Proteomic Identification of TAL1/SCL-Interacting Proteins: ETO-2 and MTGR1 Interact with TAL1 in Erythroid Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 357-357
Author(s):  
Ying Cai ◽  
Zhixiong Xu ◽  
Jingping Xie ◽  
Mark J. Koury ◽  
Scott W. Hiebert ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Bhlh Transcription Factor ◽  
Interacting Proteins ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Abstract The TAL1/SCL gene, originally identified from its involvement by a recurrent chromosomal translocation in T-cel acute lymphoblastic leukemia, encodes a basic helix-loop-helix (bHLH) transcription factor essential for hematopoietic and vascular development. Although TAL1 is believed to regulate transcription of specific sets of target genes, the mechanisms underlying TAL1-directed gene expression are poorly understood. Previous studies have shown, in fact, that it can act as either an activator or repressor depending on the coregulator(s) with which it interacts. To comprehensively identify TAL1’s interaction partners in erythroid cells, we stably expressed a tandem epitope-tagged mouse TAL1 protein in murine erythroleukemia (MEL) cells and determined the composition of affinity-purified TAL1-containing complexes by multidimensional mass spectrometry. From this analysis, we identified all known members of a TAL1-containing DNA binding complex previously characterized in erythroid cells, including TAL1, its E protein DNA-binding partners, the zinc finger transcription factor GATA-1, the LIM-only protein LMO2, and the LIM domain-binding protein Ldb1, as well as proteins described to interact with GATA-1 (FOG-1), LMO2 (ELF2A2), and Ldb1 (SSDP2 and SSDP3). In addition, we identified a number of other DNA binding proteins, chromatin modifying proteins, and transcriptional regulators, including the ETO family members ETO-2 and MTGR1. TAL1 interaction with ETO-2 and MTGR1 was verified by coimmunoprecipitation analysis in MEL cells expressing these proteins at endogenous levels, in MEL cells stably expressing an epitope-tagged TAL1 protein, and in COS cells transiently transfected with TAL1 and ETO-2 or MTGR1 expression vectors. Mapping analysis with GAL4 fusion proteins identified the bHLH domain as the region in TAL1 responsible for interaction with these ETO family proteins. Significantly, expression of MTGR1 enhanced ETO-2 interaction with TAL1-GAL4 protein. Finally, transient transfection analysis with a luciferase reporter construct linked to multiple GAL4 DNA binding sites showed greater than additive augmentation of TAL1-directed gene repression with coexpression of the two ETO-related proteins compared to that observed with ETO-2 or MTGR1 transfected individually. These results identify ETO-2 and MTGR1 as authentic TAL1 interacting proteins and suggest that a hetero-oligomeric complex of the two contributes to TAL1-directed repression in erythroid progenitors.

Incorporation of a Histone Demethylase, JARID1B, in a Novel ETS-E Box DNA-Binding Complex in Murine Erythroid Progenitors.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2335-2335
Author(s):  
Ying Cai ◽  
Lalitha Nagarajan ◽  
David J. Curtis ◽  
Stephen J. Brandt
Keyword(s):  
Dna Binding ◽  
Histone Demethylase ◽  
Mass Spectrometry Analysis ◽  
Sequence Motif ◽  
Erythroid Cells ◽  
Lim Domain ◽  
Erythroid Progenitors ◽  
E Box ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Abstract Abstract 2335 The TAL1/SCL gene, originally discovered from its involvement by a recurrent chromosomal translocation in T-cell acute lymphoblastic leukemia, is important for hematopoietic stem cell and progenitor function and is essential for hematopoietic and vascular development. A member of the basic helix-loop-helix family of transcription factors, TAL1 binds a DNA sequence motif, CANNTG, termed the E box. We and others recently identified a novel TAL1-containing DNA-binding complex that includes an ETS protein, ELF2 (also known as NERF) in erythroid cells, and recognizes a bipartite sequence element containing an adjacent ETS binding site and E box. Our work showed this complex also contains proteins common to other TAL1 DNA-binding complexes described, including a LIM domain protein, LMO2 in erythroid cells, the LIM domain binding protein Ldb1, and putative single-stranded DNA-binding proteins SSBP2 and SSBP3. As both ELF2 and histone demethylase JARID1A, and later the related JARID1B, were identified using the same methodology (yeast two-hybrid analysis) to interact with LMO2, and multiple peptides derived from Jarid1b were identified by mass spectrometry analysis of highly purified Tal1-containing complexes from murine erythroleukemia (MEL) cells, we investigated whether JARID1B was present in the TAL1- and ELF2-containing complex. First, co-immunoprecipitation analysis identified Jarid1b in Tal1- or Elf2-containing immune precipitates and Tal1 in Jarid1b-containing immune precipitates from MEL cell extracts. Further, chromatin immunoprecipitation (ChIP) and re-ChIP analysis showed that Elf2 and Jarid1b co-occupied a region in the fourth intron of the Ssbp3 gene in MEL cells, previously demonstrated to be a physiologic target of the Tal1/Elf2 complex. Finally, knockdown of Elf2 and Jarid1b in MEL cells produced the same phenotype, decreased cellular proliferation. These results suggest a role for the JARID type histone demethylase JARID1B, and by inference histone demethylation, in the function of the ETS-E box DNA-binding complex and in proliferation of late-stage murine erythroid progenitors. Disclosures: No relevant conflicts of interest to declare.

