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

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.

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Single-Stranded DNA-Binding Proteins (SSBPs) Promote the Oligomerization of LIM-Domain Binding Protein Ldb1

Blood ◽

10.1182/blood.v112.11.2437.2437 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2437-2437

Author(s):

Ying Cai ◽

Lalitha Nagarajan ◽

Stephen J. Brandt

Keyword(s):

Dna Binding ◽

Fusion Proteins ◽

Mammalian Cells ◽

Binding Protein ◽

Binding Activity ◽

Luciferase Reporter ◽

Lim Domain ◽

Single Stranded Dna ◽

Dna Binding Activity ◽

E Box

Abstract The multifunctional LIM domain-binding protein Ldb1 is important in multiple developmental programs, including hematopoiesis. An evolutionarily conserved family of proteins with single-stranded DNA-binding activity, the SSBPs, has been shown to act as Ldb1 partners and augment its biological actions. We recently established that Ssbp2 and Ssbp3 were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing the LIM-only protein Lmo2 and transcription factors Tal1, E2A, and Gata1 and showed these SSBPs stimulated E box-GATA DNA-binding activity and inhibited Ldb1 ubiquitination and subsequent proteasomal degradation (Genes & Dev.21:942–955, 2007). As its SSBP interaction domain (Ldb1/Chip conserved domain or LCCD) is adjacent to Ldb1’s N-terminal dimerization domain (DD), we sought to determine whether SSBP binding affected Ldb1 dimerization. To investigate, the Ldb1 coding region was fused to the DNA-binding domain of the yeast transcription factor GAL4 (GAL4DBD) and in a second construct to the activation domain of herpesvirus VP16 (VP16AD). These fusion proteins were then expressed in mammalian cells with a luciferase reporter linked to a promoter with iterated GAL4 binding sites. Luciferase activity became detectable with coexpression of the VP16AD-Ldb1 and GAL4DBD-Ldb1 fusions, presumably from Ldb1 dimerization, which increased markedly with simultaneous expression of SSBP2. In contrast, SSBP2 (ΔLUFS) and Ldb1 (ΔLCCD) mutants incapable of interacting with Ldb1 and SSBPs, respectively, were inactive, suggesting that SSBP2 augmentation of Ldb1 dimerization involved direct protein-protein interactions. To exclude an effect of SSBP2 on turnover of Ldb1 fusion proteins, radiolabeled full-length Ldb1 and SSBP3 were prepared by in vitro transcription/translation, mixed, and subjected to chemical crosslinking. Addition of the crosslinker bis(sulfosuccinimidyl)-suberate (BS3) to Ldb1, but not SSBP3, led to the appearance of a radiolabeled protein with mobility in denaturing polyacrylamide gels approximately twice that of Ldb1, consistent with an Ldb1 homodimer. When SSBP3 and Ldb1 were mixed together and crosslinked, a dose-related increase was noted in a more retarded species predicted to contain two molecules each of Ldb1 and SSBP3, together with a decrease in monomeric Ldb1. Finally, two well-characterized dimerization-defective Ldb1 mutants, Ldb1(200–375) and Ldb1(50–375), failed to support the formation of the higher molecular weight species or to homodimerize. Thus, the SSBPs promoted assembly of ternary complexes incorporating both SSBP and Ldb1 in a manner dependent on Ldb1 dimerization. The failure to observe Ldb1-SSBP heterodimers in cross-linking experiments suggests, further, that the SSBPs interacted with preformed Ldb1 dimers. In summary, either through an allosteric effect on Ldb1’s DD or by altering the equilibrium between monomeric and dimeric species, the SSBPs promote Ldb1 oligomerization. Together with inhibition of Ldb1 ubiquitination and turnover, this would serve to augment Ldb1 function.

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

Blood ◽

10.1182/blood.v120.21.2335.2335 ◽

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.

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A direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

10.1101/517458 ◽

2019 ◽

Cited By ~ 2

Author(s):

Christopher M. Uyehara ◽

Daniel J. McKay

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Nuclear Receptor ◽

Binding Sites ◽

Target Genes ◽

Transcriptional Responses ◽

Developmental Transitions ◽

The Impact

ABSTRACTThe ecdysone pathway was amongst the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. Here, we use the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone-response genes. However, the majority of its binding sites reside at genes with wing-specific functions. We also find that EcR binding is temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.SIGNIFICANCENuclear receptors (NRs) are sequence-specific DNA binding proteins that act as intracellular receptors for small molecules such as hormones. Prior work has shown that NRs function as ligand-dependent switches that initiate a cascade of gene expression changes. The extent to which NRs function as direct regulators of downstream genes in these hierarchies remains incompletely understood. Here, we study the role of the NR EcR in metamorphosis of the Drosophila wing. We find that EcR directly regulates many genes at the top of the hierarchy as well as at downstream genes. Further, we find that EcR binds distinct sets of target genes at different developmental times. This work helps inform how hormones elicit tissue- and temporal-specific responses in target tissues.

