RUNX1 Directly Regulates Band 3 Transcription.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1735-1735
Author(s):  
Jingping Xie ◽  
Scott W. Hiebert ◽  
Mark J. Koury ◽  
Stephen J. Brandt
Keyword(s):  
Histone Acetylation ◽  
Gene Knockout ◽  
Regulatory Region ◽  
Erythroid Differentiation ◽  
Band 3 ◽  
Upstream Region ◽  
Pcr Analysis ◽  
Upstream Regulatory Region ◽  
Terminal Erythroid Differentiation ◽  
Chip Analysis

Abstract RUNX1 (AML1 or CBFA2) regulates the expression of a number of genes important to hematopoiesis. Gene knockout studies demonstrated that a heterodimeric complex of RUNX1 and its DNA binding partner, core binding factor-beta (CBFbeta), is essential for definitive hematopoiesis. Here, we report that RUNX1 directly represses expression of the Band 3 gene prior to terminal erythroid differentiation. Band 3 is one of four major components of the erythrocyte membrane skeleton and is important for maintenance of cytoskeletal architecture and electroneutral Cl-/HCO3− exchange across the red cell membrane. Band 3 expression, like that of beta-globin, increases dramatically with terminal erythroid differentiation. In a previous study, we identified an upstream region in the mouse Band 3 gene designated as B3URE (for Band 3 upstream regulatory region) bound by multiple transcription factors, including TAL1 (also known as SCL), RUNX1, Ldb1, and GATA1, that acts as an orientation- and position-independent and tissue-specific repressive element. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 was associated with the B3URE in intact MEL cells and electrophoretic mobility shift analysis confirmed specific RUNX1 interaction with RUNX1 binding sites in the B3URE. Together with CBFbeta, RUNX1 inhibited reporter activity from a construct linking the B3URE with 1 kb of Band 3 promoter in transiently transfected MEL but not COS cells. DNA affinity precipitation analysis with wild-type and mutant oligos established that RUNX1 and CBFbeta in MEL cell nuclear extracts could interact with the B3URE in vitro and suggested that RUNX1 recruits TAL1 and Ldb1 to DNA. Northern blot and quantitative real-time PCR analysis demonstrated that enforced expression of RUNX1 dramatically inhibited dimethylsulfoxide (DMSO)-induced Band 3 gene expression. Quantitative ChIP analysis showed that histone acetylation in the B3URE increased more than 4-fold, while histone methylation decreased ~50% after 3 days of DMSO-induced differentiation. Over the same time frame, the promoter region underwent significantly less acetylation but more extensive demethylation. Finally, changes in B3URE acetylation and methylation were attenuated and inhibited, respectively, in RUNX1-transfected MEL cells relative to vector controls. In sum, these results demonstrate that the Band 3 gene is a direct target of RUNX1 in erythroid cells and indicate that the B3URE contributes to the tightly regulated expression of this gene in differentiating erythroid progenitors. One mechanism by which RUNX1 regulates Band 3 transcription may be by influencing histone acetylation/methylation in this upstream regulatory region.

TAL1 Regulates Expression of the Band 3 Gene in Erythroid Progenitors through Both Upstream Regulatory and Proximal Promoter Elements.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1604-1604
Author(s):  
Jingping Xie ◽  
Zhixiong Xu ◽  
Stephen J. Brandt
Keyword(s):  
Genomic Sequence ◽  
Regulatory Region ◽  
Band 3 ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Bhlh Transcription Factor ◽  
E Box ◽  
Murine Erythroleukemia ◽  
Chip Analysis

