor, are seemingly unconstrained across mammalian evolution. To test the possible correlation of selective constraint with enhancer activity, we used chromatin immunoprecipitation as an approach unbiased by either evolutionary constraint or prior knowledge of regulatory activity to identify DNA segments within a 66-Mb region of mouse chromosome 7 that are occupied by the erythroid transcription factor GATA1. DNA segments bound by GATA1 were identified by hybridization to high-density tiling arrays, validated by quantitative PCR, and tested for gene regulatory activity in erythroid cells. Whereas almost all of the occupied segments contain canonical WGATAR binding site motifs for GATA1, in only 45% of the cases is the motif deeply preserved (found at the orthologous position in placental mammals or more distant species). However, GATA1-bound segments with high enhancer activity tend to be the ones with an evolutionarily preserved WGATAR motif, and this relationship was confirmed by a loss-of-function assay. Thus, GATA1 binding sites that regulate gene expression during erythroid maturation are under strong selective constraint, while nonconstrained binding may have only a limited or indirect role in regulation.

FGF5 stimulates expansion of connective tissue fibroblasts and inhibits skeletal muscle development in the limb

FGF5 is expressed in the mesenchyme and skeletal muscle of developing and adult mouse limbs. However, the function of FGF5 during development of the limb and limb musculature is unknown. To elucidate the inherent participation of FGF5 during limb organogenesis, a retroviral delivery system (RCAS) was used to overexpress human FGF5 throughout developing hind limb of chicken embryos. Misexpression of the soluble growth factor severely inhibited the formation of mature myocytes. Limbs infected with RCAS‐FGF5 contained smaller presumptive muscle masses as evidenced by a decrease in MyoD and myosin heavy chain expressing cells. In contrast, ectopic expression of FGF5 significantly stimulated proliferation and expansion of the tenascin‐expressing, connective‐tissue fibroblast lineage throughout the developing limb. Histological analysis demonstrated that the increase in tenascin immunostaining surrounding the femur, ileum, and pubis in the FGF5 infected limbs corresponded to the fibroblasts forming the stacked‐cell perichondrium. Furthermore, pulse labeling experiments with the thymidine analog, BrdU, revealed that the increased size of the perichondrium was attributable to enhanced cell proliferation. These results support a model whereby FGF5 acts as a mitogen to stimulate the proliferation of mesenchymal fibroblasts that contribute to the formation of connective tissues such as the perichondrium, and inhibits the development of differentiated skeletal muscle. These results also contend that FGF5 is a candidate mediator of the exclusive spatial patterning of the hind limb connective tissue and skeletal muscle.

Inhibition of myogenin Expression by Activated Raf Is Not Responsible for the Block to Avian Myogenesis

Activated Raf is a potent inhibitor of skeletal muscle gene transcription and myocyte formation through stimulation of downstream MAPK. However, the molecular targets of elevated MAPK with regard to myogenic repression remain elusive. We examined the effects of activated Raf on myogenin gene expression in avian myoblasts. Overexpression of activated Raf in embryonic chick myoblasts prevented myogenin gene transcription and myocyte differentiation. Treatment with PD98059, an inhibitor of MAPK kinase (MEK), restored myogenin expression but did not reinstate the myogenic program. Using a panel of myogeninpromoter deletion mutants, we were unable to identify a region within the proximal 829-bp promoter that confers responsiveness to MEK. Interestingly, our experiments identified MEF2A as a target of Raf-mediated inhibition in mouse myoblasts but not in avian myogenic cells. Embryonic myoblasts overexpressing activated Raf were unable to drive transcription from a minimal myogenin promoter reporter, containing a single E-box and MEF2 site, to levels comparable with controls. Unlike mouse myoblasts, forced expression of MEF2A did not synergistically enhance transcription from the myogeninpromoter in chick myoblasts, indicating that additional molecular determinants of the block to myogenesis exist. Results of these experiments further exemplify specie differences in the mode of Raf-mediated inhibition of muscle differentiation.

Dynamics of GATA1 binding and expression response in a GATA1-induced erythroid differentiation system

During the maturation phase of mammalian erythroid differentiation, highly proliferative cells committed to the erythroid lineage undergo dramatic changes in morphology and function to produce circulating, enucleated erythrocytes. These changes are caused by equally dramatic alterations in gene expression, which in turn are driven by changes in the abundance and binding patterns of transcription factors such as GATA1. We have studied the dynamics of GATA1 binding by ChIP-seq and the global expression responses by RNA-seq in a GATA1-dependent mouse cell line model for erythroid maturation, in both cases examining seven progressive stages during differentiation. Analyses of these data should provide insights both into mechanisms of regulation (early versus late targets) and the consequences in cell physiology (e.g., distinctive categories of genes regulated at progressive stages of differentiation). The data are deposited in the Gene Expression Omnibus, series GSE36029, GSE40522, GSE49847, and GSE51338.