Linking Transcription Factor Pathways to Disease-Causing GATA1 Mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2371-2371
Author(s):  
Amy E Campbell ◽  
Gerd A. Blobel
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Erythroid Cells ◽  
Amino Terminal ◽  
Erythroid Maturation ◽  
Cofactor Recruitment ◽  
In Erythroid Cells

Abstract Abstract 2371 Missense mutations in the gene encoding hematopoietic transcription factor GATA1 cause congenital anemias and/or thrombocytopenias. Seven such mutations are reported. All of these give rise to amino acid substitutions within the amino terminal zinc finger (NF) of GATA1, producing a range of clinical phenotypes. Thus, V205M, G208R, and D218Y cause severe anemia and thrombocytopenia; G208S, R216Q, and D218G cause thrombocytopenia with minimal anemia; R216W gives rise to thrombocytopenia and congenital erythropoietic porphyria. One of these mutations, R216Q, occurs at the DNA binding interface and alters the ability of GATA1 to recognize a subset of cis motifs in vitro. Other mutations, including V205M, G208S, D218G, and D218Y, occur outside the DNA binding domain of the NF and inhibit interactions with the GATA1 cofactor FOG1 as determined by in vitro binding assays. However, these two mechanisms do not easily explain the broad spectrum of phenotypes associated with the mutations. For example, how do two substitutions of the same residue bring about disparate phenotypes? We examined the effects of each mutation on erythroid maturation, lineage-specific gene expression, in vivo target gene occupancy, and cofactor recruitment by introducing altered forms of GATA1 into murine GATA1-null proerythroblasts. The V205M, G208R, and D218Y mutations severely impaired erythroid maturation, recapitulating patient phenotypes. The G208S mutation also severely impaired erythroid maturation, causing a more pronounced defect than that expected from the clinical presentation. In contrast, R216Q and D218G produced mild effects in erythroid cells consistent with patient phenotypes. The porphyria-associated mutation R216W also produced relatively subtle effects in erythroid cells. We note that among the mutants, failure to activate gene expression strongly correlated with failure to repress gene expression. ChIP assays revealed that the V205M, G208R, and D218Y mutations impaired GATA1 target site occupancy. This indicates that despite normal DNA binding in vitro, the association with cofactor complexes is required for stable binding to chromatinized target sites in vivo. In contrast, the G208S mutant exhibited relatively normal chromatin occupancy, but reduced recruitment of FOG1 and SCL/Tal1 to GATA1-bound sites at erythroid genes. D218G also perturbed cofactor recruitment without greatly affecting GATA1 binding to its target genes. Notably, this mutation diminished SCL/Tal1 recruitment without significantly altering FOG1 occupancy. This implicates the SCL/Tal1 transcription complex in the pathogenesis of disorders caused by certain GATA1 mutations. Moreover, by uncoupling GATA1 chromatin occupancy and cofactor recruitment, G208S and D218G offer potentially useful tools for unraveling site-specific mechanisms of GATA1-regulated gene expression. Finally, both the R216Q and R216W mutants displayed relatively normal GATA1 chromatin occupancy and FOG1 and SCL/Tal1 recruitment at most sites. R216W presents as porphyria, and selective defects in the regulation of heme biosynthetic genes have yet be uncovered. Given that R216Q presents as thrombocytopenia, defects caused by this mutation may be revealed only in the context of megakaryocytes. Studies using similar rescue assays of a GATA1-null megakaryocyte-erythroid progenitor line are underway and will be discussed. In concert, our results reveal that in vivo analysis of GATA1 in its native environment provides mechanistic insights not obtainable from in vitro studies. Moreover, they demonstrate the usefulness of gene complementation assays for the dissection of transcription pathways surrounding normal and altered GATA1 to improve our understanding of disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 54-54
Author(s):  
Xianjiang Lan ◽  
Ren Ren ◽  
Ruopeng Feng ◽  
Lana C Ly ◽  
Yemin Lan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Target Gene ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Private Company ◽  
In Erythroid Cells

