Androgen Receptor Repression of GnRH Gene Transcription

Abstract Alterations in androgen levels lead to reproductive defects in both males and females, including hypogonadotropic hypogonadism, anovulation, and infertility. Androgens have been shown to down-regulate GnRH mRNA levels through an androgen receptor (AR)-dependent mechanism. Here, we investigate how androgen regulates expression from the GnRH regulatory region in the GT1-7 cell line, a model of GnRH neurons. A synthetic androgen, R1881, repressed transcription from the GnRH promoter (GnRH-P) in an AR-dependent manner, and liganded AR associated with the chromatin at the GnRH-P in live GT1-7 cells. The three known octamer-binding transcription factor-1 (Oct-1) binding sites in GnRH-P were required for AR-mediated repression, although other sequences were also involved. Although a multimer of the consensus Oct-1 binding site was not repressed, a multimer of the cluster of Oct-1, Pre-B cell leukemia transcription factor (Pbx)/Prep, and NK2 homeobox 1 (Nkx2.1) binding sites, found at −106/−91 in GnRH-P, was sufficient for repression. In fact, overexpression of any of these factors disrupted the androgen response, indicating that a balance of factors in this tripartite complex is required for AR repression. AR bound to this region in EMSA, indicating a direct interaction of AR with DNA or with other transcription factors bound to GnRH-P at this sequence. Collectively, our data demonstrate that GnRH transcription is repressed by AR via multiple sequences in GnRH-P, including three Oct-1 binding sites, and that this repression requires the complex interaction of several transcription factors.

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Phospholipid-Binding Protein EhC2A Mediates Calcium-Dependent Translocation of Transcription Factor URE3-BP to the Plasma Membrane of Entamoeba histolytica

Eukaryotic Cell ◽

10.1128/ec.00346-09 ◽

2009 ◽

Vol 9 (5) ◽

pp. 695-704 ◽

Cited By ~ 15

Author(s):

Heriberto Moreno ◽

Alicia S. Linford ◽

Carol A. Gilchrist ◽

William A. Petri

Keyword(s):

Transcription Factor ◽

Plasma Membrane ◽

Entamoeba Histolytica ◽

Binding Protein ◽

Regulatory Element ◽

Direct Interaction ◽

Immunofluorescent Staining ◽

Mrna Levels ◽

Dependent Manner ◽

Phospholipid Liposomes

ABSTRACT The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA in a Ca2+-inhibitable manner. The protein is located in both the nucleus and the cytoplasm but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. Here we identify and characterize a 22-kDa Ca2+-dependent binding partner of URE3-BP, EhC2A, a novel member of the C2-domain superfamily. Immunoprecipitations of URE3-BP and EhC2A showed that the proteins interact and that such interaction was enhanced in the presence of Ca2+. Recombinant and native EhC2A bound phospholipid liposomes in a Ca2+-dependent manner, with half-maximal binding occurring at 3.4 μM free Ca2+. A direct interaction between EhC2A and URE3-BP was demonstrated by the ability of recombinant EhC2A to recruit recombinant URE3-BP to phospholipid liposomes in a Ca2+-dependent manner. URE3-BP and EhC2A were observed to translocate to the amebic plasma membrane upon an increase in the intracellular Ca2+ concentration of trophozoites, as revealed by subcellular fractionation and immunofluorescent staining. Short hairpin RNA-mediated knockdown of EhC2A protein expression significantly modulated the mRNA levels of URE3-BP-regulated transcripts. Based on these results, we propose a model for EhC2A-mediated regulation of the transcriptional activities of URE3-BP via Ca2+-dependent anchoring of the transcription factor to the amebic plasma membrane.

