scholarly journals Cloning and characterization of a novel erythroid cell-derived CNC family transcription factor heterodimerizing with the small Maf family proteins.

1995 ◽  
Vol 15 (8) ◽  
pp. 4184-4193 ◽  
Author(s):  
K Itoh ◽  
K Igarashi ◽  
N Hayashi ◽  
M Nishizawa ◽  
M Yamamoto
Keyword(s):  
Transcription Factor ◽  
Leucine Zipper ◽  
Cdna Probe ◽  
Cdna Libraries ◽  
Erythroid Cell ◽  
Cdna Clones ◽  
Globin Genes ◽  
Beta Globin ◽  
Specific Expression

The chicken beta-globin enhancer is critical for the tissue- and developmental stage-specific expression of the beta-globin genes. This enhancer contains two indispensable cis elements, one containing two GATA sites and the other containing an NF-E2 site. To identify the putative transcription factor acting through the NF-E2 motif in the chicken beta-globin enhancer, we screened chicken cDNA libraries with a mouse p45 NF-E2 cDNA probe and isolated cDNA clones which encode a protein of 582 amino acid residues. This protein contains a region that includes the basic region-leucine zipper domain which is well conserved among members of the CNC family proteins (Cap 'n' collar, p45 NF-E2, LCR-F1, Nrf1, and Nrf2). Hence, we named this protein ECH (erythroid cell-derived protein with CNC homology). ECH is expressed abundantly in cultured erythroid cells undergoing terminal differentiation, peripheral erythrocytes, and some nonhematopoietic tissues. Since most of the cDNA clones obtained from the chicken erythrocyte cDNA library encoded ECH, ECH is likely the predominant CNC family protein present in avian peripheral erythrocytes. Like p45 NF-E2, ECH can heterodimerize with any of the small Maf family proteins and bind the NF-E2 site as a heterodimer in vitro. In a transfection assay, ECH transactivates transcription depending on the presence of NF-E2 sites on the reporter gene plasmid. These results indicate that ECH is likely a key regulator of avian erythropoiesis.

A PAR domain transcription factor is involved in the expression from a hematopoietic-specific promoter for the human LMO2 gene

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4757-4764 ◽  
Author(s):  
Scott C. Crable ◽  
Kathleen P. Anderson
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Leucine Zipper ◽  
Cell Lineage ◽  
Erythroid Cell ◽  
Protein Protein Interactions ◽  
Consensus Binding Site ◽  
Basic Leucine Zipper ◽  
Specific Expression ◽  
Dna Binding Factors

AbstractThe transcription factor LMO2 is believed to exert its effect through the formation of protein-protein interactions with other DNA-binding factors such as GATA-1 and TAL1. Although LMO2 has been shown to be critical for the formation of the erythroid cell lineage, the gene is also expressed in a number of nonerythroid tissues. In this report, we demonstrate that the more distal of the 2 promoters for the LMO2 gene is highly restricted in its pattern of expression, directing the hematopoietic-specific expression of this gene. Deletion and mutation analyses have identified a critical cis element in the first untranslated exon of the gene. This element is a consensus-binding site for a small family of basic leucine zipper proteins containing a proline and acidic amino acid–rich (PAR) domain. Although all 3 members of this family are produced in erythroid cells, only 2 of these proteins, thyrotroph embryonic factor and hepatic leukemia factor, can activate transcription from this LMO2 promoter element. These findings represent a novel mechanism in erythroid gene regulation because PAR proteins have not previously been implicated in this process.

scholarly journals Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

1995 ◽  
Vol 15 (6) ◽  
pp. 3147-3153 ◽  
Author(s):  
G A Blobel ◽  
C A Sieff ◽  
S H Orkin
Keyword(s):  
Bone Marrow ◽  
Estrogen Receptor ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Erythroid Cell ◽  
High Dose ◽  
Dependent Manner ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

scholarly journals Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

1997 ◽  
Vol 17 (3) ◽  
pp. 1642-1651 ◽  
Author(s):  
M J Weiss ◽  
C Yu ◽  
S H Orkin
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Zinc Finger ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Transactivation Domain ◽  
Stage Of Development ◽  
Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

scholarly journals Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2781-2790
Author(s):  
DE Fleenor ◽  
RE Kaufman
Keyword(s):  
Topoisomerase Ii ◽  
Globin Gene ◽  
Dnase I ◽  
Globin Genes ◽  
Beta Globin ◽  
Hypersensitive Site ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Beta Globin Gene