Abstract The TAL1 (also known as SCL and TCL-5) protein is a class II basic helix-loop-helix (bHLH) transcription factor that plays an important role during embryonic and adult hematopoiesis. We previously established that the Protein 4.2 (P4.2) gene is a physiologic target of TAL1 in erythroid cells and showed that tandem E box-GATA elements in its proximal promoter mediated TAL1-directed gene activation. Through database searches, we identified two E box-GATA consensus sequences in the proximal promoter of another erythroid membrane skeleton gene, that encoding the Band 3 protein. Identical to what was observed for P4.2, overexpression of wild-type TAL1 slightly increased while enforced expression of a DNA binding-defective TAL1 mutant severely reduced endogenous Band 3 gene expression in murine erythroleukemia (MEL) cells induced to differentiate with dimethyl sulfoxide (DMSO). Overexpression of Ldb1 significantly inhibited and a LIM protein interaction-defective Ldb1 mutant virtually ablated Band 3 mRNA accumulation in DMSO-induced MEL cells. Quantitative chromatin immunoprecipitation (ChIP) analysis with Tal1 antibody confirmed that Tal1 was associated with the proximal promoter of the Band 3 gene in MEL cells, while analysis of approximately 28 kb of genomic sequence spanning the Band 3 gene, including 5 kb 5′ and 3′ of the gene, revealed another consensus E box-GATA element in intron 16; however, no significant TAL1 binding was detected in this region by ChIP analysis. To identify potential non-conventional TAL1 binding site(s), scanning ChIP analysis was applied to the entire 28 kb Band 3 genomic region, and a region ~2.5 kb upstream of the major transcriptional start site was found to bind significantly more TAL1 protein than the proximal promoter. Fine mapping of TAL1 binding to this region by ChIP analysis using more highly sheared DNA and smaller sized amplicons narrowed TAL1 binding to a region of ~100 bp, which we designated as the Band 3 upstream regulatory region (B3URE). Luciferase reporter assays in transiently transfected MEL cells with vectors containing genomic sequence revealed the presence of an orientation- and position- independent repressor in this upstream region, with a 107 bp fragment retaining nearly all the repressing activity of a larger, 700 bp fragment. In contrast, coexpression of TAL1, E47, GATA-1, LMO2, and Ldb1 in COS cells led to transactivation of a reporter gene linked to the promoter-proximal E box-GATA element. These data suggest that TAL1 acts as a repressor on the upstream B3URE in undifferentiated cells and then, as differentiation proceeds, as an activator on the E box-GATA elements in the proximal promoter.

Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (7) ◽  
pp. 2324-2333
Author(s):  
L Sarokin ◽  
M Carlson
Keyword(s):  
Carbon Catabolite Repression ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Lacz Fusion ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Heterologous Promoter ◽  
Suc2 Gene ◽  
High Level ◽  
Repeated Motif

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.

scholarly journals The Locus Control Region Activates Serpin Gene Expression through Recruitment of Liver-Specific Transcription Factors and RNA Polymerase II

2007 ◽  
Vol 27 (15) ◽  
pp. 5286-5295 ◽  
Author(s):  
Hui Zhao ◽  
Richard D. Friedman ◽  
R. E. K. Fournier
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Protease Inhibitor ◽  
Histone Acetylation ◽  
Rna Polymerase Ii ◽  
Control Region ◽  
Regulatory Region ◽  
Locus Control Region ◽  
Upstream Regulatory Region ◽  
Serpin Gene

ABSTRACT The human serine protease inhibitor (serpin) gene cluster at 14q32.1 comprises 11 serpin genes, many of which are expressed specifically in hepatic cells. Previous studies identified a locus control region (LCR) upstream of the human α1-antitrypsin (α1AT) gene that is required for gene activation, chromatin remodeling, and histone acetylation throughout the proximal serpin subcluster. Here we show that the LCR interacts with multiple liver-specific transcription factors, including hepatocyte nuclear factor 3β (HNF-3β), HNF-6α, CCAAT/enhancer binding protein alpha (C/EBPα), and C/EBPβ. RNA polymerase II is also recruited to the locus through the LCR. Nongenic transcription at both the LCR and an upstream regulatory region was detected, but the deletion of the LCR abolished transcription at both sites. The deletion of HNF-3 and HNF-6 binding sites within the LCR reduced histone acetylation at both the LCR and the upstream regulatory region and decreased the transcription of the α1AT, corticosteroid binding globulin, and protein Z-dependent protease inhibitor genes. These results suggest that the LCR activates genes in the proximal serpin subcluster by recruiting liver-specific transcription factors and components of the general transcription machinery to regulatory regions upstream of the α1AT gene.

scholarly journals Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2324-2333 ◽  
Author(s):  
L Sarokin ◽  
M Carlson
Keyword(s):  
Carbon Catabolite Repression ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Lacz Fusion ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Heterologous Promoter ◽  
Suc2 Gene ◽  
High Level ◽  
Repeated Motif

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.

scholarly journals Molecular architecture of the hsp70 promoter after deletion of the TATA box or the upstream regulation region.

1997 ◽  
Vol 17 (7) ◽  
pp. 3799-3808 ◽  
Author(s):  
J A Weber ◽  
D J Taxman ◽  
Q Lu ◽  
D S Gilmour
Keyword(s):  
Transcriptional Activation ◽  
Regulatory Region ◽  
Dnase I ◽  
Tata Box ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Gaga Factor ◽  
Transcription Start ◽  
Hsp70 Promoter ◽  
Promoter Construct

