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.

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.

The role of NeuroD1 in the upregulation of SMYD3 by HBx.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 183-183
Author(s):  
Junyao Xu ◽  
Qingqi Hong ◽  
Chuanchao He ◽  
Jie Wang
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Promoter Region ◽  
Gene Transfection ◽  
Histone Methyltransferase ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Cis Element ◽  
E Box

183 Background: SET and MYND Domain-Containing Protein 3 (SMYD3) is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis B virus x protein (HBx) is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Methods: Immunohistochemical staining was used to detect the expression of HBx and SMYD3 in HCC tumor tissues. HBx gene transfection, RNAi, and histone methyltransferase(H3-K4) activity assay were performed to reveal the transcrpitionally activation of HBx on functional SMYD3 gene expression. Chromatin immunoprecipitation (ChIP), Co-immunoprecipitation (Co-IP), Electrophoretic mobility shift assay (EMSA) were applied to investigate the underlying mechanism. Dual-luciferase reporter assay was used to search for the HBx responsive cis-element of SMYD3 gene. Results: Immunohistochemistry identified the positive correlation between HBx and SMYD3 expression in 42 HCC tissues. Up-regulation of HBx on SMYD3 expression was validated through experiments involving overexpression or knock-down of HBx in different HCC cell lines. And up-regulated SMYD3 is functionally active as histone methyltransferase. Next we found that HBx transcriptionally regulated SMYD3 gene expression by interacting with RNA polymerase IIand altering its binding site to a proximal promoter region(SD2) from a distant promoter region(SD6) of SMYD3. Truncated and mutant reporter assays revealed that the cis-element mapped in -178~-203bp in SMYD3 promotor is responsive for HBx-transactivation. And this 25bp cis-element contains a E-box 3 unit, which is a binding site for the transcriptional factor Neurogenic differentiation 1(NeuroD1). EMSA and Chip showed that HBx increased NeuroD1 binding to SMYD3 proximal promotor, however transcient expression of antisense NeuroD1 abolished HBx-induced SMYD3 expression. Conclusions: HBx transcriptionally up-regulates SMYD3 and that this process is mediated by NeuroD1 through binding to the E-box 3 site of SMYD3 promotor.

scholarly journals Identification of a TAL1 Target Gene Reveals a Positive Role for the LIM Domain-Binding Protein Ldb1 in Erythroid Gene Expression and Differentiation

2003 ◽  
Vol 23 (21) ◽  
pp. 7585-7599 ◽  
Author(s):  
Zhixiong Xu ◽  
Suming Huang ◽  
Long-Sheng Chang ◽  
Alan D. Agulnick ◽  
Stephen J. Brandt
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Target Genes ◽  
Binding Protein ◽  
Proximal Promoter ◽  
Lim Domain ◽  
Positive Role ◽  
E Box ◽  
Murine Erythroleukemia

ABSTRACT The TAL1 (or SCL) gene, originally identified from its involvement by a recurrent chromosomal translocation, encodes a basic helix-loop-helix transcription factor essential for erythropoiesis. Although presumed to regulate transcription, its target genes are largely unknown. We show here that a nuclear complex containing TAL1, its DNA-binding partner E47, zinc finger transcription factor GATA-1, LIM domain protein LMO2, and LIM domain-binding protein Ldb1 transactivates the protein 4.2 (P4.2) gene through two E box GATA elements in its proximal promoter. Binding of this complex to DNA was dependent on the integrity of both E box and GATA sites and was demonstrated to occur on the P4.2 promoter in cells. Maximal transcription in transiently transfected cells required both E box GATA elements and expression of all five components of the complex. This complex was shown, in addition, to be capable of linking in solution double-stranded oligonucleotides corresponding to the two P4.2 E box GATA elements. This DNA-linking activity required Ldb1 and increased with dimethyl sulfoxide-induced differentiation of murine erythroleukemia (MEL) cells. In contrast, enforced expression in MEL cells of dimerization-defective mutant Ldb1, as well as wild-type Ldb1, significantly decreased E box GATA DNA-binding activities, P4.2 promoter activity, and accumulation of P4.2 and β-globin mRNAs. These studies define a physiologic target for a TAL1- and GATA-1-containing ternary complex and reveal a positive role for Ldb1 in erythroid gene expression and differentiation.