Transcription factors typically regulate a large number of genes. Here we found that transcription factor ZNF410 binds and activates the expression of a single direct target gene, CHD4, to enforce the silencing of the fetal hemoglobin genes (HBG1 and HBG2) in adult erythroid cells. ZNF410 is a pentadactyl DNA binding protein that emerged from a DNA binding domain-focused CRISPR-Cas9 screen aimed at the identification of new regulators of fetal hemoglobin silencing. Depletion of ZNF410 specifically diminished CHD4 expression, leading to reactivation of the normally silent fetal globin genes in both human erythroid culture systems and a human-to-mouse xenotransplant model. Combining RNA-seq and ChIP-seq analyses revealed that CHD4 is the sole direct ZNF410 target gene in erythroid cells, which was further validated by rescue of fetal hemoglobin silencing and other transcriptional changes upon CHD4 restoration in ZNF410-deficient cells. ZNF410 ChIP-seq detected only eight high-confidence peaks with seven associated genes including CHD4. Most strikingly, the two most predominant peaks are located at the CHD4 locus, which contains two highly conserved, dense clusters of ZNF410 binding motifs. The two motif clusters appear to be unique in the human and mouse genomes. Moreover, among the seven ZNF410-bound genes, CHD4 was the only one whose expression was down-regulated upon ZNF410 depletion, indicating that CHD4 is the sole target of ZNF410. Electrophoretic mobility shift assays (EMSAs) showed that the zinc finger (ZF) domain of ZNF410 is necessary and sufficient for DNA binding. When overexpressed, the DNA binding profile of ZF domain alone is very similar to full length ZNF410. Indeed, forced expression of the ZF domain displaced endogenous ZNF410 at all binding sites, including the CHD4 locus. This reduced CHD4 expression to levels comparable to those in ZNF410 deficient cells (and activated the fetal globin genes) but had no effect on the other ZNF410 bound genes, again confirming target specificity. ZNF410 depletion or expression of the dominant negative acting ZF domain lowered CHD4 only by ~65%-70%, which is very well tolerated by erythroid cells, as determined by morphology, cell surface phenotyping, and gene expression profiling. This exposes the fetal globin genes as highly sensitive to CHD4 levels. Lastly, we solved the crystal structure of the ZF domain-DNA complex at 2.75Å resolution pinpointing the protein-DNA contacts and showing that each of the five ZFs make specific DNA contacts. In sum, to our knowledge, ZNF410 is the only transcription factor with just one direct functional target gene in erythroid cells. Given the strong impetus to reactivate fetal globin gene expression in patients with sickle cell disease and some forms of b-thalassemia, it might be possible to exploit the exceptionally high transcriptional selectivity of ZNF410 to raise fetal hemoglobin expression for the treatment of these hemoglobinopathies. Disclosures Weiss: Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company. Blobel:Fulcrum Therapeutics: Consultancy; Pfizer: Research Funding.

trans-Activation of a globin promoter in nonerythroid cells

1991 ◽  
Vol 11 (2) ◽  
pp. 843-853
Author(s):  
T Evans ◽  
G Felsenfeld
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Gene Activation ◽  
Specific Factor ◽  
Erythroid Cells ◽  
Specific Manner ◽  
Alpha Globin ◽  
A Cell ◽  
Globin Promoter ◽  
In Erythroid Cells

We show that expression in fibroblasts of a single cDNA, encoding the erythroid DNA-binding protein Eryf1 (GF-1, NF-E1), very efficiently activates transcription of a chicken alpha-globin promoter, trans-Activation in these cells occurred when Eryf1 bound to a single site within a minimal globin promoter. In contrast, efficient activation in erythroid cells required multiple Eryf1 binding sites. Our results indicate that mechanisms exist that are capable of modulating the trans-acting capabilities of Eryf1 in a cell-specific manner, without affecting DNA binding. The response of the minimal globin promoter to Eryf1 in fibroblasts was at least as great as for optimal constructions in erythroid cells. Therefore, the assay provides a very simple and sensitive system with which to study gene activation by a tissue-specific factor.