GAGA factor, TFIID, and paused polymerase are present on the hsp70 promoter in Drosophila melanogaster prior to transcriptional activation. In order to investigate the interplay between these components, mutant constructs were analyzed after they had been transformed into flies on P elements. One construct lacked the TATA box and the other lacked the upstream regulatory region where GAGA factor binds. Transcription of each mutant during heat shock was at least 50-fold less than that of a normal promoter construct. Before and after heat shock, both mutant promoters were found to adopt a DNase I hypersensitive state that included the region downstream from the transcription start site. High-resolution analysis of the DNase I cutting pattern identified proteins that could be contributing to the hypersensitivity. GAGA factor footprints were clearly evident in the upstream region of the TATA deletion construct, and a partial footprint possibly caused by TFIID was evident on the TATA box of the upstream deletion construct. Permanganate treatment of intact salivary glands was used to further characterize each promoter construct. Paused polymerase and TFIID were readily detected on the normal promoter construct, whereas both deletions exhibited reduced levels of each of these factors. Hence both the TATA box and the upstream region are required to efficiently recruit TFIID and a paused polymerase to the promoter prior to transcriptional activation. In contrast, GAGA factor appears to be capable of binding and establishing a DNase I hypersensitive region in the absence of TFIID and polymerase. Interestingly, purified GAGA factor was found to bind near the transcription start site, and the strength of this interaction was increased by the presence of the upstream region. GAGA factor alone might be capable of establishing an open chromatin structure that encompasses the upstream regulatory region as well as the core promoter region, thus facilitating the binding of TFIID.

scholarly journals Control of Transposon-mediated Activation of theglpFKOperon ofEscherichia coliby two DNA binding Proteins

2016 ◽  
Author(s):  
Zhongge Zhang ◽  
Milton H. Saier
Keyword(s):  
Insertion Sequence ◽  
Gene Activation ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Wild Type ◽  
Transposon Insertion ◽  
Glycerol Utilization ◽  
Camp Receptor

AbstractEscherichia colicells deleted for the cyclic AMP (cAMP) receptor protein (Crp) gene (Δcrp) cannot utilize glycerol because cAMP-Crp is a required positive activator of glycerol utilization operonglpFK. We have previously shown that a transposon, Insertion Sequence 5 (IS5), can reversibly insert into the upstream regulatory region of the operon so as to activateglpFKand enable glycerol utilization. GlpR, which repressesglpFKtranscription, binds to theglpFKupstream region near the site of IS5insertion, and prevents insertion. We here show that the cAMP-Crp complex, which also binds to theglpFKupstream regulatory region, also inhibits IS5hopping into the activating site. This finding allowed us to identify conditions under which wild type cells can acquireglpFK-activating IS5insertions. Maximal rates of IS5insertion into the activating site require the presence of glycerol as well as a non-metabolizable sugar analogue that lowers cytoplasmic cAMP concentrations. Under these conditions, IS5insertional mutants accumulate and outcompete the wild type cells. Because of the widespread distribution of glucose analogues in nature, this mechanism of gene activation could have evolved by natural selection.

Marked difference in membrane-protein-binding properties of the two isoforms of protein 4.1R expressed at early and late stages of erythroid differentiation

2008 ◽  
Vol 417 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Wataru Nunomura ◽  
Marilyn Parra ◽  
Miwa Hebiguchi ◽  
Ken-Ichi Sawada ◽  
Narla Mohandas ◽  
...  
Keyword(s):  
Principal Component ◽  
Erythroid Differentiation ◽  
Band 3 ◽  
Binding Activity ◽  
Binding Affinities ◽  
Order Of Magnitude ◽  
Protein 4.1R ◽  
Terminal Erythroid Differentiation ◽  
Late Stages

Two major isoforms of protein 4.1R, a 135 kDa isoform (4.1R135) and an 80 kDa isoform (4.1R80), are expressed at distinct stages of terminal erythroid differentiation. The 4.1R135 isoform is exclusively expressed in early erythroblasts and is not present in mature erythrocytes, whereas the 4.1R80 isoform is expressed at late stages of erythroid differentiation and is the principal component of mature erythrocytes. These two isoforms differ in that the 4.1R135 isoform includes an additional 209 amino acids designated as the HP (head-piece) at the N-terminus of 4.1R80. In the present study, we performed detailed characterization of the interactions of the two 4.1R isoforms with various membrane-binding partners and identified several isoform-specific differences. Although both 4.1R135 and 4.1R80 bound to cytoplasmic domains of GPC (glycophorin C) and band 3, there is an order of magnitude difference in the binding affinities. Furthermore, although both isoforms bound CaM (calmodulin), the binding of 4.1R80 was Ca2+-independent, whereas the binding of 4.1R135 was strongly Ca2+-dependent. The HP of 4.1R135 mediates this Ca2+-dependent binding. Ca2+-saturated CaM completely inhibited the binding of 4.1R135 to GPC, whereas it strongly reduced the affinity of its binding to band 3. Interestingly, in spite of the absence of spectrin-binding activity, the 4.1R135 isoform was able to assemble on to the membrane of early erythroblasts suggesting that its ability to bind to membrane proteins is sufficient for its membrane localization. These findings enable us to offer potential new insights into the differential contribution of 4.1R isoforms to membrane assembly during terminal erythroid differentiation.

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