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.

scholarly journals Constitutive expression of GATA-1, EPOR, alpha-globin, and gamma-globin genes in myeloid clonogenic cells from juvenile chronic myelocytic leukemia

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 323-328 ◽  
Author(s):  
E Privitera ◽  
R Schiro ◽  
D Longoni ◽  
A Ronchi ◽  
A Rambaldi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemic Cells ◽  
Globin Genes ◽  
Chronic Myelocytic Leukemia ◽  
Beta Globin ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Myelocytic Leukemia ◽  
Alpha Globin

Juvenile chronic myelocytic leukemia (JCML) is a rare disorder of early childhood. Characteristic of JCML are the progressive appearance of high levels of fetal hemoglobin (HbF), reflecting a true reversion to a fetal type of erythropoiesis, and the presence of colony-forming cells able to grow in vitro spontaneously in the absence of growth factors. To better understand the relationship between the erythroid abnormalities and the leukemic process, we analyzed the expression pattern of specific genes related to erythroid differentiation--GATA-1, EPOR, alpha-globin, beta-globin, and gamma-globin genes--in JCML peripheral blood (PB) cells and in vitro-derived colonies. Northern blot analysis of PB cells from five JCML patients indicated levels of GATA-1 transcripts much higher than those usually found in other types of leukemic cells, and S1 nuclease protection assay detected significantly increased expression of gamma-globin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single granulocyte-macrophage colony-forming unit (CFU-GM) colonies, obtained in vitro in the absence of added growth factors from four JCML patients, detected GATA-1, EPOR, and globin (alpha and gamma) transcripts in most of the colonies tested, in contrast with control CFU-GM from normal bone marrow, which were positive only for GATA-1. Single JCML colonies were tested for the presence of two different transcripts; whereas alpha- and gamma-globin genes appeared mostly coexpressed, beta-globin mRNA was detected only in a minority of the gamma-globin-positive colonies, indicating that the leukemic pattern of hemoglobin synthesis is mainly fetal. In addition, the leukemic cells occurring during blast crisis of one of our patients displayed the typical features of a stem cell leukemia (CD34+, CD19-, CD2-, myeloperoxidase-). In this sorted CD34+ population, we detected the presence of a marker chromosome, der(12)t(3;12), previously identified in bone marrow cells at diagnosis and an expression pattern superimposable to that of the JCML colonies, consistently displaying a high gamma-globin:beta-globin mRNA ratio. The expression of erythroid markers within populations of leukemic cells, both in vivo and in vitro, supports the hypothesis that abnormal JCML erythroid cells may originate from the same mutated progenitor that sustains the growth of the leukemic cells.

scholarly journals The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor.

1994 ◽  
Vol 14 (1) ◽  
pp. 373-381 ◽  
Author(s):  
D E Zhang ◽  
C J Hetherington ◽  
H M Chen ◽  
D G Tenen
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Transcription Initiation ◽  
Colony Stimulating Factor ◽  
Specific Expression ◽  
Tissue Specific ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The macrophage colony-stimulating factor (M-CSF) receptor is expressed in a tissue-specific fashion from two distinct promoters in monocytes/macrophages and the placenta. In order to further understand the transcription factors which play a role in the commitment of multipotential progenitors to the monocyte/macrophage lineage, we have initiated an investigation of the factors which activate the M-CSF receptor very early during the monocyte differentiation process. Here we demonstrate that the human monocytic M-CSF receptor promoter directs reporter gene activity in a tissue-specific fashion. Since one of the few transcription factors which have been implicated in the regulation of monocyte genes is the macrophage- and B-cell-specific PU.1 transcription factor, we investigated whether PU.1 binds and activates the M-CSF receptor promoter. Here we demonstrate that both in vitro-translated PU.1 and PU.1 from nuclear extracts bind to a specific site in the M-CSF receptor promoter just upstream from the major transcription initiation site. Mutations in this site which eliminate PU.1 binding decrease M-CSF receptor promoter activity significantly in macrophage cell lines only. Furthermore, PU.1 transactivates the M-CSF receptor promoter in nonmacrophage cells. These results suggest that PU.1 plays a major role in macrophage gene regulation and development by directing the expression of a receptor for a key macrophage growth factor.