scholarly journals An androgen-independent mechanism underlying the androgenic effects of 3-methylcholanthrene, a potent aryl hydrocarbon receptor agonist

2020 ◽  
Vol 9 (3) ◽  
pp. 271-282
Author(s):  
Noriko Sanada ◽  
Yuka Gotoh-Kinoshita ◽  
Naoya Yamashita ◽  
Ryoichi Kizu
Keyword(s):  
Aryl Hydrocarbon Receptor ◽  
Regulatory Region ◽  
Specific Inhibitor ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Distal Enhancer ◽  
Aryl Hydrocarbon ◽  
Aryl Hydrocarbon Receptor Agonist ◽  
Significant Difference ◽  
Androgen Independent

Abstract Aryl hydrocarbon receptor (AhR) and androgen receptor (AR) are ligand-activated transcription factors with profound cross-talk between their signal transduction pathways. Previous studies have shown that AhR agonists activate the transcription of AR-regulated genes in an androgen-independent manner; however, the underlying mechanism remains unclear. To decipher this mechanism, we evaluated the effects of 3-methylcholanthrene (3MC), a potent AhR agonist, on the transcription of AR-regulated genes in three AR-expressing cell lines. 3MC induced the expression of not only three representative AR-regulated chromosomal genes but also the exogenous AR-responsive luciferase reporter gene. No significant difference in the 3MC-induced luciferase activity was detected in the presence of SKF-525A, a non-specific inhibitor of CYP enzymes. The androgenic effects of 3MC were diminished by AhR and AR knockdown. Following 3MC treatment, the amount of nuclear AhR and AR increased synchronously. Co-immunoprecipitation revealed that AhR and AR formed a complex in the nucleus of cells treated with 3MC. AR was recruited to the proximal promoter and distal enhancer regions of the PSA gene upon the addition of 3MC. We propose that AhR activated by 3MC forms a complex with unliganded AR which translocates from the cytoplasm to the nucleus. Nuclear AR now binds the transcriptional regulatory region of AR-regulated genes and activates the transcription.

Promoter I of the ovine acetyl-CoA carboxylase-α gene: an E-box motif at −114 in the proximal promoter binds upstream stimulatory factor (USF)-1 and USF-2 and acts as an insulin-response sequence in differentiating adipocytes

2001 ◽  
Vol 359 (2) ◽  
pp. 273-284 ◽  
Author(s):  
Maureen T. TRAVERS ◽  
Amanda J. VALLANCE ◽  
Helen T. GOURLAY ◽  
Clare A. GILL ◽  
Izabella KLEIN ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Binding Protein ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Acetyl Coa Carboxylase ◽  
Upstream Stimulatory Factor ◽  
Acetyl Coa ◽  
Rnase Protection ◽  
E Box ◽  
Stimulatory Factor

