Chromatin topology and the timing of enhancer function at the hoxd locus

ABSTRACTThe HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different sub-groups of Hoxd genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct TADs flanking HoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory sub-modules. To understand the importance of this particular regulatory topology to control Hoxd gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of Hoxd genes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancers sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavourable orientation of CTCF sites.SIGNIFICANCE STATEMENTMany genes important for vertebrate development are surrounded by series of remote enhancer sequences. Such regulatory landscapes and their target genes are usually located within the same chromatin domains, which appears to constrain the action of these regulatory sequences and hence to facilitate enhancer-promoter recognition and gene expression. We used the HoxD locus to assess the impact of modifying the regulatory topology upon gene activation in space and time. A series of chromosomal re-arrangements involving deletions and inversions reveals that the enhancer topology plays a role in the timing of gene activation. However, gene expression was often recovered, subsequently, illustrating the intrinsic capacity of some enhancers to find their target promoters despite an apparently adverse chromatin topology.

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Chromatin topology and the timing of enhancer function at theHoxDlocus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2015083117 ◽

2020 ◽

Vol 117 (49) ◽

pp. 31231-31241

Author(s):

Eddie Rodríguez-Carballo ◽

Lucille Lopez-Delisle ◽

Andréa Willemin ◽

Leonardo Beccari ◽

Sandra Gitto ◽

...

Keyword(s):

Binding Sites ◽

Target Genes ◽

Gene Activation ◽

Ctcf Binding ◽

Topologically Associating Domains ◽

Regulatory Signal ◽

Key Factor ◽

Enhancer Function ◽

Natural Target ◽

Enhancer Sequences

TheHoxDgene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different subgroups ofHoxdgenes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct topologically associating domains (TADs) flankingHoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory submodules. To understand the importance of this particular regulatory topology to controlHoxdgene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites, or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation ofHoxdgenes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancer sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavorable orientation of CTCF sites.

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Dissecting the Molecular Pathways That Underlie Disease-Causing GATA1 Mutations

Blood ◽

10.1182/blood.v120.21.3439.3439 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3439-3439

Author(s):

Amy E Campbell ◽

Lorna Wilkinson-White ◽

Joel P Mackay ◽

Jacqueline M Matthews ◽

Gerd A Blobel

Keyword(s):

Gene Expression ◽

Target Genes ◽

Gene Activation ◽

In Vitro Studies ◽

Clinical Severity ◽

Binding Studies ◽

Amino Terminal ◽

The Impact

Abstract Abstract 3439 Missense mutations in the gene encoding hematopoietic transcription factor GATA1 cause congenital anemias and/or thrombocytopenias. All seven reported mutations give rise to amino acid substitutions within the amino-terminal zinc finger (NF), but produce a range of phenotypes. The clinical severity depends on the site and type of substitution, and different substitutions of the same residue can produce disparate phenotypes. We combined structural, biochemical, in vivo conditional rescue approaches, and genomic analyses to systematically characterize all known GATA1 mutations with the goal of determining how they alter GATA1 function to result in disease. Introducing mutant forms of GATA1 into GATA1-null erythroid or bipotential erythromegakaryocytic cell lines essentially recapitulated patient phenotypes. The V205M, G208S, G208R, and D218Y mutations severely impaired both erythroid and megakaryocyte maturation, while the R216Q, R216W, and D218G mutations had only a mild effect on the maturation of these lineages. Global differentiation defects were reproduced at the level of individual GATA1 target genes. Moreover, the former mutants greatly impaired both the transcriptional activation and repression functions of GATA1, while the latter moderately impaired gene activation but had no effect on repression. It had been suggested previously that GATA1 mutations could be categorized into two classes, those that impair binding of the NF to the essential GATA1 cofactor FOG1 (V205M, G208S, G208R) and those that diminish binding of the NF to DNA (R216Q and R216W). The impact of the final two mutations (D218G and D218Y) remained uncertain, as this residue is not part of any known interaction face. Our work led to the following novel conclusions: Binding studies using isothermal titration calorimetry (ITC) and chromatin immunoprecipitation (ChIP) produced concurrent results showing that the V205M, G208S, G208R, and D218Y mutations diminish the GATA1-FOG1 interaction in vitro and FOG1 recruitment to GATA1 target genes in vivo. Interestingly, in contrast to D218Y, D218G did not affect FOG1 binding in vitro or in vivo. Furthermore, G208S had a less pronounced impact on FOG1 binding than the other three mutations, thus correlating the severity of the clinical presentation with the degree of FOG1 disruption. This confirms and extends previous work linking impaired FOG1 binding to the disease phenotypes associated with this class of mutations.ITC showed that R216Q and R216W disrupt DNA binding in vitro, consistent with previous in vitro studies. However, remarkably, ChIP assays revealed that neither mutation impaired in vivo GATA1 target site occupancy at any examined simple or palindromic GATA elements, suggesting that failure to bind DNA does not account for the associated clinical phenotypes.Notably, the R216Q and D218G mutations selectively diminished recruitment of Tal1/SCL without affecting the interaction with FOG1 or DNA. This implicates for the first time the Tal1/SCL complex in the pathogenesis of disorders caused by GATA1 mutations. Since the Tal1/SCL complex functions mostly during GATA1 gene activation, this also explains the observation that these GATA1 mutants largely retain their ability to repress transcription. Moreover, changes in the gene expression profiles of R216Q and D218G expressing cells are highly correlated with each other but clearly distinct from the gene expression changes associated with different substitutions at the same residues (R216W or D218Y), revealing a specific subset of genes that are most sensitive to disruption of the GATA1-Tal1/SCL interaction.An unexpected finding from our studies is that different substitutions of the same residue can disrupt binding to distinct cofactors (e.g. D218G impairs Tal1/SCL binding while D218Y impairs FOG1 binding), thus accounting for variable disease presentation. In concert, our work on GATA1 mutations in their native environment reveals critical new insights not obtainable from in vitro studies. This highlights the usefulness of gene complementation studies in the relevant lineages for the dissection of transcription pathways to better understand and ultimately diagnose and treat hematologic disease. Disclosures: No relevant conflicts of interest to declare.

