HOX paralogs selectively convert binding of ubiquitous transcription factors into tissue-specific patterns of enhancer activation

Gene expression programs determine cell fate in embryonic development and their dysregulation results in disease. Transcription factors (TFs) control gene expression by binding to enhancers, but how TFs select and activate their target enhancers is still unclear. HOX TFs share conserved homeodomains with highly similar sequence recognition properties, yet they impart the identity of different animal body parts. To understand how HOX TFs control their specific transcriptional programs in vivo, we compared HOXA2 and HOXA3 binding profiles in the mouse embryo. HOXA2 and HOXA3 directly cooperate with TALE TFs and selectively target different subsets of a broad TALE chromatin platform. Binding of HOX and tissue-specific TFs convert low affinity TALE binding into high confidence, tissue-specific binding events, which bear the mark of active enhancers. We propose that HOX paralogs, alone and in combination with tissue-specific TFs, generate tissue-specific transcriptional outputs by modulating the activity of TALE TFs at selected enhancers.

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HOX paralogs selectively convert binding of ubiquitous transcription factors into tissue-specific patterns of enhancer activation

10.1101/871640 ◽

2019 ◽

Author(s):

Laure Bridoux ◽

Peyman Zarrineh ◽

Joshua Mallen ◽

Mike Phuycharoen ◽

Victor Latorre ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Cell Fate ◽

Specific Binding ◽

Body Parts ◽

Similar Sequence ◽

Tissue Specific ◽

Sequence Recognition ◽

Control Gene Expression

SummaryGene expression programs determine cell fate in embryonic development and their dysregulation results in disease. Transcription factors (TFs) control gene expression by binding to enhancers, but how TFs select and activate their target enhancers is still unclear. HOX TFs share conserved homeodomains with highly similar sequence recognition properties, yet they impart the identity of different animal body parts. To understand how HOX TFs control their specific transcriptional programs in vivo, we compared HOXA2 and HOXA3 binding profiles in the mouse embryo. HOXA2 and HOXA3 directly cooperate with TALE TFs and selectively target different subsets of a broad TALE chromatin platform. Binding of HOX and tissue-specific TFs convert low affinity TALE binding into high confidence, tissue-specific binding events, which bear the mark of active enhancers. We propose that HOX paralogs, alone and in combination with tissue-specific TFs, generate tissue-specific transcriptional outputs by modulating the activity of TALE TFs at selected enhancers.

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Engineering the smallest transcription factor: accelerated evolution of a 63-amino acid peptide dual activator-repressor

10.1101/725739 ◽

2019 ◽

Author(s):

Andreas K. Brödel ◽

Rui Rodrigues ◽

Alfonso Jaramillo ◽

Mark Isalan

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Protein Gene ◽

Control Gene ◽

Bacteriophage Λ ◽

Short Peptide ◽

Amino Acid Peptide ◽

Control Gene Expression ◽

Accelerated Evolution

Transcription factors control gene expression in all life. This raises the question of what is the smallest protein that can support such activity. In nature, Cro from bacteriophage λ is the smallest known repressor (66 amino acids; a.a.) but activators are typically much larger (e.g. λ cI, 237 a.a.). Indeed, previous efforts to engineer a minimal activator from Cro resulted in no activity in vivo. In this study, we show that directed evolution results in a new Cro activator-repressor that functions as efficiently as λ cI, in vivo. To achieve this, we develop Phagemid-Assisted Continuous Evolution: PACEmid. We find that a peptide as small as 63-a.a. functions efficiently as an activator and/or repressor. To our knowledge, this is the smallest protein gene regulator reported to date, highlighting the capacity of transcription factors to evolve from very short peptide sequences.

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A comprehensive manually-curated Compendium of Bovine Transcription Factors

10.1101/254011 ◽

2018 ◽

Author(s):

Marcela M de Souza ◽

Juan M Vaquerizas ◽

Adhemar Zerlotini ◽

Ludwig Geistlinger ◽

Benjamín Hernández-Rodríguez ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Regulation Of Gene Expression ◽

Regulatory Proteins ◽

Regulatory Mechanisms ◽

Domain Family ◽

Specific Expression ◽

Tissue Specific ◽

Control Gene Expression ◽

Specific Manner

ABSTRACTTranscription factors (TFs) are pivotal regulatory proteins that control gene expression in a context-dependent and tissue-specific manner. In contrast to human, where comprehensive curated TF collections exist, bovine TFs are only rudimentary recorded and characterized. In this article, we present a manually-curated compendium of 865 sequence-specific DNA-binding bovines TFs, which we analyzed for domain family distribution, evolutionary conservation, and tissue-specific expression. In addition, we provide a list of putative transcription cofactors derived from known interactions with the identified TFs. Since there is a general lack of knowledge concerning the regulation of gene expression in cattle, the curated list of TF should provide a basis for an improved comprehension of regulatory mechanisms that are specific to the species.