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Max is acetylated by p300 at several nuclear localization residues

Biochemical Journal ◽

10.1042/bj20061593 ◽

2007 ◽

Vol 403 (3) ◽

pp. 397-407 ◽

Cited By ~ 13

Author(s):

Francesco Faiola ◽

Yi-Ting Wu ◽

Songqin Pan ◽

Kangling Zhang ◽

Anthony Farina ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Nuclear Localization ◽

Mammalian Cells ◽

Target Genes ◽

Apoptotic Cell ◽

Central Component ◽

Post Translational Modification ◽

E Box

Max is a ubiquitous transcription factor with a bHLHZip [basic HLH (helix–loop–helix) leucine zipper] DNA-binding/dimerization domain and the central component of the Myc/Max/Mad transcription factor network that controls cell growth, proliferation, differentiation and apoptotic cell death in metazoans. Max is the obligatory DNA-binding and dimerization partner for all the bHLHZip regulators of the Myc/Max/Mad network, including the Myc family of oncoproteins and the Mad family of Myc antagonists, which recognize E-box DNA elements in the regulatory regions of target genes. Max lacks a transcription regulatory domain and is the only member of the network that efficiently homodimerizes. Binding of Max homodimers to E-box elements suppresses the transcription regulatory functions of its network partners and of other non-network E-box-binding regulators. In contrast with its highly regulated partners, Max is a constitutively expressed and phosphorylated protein. Phosphorylation is, however, the only Max post-translational modification identified so far. In the present study, we have analysed Max posttranslational modifications by MS. We have found that Max is acetylated at several lysine residues (Lys-57, Lys-144 and Lys-145) in mammalian cells. Max acetylation is stimulated by inhibitors of histone deacetylases and by overexpression of the p300 co-activator/HAT (histone acetyltransferase). The p300 HAT also directly acetylates Max in vitro at these three residues. Interestingly, the three Max residues acetylated in vivo and in vitro by p300 are important for Max nuclear localization and Max-mediated suppression of Myc transactivation. These results uncover novel post-translational modifications of Max and suggest the potential regulation of specific Max complexes by p300 and reversible acetylation.

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Regulation of Ldb1 Stability and Transcriptional Complex Assembly by Single-Stranded DNA-Binding Protein SSDP2.

Blood ◽

10.1182/blood.v106.11.1739.1739 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1739-1739

Author(s):

Zhixiong Xu ◽

Xianzhang Meng ◽

Ying Cai ◽

Lalitha Nagarajan ◽

Stephen J. Brandt

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Binding Activity ◽

Lim Domain ◽

Complex Assembly ◽

Single Stranded Dna ◽

Dna Binding Activity ◽

E Box ◽

Transcriptional Complex

Abstract The LIM domain-binding protein Ldb1 is known to form higher order complexes with LIM-homeodomain and LIM-only (LMO) proteins to regulate diverse developmental programs, including hematopoiesis. The level of Ldb1 is critical for its cellular roles, and its turnover is regulated by the E3 ubiquitin ligase RLIM. Single-stranded DNA-binding protein (SSDP), an Ldb1-interacting partner, is an essential gene for embryonic development and has been shown to regulate axis formation in Xenopus and wing development in Drosophila through Ldb1; however, the mechanisms by which SSDPs regulate these and other developmental programs are still obscure. We previously reported that a DNA-binding complex containing the basic helix-loop-helix protein TAL1/SCL, its DNA-binding partner E47, zinc finger protein GATA-1, LIM domain protein LMO2, and Ldb1 stimulates Protein 4.2 (P4.2) transcription in erythroid progenitors through tandem E box-GATA elements in the gene’s proximal promoter. We have now established that SSDP2 is associated with this complex (by supershift analysis) and occupies the promoter of this gene (by chromatin immunoprecipitation analysis) in murine erythroleukemia (MEL) cells. Further, enforced expression of SSDP2 in these cells stimulated P4.2 reporter activity and accumulation of P4.2 and beta-globin mRNAs, and cotransfection of SSDP2 with the five originally identified components of this complex further increased promoter activity in reporter analysis. Importantly, overexpression of SSDP2 in MEL cells significantly increased Ldb1 protein half-life and steady-state levels of Ldb1 and LMO2 protein. This effect on Ldb1 stability required the Ldb1-interacting domain of SSDP2, consisting of its first 94 amino acids (SSDP2(1–94)), and was also observed in Cos7L and CHO cells. We showed, in addition, that SSDP2 or SSDP2(1–94), but not an Ldb1 interaction-defective mutant, prevented RLIM-mediated degradation of both Ldb1 and LMO2 in transfected cells, that SSDP2 protection of LMO2 degradation required Ldb1, and that SSDP2 directly inhibited RLIM-mediated ubiquitination of Ldb1. Immunoprecipitation analysis revealed that overexpression of SSDP2 or SSDP2(1–94) significantly decreased interaction between Ldb1 and RLIM. Finally, SSDP2 protein expression in differentiating MEL cells paralleled this multi-protein DNA-binding activity and overexpression of SSDP2 in these cells dramatically increased E box-GATA DNA-binding activity, with maximal formation of the ternary complex requiring coexpression of SSDP2, Ldb1, and LMO2. Together, these studies reveal a positive role for SSDP2 in erythroid gene expression and identify a biochemical function for SSDP2 in regulating Ldb1 stability and transcriptional complex assembly. The mechanism mediating Ldb1 stabilization appears to involve competitive inhibition of RLIM interaction with Ldb1.

