Electrostatic repulsion causes anticooperative DNA binding between tumor suppressor ETS transcription factors and JUN-FOS at composite DNA sites

Mapping Intimacies ◽

10.1101/318048 ◽

2018 ◽

Author(s):

Bethany J. Madison ◽

Kathleen A. Clark ◽

Niraja Bhachech ◽

Peter C. Hollenhorst ◽

Barbara J. Graves ◽

...

Keyword(s):

Prostate Cancer ◽

Transcription Factors ◽

Dna Binding ◽

Dna Sequences ◽

Binding Sites ◽

Epithelial Cell Line ◽

Electrostatic Repulsion ◽

Ets Transcription Factors ◽

Positively Charged

AbstractMany transcription factors regulate gene expression in a combinatorial fashion often by binding in close proximity on composite cis-regulatory DNA elements. Here we investigate the molecular basis by which ETS transcription factors bind with AP1 transcription factors JUN-FOS at composite DNA-binding sites. The ability to bind to DNA with JUN-FOS correlates with the phenotype of these proteins in prostate cancer: the oncogenic ERG and ETV1/4/5 subfamilies co-occupy ETS-AP1 sites with JUN-FOS in vitro, whereas JUN-FOS robustly inhibits DNA binding by the tumor suppressors EHF and SPDEF. EHF binds to ETS-AP1 DNA with tighter affinity than ERG in the absence of JUN-FOS, which may enable EHF to compete with ERG and JUN-FOS for binding to ETS-AP1 sites. Genome-wide mapping of EHF and ERG binding sites in a prostate epithelial cell line reveal that EHF is preferentially excluded from closely spaced ETS-AP1 DNA sequences. Structural modeling and mutational analyses indicate that adjacent positively-charged surfaces from EHF and JUN-FOS disfavor simultaneous DNA binding due to electrostatic repulsion. The conservation of positively charged residues on the JUN-FOS interface identified ELF1 as an additional ETS factor that exhibits anticooperative DNA binding, and we present evidence that ELF1 is frequently downregulated in prostate cancer. In summary, the divergence of electrostatic features of ETS factors at their JUN-FOS interface enables distinct binding events at ETS-AP1 DNA sequences. We propose that this mechanism can drive unique targeting of ETS transcription factors, thereby facilitating distinct transcriptional programs.

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In vitro DNA-binding profile of transcription factors: methods and new insights

Journal of Endocrinology ◽

10.1530/joe-11-0010 ◽

2011 ◽

Vol 210 (1) ◽

pp. 15-27 ◽

Cited By ~ 20

Author(s):

Jinke Wang ◽

Jie Lu ◽

Guangming Gu ◽

Yingxun Liu

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Dna Sequences ◽

Binding Sites ◽

Target Genes ◽

Binding Specificity ◽

Cell Physiology ◽

Binding Profile ◽

Dna Binding Specificity

The DNA-binding specificity of transcription factors (TFs) has broad impacts on cell physiology, cell development and in evolution. However, the DNA-binding specificity of most known TFs still remains unknown. The specificity of a TF protein is determined by its relative affinity to all possible binding sites. In recent years, the development of several in vitro techniques permits high-throughput determination of relative binding affinity of a TF to all possible k bp-long DNA sequences, thus greatly promoting the characterization of DNA-binding specificity of many known TFs. All DNA sequences that can be bound by a TF with various binding affinities form their DNA-binding profile (DBP). The DBP is important to generate an accurate DNA-binding model, identify all DNA-binding sites and target genes of TFs in the whole genome, and build transcription regulatory network. This study reviewed these techniques, especially two master techniques: double-stranded DNA microarray and systematic evolution of ligands by exponential enrichment in combination with parallel DNA sequencing techniques (SELEX-seq).