Acetyl-CoA carboxylase-α (ACC-α) plays a central role in co-ordinating de novo fatty acid synthesis in animal tissues. We have characterized the regulatory region of the ovine ACC-α gene. Three promoters, PI, PII and PIII, are dispersed throughout 50kb of genomic DNA. Expression from PI is limited to adipose tissue and liver. Sequence comparison of the proximal promoters of ovine and mouse PIs demonstrates high nucleotide identity and that they are characterized by a TATA box at −29, C/EBP (CCAAT enhancer-binding protein)-binding motifs and multiple E-box motifs. A 4.3kb ovine PI-luciferase reporter construct is insulin-responsive when transfected into differentiated ovine adipocytes, whereas when this construct is transfected into ovine preadipocytes and HepG2 cells the construct is inactive and is not inducible by insulin. By contrast, transfection of a construct corresponding to 132bp of the proximal promoter linked to a luciferase reporter is active and inducible by insulin in all three cell systems. Insulin signalling to the −132bp construct in differentiated ovine adipocytes involves, in part, an E-box motif at −114. Upstream stimulatory factor (USF)-1 and USF-2, but not sterol regulatory element-binding protein 1 (SREBP-1), are major components of protein complexes that bind this E-box motif. Activation of the 4.3kb PI construct in differentiated ovine adipocytes is associated with endogenous expression of PI transcripts throughout differentiation; PI transcripts are not detectable by RNase-protection assay in ovine preadipocytes, HepG2 cells or 3T3-F442A adipocytes. These data indicate the presence of repressor motifs in PI that are required to be de-repressed during adipocyte differentiation to allow induction of the promoter by insulin.

scholarly journals Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Seung-Hun Kim ◽  
Kwang-Hwan Choi ◽  
Dong-Kyung Lee ◽  
Mingyun Lee ◽  
Jae Yeon Hwang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Promoter Region ◽  
Regulatory Region ◽  
Embryonic Stem ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Fluorescence Signal ◽  
Reporter Assay ◽  
Reporter System

OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.

Glucocorticoid regulation of branched-chain α-ketoacid dehydrogenase E2 subunit gene expression

2000 ◽  
Vol 347 (2) ◽  
pp. 449-457 ◽  
Author(s):  
Paul A. COSTEAS ◽  
Jeffrey M. CHINSKY
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Genomic Sequence ◽  
Deletion Analysis ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Complex Activity ◽  
Branched Chain ◽  
Ketoacid Dehydrogenase

Regulation of the mammalian branched-chain α-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, α-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1α subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5ʹ proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5ʹ upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors.

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.

The TAL1/SCL Transcription Factor Regulates Cell-Cycle Progression in Murine Monocyte-Macrophage Precursors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 570-570
Author(s):  
Soumyadeep Dey ◽  
David J. Curtis ◽  
Stephen Jane ◽  
Stephen J. Brandt
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cell Cycle Progression ◽  
Precursor Cells ◽  
Upstream Region ◽  
Mrna Levels ◽  
Cycle Progression ◽  
E Box ◽  
P16 Ink4a ◽  
Chip Analysis