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Accelerated evolution of a minimal 63–amino acid dual transcription factor

Science Advances ◽

10.1126/sciadv.aba2728 ◽

2020 ◽

Vol 6 (24) ◽

pp. eaba2728 ◽

Cited By ~ 2

Author(s):

Andreas K. Brödel ◽

Rui Rodrigues ◽

Alfonso Jaramillo ◽

Mark Isalan

Keyword(s):

Gene Expression ◽

Amino Acids ◽

Transcription Factors ◽

Control Gene ◽

Bacteriophage Λ ◽

Short Peptide ◽

Protein Activator ◽

Control Gene Expression ◽

Accelerated Evolution

Transcription factors control gene expression in all life. This raises the question of what is the smallest protein that can support such activity. In nature, Cro from bacteriophage λ is one of the smallest known repressors (66 amino acids), and activators are typically much larger (e.g., λ cI, 237 amino acids). Previous efforts to engineer a minimal activator from λ Cro resulted in no activity in vivo in cells. In this study, we show that directed evolution results in a new Cro activator-repressor that functions as efficiently as λ cI in vivo. To achieve this, we develop phagemid-assisted continuous evolution (PACEmid). We find that a peptide as small as 63 amino acids functions efficiently as an activator and/or repressor. To our knowledge, this is the smallest protein activator that enables polymerase recruitment, highlighting the capacity of transcription factors to evolve from very short peptide sequences.

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Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

Biochemistry and Cell Biology ◽

10.1139/o05-062 ◽

2005 ◽

Vol 83 (4) ◽

pp. 535-547 ◽

Cited By ~ 7

Author(s):

Gareth N Corry ◽

D Alan Underhill

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activation ◽

Current Knowledge ◽

Nuclear Architecture ◽

Subnuclear Localization ◽

Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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Single molecule tracking of Ace1p in Saccharomyces cerevisiae defines a characteristic residence time for non-specific interactions of transcription factors with chromatin

Nucleic Acids Research ◽

10.1093/nar/gkw744 ◽

2016 ◽

Vol 44 (21) ◽

pp. e160-e160 ◽

Cited By ~ 23

Author(s):

David A Ball ◽

Gunjan D Mehta ◽

Ronit Salomon-Kent ◽

Davide Mazza ◽

Tatsuya Morisaki ◽

...

Keyword(s):

Transcription Factors ◽

Residence Time ◽

Single Molecule ◽

Specific Binding ◽

Underlying Mechanism ◽

Specific Interactions ◽

Single Molecule Tracking ◽

Reliable Performance ◽

Transient Interactions

Abstract In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12–0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.

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Molecular Characterization of HOXA2 and HOXA3 Binding Properties

Journal of Developmental Biology ◽

10.3390/jdb9040055 ◽

2021 ◽

Vol 9 (4) ◽

pp. 55

Author(s):

Joshua Mallen ◽

Manisha Kalsan ◽

Peyman Zarrineh ◽

Laure Bridoux ◽

Shandar Ahmad ◽

...

Keyword(s):

Transcription Factors ◽

Molecular Characterization ◽

Sequence Motif ◽

Body Parts ◽

Binding Affinities ◽

Binding Properties ◽

Functional Consequences

The highly conserved HOX homeodomain (HD) transcription factors (TFs) establish the identity of different body parts along the antero–posterior axis of bilaterian animals. Segment diversification and the morphogenesis of different structures is achieved by generating precise patterns of HOX expression along the antero–posterior axis and by the ability of different HOX TFs to instruct unique and specific transcriptional programs. However, HOX binding properties in vitro, characterised by the recognition of similar AT-rich binding sequences, do not account for the ability of different HOX to instruct segment-specific transcriptional programs. To address this problem, we previously compared HOXA2 and HOXA3 binding in vivo. Here, we explore if sequence motif enrichments observed in vivo are explained by binding affinities in vitro. Unexpectedly, we found that the highest enriched motif in HOXA2 peaks was not recognised by HOXA2 in vitro, highlighting the importance of investigating HOX binding in its physiological context. We also report the ability of HOXA2 and HOXA3 to heterodimerise, which may have functional consequences for the HOX patterning function in vivo.