scholarly journals A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1384-1392 ◽  
Author(s):  
I Plavec ◽  
T Papayannopoulou ◽  
C Maury ◽  
F Meyer
Keyword(s):  
Bone Marrow Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Abstract Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

scholarly journals Dynamics of GATA transcription factor expression during erythroid differentiation

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1071-1079 ◽  
Author(s):  
M Leonard ◽  
M Brice ◽  
JD Engel ◽  
T Papayannopoulou
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Erythroid Differentiation ◽  
Regulatory Control ◽  
Leukemic Cells ◽  
Erythroid Cells ◽  
Gata Factor ◽  
Erythroid Progenitors ◽  
Factor Expression

Abstract Although the formation of terminally differentiated erythroid cells has been shown to require the presence of a functional GATA-1 gene in vivo, the role of this transcription factor and other members of the GATA family at earlier stages of erythroid differentiation is unclear. In this report, the expression of GATA-1, GATA-2, and GATA-3 has been examined in enriched peripheral blood progenitors before and after culture in a well-characterized liquid culture system. In addition primary leukemic cells as well as several erythroleukemic and nonerythroid cell lines were analyzed for GATA factor expression. The results show that the profile of GATA factor expression in erythroid cells is distinct from that of myeloid or lymphoid lineages. Erythroleukemic cell lines express little or no GATA-3, but high levels of GATA-1 and GATA-2. When they are induced to display the terminal erythroid phenotype, little change in the level of GATA-1 is detected but a significant decline in the levels of GATA-2 is observed commensurate with the degree of maturation achieved by the cells. Enrichment of erythroid progenitors from peripheral blood leads to selection of cells that express both GATA-1 and GATA-2. As the enriched populations are cultured in suspension in the presence of multiple cytokines, the levels of both GATA-1 and GATA-2 initially increase. However, in cultures containing only erythropoietin, which show exclusive erythroid differentiation, the levels of GATA-1 continue to increase, whereas GATA-2 expression declines as erythroid maturation progresses. In contrast, cultures lacking Epo (ie, with interleukin-3 and kit ligand) display limited progression towards both the myeloid and erythroid pathways, and high levels of expression of both GATA-1 and GATA-2 are maintained. Despite the initial upregulation of GATA-1 expression in the latter cultures, terminal erythroid differentiation does not occur in the absence of erythropoietin. These results indicate that GATA-1 upregulation is associated with both the initiation and the maintenance of the erythroid program, but that these two processes appear to be under separate regulatory control. Thus, the dynamic changes in the levels of different GATA factors that occur during primary erythroid differentiation suggest that the levels of these factors may influence the progression to specific hematopoietic pathways.

Regulation of SCL Expression by the Homeodomain Protein Otx-1 and the Erythroid Transcription Factor GATA-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1598-1598
Author(s):  
Richard Martin ◽  
Virginie Sanguin-Gendreau ◽  
Mathieu Tremblay ◽  
Elena Levantini ◽  
Christina Magli ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fold Increase ◽  
Proximal Promoter ◽  
Physical Interaction ◽  
Homeodomain Protein ◽  
Erythroid Cells ◽  
Gain Of Function ◽  
Erythroid Progenitor ◽  
Left Right Asymmetry ◽  
In Erythroid Cells

Abstract Members of the bicoid homeodomain-containing proteins are important in establishing left-right asymmetry and the antero-posterior axis, suggesting that they could also be involved in asymmetric determination within the hematopoietic system. We have previously shown that Otx1, a member of the bicoid homeodomain-containing proteins, is co-expressed with the SCL transcription factor in hematopoietic pluripotent and erythroid progenitor cells and Otx1-deficiency impairs the erythroid compartment in mice, associated with decreased SCL levels. In the present study, we provide molecular and functional evidence that SCL is a direct transcriptional target of Otx1. First, we show by chromatin immunoprecipitation that Otx1 and GATA-1 are specifically bound to the SCL proximal promoter in erythroid cells. Second, Otx-1 synergizes with GATA-1 to activate transcription from the SCL proximal promoter and this activity depends on the integrity of the proximal GATA site of the SCL promoter 1a. At the molecular level, we show that this synergy occurs via a physical interaction between Otx-1 and GATA-1 in erythroid cells, which maps to the homeodomain of Otx-1. Furthermore, a gain of function of Otx1 in primary hematopoietic cells gives rise to a 6-fold increase in endogenous SCL levels, an increase in TER119-positive erythroid cells and a decrease in the number of CD11b-positive myeloid cells. Finally, a gain of function of SCL rescues the erythroid deficiency in Otx1−/− mice, consistent with the view that SCL operates downstream of Otx1. Taken together, our observations indicate that Otx1, GATA-1 and SCL operate within the same genetic pathway to specify the erythroid fate during hematopoiesis.

