scholarly journals Loading of DNA-Binding Factors to an Erythroid Enhancer

2000 ◽  
Vol 20 (6) ◽  
pp. 1993-2003 ◽  
Author(s):  
Shau-Ching Wen ◽  
Karim Roder ◽  
Kuang-Yu Hu ◽  
Irene Rombel ◽  
Narender R. Gavva ◽  
...  
Keyword(s):  
Regulatory Element ◽  
K562 Cells ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Binding Studies ◽  
Mobility Shift ◽  
Specific Expression ◽  
Factor Binding ◽  
Globin Promoter

ABSTRACT The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage- and erythroid lineage-specific expression of the human α-like globin genes, the embryonic ζ and the adult α2/α/1. A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3′-NA, modulates the capability of HS-40 to activate the embryonic ζ-globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecular basis of this model. First, human erythroid K562 cells stably integrated with various HS-40 mutants cis linked to a human α-globin promoter-growth hormone hybrid gene were analyzed by genomic footprinting and expression analysis. By the assay, we demonstrate that factors bound at different motifs of HS-40 indeed act in concert to build a fully functional enhanceosome. Thus, modification of factor binding at a single motif could drastically change the configuration and function of the HS-40 enhanceosome. Second, a specific 1-bp, GC→TA mutation in the 3′-NA motif of HS-40, 3′-NA(II), has been shown previously to cause significant derepression of the embryonic ζ-globin promoter activity in erythroid cells. This derepression was hypothesized to be regulated through competitive binding of different nuclear factors, in particular AP1 and NF-E2, to the 3′-NA motif. By gel mobility shift and transient cotransfection assays, we now show that 3′-NA(II) mutation completely abolishes the binding of small MafK homodimer. Surprisingly, NF-E2 as well as AP1 can still bind to the 3′-NA(II) sequence. The association constants of both NF-E2 and AP1 are similar to their interactions with the wild-type 3′-NA motif. However, the 3′-NA(II) mutation causes an approximately twofold reduction of the binding affinity of NF-E2 factor to the 3′-NA motif. This reduction of affinity could be accounted for by a twofold-higher rate of dissociation of the NF-E2–3′-NA(II) complex. Finally, we show by chromatin immunoprecipitation experiments that only binding of NF-E2, not AP1, could be detected in vivo in K562 cells around the HS-40 region. These data exclude a role for AP1 in the developmental regulation of the human α-globin locus via the 3′-NA motif of HS-40 in embryonic/fetal erythroid cells. Furthermore, extrapolation of the in vitro binding studies suggests that factors other than NF-E2, such as the small Maf homodimers, are likely involved in the regulation of the HS-40 function in vivo.

Ferritin Heavy Chain, a Repressor of the Human β-Globin Gene That Binds a Conserved Cagtgc Motif, Is Bound to the Repressed β-Globin Promoter In Vivo in K562 Cells and Specifically Binds an Analogous Site in the Mouse βMajor-Globin Promoter.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1223-1223
Author(s):  
Robert H. Broyles ◽  
Visar Belegu ◽  
Charles A. Stewart ◽  
Quentin N. Pye ◽  
Austin C. Roth ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Chromatin Immunoprecipitation ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cells ◽  
Ferritin Heavy Chain ◽  
Polyclonal Antisera ◽  
Globin Promoter ◽  
F1 Generation

Abstract Ferritin heavy chain (FH), an embryonically-expressed protein in the erythroid lineage, localizes to the nucleus and represses the human adult β-globin promoter in transient expression assays (Broyles et al., PNAS98: 9145, 2001). Recently, we have performed chromatin immunoprecipitation (ChIP) assays with cross-linked chromatin of K562 cells in which the β-globin gene is repressed, using anti-FH polyclonal antisera. These results strongly indicate that FH occupies the repression site (a CAGTGC motif) in vivo. Binding to this -150 site has been previously demonstrated to be required for β-promoter repression in co-transfections. EMSA assays (competitive gel shifts) have revealed that the mouse βMajor-globin promoter has an analogous CAGTGN motif at -160 bp from the cap site that competes specifically with the human CAGTGC site for FH binding. The mouse βMinor-globin promoter lacks the -150/-160 CAGTGN motif and, therefore, the FH binding site. Thus, a human FH transgenic mouse, in which the FH gene is driven by a truncated β-promoter lacking the CAGTGN motif, should express human FH in definitive erythroid cells where the FH would be predicted to repress βMajor-globin but not βMinor-globin. Such a mouse would be predicted to survive but be born with a mild β-thalassemia due to the decreased βMajor/βMinor ratio in its definitive erythroid cells. Preliminary results from the litters of F1 generation FH-tg mice indicate that such is indeed the case, i.e., that human FH functions as a βMajor-globin repressor in vivo.