Heme-Regulated Inhibitor (HRI) Activates Transcription Factor ATF4 to Promote BCL11A Transcription and Fetal Hemoglobin Silencing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 814-814
Author(s):  
Peng Huang ◽  
Scott A. Peslak ◽  
Xianjiang Lan ◽  
Eugene Khandros ◽  
Malini Sharma ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Research Funding ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Major Pathway ◽  
Specific Regulation

Reactivation of fetal hemoglobin in adult red blood cells benefits patients with sickle cell disease and β-thalassemia. BCL11A is one of the predominant repressors of fetal γ-globin transcription and stands as an appealing target for therapeutic genome manipulation. However, pharmacologic perturbation of BCL11A function or its co-regulators remains an unmet challenge. Previously, we reported the discovery of the erythroid-enriched protein kinase HRI as a novel regulator of γ-globin transcription and found that HRI functions in large part via controlling the levels of BCL11A transcription (Grevet et al., Science, 2018). However, the specific mechanisms underlying HRI-mediated modulation of BCL11A levels remain unknown. To identify potential HRI-controlled transcription factors that regulate BCL11A, we performed a domain-focused CRISPR screen that targeted the DNA binding domains of 1,447 genes in the human erythroid cell line HUDEP2. Activating transcription factor 4 (ATF4) emerged as a novel γ-globin repressor. Prior studies reported that ATF4 production is under positive influence of HRI. Specifically, HRI phosphorylates translation factor EIF2α which in turn augments translation of ATF4 mRNA. As expected, HRI deficiency reduced ATF4 protein amounts in HUDEP2 and primary erythroid cells. We further found that the degree of γ-globin reactivation was similar in ATF4 and HRI-depleted cells. ATF4 ChIP-seq in both HUDEP2 and primary erythroblast identified 4,547 and 3,614 high confidence binding sites, respectively. Notably, we did not observe significant enrichment of ATF4 binding or even the presence of an ATF4 consensus motif at the γ-globin promoters, suggesting that ATF4 regulates the γ-globin genes indirectly. However, ATF4 specifically bound to one of the three major BCL11A erythroid enhancers (+55) in both cell types. This was the sole binding site within the ~0.5Mb topologically associating domain that contains the BCL11A gene. Eliminating this ATF4 motif via CRISPR guided genome editing lowered BCL11A mRNA levels and increased γ-globin transcription. Capture-C showed that ATF4 knock-out or removal of the ATF4 site at the BCL11A (+55) enhancer decreased chromatin contacts with the BCL11A promoter. Forced expression of BCL11A largely restored γ-globin silencing in cells deficient for ATF4 or lacking the ATF4 motif in the BCL11A (+55) enhancer. An unexplained observation from our prior study was that HRI loss did not significantly lower Bcl11a levels in murine erythroid cells. Therefore, we mutated the analogous ATF4 motif in the Bcl11a enhancer in the murine erythroid cell line G1E. Unlike in human cells, Bcl11a mRNA synthesis was decreased only very modestly, and there was no effect on the murine embryonic globin genes whose silencing requires Bcl11a. This suggests that the species specific regulation of BCL11A by HRI results from divergent functional roles of ATF4 binding at the BCL11A (+55) enhancer. In sum, our studies uncover a major pathway that extends linearly from HRI to ATF4 to BCL11A to γ-globin. Moreover, these results further support HRI as a pharmacologic target for the selective regulation of BCL11A and γ-globin. Disclosures Blobel: Pfizer: Research Funding; Bioverativ: Research Funding.