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Integrating binding and expression data to predict transcription factors combined function

BMC Genomics ◽

10.1186/s12864-020-06977-1 ◽

2020 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Mahmoud Ahmed ◽

Do Sik Min ◽

Deok Ryong Kim

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Binding Sites ◽

Regulatory Region ◽

Transcription Factor Binding ◽

Expression Data ◽

Dominant Type ◽

Factor Binding ◽

Two Factors

Abstract Background Transcription factor binding to the regulatory region of a gene induces or represses its gene expression. Transcription factors share their binding sites with other factors, co-factors and/or DNA-binding proteins. These proteins form complexes which bind to the DNA as one-units. The binding of two factors to a shared site does not always lead to a functional interaction. Results We propose a method to predict the combined functions of two factors using comparable binding and expression data (target). We based this method on binding and expression target analysis (BETA), which we re-implemented in R and extended for this purpose. target ranks the factor’s targets by importance and predicts the dominant type of interaction between two transcription factors. We applied the method to simulated and real datasets of transcription factor-binding sites and gene expression under perturbation of factors. We found that Yin Yang 1 transcription factor (YY1) and YY2 have antagonistic and independent regulatory targets in HeLa cells, but they may cooperate on a few shared targets. Conclusion We developed an R package and a web application to integrate binding (ChIP-seq) and expression (microarrays or RNA-seq) data to determine the cooperative or competitive combined function of two transcription factors.

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Transcription Factors PU.1 and EGR2 Inhibits the Oncogenic Microrna Cluster Mir-17-92 during Macrophage Differentiation

Blood ◽

10.1182/blood.v112.11.473.473 ◽

2008 ◽

Vol 112 (11) ◽

pp. 473-473

Author(s):

Vit Pospisil ◽

Karin Vargova ◽

Pavel Burda ◽

Juraj Kokavec ◽

Emanuel Necas ◽

...

Keyword(s):

Transcription Factor ◽

Mononuclear Cells ◽

Regulatory Region ◽

Ectopic Expression ◽

Mature Mirnas ◽

Mrna Levels ◽

Dependent Manner ◽

Differentiation Markers ◽

Macrophage Differentiation ◽

Peripheral Blood Mononuclear

Abstract PU.1 (Sfpi1) is an ets family transcription factor required for the proper generation of both myeloid and lymphoid lineages. Distinct nuclear levels of PU.1 are required for macrophage differentiation (DeKoter 2000). MicroRNAs are a novel class of 22nt long non-coding post-transcriptional regulators, associated with differentiation, development and carcinogenesis. To identify miRNAs involved in PU.1 dependent hematopoietic differentiation we used PU.1-null hematopoietic progenitor cells stably expressing PU.1 fused to the estrogen receptor ligand-binding domain inducible by tamoxifen (PUER) (Walsh 2002). PU.1 affected expression approximately 100 mature miRNA. Among them, we have observed that PU.1 downregulates members of miR-17~92 cluster and its paralog miR-106b~25 previously associated with macrophage differentiation, apoptosis and hematologic and other malignancies (Ota 2004, Hayashita 2005, Fontana 2008). During macrophage differentiation miR-17~92 becomes downregulated and its sustained expression is associated with the differentiation block (Fontana 2007). In tamoxifen-stimulated PUER cells, PU.1 efficiently downregulates mature miRNAs of the miR-17~92 (-17.5p, -20a, -92) cluster and its paralog miR-106b~25 (but not miR-106a~363) within 96 hours and in dose dependent manner. PU.1 in the same cells activates expression of Egr2, myeloid transcription factor promoting macrophage transcriptional program a putative 8-mer-seed sequence-containing target of miR-17~92 cluster (predicted by TargetScan). We tested whether PU.1 represses expression of miR-17~92 cluster by inducing Egr2. We demonstrate that ectopic expression of Egr2 in unstimulated PUER cells inhibited expression of miR-17~92 cluster, both at the levels of mature miRs and pri-miRNAs, and induced expression of macrophage differentiation markers (Cd14, Csf1r) coupled with cell cycle arrest. Trichostatin-dependent derepression of miR-17~92 indicated that its expression is regulated at the chromatin level. Chromatin immunoprecipitation revealed specific Egr2 occupancy within the regulatory region upstream of the miR-17~92 cluster. Our data indicate that in differentiating cells, a mechanism exists involving direct repression of the miR-17~92 cluster that in turn has the potential to inhibit Egr2 expression. We have tested this mechanisms in peripheral blood mononuclear cells isolated from acute myeloid leukemia (AML) patients (N=9) where levels of PU.1 and Egr2 are significantly lower that in normal controls. 5 out of 9 AML patients significantly overexpressed miR- 17.5p, miR-20a, miR-92 coinciding with downregulation of its established targets p21 and BIM. Rescue of Egr2 in AML cells (N=2) led to downregulation of miR-17~92 cluster and upregulation of BIM mRNA levels coincident with induction of apoptosis. We conclude that PU.1 regulates transcription of the miR-17~92 cluster by inducing Egr2 and demonstrate this mechanism in differentiating macrophages and its disruption in AML cells.

