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Human globin gene production transcriptionally "switches" from fetal to adult synthesis shortly after birth and is controlled by macromolecular complexes that enhance or suppress transcription by cis elements scattered throughout the locus. The DRED (direct repeat erythroid-definitive) repressor is recruited to the ε-globin and γ-globin promoters by the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2) to engender their silencing in adult erythroid cells. Here we found that nuclear receptor corepressor-1 (NCoR1) is a critical component of DRED that acts as a scaffold to unite the DNA-binding and epigenetic enzyme components (e.g., DNA methyltransferase 1 [DNMT1] and lysine-specific demethylase 1 [LSD1]) that elicit DRED function. We also describe a potent new regulator of γ-globin repression: The deubiquitinase BRCA1-associated protein-1 (BAP1) is a component of the repressor complex whose activity maintains NCoR1 at sites in the β-globin locus, and BAP1 inhibition in erythroid cells massively induces γ-globin synthesis. These data provide new mechanistic insights through the discovery of novel epigenetic enzymes that mediate γ-globin gene repression., (© 2018 Yu et al.; Published by Cold Spring Harbor Laboratory Press.)

Protein abundance must be precisely regulated throughout life, and nowhere is the stringency of this requirement more evident than during T-cell development: A twofold increase in the abundance of transcription factor GATA3 results in thymic lymphoma, while reduced GATA3 leads to diminished T-cell production. GATA3 haploinsufficiency also causes human HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome, often accompanied by immunodeficiency. Here we show that loss of one Gata3 allele leads to diminished expansion (and compromised development) of immature T cells as well as aberrant induction of myeloid transcription factor PU.1. This effect is at least in part mediated transcriptionally: We discovered that Gata3 is monoallelically expressed in a parent of origin-independent manner in hematopoietic stem cells and early T-cell progenitors. Curiously, half of the developing cells switch to biallelic Gata3 transcription abruptly at midthymopoiesis. We show that the monoallelic-to-biallelic transcriptional switch is stably maintained and therefore is not a stochastic phenomenon. This unique mechanism, if adopted by other regulatory genes, may provide new biological insights into the rather prevalent phenomenon of monoallelic expression of autosomal genes as well as into the variably penetrant pathophysiological spectrum of phenotypes observed in many human syndromes that are due to haploinsufficiency of the affected gene., (© 2015 Ku et al.; Published by Cold Spring Harbor Laboratory Press.)

When the orphan nuclear receptors TR2 and TR4, the DNA-binding subunits of the DRED repressor complex, are forcibly expressed in erythroid cells of transgenic mice, embryos exhibit a transient mid-gestational anemia as a consequence of a reduction in the number of primitive erythroid cells. GATA-1 mRNA is specifically diminished in the erythroid cells of these TR2/TR4 transgenic embryos as it is in human CD34(+) progenitor cells transfected with forcibly expressed TR2/TR4. In contrast, in loss-of-function studies analyzing either Tr2- or Tr4-germline-null mutant mice or human CD34(+) progenitor cells transfected with force-expressed TR2 and TR4 short hairpin RNAs (shRNAs), GATA-1 mRNA is induced. An evolutionarily conserved direct repeat (DR) element, a canonical binding site for nuclear receptors, was identified in the GATA1 hematopoietic enhancer (G1HE), and TR2/TR4 binds to that site in vitro and in vivo. Mutation of that DR element led to elevated Gata1 promoter activity, and reduced promoter responsiveness to cotransfected TR2/TR4. Thus, TR2/TR4 directly represses Gata1/GATA1 transcription in murine and human erythroid progenitor cells through an evolutionarily conserved binding site within a well-characterized, tissue-specific Gata1 enhancer, thereby providing a mechanism by which Gata1 can be directly silenced during terminal erythroid maturation.