The SCL Transcription Factor Supports Erythroid Cell Survival and Differentiation Downstream of the Erythropoietin Receptor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 818-818
Author(s):  
Rachid Lahlil ◽  
Richard Martin ◽  
Peter D. Aplan ◽  
C. Glenn Begley ◽  
Jacqueline E. Damen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Survival ◽  
Cell Fate ◽  
Genetic Interaction ◽  
Fetal Liver ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Loss Of Function

Abstract Erythroid cell development critically depends on the SCL/Tal1 transcription factor and on erythropoietin signalling. In the present study, we have taken several approaches to show that the two genes operate within the same pathway to consolidate the erythroid lineage. Signaling through the erythropoietin receptor (EpoR) upregulates SCL protein levels in a clonal cell line (TF-1) in vitro, and in murine fetal liver cells in vivo, when Epor−/− cells were compared to those of wild type littermates at E12.5. In addition, we provide functional evidence for a linear pathway from EpoR to SCL that regulates erythropoiesis. Interfering with SCL induction or SCL function prevents the anti-apoptotic effect of Epo in TF-1 cells and conversely, ectopic SCL expression is sufficient to substitute for Epo to transiently maintain cell survival. In vivo, SCL gain of function complements the cellular defects in Epor−/− embryos to support cell survival and maturation during primitive and definitive erythropoiesis, as assessed by cellular and histological analyses of Epor−/− SCLtg embryos. Moreover, several erythroid specific genes that are decreased in Epor−/− embryos are rescued by the SCL transgene including glycophorinA, bH1 and bmaj globin, providing molecular confirmation of the functional and genetic interaction between Epor and SCL. Conversely, erythropoiesis becomes deficient in compound Epor+/−SCL+/− heterozygote mice, indicating that the genetic interaction between EpoR and SCL is synthetic. Finally, using EpoR mutants that harbour well defined signalling deficiencies, combined with gain and loss of function approaches for specific kinases, we identify MAPK as the major signal transduction pathway downstream of EpoR that upregulates SCL function, necessary for erythroid cell survival and differentiation. Taken together, our observations are consistent with the view that cytokines can influence cell fate by altering the dosage of lineage transcriptional regulators.

Inhibitory Effect of the Transcription Factor Encoded by themi Mutant Allele in Cultured Mast Cells of Mice

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1189-1196 ◽  
Author(s):  
Akihiko Ito ◽  
Eiichi Morii ◽  
Dae-Ki Kim ◽  
Tatsuki R. Kataoka ◽  
Tomoko Jippo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mast Cells ◽  
Leucine Zipper ◽  
Tryptophan Hydroxylase ◽  
Cdna Probe ◽  
Inhibitory Effect ◽  
Northern Analysis ◽  
Helix Loop Helix ◽  
Genes Encoding ◽  
Mouse Mast Cell

The mi locus of mice encodes a transcription factor of the basic-helix-loop-helix-leucine zipper protein family (MITF). The MITF encoded by the mutant mi allele (mi-MITF) deletes 1 of 4 consecutive arginines in the basic domain. The mice of mi/migenotype express mi-MITF, whereas the mice of tg/tggenotype have a transgene at the 5′ flanking region of themi gene and do not express any MITF. To investigate the function of mi-MITF in cultured mast cells (CMCs), we took two approaches. First, mRNA obtained from mi/mi CMCs ortg/tg CMCs was subtracted from complementary (c) DNA library of normal (+/+) CMCs, and the (+/+-mi/mi) and (+/+-tg/tg) subtraction libraries were obtained. When the number of clones that hybridized more efficiently with +/+ CMC cDNA probe than with mi/mi or tg/tg CMC cDNA probe was compared using Southern analysis, the number was larger in the (+/+-mi/mi) library than in the (+/+-tg/tg) library. Second, we compared mRNA expression of six genes betweenmi/mi and tg/tg CMCs by Northern analysis. The transcription of three genes encoding mouse mast cell proteases was impaired in both mi/mi and tg/tg CMCs. On the other hand, the transcription of three genes encoding c-kit receptor, tryptophan hydroxylase, and granzyme B was markedly reduced inmi/mi CMCs, but the reduction was significantly smaller intg/tg CMCs. These results indicated the inhibitory effect ofmi-MITF on the transactivation of particular genes in CMCs.

scholarly journals In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

1985 ◽  
Vol 5 (8) ◽  
pp. 1887-1893 ◽  
Author(s):  
D Wolf ◽  
Z Laver-Rudich ◽  
V Rotter
Keyword(s):  
Cdna Clone ◽  
P53 Protein ◽  
Cdna Probe ◽  
P53 Gene ◽  
Heteroduplex Analysis ◽  
Cdna Clones ◽  
Free System ◽  
Human Cdna ◽  
Mrna Molecule

The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons.

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