C-MYB and C/EBP Family Transcription Factors Regulate the Gfi-1 Promoter.

Abstract Gfi-1 −/− mice generate abnormal immature myeloid cells exhibiting characteristics of both monocytes and granulocytes. One of Gfi-1’s critical functions is to downregulate monocyte specific genes in order for granulocytes to develop properly. Since the transcription factors C/EBP alpha and C/EBP epsilon are needed for granulocyte development we hypothesized that these factors may regulate Gfi-1 expression. The Gfi-1 promoter contains several putative C/EBP binding sites and we show by electrophoretic mobility shift and chromatin immunoprecipitation that C/EBP family members can bind to some of these sites. However we were unable to see activation of the Gfi-1 promoter by C/EBP proteins in transient transfection reporter assays. Other groups have shown that C/EBP proteins can synergize with the transcription factor c-myb. We observed that the Gfi-1 promoter contains sites for the hematopoietic transcription factor c-myb. Sevral of these c-myb binding sites are adjacent to C/EBP binding sites. In reporter assays in non-hematopoietic cells c-myb activated the Gfi-1 promoter by itself and this activity was enhanced when we included either C/EBP alpha or epsilon in the transfection. Our data suggests that C/EBP proteins and c-myb regulate the transcription of Gfi-1 in myeloid cells.

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NRF-1 is the major transcription factor regulating the expression of the human TOMM34 gene

Biochemistry and Cell Biology ◽

10.1139/o07-151 ◽

2008 ◽

Vol 86 (1) ◽

pp. 46-56 ◽

Cited By ~ 22

Author(s):

José R. Blesa ◽

Jesús A. Prieto-Ruiz ◽

Beth A. Abraham ◽

Bridget L. Harrison ◽

Anita A. Hegde ◽

...

Keyword(s):

Transcription Factor ◽

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Colorectal Tumors ◽

Methylation Analysis ◽

Mobility Shift ◽

Nuclear Respiratory Factors ◽

Hela S3 ◽

Electrophoretic Mobility Shift Assays

The human TOMM34 gene encodes a cytosolic protein with chaperone-like activity that helps import some preproteins to the mitochondria by keeping them in an unfolded, import-compatible state. TOMM34 was found to be upregulated frequently in colorectal tumors, suggesting that it also has a role in the growth of cancer cells. In this context, TOMM34 is a potential target for novel anticancer drugs, and it might also be used in the diagnosis of colorectal cancer. Nuclear respiratory factors (NRFs) play an important role in governing the nuclear–mitochondrial interactions implicated in mitochondrial biogenesis. Our previous studies revealed that NRFs promote the expression of the major members of the mitochondrial transport machinery, TOMM70 and TOMM20. Here we report the existence of binding sites for NRF-1, Sp1, and NRF-2 in the 5′ region of the human TOMM34 gene. We determined the effects of mutations at these sites on promoter activity in HeLa S3 and A204 cells, in conjunction with chromatin immunoprecipitation experiments, electrophoretic mobility shift assays, and in vivo methylation analysis of the promoter region. We conclude that NRF-1 is the main transcription factor regulating the expression of TOMM34. Sp1 interacts with NRF-1 to stimulate the promoter's full activity.

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GSK3β inactivation induces apoptosis of leukemia cells by repressing the function of c-Myb

Molecular Biology of the Cell ◽

10.1091/mbc.e11-06-0483 ◽

2011 ◽

Vol 22 (18) ◽

pp. 3533-3540 ◽

Cited By ~ 34

Author(s):

Fangfang Zhou ◽

Long Zhang ◽

Theo van Laar ◽

Hans van Dam ◽

Peter ten Dijke

Keyword(s):

Transcription Factor ◽

Cell Survival ◽

Chromatin Immunoprecipitation ◽

Kinase Activity ◽

Glycogen Synthase ◽

Leukemia Cells ◽

Physiological Processes ◽

Different Types ◽

Reporter Assays ◽

Factor C

Glycogen synthase kinase 3β (GSK3β) regulates diverse physiological processes, including metabolism, development, oncogenesis, and neuroprotection. GSK3β kinase activity has been reported to be critical for various types of cancer cells, but the mechanism has remained elusive. In this study we examine the mechanism by which GSK3β regulates the survival of leukemia cells. We demonstrate that upon GSK3β kinase inhibition different types of leukemia cells show severe proliferation defects as a result of apoptosis. The transcription factor c-Myb is found to be the main target of GSK3β inhibition in cell survival. GSK3β inactivation reduces the expression of c-Myb by promoting its ubiquitination-mediated degradation, thereby inhibiting the expression of c-Myb–dependent antiapoptotic genes Bcl2 and survivin. Coimmunoprecipitation, reporter assays, chromatin immunoprecipitation, and knockdown studies show that c-Myb needs to interact and cooperate with transcription factor LEF-1 in the activation of Bcl2 and survivin and that both transcription factors are required for cell survival. These data reveal an as-yet-unknown mechanism by which GSK3β controls cell survival.