We explored the mechanism of definitive-stage epsilon-globin transcriptional inactivity within a human beta-globin YAC expressed in transgenic mice. We focused on the globin CAC and CAAT promoter motifs, as previous laboratory and clinical studies indicated a pivotal role for these elements in globin gene activation. A high-affinity CAC-binding site for the erythroid krüppel-like factor (EKLF) was placed in the epsilon-globin promoter at a position corresponding to that in the adult beta-globin promoter, thereby simultaneously ablating a direct repeat (DR) element. This mutation led to EKLF-independent epsilon-globin transcription during definitive erythropoiesis. A second 4-bp substitution in the epsilon-globin CAAT sequence, which simultaneously disrupts a second DR element, further enhanced ectopic definitive erythroid activation of epsilon-globin transcription, which surprisingly became EKLF dependent. We finally examined factors in nuclear extracts prepared from embryonic or adult erythroid cells that bound these elements in vitro, and we identified a novel DR-binding protein (DRED) whose properties are consistent with those expected for a definitive-stage epsilon-globin repressor. We conclude that the suppression of epsilon-globin transcription during definitive erythropoiesis is mediated by the binding of a repressor that prevents EKLF from activating the epsilon-globin gene.

Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes. Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response. We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism. The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.

The small Maf proteins (MafG, MafK, and MafF), which serve as heterodimeric partner molecules of CNC family proteins for binding in vitro to MARE sites, have been implicated in the regulation of both transcription and chromatin structure, but there is no current evidence that the proteins fulfill these functions in vivo. To elucidate possible contributions of the small Maf proteins to gene regulation, we have ablated the mafG and mafK genes in mice by replacing their entire coding sequences with the Escherichia coli lacZ gene. mafG homozygous mutant animals exhibit impaired platelet formation accompanied by megakaryocyte proliferation, as well as behavioral abnormalities, whereas mafK-null mutant mice are phenotypically normal. Characterization of the mafG and mafK embryonic expression patterns show that their developmental programs are distinct and intersecting, but not entirely overlapping. These results provide direct evidence that the small Maf transcription factors are vital participants in embryonic development and cellular differentiation.

Proper tissue- and developmental stage-specific transcriptional control over the five genes of the human beta-globin locus is elicited in part by the locus control region (LCR), but the molecular mechanisms that dictate this determined pattern of gene expression during human development are still controversial. By use of homologous recombination in yeast to generate mutations in the LCR within a yeast artificial chromosome (YAC) bearing the entire human beta-globin gene locus, followed by injection of each of the mutated YACs into murine ova, we addressed the function of LCR hypersensitive site (HS) elements 3 and 4 in human beta-globin gene switching. The experiments revealed a number of unexpected properties that are directly attributable to LCR function. First, deletion of either HS3 or HS4 core elements from an otherwise intact YAC results in catastrophic disruption of globin gene expression at all erythroid developmental stages, despite the presence of all other HS elements in the YAC transgenes. If HS3 is used to replace HS4, gene expression is normal at all developmental stages. Conversely, insertion of the HS4 element in place of HS3 results in significant expression changes at every developmental stage, indicating that individual LCR HS elements play distinct roles in stage-specific beta-type globin gene activation. Although the HS4 duplication leads to alteration in the levels of epsilon- and gamma-globin mRNAs during embryonic erythropoiesis, total beta-type globin mRNA synthesis is balanced, thereby leading to the conclusion that all of the human beta-locus genes are competitively regulated. In summary, the human beta-globin HS elements appear to form a single, synergistic functional entity called the LCR, and HS3 and HS4 appear to be individually indispensable to the integrity of this macromolecular complex.

The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains. One enigmatic aspect of GATA factor expression is that several GATA proteins, which ostensibly share the same DNA-binding site specificity, are coexpressed in erythroid cells. To elucidate the roles of individual GATA factors in erythropoiesis, conditional alleles of GATA-1, GATA-2, and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor (ER). These GATA/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays. When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells, exogenous GATA-2/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner. These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal/ER or GATA-3/ER do not display a hormone-responsive block in differentiation. Thus, the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells.