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The ETS Protein ELF2 Contributes to a Novel TAL1/SCL-Containing DNA-Binding Complex That Positively Regulates Erythroid Gene Expression and Cell Differentiation,

Blood ◽

10.1182/blood.v118.21.3389.3389 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3389-3389

Author(s):

Ying Cai ◽

Matthew P. McCormack ◽

David J. Curtis ◽

Lalitha Nagarajan ◽

Stephen J. Brandt

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

Dna Binding ◽

Complex Formation ◽

Binding Activity ◽

Mass Spectrometry Analysis ◽

Sequence Motif ◽

Dna Binding Activity ◽

E Box ◽

Murine Erythroleukemia

Abstract Abstract 3389 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 erythroid, megakaryocyte, and macrophage progenitor function and is essential for hematopoietic and vascular development. Although Tal1 can bind DNA with one of its E protein DNA binding partners, its function in erythroid cells has been thought to be mediated by a larger complex recognizing an E box-GATA or GATA sequence motif. In independent lines of investigation, a novel DNA-binding motif for TAL1 comprised of an ETS site immediately adjacent to an E box was identified by site selection analysis in K562 cells, and multiple peptides derived from a single ETS protein, E74-like factor 2 (Elf2, also known as NERF), were identified by tandem mass spectrometry analysis of Tal1-containing protein complexes purified from murine erythroleukemia (MEL) cells. To determine whether Elf2, previously shown to interact with Tal1's interaction partner LMO2, recognized this sequence element, electrophoretic mobility shift analysis was carried out using MEL cell nuclear extracts. This revealed the existence of a novel multiprotein ETS-E box DNA-binding complex containing Tal1, Elf2, Lmo2, the LIM domain-binding protein Ldb1, and Ldb1-interacting single-stranded DNA binding proteins Ssbp2 and Ssbp3. Structure-function analysis of this DNA-binding activity, which increased with chemical induction of MEL cell differentiation, showed that both the E box and ETS site were required for ternary complex formation, that preference existed for specific ETS flanking sequences, and that precise spacing between the two transcription factor binding sites was critical for complex formation. Finally, a physiological target for this complex was identified in the Ssbp3 gene, with Tal1, Elf2, and Ldb1 found to specifically co-occupy the third intron of this gene by chromatin immunoprecipitation analysis but not a similar ETS-E box element in the 11th intron. In accord, Ssbp3 expression, ETS-E box DNA-binding activity, and hemoglobin concentration increased with enforced expression of Elf2 in MEL cells, while Ssbp3 protein abundance, ETS-E box DNA-binding activity, and cellular differentiation decreased with Elf2 knockdown. These studies identify a novel TAL1- and ELF2-containing DNA-binding complex in erythroid progenitors that positively regulates gene expression and differentiation. In addition, they reveal a previously unknown role for the ETS protein ELF2 in erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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Efg1, a Morphogenetic Regulator in Candida albicans, Is a Sequence-Specific DNA Binding Protein

Journal of Bacteriology ◽

10.1128/jb.183.13.4090-4093.2001 ◽

2001 ◽

Vol 183 (13) ◽

pp. 4090-4093 ◽

Cited By ~ 54

Author(s):

Ping Leng ◽

Philip R. Lee ◽

Hong Wu ◽

Alistair J. P. Brown

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Human Pathogen ◽

Hyphal Development ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box

ABSTRACT Efg1 is essential for hyphal development in the human pathogenCandida albicans under most conditions. Efg1 is related to basic helix-loop-helix regulators, and therefore most workers presume that Efg1 is a transcription factor. Here we confirm that Efg1 is a DNA binding protein that can interact specifically with the E box.