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Functional effects of variation in transcription factor binding highlight long-range gene regulation by epromoters

10.1101/620062 ◽

2019 ◽

Author(s):

Joanna Mitchelmore ◽

Nastasiya Grinberg ◽

Chris Wallace ◽

Mikhail Spivakov

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Statistical Power ◽

Target Genes ◽

Allelic Variation ◽

Regulatory Function ◽

Regulatory Sequences ◽

Distal Enhancer ◽

Association Testing ◽

Gene Regulatory

AbstractIdentifying DNA cis-regulatory modules (CRMs) that control the expression of specific genes is crucial for deciphering the logic of transcriptional control. Natural genetic variation can point to the possible gene regulatory function of specific sequences through their allelic associations with gene expression. However, comprehensive identification of causal regulatory sequences in brute-force association testing without incorporating prior knowledge is challenging due to limited statistical power and effects of linkage disequilibrium. Sequence variants affecting transcription factor (TF) binding at CRMs have a strong potential to influence gene regulatory function, which provides a motivation for prioritising such variants in association testing. Here, we generate an atlas of CRMs showing predicted allelic variation in TF binding affinity in human lymphoblastoid cell lines (LCLs) and test their association with the expression of their putative target genes inferred from Promoter Capture Hi-C and immediate linear proximity. We reveal over 1300 CRM TF-binding variants associated with target gene expression, the majority of them undetected with standard association testing. A large proportion of CRMs showing associations with the expression of genes they contact in 3D localise to the promoter regions of other genes, supporting the notion of ‘epromoters’: dual-action CRMs with promoter and distal enhancer activity.

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RNAseq Studies Reveal Distinct Transcriptional Response to Vitamin a (VA) Deficiency in Small Intestine (SI) versus Colon, Discovering Novel VA-regulated Genes (P15-002-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz037.p15-002-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Zhi Chai ◽

Yafei Lyu ◽

Qiuyan Chen ◽

Cheng-Hsin Wei ◽

Lindsay Snyder ◽

...