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The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila

10.1101/373308 ◽

2018 ◽

Author(s):

Arya Zandvakili ◽

Juli Uhl ◽

Ian Campbell ◽

Yuntao Charlie Song ◽

Brian Gebelein

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Dna Sequences ◽

Binding Sites ◽

Target Genes ◽

Hox Genes ◽

Regulatory Elements ◽

Protease Gene ◽

Precise Integration

AbstractHox genes encode a family of transcription factors that, despite having similar in vitro DNA binding preferences, regulate distinct genetic programs along the metazoan anterior-posterior axis. To better define mechanisms of Hox specificity, we compared and contrasted the ability of abdominal Hox factors to regulate two cis-regulatory elements within the Drosophila embryo. Both the Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox factors form cooperative complexes with the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the distal-less leg selector gene via the DCRE, whereas only Abd-A interacts with Exd and Hth on the RhoA element to activate a rhomboid serine protease gene that stimulates Epidermal Growth Factor secretion. By swapping binding sites between these elements, we found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can also convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE, but only in one orientation. We further show that the orientation and spacing of Hox sites relative to additional transcription factor binding sites within the RhoA and DCRE elements is critical to mediate appropriate cell- and segment-specific output. These results indicate that the interaction between Hox, Exd, and Hth neither determines activation vs repression specificity nor defines Ubx vs Abd-A specificity. Instead the precise integration of Hox sites with additional TF inputs is required for accurate transcriptional output. Taken together, these studies provide new insight into the mechanisms of Hox target and regulatory specificity as well as the constraints placed on regulatory elements to convey appropriate outputs.Author SummaryThe Hox genes encode a family of transcription factors that give cells within each region along the developing body plan a unique identity in animals from worms to mammals. Surprisingly, however, most of the Hox factors bind the same or highly similar DNA sequences. These findings raise a paradox: How can proteins that have highly similar DNA binding properties perform different functions in the animal by regulating different sets of target genes? In this study, we address this question by studying how two Hox factors regulate the expression of target genes that specify leg development and the making of liver-like cells in the developing fly. By comparing and contrasting how Hox target genes are activated and/or repressed, we found that the same Hox binding sites can mediate either activation or repression in a manner that depends upon context. In addition, we found that a Hox binding site that is normally regulated by only one Hox factor, can also be used by more than one Hox factor swapped into another target gene. These findings indicate that the specificity of a Hox factor to regulate target genes does not rely solely upon DNA binding specificity but also requires regulatory specificity.

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Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.964-971.1985 ◽

1985 ◽

Vol 5 (5) ◽

pp. 964-971

Author(s):

R M Gronostajski ◽

S Adhya ◽

K Nagata ◽

R A Guggenheimer ◽

J Hurwitz

Keyword(s):

Dna Binding ◽

Hela Cell ◽

Dna Sequences ◽

Binding Sites ◽

Consensus Sequence ◽

Nuclear Factor ◽

Site Specific ◽

Factor I ◽

Nuclear Factor I

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.

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Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1405 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1405-1421 ◽

Cited By ~ 197

Author(s):

C C Adams ◽

J L Workman

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Binding Sites ◽

Specific Binding ◽

General Mechanism ◽

Nf Kappa B ◽

Nucleosomal Dna ◽

Nucleosome Binding

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

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Genome reading by the NF-κB transcription factors

Nucleic Acids Research ◽

10.1093/nar/gkz739 ◽

2019 ◽

Vol 47 (19) ◽

pp. 9967-9989 ◽

Cited By ~ 12

Author(s):

Maria Carmen Mulero ◽

Vivien Ya-Fan Wang ◽

Tom Huxford ◽

Gourisankar Ghosh

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Dna Sequences ◽

Target Genes ◽

Target Selection ◽

Structural Features ◽

Response Elements ◽

Target Binding

Abstract The NF-κB family of dimeric transcription factors regulates transcription by selectively binding to DNA response elements present within promoters or enhancers of target genes. The DNA response elements, collectively known as κB sites or κB DNA, share the consensus 5′-GGGRNNNYCC-3′ (where R, Y and N are purine, pyrimidine and any nucleotide base, respectively). In addition, several DNA sequences that deviate significantly from the consensus have been shown to accommodate binding by NF-κB dimers. X-ray crystal structures of NF-κB in complex with diverse κB DNA have helped elucidate the chemical principles that underlie target selection in vitro. However, NF-κB dimers encounter additional impediments to selective DNA binding in vivo. Work carried out during the past decades has identified some of the barriers to sequence selective DNA target binding within the context of chromatin and suggests possible mechanisms by which NF-κB might overcome these obstacles. In this review, we first highlight structural features of NF-κB:DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation. We then discuss how native NF-κB dimers identify DNA binding targets in the nucleus with support from additional factors and how post-translational modifications enable NF-κB to selectively bind κB sites in vivo.