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Regulation of IκBβ Expression in Testis

Molecular Biology of the Cell ◽

10.1091/mbc.01-07-0373 ◽

2002 ◽

Vol 13 (12) ◽

pp. 4179-4194 ◽

Cited By ~ 30

Author(s):

Lucy M. Budde ◽

Chun Wu ◽

Christopher Tilman ◽

Iris Douglas ◽

Sankar Ghosh

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Reporter Gene ◽

Binding Sites ◽

Potential Role ◽

Nuclear Factor Κb ◽

Transient Transfection ◽

Nuclear Factor ◽

Transcription Factor Family ◽

Novel Target

IκBα and IκBβ are regulators of the nuclear factor-κB (NF-κB) transcription factor family. Both IκBs bind to the same NF-κB dimers and are widely expressed in different cells and tissues. To better understand how these two IκB isoforms differ biologically, we have characterized the expression of IκBβ in testis, a tissue in which IκBα is only minimally expressed. We have found that IκBβ expression is localized within the haploid spermatid stages of spermatogenesis and follows the expression of nuclear NF-κB. IκBβ expression in haploid spermatids is likely regulated by Sox family proteins, members of which are also expressed within spermatids. We have shown that both SRY and Sox-5 can bind to multiple Sox binding sites found within the IκBβ promoter and can enhance transcription of a reporter gene in transient transfection assays. We also demonstrate that IκBβ mRNA is strongly expressed in developing male gonads. These results therefore suggest that IκBβ may be a novel target for transcription factors of the HMG-box SRY/Sox family and imply a potential role for NF-κB/IκBβ in spermatogenesis.

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The AML-associated K313 mutation enhances C/EBPα activity by leading to C/EBPα overexpression

Cell Death and Disease ◽

10.1038/s41419-021-03948-6 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Ian Edward Gentle ◽

Isabel Moelter ◽

Mohamed Tarek Badr ◽

Konstanze Döhner ◽

Michael Lübbert ◽

...

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activity ◽

Target Gene ◽

Wild Type ◽

Elevated Expression ◽

Factor C ◽

Acute Myeloid

AbstractMutations in the transcription factor C/EBPα are found in ~10% of all acute myeloid leukaemia (AML) cases but the contribution of these mutations to leukemogenesis is incompletely understood. We here use a mouse model of granulocyte progenitors expressing conditionally active HoxB8 to assess the cell biological and molecular activity of C/EBPα-mutations associated with human AML. Both N-terminal truncation and C-terminal AML-associated mutations of C/EBPα substantially altered differentiation of progenitors into mature neutrophils in cell culture. Closer analysis of the C/EBPα-K313-duplication showed expansion and prolonged survival of mutant C/EBPα-expressing granulocytes following adoptive transfer into mice. C/EBPα-protein containing the K313-mutation further showed strongly enhanced transcriptional activity compared with the wild-type protein at certain promoters. Analysis of differentially regulated genes in cells overexpressing C/EBPα-K313 indicates a strong correlation with genes regulated by C/EBPα. Analysis of transcription factor enrichment in the differentially regulated genes indicated a strong reliance of SPI1/PU.1, suggesting that despite reduced DNA binding, C/EBPα-K313 is active in regulating target gene expression and acts largely through a network of other transcription factors. Strikingly, the K313 mutation caused strongly elevated expression of C/EBPα-protein, which could also be seen in primary K313 mutated AML blasts, explaining the enhanced C/EBPα activity in K313-expressing 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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Dynamic Association of the Replication Initiator and Transcription Factor DnaA with the Bacillus subtilis Chromosome during Replication Stress

Journal of Bacteriology ◽

10.1128/jb.01294-08 ◽

2008 ◽

Vol 191 (2) ◽

pp. 486-493 ◽

Cited By ~ 22

Author(s):

Adam M. Breier ◽

Alan D. Grossman

Keyword(s):

Transcription Factor ◽

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Replication Fork ◽

Replication Stress ◽

Replication Initiation ◽

Pass Through ◽

Bacillus Subtilis Chromosome ◽

Rapid Signaling ◽

Replication Initiator

ABSTRACT DnaA functions as both a transcription factor and the replication initiator in bacteria. We characterized the DNA binding dynamics of DnaA on a genomic level. Based on cross-linking and chromatin immunoprecipitation data, DnaA binds at least 17 loci, 15 of which are regulated transcriptionally in response to inhibition of replication (replication stress). Six loci, each of which has a cluster of at least nine potential DnaA binding sites, had significant increases in binding by DnaA when replication was inhibited, indicating that the association of DnaA with at least some of its target sites is altered after replication stress. When replication resumed from oriC after inhibition of replication initiation, these high levels of binding decreased rapidly at origin-proximal and origin-distal regions, well before a replication fork could pass through each of the regulated regions. These findings indicate that there is rapid signaling to decrease activation of DnaA during replication and that interaction between DnaA bound at each site and the replication machinery is not required for regulation of DnaA activity in response to replication stress.

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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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