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GATA transcription factors as potentiators of gut endoderm differentiation

Development ◽

10.1242/dev.125.24.4909 ◽

1998 ◽

Vol 125 (24) ◽

pp. 4909-4917 ◽

Cited By ~ 8

Author(s):

P. Bossard ◽

K.S. Zaret

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Site Occupancy ◽

Gata Factor ◽

Transcriptional Enhancer ◽

Gata Factors ◽

Endoderm Differentiation ◽

Albumin Gene ◽

Gut Endoderm

Gene inactivation studies have shown that members of the GATA family of transcription factors are critical for endoderm differentiation in mice, flies and worms, yet how these proteins function in such a conserved developmental context has not been understood. We use in vivo footprinting of mouse embryonic endoderm cells to show that a DNA-binding site for GATA factors is occupied on a liver-specific, transcriptional enhancer of the serum albumin gene. GATA site occupancy occurs in gut endoderm cells at their pluripotent stage: the cells have the potential to initiate tissue development but they have not yet been committed to express albumin or other tissue-specific genes. The GATA-4 isoform accounts for about half of the nuclear GATA-factor-binding activity in the endoderm. GATA site occupancy persists during hepatic development and is necessary for the activity of albumin gene enhancer. Thus, GATA factors in the endoderm are among the first to bind essential regulatory sites in chromatin. Binding occurs prior to activation of gene expression, changes in cell morphology or functional commitment that would indicate differentiation. We suggest that GATA factors at target sites in chromatin may generally help potentiate gene expression and tissue specification in metazoan endoderm development.

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P0511ENHANCER AND SUPER-ENHANCER DYNAMICS IN REPAIR AFTER ISCHEMIC ACUTE KIDNEY INJURY

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa143.p0511 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Julia Wilflingseder ◽

Michaela Willi ◽

Hye Kyung Lee ◽

Hannes Olauson ◽

Jakub Jankowsky ◽

...

Keyword(s):

Acute Kidney Injury ◽

Transcription Factors ◽

Small Molecule ◽

Ischemia Reperfusion Injury ◽

Specific Binding ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Kidney Cortex ◽

Super Enhancer

Abstract Background and Aims The endogenous repair process of the mammalian kidney allows rapid recovery after acute kidney injury (AKI) through robust proliferation of tubular epithelial cells. There is currently limited understanding of which transcriptional regulators activate these repair programs and how transcriptional dysregulation leads to maladaptive repair. Here we investigate the existence of enhancer dynamics in the regenerating mouse kidney. Method RNA-seq and ChIP-seq (H3K27ac, H3K4m3, BRD4, POL2 and selected transcription factors) were performed on samples from repairing kidney cortex 2 days after ischemia/reperfusion injury (IRI) to identify activated genes, transcription factors, enhancer and super-enhancers associated with kidney repair. Further we investigated the role of super-enhancer activation in kidney repair through pharmacological BET inhibition using the small molecule JQ1 in vitro and in acute kidney injury models in vivo. Results Response to kidney injury leads to genome-wide alteration in enhancer repertoire in-vivo. We identified 16,781 enhancer sites (H3K27ac and BRD4 positive, H3K4me3 negative binding) active in SHAM and IRI samples; 6,512 lost and 9,774 gained after IRI. The lost and gained enhancer sites can be annotated to 62% and 63% of down- and up-regulated transcripts at day 2 after kidney injury, respectively. Super-enhancer analysis revealed 164 lost and 216 gained super-enhancer sites at IRI day 2. 385 super-enhancers maintain activity before and after injury. ChIP-seq profiles of selected transcription factors based on motif analysis show specific binding at corresponding enhancer sites. We observed lost enhancer binding of HNF4A and GR mainly at kidney related enhancer elements. In contrast, STAT3 showed increased binding at injury induces enhancer elements. No dynamic was observed for STAT5. Both transcription factor groups show corresponding mRNA changes after injury. Pharmacological inhibition of enhancer and super-enhancer activity by BRD4 inhibition (JQ1: 50mg/kg/day) before IRI leads to suppression of 40% of injury-induced transcripts associated with cell cycle regulation and significantly increased mortality between days 2 and 3 after AKI. Conclusion This is the first demonstration of enhancer and super-enhancer function in the repairing kidney. In addition, our data call attention to potential caveats for use of small molecule inhibitors of BET proteins that are currently being tested in clinical trials in cancer patients who are at risk for AKI. Our analyses of enhancer dynamics after kidney injury in vivo have the potential to identify new targets for therapeutic intervention.

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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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