Abstract Abstract 570 The TAL1 (or SCL) gene, first identified through its involvement by a chromosomal translocation in T-cell acute lymphoblastic leukemia, encodes a basic helix-loop-helix transcription factor critical for embryonic and adult hematopoiesis and vascular remodeling. We reported that Tal1 was expressed at all stages of monocytic differentiation from the common myeloid progenitor to the mature (and activated) macrophage and that ex vivo knockout of this gene in BM macrophage (BMM) precursors from Tal1loxP/Lacz mice profoundly reduced cellular proliferation without significantly affecting survival or differentiation. To characterize the cell-cycle status of Tal1-deficient BMM precursors, we carried out flow cytometry-based BrdU pulse-chase analysis. This revealed delayed progression from G1 to S phase and S to G2 in Tal1-/- relative to control Tal1+/- cells. Moreover, when Tal1-/- cells were cultured with BrdU for extended times (48 hr), a significantly larger fraction of Tal1-/- cells (27.5%) failed to incorporate BrdU compared to Tal1+/- cells (7.6%), indicating an additional defect in exiting G0/G1. To determine whether Tal1's function in proliferation was gene dose-dependent, cell numbers were enumerated in cultures of Tal1-transduced, Tal1+/+, Tal1+/-, and Tal1-/- BMM precursor cells. Cells over-expressing Tal1 accumulated in higher numbers than Tal1+/+ cells, which had a proliferative advantage over Tal1+/- cells that was evident only late in culture. To address the mechanism by which TAL1 regulates cell-cycle progression in this lineage, we investigated the effect of increasing and decreasing its expression on expression of the cyclin-dependent kinase inhibitor p16(Ink4a), which was suggested in transient transfection experiments to be a target of TAL1-mediated repression. Indeed, p16(Ink4a) mRNA levels were ∼4-fold higher in Tal1-/- relative to Tal1+/+ cells and were reduced, although less significantly, in Tal1-over-expressing compared to Tal1+/+ cells. Quantitative chromatin immunoprecipitation (ChIP) analysis in wild-type BMM precursor cells showed direct association of Tal1 and its E protein DNA-binding partner E47 with 3 consensus E-box elements in the p16(Ink4a) upstream region that was greater in day 4 than day 7 cells and correlated inversely with p16(Ink4a) mRNA expression. In addition, we confirmed the previously reported increase in p16(Ink4a) mRNA abundance and decrease in Tal1 mRNA in M1 monocytic leukemia cells induced to differentiate into macrophages with interleukin-6 (IL-6). Quantitative ChIP analysis with sonicated chromatin from IL-6 treated (48 hr) and untreated M1 cells showed Tal1 occupancy at the same E-box elements in untreated cells as in primary BMM precursors that was completely abolished by IL-6 treatment. In contrast E2A occupancy was detected in both treated and untreated cells, consistent with relief of Tal1-directed repression of p16(Ink4a) in M1 cells upon differentiation. In summary, these studies have uncovered a critical role for Tal1 in cell cycle regulation during monocytopoiesis and suggest that TAL1 repression of p16(Ink4a) transcription likely contributes. Further, the ChIP and expression studies provide more definitive evidence for p16(Ink4a) as a target gene of TAL1. Finally, the results in Tal1-over-expressing BMM precursors may be relevant to TAL1's actions in T-lymphoid and, in particular, myeloid leukemias. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of a non-canonical E-box motif as a regulatory element in the proximal promoter region of the apolipoprotein E gene

2003 ◽  
Vol 370 (3) ◽  
pp. 979-986 ◽  
Author(s):  
Enrique SALERO ◽  
Cecilio GIMÉNEZ ◽  
Francisco ZAFRA
Keyword(s):  
Transcription Factors ◽  
Apolipoprotein E ◽  
Cell Lines ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Cdna Clones ◽  
E Gene ◽  
Basal Activity ◽  
Apolipoprotein E Gene ◽  
E Box

We have used the yeast one-hybrid system to identify transcription factors with binding capability to specific sequences in proximal regions of the apolipoprotein E gene (APOE) promoter. The sequence between −113 and −80nt, which contains regulatory elements in various cell types, was used as a bait to screen a human brain cDNA library. Four cDNA clones that encoded portions of the human upstream-stimulatory-factor (USF) transcription factor were isolated. Electrophoretic-mobility-shift assays ('EMSAs') using nuclear extracts from various human cell lines as well as from rat brain and liver revealed the formation of two DNA—protein complexes within the sequence CACCTCGTGAC (region −101/-91 of the APOE promoter) that show similarity to the E-box element. The retarded complexes contained USF1, as deduced from competition and supershift assays. Functional experiments using different APOE promoter—luciferase reporter constructs transiently transfected into U87, HepG2 or HeLa cell lines showed that mutations that precluded the formation of complexes decreased the basal activity of the promoter by about 50%. Overexpression of USF1 in U87 glioblastoma cells led to an increased activity of the promoter that was partially mediated by the atypical E-box. The stimulatory effect of USF1 was cell-type specific, as it was not observed in hepatoma HepG2 cells. Similarly, overexpression of a USF1 dominant-negative mutant decreased the basal activity of the promoter in glioblastoma, but not in hepatoma, cells. These data indicated that USF, and probably other related transcription factors, might be involved in the basal transcriptional machinery of APOE by binding to a non-canonical E-box motif within the proximal promoter.

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