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The interaction of proteins encoded by Drosophila homeotic and segmentation genes with specific DNA sequences

Development ◽

10.1242/dev.104.supplement.75 ◽

1988 ◽

Vol 104 (Supplement) ◽

pp. 75-83 ◽

Cited By ~ 2

Author(s):

Allen Laughon ◽

William Howell ◽

Matthew P. Scott

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Binding Sites ◽

Binding Activity ◽

Regulatory Function ◽

Temperature Sensitive ◽

Structural Domain ◽

Dna Binding Activity ◽

Dna Restriction Fragments

The ANT-C gene cluster is part of a network of genes that govern pattern formation in the development of Drosophila. The ANT-C genes encode proteins that contain a conserved 60 amino acid sequence, the homeodomain. Here we show that the homeodomains encoded by two of the ANT-C loci confer sequencespecific DNA-binding activity. The DNA sequence specificities of the Dfd and ftz homeodomains appear to overlap completely in vitro, indicating that differences in regulatory specificity among ANT-C and BX-C proteins (assuming that differences exist) must be a consequence of the nonconserved protein sequences found outside of the homeodomains. Deletions that remove sequences from either end of the ftz homeodomain abolish DNA-binding activity, consistent with the commonly held assumption that the homeodomain is a structural domain. The relevance of in vitro DNA-binding experiments to the regulatory function of ftz is supported by our finding that a temperature-sensitive ftz mutation that causes a pairwise fusion of embryonic segments also reduces the affinity of the ftz homeodomain for DNA. Restriction fragments containing ftz homeodomain binding sites were identified within a 90 kb stretch of DNA extending the Antp P1 and P2 promoters. Binding sites appear to be clustered near the P1 promoter but also occur near P2 and in the region between the two. The task remains of determining which of these sequences mediate regulation of Antp by ftz or by other genes that encode closely related homeodomains.

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Archaeal Nucleosome Positioning by CTG Repeats

Journal of Bacteriology ◽

10.1128/jb.181.3.1035-1038.1999 ◽

1999 ◽

Vol 181 (3) ◽

pp. 1035-1038 ◽

Cited By ~ 14

Author(s):

Kathleen Sandman ◽

John N. Reeve

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Binding Sites ◽

Shape Recognition ◽

Nucleosome Positioning ◽

Dna Binding Proteins ◽

Ctg Repeats ◽

Dna Shape ◽

Ctg Repeat

ABSTRACT DNA shape recognition determines the preferred binding sites for sequence-independent DNA binding proteins, and here we document that archaeal histones assemble archaeal nucleosomes in vitro centered preferentially within (CTG)6 and (CTG)8repeats, close to junctions with flanking mixed-sequence DNA. Archaeal nucleosomes were not positioned by (CTG)4-, (CTG)5-, or (CTG)3AA(CTG)3-containing DNA sequences. The features of CTG repeat-containing sequences that direct eucaryal nucleosome positioning may also be similarly recognized by archaeal histones.

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Upregulation of PACE4 in prostate cancer is not dependent on E2F transcription factors

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2019-0668 ◽

2020 ◽

Vol 98 (7) ◽

pp. 477-481

Author(s):

Anita Bakrania ◽

Mélanie Aubé ◽

Roxane Desjardins ◽

Robert Sabbagh ◽

Robert Day

Keyword(s):

Prostate Cancer ◽

Transcription Factors ◽

Therapeutic Target ◽

Binding Sites ◽

Mammalian Cells ◽

Potential Binding ◽

Xenograft Models ◽

E2f Transcription Factors ◽

Cycle Regulation

Recent studies in prostate cancer have identified PACE4, a proprotein convertase enzyme, as an emerging therapeutic target. Inhibition of PACE4-altCT, an oncogenic isoform of PACE4, using molecular or pharmacological approaches results in decreased cell proliferation and tumor progression in xenograft models. Although several validations have confirmed PACE4-altCT as a novel therapeutic target, the transcriptional regulation of PACE4 isoforms and mechanism of action remain a challenge. Previously, it has been reported that the human PACE4 promoter possesses potential binding sites for the E2F family of transcription factors, all of which are involved in cell cycle regulation and synthesis of DNA in mammalian cells. Therefore, we attempted to conduct in-depth evaluation of E2Fs on PACE4 and PACE4 isoform expression in prostate cancer. We conducted in vitro molecular silencing studies in various prostate cancer cell lines and determined the change in PACE4 expression levels. The results clearly show that the E2Fs alone do not alter PACE4 expression.

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