Biochemical and genetic analysis of the embryonic to adult beta-like globin gene switch in chickens has led to the hypothesis that competition between the promoters of the cis-linked epsilon- and beta-globin genes for interaction with a shared enhancer mediates the developmental changes in expression of beta-globin protein isotypes. To test specific predictions of this promoter competition model, a sensitive RNA/polymerase chain reaction assay has been used to investigate the effects of individual beta-globin promoter mutations on expression of the two linked genes in transiently transfected erythroid cells. Mutations that attenuated adult beta-globin transcription resulted concomitantly in a proportional increase in expression of the embryonic epsilon-globin gene. Consistent with the model, mutations disrupting the binding sites for either of two adult stage-specific transcription factors (NF-E4 and beta CTF) indicate that these sites are essential both for induction of beta-globin gene expression and for indirect suppression (through promoter competition) of epsilon-globin transcription in definitive (adult) erythroid cells. These results provide direct evidence that stage-specific transcription factors affect the equilibrium existing between multiple interacting globin cis-regulatory elements. We conclude that promoter competition is an important mechanism through which developmental regulation of chicken beta-globin gene switching is achieved and that such competitive interactions may prove to be generally applicable to the regulation of a variety of other temporally or spatially restricted gene expression patterns.

NF-E1, a DNA-binding protein that recognizes the general consensus motif WGATAR, is the first tissue-specific factor to be identified in erythroid cells. Using a probe from the murine GF-1 (NF-E1) cDNA clone, we isolated three homologous chicken cDNAs: One of these corresponds to an mRNA (NF-E1a) that is abundantly and exclusively expressed in erythroid cells; a second mRNA (NF-E1b) is also expressed in all developmental stages of erythroid cells but is additionally found in a limited subset of other chicken tissues; mRNA representative of a third gene (NF-E1c) is expressed only in definitive (adult) erythrocytes within the red cell lineage but is also abundantly expressed in T lymphocytes and brain. All NF-E1 proteins are highly conserved within the DNA-binding domain and bind to the consensus motif with similar affinities in vitro; they are also all stimulatory trans-acting factors in vivo. The factors differ quantitatively in their ability to trans-activate reporter genes in which the number and position of cognate binding sites is varied relative to the transcriptional initiation site. These data suggest that the NF-E1 consensus motif directs a broader and more complicated array of developmental transcriptional regulatory processes than has been assumed and that NF-E1c may play a unique regulatory role in the developing chicken brain and in T lymphocytes.

The isolation and characterization of a temperature-sensitive mutant (ts1 S13) of the avian erythroblastosis virus, S13, is described. The temperature-sensitive lesion in ts1 S13 was identified as affecting the tyrosine kinase activity but not the plasma membrane localization of the ts1 S13 v-sea gene product. Erythroblasts transformed by ts1 S13 can be induced to synchronously differentiate into erythrocytes in an erythropoietin (EPO)-dependent fashion. Analysis of erythrocyte-specific gene expression in ts1 S13 erythroblasts reveals that the transformed, self-renewing erythroblasts obtained at permissive temperature already express all erythrocyte genes tested for, although at a low level. Upon differentiation induction, expression of erythrocyte-specific genes is not coordinately regulated but rather involves complex regulatory mechanisms that appear to be specific for the individual genes.

The analysis of transcriptional regulatory proteins is often hampered because such factors are present in cells in only sparing abundance. Although direct biochemical purification has been successfully applied to the analysis of many of these factors, such methods are labor intensive and expensive. We have developed an alternative strategy to identify and characterize such trans-acting factors and have used it to analyze the proteins that interact with the chicken adult beta-globin gene enhancer and promoter. The methodology involves (1) a sensitive 'reverse' radioimmunoassay used for the identification of antibodies to sequence-specific DNA-binding proteins, and (2) a monoclonal antibody-based DNase I footprint selection technique, which unambiguously identifies proteins responsible for particular footprints. Because this methodology relies on the isolation of antibodies to sequence-specific DNA-binding proteins, it should be of general utility in studying any trans-acting regulatory factor for which a specific DNA-binding sequence can be identified. In the present analysis, we report the identification of a 65-kD protein that is present only in mature definitive (adult) chicken erythroid cells. We show that this protein (termed NF-E4) binds to closely related sequences present in both the beta-globin promoter and enhancer. Biochemical analysis of extracts prepared from both nonerythroid and a variety of erythroid cell types suggests that NF-E4 is the trans-acting factor that confers definitive erythrocyte stage-specific transcriptional activation to the adult beta-globin gene.