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Persistent Interactions of Core Histone Tails with Nucleosomal DNA following Acetylation and Transcription Factor Binding

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6293 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6293-6304 ◽

Cited By ~ 96

Author(s):

Vesco Mutskov ◽

Delphine Gerber ◽

Dimitri Angelov ◽

Juan Ausio ◽

Jerry Workman ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Molecular Biology ◽

Binding Sites ◽

Cross Linking ◽

Uv Laser ◽

Histone Tail ◽

Histone Tails ◽

Nucleosomal Dna ◽

Core Histones

ABSTRACT In this study, we examined the effect of acetylation of the NH2 tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH2 tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH2 tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.

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Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2514-2524.1992 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2514-2524 ◽

Cited By ~ 1

Author(s):

Z S Guo ◽

M L DePamphilis

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Dna Replication ◽

Binding Sites ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Activation Domains ◽

Auxiliary Component ◽

Cell Extracts

The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites. To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor. Sp1 and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided approximately 75 and approximately 20%, respectively, of aux-2 activity when transfected into monkey cells. In cell extracts, only T-ag sites were active. AP1 binding sites could replace completely either SV40 or Py aux-2. Mutations that eliminated AP1 binding also eliminated AP1 stimulation of replication. Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun. These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori. However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity. We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity.

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HOT or not: examining the basis of high-occupancy target regions

10.1101/107680 ◽

2017 ◽

Cited By ~ 3

Author(s):

Katarzyna Wreczycka ◽

Vedran Franke ◽

Bora Uyar ◽

Ricardo Wurmus ◽

Altuna Akalin

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Binding Sites ◽

Cell Types ◽

Data Sets ◽

Gene Promoters ◽

Quadruplex Dna ◽

Golden Standard ◽

Multiple Cell ◽

Multiple Species

AbstractHigh-occupancy target (HOT) regions are the segments of the genome with unusually high number of transcription factor binding sites. These regions are observed in multiple species and thought to have biological importance due to high transcription factor occupancy. Furthermore, they coincide with house-keeping gene promoters and the associated genes are stably expressed across multiple cell types. Despite these features, HOT regions are solemnly defined using ChIP-seq experiments and shown to lack canonical motifs for transcription factors that are thought to be bound there. Although, ChIP-seq experiments are the golden standard for finding genome-wide binding sites of a protein, they are not noise free. Here, we show that HOT regions are likely to be ChIP-seq artifacts and they are similar to previously proposed “hyper-ChIPable” regions. Using ChIP-seq data sets for knocked-out transcription factors, we demonstrate presence of false positive signals on HOT regions. We observe sequence characteristics and genomic features that are discriminatory of HOT regions, such as GC/CpG-rich k-mers and enrichment of RNA-DNA hybrids (R-loops) and DNA tertiary structures (G-quadruplex DNA). The artificial ChIP-seq enrichment on HOT regions could be associated to these discriminatory features. Furthermore, we propose strategies to deal with such artifacts for the future ChIP-seq studies.