Chromatin Conformation and a Distal Regulatory Element Activate the Human Erythroid Ankyrin-1 Promoter.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 803-803
Author(s):  
Ashley N. Owen ◽  
Robert I. Liem ◽  
Andre M. Pilon ◽  
Patrick G. Gallagher ◽  
David M. Bodine
Keyword(s):  
Reporter Gene ◽  
Regulatory Element ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Dnase I ◽  
Luciferase Reporter ◽  
Erythroid Cells ◽  
Luciferase Reporter Gene ◽  
Human And Mouse ◽  
In Erythroid Cells

Abstract Ankyrin forms the bridge between the spectrin/actin network of the erythrocyte membrane skeleton and the red cell membrane by binding to both β-spectrin and band 3. The erythrocyte ankyrin promoter (Ank-1E) is active only in erythroid cells, while two other Ank-1 promoters located 20 kb downstream and 40 kb upstream of Ank-1E are active in the cerebellum and muscle cells respectively. We have been studying the mechanism by which the Ank-1E promoter becomes active in erythroid cells by studying the cis acting regulatory elements and the chromatin structure of the Ank-1 promoter region. We have previously shown that the sequences between −296 and −15 of the Ank-1E promoter are fully sufficient for erythroid specific, copy number dependent uniform expression of reporter genes in transgenic mice. We have also mapped a DNase I Hypersensitive site (5′HS) between −300 and −100 of the human and mouse Ank-1E promoters in human K562 and mouse fetal liver cells. Both the mouse and human 5′HS are capable of preventing the silencing of a β-globin/GFP reporter gene in K562 cells, establishing that they function as barrier elements. Consistent with this observation, the human and mouse 5′HS are hyperacetylated in erythroid cells. The chromatin 10 kb 5′ to the 5′HS is DNase I resistant (associated with inactive chromatin) in human and mouse erythroid and non-erythroid cells. Approximately 6 kb 3′ to 5′HS are two adjacent HS (3′HS1, 3′HS2). Beyond 3′HS2 the chromatin is also DNase I resistant in both human and mouse erythroid and non-erythroid cells. Between 5′HS and 3′HS1 the 6kb region is DNase I sensitive (active) in erythroid cells but not in other cell types. We hypothesized that this 6 kb region contains regulatory elements that activate the Ank-1E promoter. To screen for regulatory elements we isolated overlapping segments of a 10 kb region extending from 2 kb upstream of 5′HS to 2 kb downstream of 3′HS2. We inserted these fragments into a plasmid vector containing the Ank-1E promoter linked to a luciferase reporter gene and transfected these constructs into K562 cells. A single region up regulated Ank-1E/luciferase expression. This region mapped to a 211bp segment that included 3′HS1, but did not include 3′HS2. A fragment containing only 3′HS2 did not up regulate an Ank-1E/luciferase reporter gene, but 3′HS2 was capable of preventing the silencing of a β-globin/Green Fluorescent Protein reporter gene in K562 cells, demonstrating barrier activity. The region around 3′HS1 and 2 was also a site of histone hyperacetylation. The sequence of the 211 bp fragment containing 3′HS1 does not contain consensus sequences for any known erythroid-specific transcription factors, but does contain potential binding sites fro Sp1, AP-1 and E-box binding proteins. Using the Chromatin Conformation Capture assay we demonstrated that 5′HS and 3′HS1 and 2 are in close proximity in K562 chromatin, but are not closely associated in chromatin from other cell types. We propose that an erythroid-specific chromatin loop brings 3′HS1 and 2 into proximity with 5′HS, adjacent to the Ank-1E promoter. This interaction translocates the positive regulatory element in 3′HS1 to the Ank-1E promoter allowing the Ank-1E promoter to become active in erythroid cells.

scholarly journals cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein

1999 ◽  
Vol 339 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Takashi TANAKA ◽  
Tetsuya INAZU ◽  
Kazuya YAMADA ◽  
Zaw MYINT ◽  
Vincent W. KENG ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Rat Liver ◽  
Functional Characterization ◽  
Homeobox Gene ◽  
Hepatoma Cells ◽  
Cloning And Expression ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Cdna Clones

We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of α-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.

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