Keyword(s):

Gene Expression ◽

Small Intestine ◽

Vitamin A ◽

Target Genes ◽

Expression Profiles ◽

Expression Patterns ◽

Differentially Expressed Gene ◽

Gene Expression Profiles ◽

Illumina Hiseq ◽

The Impact

Abstract Objectives To characterize and compare the impact of vitamin A (VA) deficiency on gene expression patterns in the small intestine (SI) and the colon, and to discover novel target genes in VA-related biological pathways. Methods vitamin A deficient (VAD) mice were generated by feeding VAD diet to pregnant C57/BL6 dams and their post-weaning offspring. Total mRNA extracted from SI and colon were sequenced using Illumina HiSeq 2500 platform. Differentially Expressed Gene (DEG), Gene Ontology (GO) enrichment, and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to characterize expression patterns and co-expression patterns. Results The comparison between vitamin A sufficient (VAS) and VAD groups detected 49 and 94 DEGs in SI and colon, respectively. According to GO information, DEGs in the SI demonstrated significant enrichment in categories relevant to retinoid metabolic process, molecule binding, and immune function. Immunity related pathways, such as “humoral immune response” and “complement activation,” were positively associated with VA in SI. On the contrary, in colon, “cell division” was the only enriched category and was negatively associated with VA. WGCNA identified modules significantly correlated with VA status in SI and in colon. One of those modules contained five known retinoic acid targets. Therefore we have prioritized the other module members (e.g., Mbl2, Mmp9, Mmp13, Cxcl14 and Pkd1l2) to be investigated as candidate genes regulated by VA. Comparison of co-expression modules between SI and colon indicated distinct VA effects on these two organs. Conclusions The results show that VA deficiency alters the gene expression profiles in SI and colon quite differently. Some immune-related genes (Mbl2, Mmp9, Mmp13, Cxcl14 and Pkd1l2) may be novel targets under the control of VA in SI. Funding Sources NIH training grant and NIH research grant. Supporting Tables, Images and/or Graphs

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Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1746 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1746-1758 ◽

Cited By ~ 10

Author(s):

D J DeAngelo ◽

J DeFalco ◽

G Childs

Keyword(s):

Gene Expression ◽

Molecular Weight ◽

Transcriptional Activation ◽

Target Genes ◽

Gene Activation ◽

Upstream Sequence ◽

Blastula Stage ◽

Sequence Element ◽

Enhancer Element ◽

Beta Gene

We have demonstrated that a highly conserved segment of DNA between positions -288 and -317 (upstream sequence element IV [USE IV]) is largely responsible for the transcriptional activation of the sea urchin H1-beta histone gene during the blastula stage of embryogenesis. This sequence is capable of acting as an embryonic enhancer element, activating target genes in a stage-specific manner. Nuclear extracts prepared from developmentally-staged organisms before and after the gene is activated all contain a factor which specifically binds to the enhancer. We have purified a 43-kDa polypeptide which binds to and footprints the USE IV enhancer element. We refer to this protein as stage-specific activator protein 1 (SSAP-1). Early in development before the enhancer is active, SSAP appears as a 43-kDa monomer, but it undergoes a change in its molecular weight beginning at about 12 h postfertilization (early blastula) which precisely parallels the increase in H1-beta gene expression. Modified SSAP has an apparent molecular mass of approximately 90 to 100 kDa and contains at least one 43-kDa SSAP polypeptide. Thus, it is the disappearance of the 43-kDa species and the appearance of the 90- to 100-kDa species which coincide with the H1-beta gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-beta gene expression strongly suggests that this higher-molecular-weight form of SSAP is directly responsible for the blastula stage-specific transcriptional activation of the late H1 gene.

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RegVar: Tissue-specific Prioritization of Noncoding Regulatory Variants

10.1101/2021.04.17.440295 ◽

2021 ◽

Author(s):

Hao Lu ◽

Luyu Ma ◽

Lei Li ◽

Cheng Quan ◽

Yiming Lu ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Human Genetics ◽

Computational Framework ◽

Tissue Specific ◽

Regulatory Variants ◽

Regulatory Impact ◽

Genome Variations ◽

The Impact ◽

Specific Impact

Noncoding genomic variants constitute the majority of trait-associated genome variations; however, identification of functional noncoding variants is still a challenge in human genetics, and a method systematically assessing the impact of regulatory variants on gene expression and linking them to potential target genes is still lacking. Here we introduce a deep neural network (DNN)-based computational framework, RegVar, that can accurately predict the tissue-specific impact of noncoding regulatory variants on target genes. We show that, by robustly learning the genomic characteristics of massive variant-gene expression associations in a variety of human tissues, RegVar vastly surpasses all current noncoding variants prioritization methods in predicting regulatory variants under different circumstances. The unique features of RegVar make it an excellent framework for assessing the regulatory impact of any variant on its putative target genes in a variety of tissues. RegVar is available as a webserver at http://regvar.cbportal.org/.