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TFutils: Data structures for transcription factor bioinformatics

F1000Research ◽

10.12688/f1000research.17976.1 ◽

2019 ◽

Vol 8 ◽

pp. 152

Author(s):

Benjamin J. Stubbs ◽

Shweta Gopaulakrishnan ◽

Kimberly Glass ◽

Nathalie Pochet ◽

Celine Everaert ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Data Structures ◽

Binding Sites ◽

Genome Wide Association Study ◽

Genome Wide Association ◽

Bioconductor Package ◽

Genome Wide ◽

Study Results ◽

Integrative Analyses

DNA transcription is intrinsically complex. Bioinformatic work with transcription factors (TFs) is complicated by a multiplicity of data resources and annotations. The Bioconductor package TFutils includes data structures and functions to enhance the precision and utility of integrative analyses that have components involving TFs. TFutils provides catalogs of human TFs from three reference sources (CISBP, HOCOMOCO, and GO), a catalog of TF targets derived from MSigDb, and multiple approaches to enumerating TF binding sites. Aspects of integration of TF binding patterns and genome-wide association study results are explored in examples.

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Virtual ChIP-seq: predicting transcription factor binding by learning from the transcriptome

10.1101/168419 ◽

2018 ◽

Cited By ~ 10

Author(s):

Mehran Karimzadeh ◽

Michael M. Hoffman

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Binding Sites ◽

Cell Types ◽

Transcription Factor Binding ◽

Regulatory Function ◽

Factor Binding ◽

Link Type ◽

Genomic Regions ◽

Factor Sequence

AbstractMotivationIdentifying transcription factor binding sites is the first step in pinpointing non-coding mutations that disrupt the regulatory function of transcription factors and promote disease. ChIP-seq is the most common method for identifying binding sites, but performing it on patient samples is hampered by the amount of available biological material and the cost of the experiment. Existing methods for computational prediction of regulatory elements primarily predict binding in genomic regions with sequence similarity to known transcription factor sequence preferences. This has limited efficacy since most binding sites do not resemble known transcription factor sequence motifs, and many transcription factors are not even sequence-specific.ResultsWe developed Virtual ChIP-seq, which predicts binding of individual transcription factors in new cell types using an artificial neural network that integrates ChIP-seq results from other cell types and chromatin accessibility data in the new cell type. Virtual ChIP-seq also uses learned associations between gene expression and transcription factor binding at specific genomic regions. This approach outperforms methods that predict TF binding solely based on sequence preference, pre-dicting binding for 36 transcription factors (Matthews correlation coefficient > 0.3).AvailabilityThe datasets we used for training and validation are available at https://virchip.hoffmanlab.org. We have deposited in Zenodo the current version of our software (http://doi.org/10.5281/zenodo.1066928), datasets (http://doi.org/10.5281/zenodo.823297), predictions for 36 transcription factors on Roadmap Epigenomics cell types (http://doi.org/10.5281/zenodo.1455759), and predictions in Cistrome as well as ENCODE-DREAM in vivo TF Binding Site Prediction Challenge (http://doi.org/10.5281/zenodo.1209308).

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Holocentromeres are dispersed point centromeres localized at transcription factor hotspots

eLife ◽

10.7554/elife.02025 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 61

Author(s):

Florian A Steiner ◽

Steven Henikoff

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Caenorhabditis Elegans ◽

Binding Sites ◽

Specific Binding ◽

Basic Unit ◽

Centromeric Chromatin ◽

Sequence Composition ◽

Specific Binding Sites ◽

Mechanistic Basis

Centromeres vary greatly in size and sequence composition, ranging from ‘point’ centromeres with a single cenH3-containing nucleosome to ‘regional’ centromeres embedded in tandemly repeated sequences to holocentromeres that extend along the length of entire chromosomes. Point centromeres are defined by sequence, whereas regional and holocentromeres are epigenetically defined by the location of cenH3-containing nucleosomes. In this study, we show that Caenorhabditis elegans holocentromeres are organized as dispersed but discretely localized point centromeres, each forming a single cenH3-containing nucleosome. These centromeric sites co-localize with kinetochore components, and their occupancy is dependent on the cenH3 loading machinery. These sites coincide with non-specific binding sites for multiple transcription factors (‘HOT’ sites), which become occupied when cenH3 is lost. Our results show that the point centromere is the basic unit of holocentric organization in support of the classical polycentric model for holocentromeres, and provide a mechanistic basis for understanding how centromeric chromatin might be maintained.

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