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Molecular Clues From Testis Involve NHLH2, a DNA Binding Transcription Factor That Controls the Onset of Mini-Puberty via Its Neuronal Activity, in Curative GnRH Treatment of Cryptorchidism Dependent Infertility

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1365 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A669-A669

Author(s):

Faruk Hadziselimovic

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Neuronal Activity ◽

Target Genes ◽

Hypogonadotropic Hypogonadism ◽

Hormone Treatment ◽

Mrna Levels ◽

Expression Levels ◽

Rna Profiling

Abstract Introduction: GnRHa treatment following surgery to correct cryptorchidism restores mini-puberty via endocrinological and transcriptional effects and prevents adult infertility in most cases. A large group of genes are important for central hypogonadotropic hypogonadism in mammals, including many that are transcribed both in brain and testis. However, the expression of these genes in prepubertal gonads has not been systematically studied and little is known about the effect of hormone therapy on their testicular and neuronal expression levels. Here, we interpret histological sections, data on hormone levels and RNA profiling data from adult normal testis in comparison to pre-pubertal low infertility risk (LIR) and high infertility risk (HIR) patients randomized for treatment with surgery in combination with GnRHa or only surgery. Major Results: We organize 31 target genes relevant for hypogonadotropic hypogonadism and cryptorchidism into five classes depending on their expression levels in HIR versus LIR samples and their response to GnRHa treatment. Only the mRNA encoding the DNA binding transcription factor NHLH2 is decreased in HIR as compared to LIR samples, increased after GnRHa treatment, and expressed in both brain and testis. GnRHa treatment of cryptorchidism that restores mini-puberty and rescues adult fertility increases NHLH2 mRNA levels in testis from HIR patients. Conclusion: This phenomenon may reflect a broader effect of hormone treatment on gene expression in both testicular and central nervous tissues, which could explain why the hypothalamus-pituitary-testicular axis is permanently restored by the administration of GnRHa.

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The E-box DNA binding protein Sgc1p suppresses thegcr2mutation, which is involved in transcriptional activation of glycolytic genes inSaccharomyces cerevisiae

FEBS Letters ◽

10.1016/s0014-5793(99)01654-3 ◽

1999 ◽

Vol 463 (3) ◽

pp. 307-311 ◽

Cited By ~ 25

Author(s):

Takashi Sato ◽

M.Cecilia Lopez ◽

Shigemi Sugioka ◽

Yoshifumi Jigami ◽

Henry V. Baker ◽

...

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Protein ◽

Dna Binding Protein ◽

E Box

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Repression of ESR1 through Actions of Estrogen Receptor Alpha and Sin3A at the Proximal Promoter

Molecular and Cellular Biology ◽

10.1128/mcb.00383-09 ◽

2009 ◽

Vol 29 (18) ◽

pp. 4949-4958 ◽

Cited By ~ 47

Author(s):

Stephanie J. Ellison-Zelski ◽

Natalia M. Solodin ◽

Elaine T. Alarid

Keyword(s):

Gene Expression ◽

Estrogen Receptor ◽

Transcription Factor ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Estrogen Receptor Alpha ◽

Proximal Promoter ◽

Receptor Alpha ◽

Expression Of Genes ◽

Activation Of Transcription

ABSTRACT Gene expression results from the coordinated actions of transcription factor proteins and coregulators. Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that can both activate and repress the expression of genes. Activation of transcription by estrogen-bound ERα has been studied in detail, as has antagonist-induced repression, such as that which occurs by tamoxifen. How estrogen-bound ERα represses gene transcription remains unclear. In this report, we identify a new mechanism of estrogen-induced transcriptional repression by using the ERα gene, ESR1. Upon estrogen treatment, ERα is recruited to two sites on ESR1, one distal (ENH1) and the other at the proximal (A) promoter. Coactivator proteins, namely, p300 and AIB1, are found at both ERα-binding sites. However, recruitment of the Sin3A repressor, loss of RNA polymerase II, and changes in histone modifications occur only at the A promoter. Reduction of Sin3A expression by RNA interference specifically inhibits estrogen-induced repression of ESR1. Furthermore, an estrogen-responsive interaction between Sin3A and ERα is identified. These data support a model of repression wherein actions of ERα and Sin3A at the proximal promoter can overcome activating signals at distal or proximal sites and ultimately decrease gene expression.

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