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Interpretable deep learning for chromatin-informed inference of transcriptional programs driven by somatic alterations across cancers

10.1101/2021.09.07.459263 ◽

2021 ◽

Author(s):

Yifeng Tao ◽

Xiaojun Ma ◽

Georgios I. Laliotis ◽

Adler Guerrero Zuniga ◽

Drake Palmer ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Cancer Cell Line ◽

Attention Mechanism ◽

The Cancer Genome Atlas ◽

Patient Specific ◽

Open Chromatin ◽

Therapeutic Decisions ◽

Somatic Alterations ◽

The Impact

AbstractCancer is a disease of gene dysregulation, where cells acquire somatic and epigenetic alterations that drive aberrant cellular signaling. These alterations adversely impact transcriptional programs and cause profound changes in gene expression. Ultimately, interpreting patient somatic alterations within context-specific regulatory programs will facilitate personalized therapeutic decisions for each individual. Towards this goal, we develop a partially interpretable neural network model with encoder-decoder architecture, called Chromatin-informed Inference of Transcriptional Regulators Using Self-attention mechanism (CITRUS), to model the impact of somatic alterations on cellular states and further onto downstream gene expression programs. The encoder module employs a self-attention mechanism to model the contextual impact of somatic alterations in a tumor-specific manner. Furthermore, the model uses a layer of hidden nodes to explicitly represent the state of transcription factors (TFs), and the decoder learns the relationships between TFs and their target genes guided by the sparse prior based on TF binding motifs in the open chromatin regions of tumor samples. We apply CITRUS to genomic, mRNA sequencing and ATAC-seq data from tumors of 17 cancer types profiled by The Cancer Genome Atlas. Our computational framework enables us to share information across tumors to learn patient-specific TF activities, revealing regulatory program similarities and differences between and within tumor types. We show that CITRUS not only outperforms the competing models in predicting RNA expression, but also yields biological insights in delineating TFs associated with somatic alterations in individual tumors. We also validate the differential activity of TFs associated with mutant PIK3CA in breast cancer cell line and xenograft models using a panel of PI3K pathway inhibitors.

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The transcription factor Schnurri plays a dual role in mediating Dpp signaling during embryogenesis

Development ◽

10.1242/dev.128.9.1657 ◽

2001 ◽

Vol 128 (9) ◽

pp. 1657-1670 ◽

Cited By ~ 1

Author(s):

J. Torres-Vazquez ◽

S. Park ◽

R. Warrior ◽

K. Arora

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Cell Fate ◽

Target Genes ◽

Gene Activation ◽

Nuclear Accumulation ◽

Embryonic Patterning ◽

Specific Analysis ◽

Positive Input ◽

Affect Gene Expression

Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenic protein 2/4, is crucial for embryonic patterning and cell fate specification in Drosophila. Dpp signaling triggers nuclear accumulation of the Smads Mad and Medea, which affect gene expression through two distinct mechanisms: direct activation of target genes and relief of repression by the nuclear protein Brinker (Brk). The zinc-finger transcription factor Schnurri (Shn) has been implicated as a co-factor for Mad, based on its DNA-binding ability and evidence of signaling dependent interactions between the two proteins. A key question is whether Shn contributes to both repression of brk as well as to activation of target genes. We find that during embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated repression of brk, the requirement for shn is stage specific. Analysis of brk; shn double mutants reveals that upregulation of brk does not account for all aspects of the shn mutant phenotype. Several Dpp target genes are expressed at intermediate levels in double mutant embryos, demonstrating that shn also provides a brk-independent positive input to gene activation. We find that Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of Dpp target gene expression in the embryo.

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FLT3ITD Target Enhancers Are Primed but Inaccessible in Fetal Hematopoietic Progenitors

Blood ◽

10.1182/blood-2019-126201 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1228-1228

Author(s):