scholarly journals Alterations in Expression and Chromatin Configuration of the Alpha Hemoglobin-Stabilizing Protein Gene in Erythroid Krüppel-Like Factor-Deficient Mice

2006 ◽  
Vol 26 (11) ◽  
pp. 4368-4377 ◽  
Author(s):  
Andre M. Pilon ◽  
Douglas G. Nilson ◽  
Dewang Zhou ◽  
Jose Sangerman ◽  
Tim M. Townes ◽  
...  
Keyword(s):  
Target Genes ◽  
Zinc Finger Protein ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Wild Type ◽  
Consensus Binding Site ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Globin Promoter

ABSTRACT Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the β-globin promoter, where it establishes local chromatin structure permitting β-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the β-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and β-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements

1993 ◽  
Vol 13 (12) ◽  
pp. 7457-7468
Author(s):  
W L Trepicchio ◽  
M A Dyer ◽  
M H Baron
Keyword(s):  
Developmental Regulation ◽  
Regulatory Element ◽  
Globin Gene ◽  
Negative Control ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Specific Expression ◽  
Systematic Analysis ◽  
Specific Regulation

The stage-specific regulation of mammalian embryonic globin genes has been an experimentally elusive problem, in part because of the developmentally early timing of their expression. We have carried out a systematic analysis of truncation and internal deletion mutations within the 5'-flanking region of the human embryonic beta-like globin gene (epsilon) in erythroid and nonerythroid cell lines. Within a 670-bp region upstream from the constitutive promoter are multiple positive and negative control elements. Of these, a positive regulatory element (epsilon-PRE II) which is active only in embryonic erythroid cells is of particular interest. Remarkably, although it is inactive on its own, in the presence of other sequences located further upstream, it confers tissue- and developmental stage-specific expression on a constitutive epsilon-globin or heterologous promoter. The activity of epsilon-PRE II is also modulated by another positive regulatory domain located further downstream to direct erythroid cell-specific, but little or no embryonic stage-specific, transcription. A nuclear factor highly enriched in embryonic erythroid cells binds specifically within a 19-bp region of epsilon-PRE II. Nuclei from adult erythroid cells also contain a factor that binds to this region but forms a complex of faster electrophoretic mobility. We speculate that interactions between epsilon-PRE II and other upstream control elements play an important role in the developmental regulation of the human embryonic beta-like globin gene.

scholarly journals Identification of a major positive regulatory element located 5' to the human zeta-globin gene

Blood ◽  
1995 ◽  
Vol 85 (9) ◽  
pp. 2587-2597 ◽  
Author(s):  
DE Sabath ◽  
KM Koehler ◽  
WQ Yang ◽  
K Patton ◽  
G Stamatoyannopoulos
Keyword(s):  
Point Mutation ◽  
Promoter Activity ◽  
Regulatory Element ◽  
Globin Gene ◽  
K562 Cells ◽  
Luciferase Expression ◽  
Globin Mrna ◽  
Mobility Shift ◽  
Alpha Globin ◽  
Globin Promoter

The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive luciferase expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta-globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-luciferase constructs and the alpha-luciferase constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta-globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5′ to the zeta-globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5′ to the zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This element requires GATA-1 and additional unknown factors for maximal activity.

scholarly journals Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements.

1993 ◽  
Vol 13 (12) ◽  
pp. 7457-7468 ◽  
Author(s):  
W L Trepicchio ◽  
M A Dyer ◽  
M H Baron
Keyword(s):  
Developmental Regulation ◽  
Regulatory Element ◽  
Globin Gene ◽  
Negative Control ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Specific Expression ◽  
Systematic Analysis ◽  
Specific Regulation

The stage-specific regulation of mammalian embryonic globin genes has been an experimentally elusive problem, in part because of the developmentally early timing of their expression. We have carried out a systematic analysis of truncation and internal deletion mutations within the 5'-flanking region of the human embryonic beta-like globin gene (epsilon) in erythroid and nonerythroid cell lines. Within a 670-bp region upstream from the constitutive promoter are multiple positive and negative control elements. Of these, a positive regulatory element (epsilon-PRE II) which is active only in embryonic erythroid cells is of particular interest. Remarkably, although it is inactive on its own, in the presence of other sequences located further upstream, it confers tissue- and developmental stage-specific expression on a constitutive epsilon-globin or heterologous promoter. The activity of epsilon-PRE II is also modulated by another positive regulatory domain located further downstream to direct erythroid cell-specific, but little or no embryonic stage-specific, transcription. A nuclear factor highly enriched in embryonic erythroid cells binds specifically within a 19-bp region of epsilon-PRE II. Nuclei from adult erythroid cells also contain a factor that binds to this region but forms a complex of faster electrophoretic mobility. We speculate that interactions between epsilon-PRE II and other upstream control elements play an important role in the developmental regulation of the human embryonic beta-like globin gene.