Yanan Li ◽

Riddhi M Patel ◽

Emily Casey ◽

Jeffrey A. Magee

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Target Gene ◽

Target Genes ◽

Conflicts Of Interest ◽

Gene Activation ◽

Chromatin Accessibility ◽

Luciferase Reporter ◽

Enhancer Activity ◽

Target Gene Expression

The FLT3 Internal Tandem Duplication (FLT3ITD) is common somatic mutation in acute myeloid leukemia (AML). We have previously shown that FLT3ITD fails to induce changes in HSC self-renewal, myelopoiesis and leukemogenesis during fetal stages of life. FLT3ITD signal transduction pathways are hyperactivated in fetal progenitors, but FLT3ITD target genes are not. This suggests that postnatal-specific transcription factors may be required to help induce FLT3ITD target gene expression. Alternatively, repressive histone modifications may impose a barrier to FLT3ITD target gene activation in fetal HPCs that is relaxed during postnatal development. To resolve these possibilities, we used ATAC-seq, as well as H3K4me1, H3K27ac and H3K27me3 ChIP-seq, to identify cis-elements that putatively control FLT3ITD target gene expression in fetal and adult hematopoietic progenitor cells (HPCs). We identified many enhancer elements (ATAC-seq peaks with H3K4me1 and H3K27ac) that exhibited increased chromatin accessibility and activity in FLT3ITD adult HPCs relative to wild type adult HPCs. These elements were enriched near FLT3ITD target genes. HOMER analysis showed enrichment for STAT5, ETS, RUNX1 and IRF binding motifs within the FLT3ITD target enhancers, but motifs for temporally dynamic transcription factors were not identified. We cloned a subset of the enhancers and confirmed that they could synergize with their promoter to activate a luciferase reporter. For representative enhancers, STAT5 binding sites were required to activate the enhancer - as anticipated - and RUNX1 repressed enhancer activity. We tested whether accessibility or priming changed between fetal and adult stages of HPC development. FLT3ITD-dependent changes in chromatin accessibility were not observed in fetal HPCs, though the enhancers were primed early in development as evidenced by the presence of H3K4me1. Repressive H3K27me3 were not present at FLT3ITD target enhancers in either or adult HPCs. The data show that FLT3ITD target enhancers are demarcated early in hematopoietic development, long before they become responsive to FLT3ITD signaling. Repressive marks do not appear to create an epigenetic barrier to enhancer activation in the fetal stage. Instead, age-specific transcription factors are likely required to pioneer enhancer elements so that they can respond to STAT5 and other FLT3ITD effectors. Disclosures No relevant conflicts of interest to declare.

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Induction of Mxi1-SRα by FOXO3a Contributes to Repression of Myc-Dependent Gene Expression

Molecular and Cellular Biology ◽

10.1128/mcb.01789-06 ◽

2007 ◽

Vol 27 (13) ◽

pp. 4917-4930 ◽

Cited By ~ 111

Author(s):

Oona Delpuech ◽

Beatrice Griffiths ◽

Philip East ◽

Abdelkader Essafi ◽

Eric W.-F. Lam ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle Progression ◽

Dna Microarrays ◽

Target Genes ◽

Gene Activation ◽

Transcriptional Response ◽

Inhibitory Effect ◽

Pi3 Kinase ◽

Promoter Regions ◽

Inhibition Of Proliferation

ABSTRACT Forkhead transcription factors of the O class (FOXOs) are important targets of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. FOXOs have been implicated in the regulation of cell cycle progression, oxidative stress resistance, and apoptosis. Using DNA microarrays, we analyzed the transcriptional response to FOXO3a activation by gene expression analysis in DLD-1 colon cancer cells stably expressing a FOXO3a.A3-ER fusion protein. We found that activation of FOXO3a resulted in repression of a number of previously identified Myc target genes. Furthermore, FOXO3a activation induced expression of several members of the Mad/Mxd family of transcriptional repressors, most notably Mxi1. The induction of Mxi1 by FOXO3a was specific to the Mxi1-SRα isoform and was mediated by three highly conserved FOXO binding sites within the first intron of the gene. Activation of FOXO3a in response to inhibition of Akt also resulted in activation of Mxi1-SRα expression. Silencing of Mxi1 by small interfering RNA (siRNA) reduced FOXO3a-mediated repression of a number of Myc target genes. We also observed that FOXO3a activation induced a switch in promoter occupancy from Myc to Mxi1 on the E-box containing promoter regions of two Myc target genes, APEX and FOXM1. siRNA-mediated transient silencing of Mxi1 or all Mad/Mxd proteins reduced exit from S phase in response to FOXO3a activation, and stable silencing of Mxi1 or Mad1 reduced the growth inhibitory effect of FOXO3a. We conclude that induction of Mad/Mxd proteins contributes to the inhibition of proliferation in response to FOXO3a activation. Our results provide evidence of direct regulation of Mxi1 by FOXO3a and imply an additional mechanism through which the PI3-kinase/Akt/FOXO pathway can modulate Myc function.

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