Activation of octamer-containing promoters by either octamer-binding transcription factor 1 (OTF-1) or OTF-2 and requirement of an additional B-cell-specific component for optimal transcription of immunoglobulin promoters

1990 ◽  
Vol 10 (12) ◽  
pp. 6204-6215
Author(s):  
A Pierani ◽  
A Heguy ◽  
H Fujii ◽  
R G Roeder
Keyword(s):  
B Cell ◽  
Regulatory Element ◽  
Immunoglobulin Gene ◽  
Mobility Shift ◽  
Specific Expression ◽  
Tissue Specific ◽  
Activation Domains ◽  
Cell Extracts ◽  
Octamer Motif ◽  
High Level

Several distinct octamer-binding transcription factors (OTFs) interact with the sequence ATTTGCAT (the octamer motif), which acts as a transcription regulatory element for a variety of differentially controlled genes. The ubiquitous OTF-1 plays a role in expression of the cell cycle-regulated histone H2b gene as well as several other genes, while the tissue-specific OTF-2 has been implicated in the tissue-specific expression of immunoglobulin genes. In an attempt to understand the apparent transcriptional selectivity of these factors, we have investigated the physical and functional characteristics of OTF-1 purified from HeLa cells and both OTF-1 and OTF-2 purified from B cells. High-resolution footprinting and mobility shift-competition assays indicated that these factors were virtually indistinguishable in binding affinities and DNA-protein contacts on either the H2b or an immunoglobulin light-chain (kappa) promoter. In addition, each of the purified factors showed an equivalent intrinsic capacity to activate transcription from either immunoglobulin promoters (kappa and heavy chain) or the H2b promoter in OTF-depleted HeLa and B-cell extracts. However, with OTF-depleted HeLa extracts, neither factor could restore immunoglobulin gene transcription to the relatively high level observed in unfractionated B-cell extracts. Restoration of full immunoglobulin gene activity appears to require an additional B-cell regulatory component which interacts with the OTFs. The additional B-cell factor could act either by facilitating interaction of OTF activation domains with components of the general transcriptional machinery or by contributing a novel activation domain.

scholarly journals AUF-1 and YB-1 are critical determinants of β-globin mRNA expression in erythroid cells

Blood ◽  
2012 ◽  
Vol 119 (4) ◽  
pp. 1045-1053 ◽  
Author(s):  
Sebastiaan van Zalen ◽  
Grace R. Jeschke ◽  
Elizabeth O. Hexner ◽  
J. Eric Russell
Keyword(s):  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Affinity Enrichment

Abstract The normal accumulation of β-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate β-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of β-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted β-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34+ cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the β-globin 3′untranslated region that has recently been implicated as a determinant of β-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of β-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of β-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of β-globin gene expression.

Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins

1993 ◽  
Vol 13 (11) ◽  
pp. 6690-6701
Author(s):  
H Koizumi ◽  
M F Horta ◽  
B S Youn ◽  
K C Fu ◽  
B S Kwon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Site ◽  
Protein Interactions ◽  
Regulatory Element ◽  
Killer Cell ◽  
Mobility Shift ◽  
Specific Expression ◽  
Native Molecular Mass ◽  
Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice

1992 ◽  
Vol 12 (9) ◽  
pp. 3978-3990
Author(s):  
B Liu ◽  
G D Hammer ◽  
M Rubinstein ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Intermediate Lobe ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dna Elements ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

scholarly journals A protein factor binding to an octamer motif in the γ-globin promoter disappears upon induction of differentiation and hemoglobin synthesis in K562 cells

1987 ◽  
Vol 15 (22) ◽  
pp. 9349-9364 ◽  
Author(s):  
Roberto Mantovani ◽  
Nicoletta Malgaretti ◽  
Barbara Giglioni ◽  
Paola Comi ◽  
Nica Cappellini ◽  
...  
Keyword(s):  
K562 Cells ◽  
Hemoglobin Synthesis ◽  
Factor Binding ◽  
Protein Factor ◽  
Octamer Motif ◽  
Induction Of Differentiation ◽  
Globin Promoter
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