scholarly journals Interrogation of Enhancer Function by Enhanced CRISPR Epigenetic Editing

2019 ◽  
Author(s):  
Kailong Li ◽  
Yuxuan Liu ◽  
Hui Cao ◽  
Yuannyu Zhang ◽  
Zhimin Gu ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Cancer Progression ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Enhancer Activity ◽  
Super Enhancer ◽  
Allele Specific ◽  
Enhancer Function ◽  
Epigenetic Editing

ABSTRACTTissue-specific gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe enhanced CRISPR/dCas9-based epigenetic editing systems, enCRISPRa and enCRISPRi, for multiplexed analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the new systems display more robust perturbation of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Multiplexed perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoietic lineage specification. Hence, enhanced CRSIPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.

scholarly journals Integrative epigenomic and high-throughput functional enhancer profiling reveals determinants of enhancer heterogeneity in gastric cancer

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Taotao Sheng ◽  
Shamaine Wei Ting Ho ◽  
Wen Fong Ooi ◽  
Chang Xu ◽  
Manjie Xing ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Histone Modification ◽  
Cell Fate ◽  
Copy Number ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Functional Assay ◽  
Enhancer Activity

Abstract Background Enhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity—however, most predicted enhancer regions remain to be functionally tested. Methods We analyzed 132 epigenomic histone modification profiles of 18 primary gastric cancer (GC) samples, 18 normal gastric tissues, and 28 GC cell lines using Nano-ChIP-seq technology. We applied Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to assess functional enhancer activity. An Activity-by-contact (ABC) model was employed to explore the effects of histone acetylation and CapSTARR-seq levels on enhancer-promoter interactions. Results We report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are GC-associated in vivo (> 50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying CapSTARR-seq to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers, even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. We identified cancer-relevant genes (ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity. Conclusions Our results indicate that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, providing insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.

scholarly journals Systematic dissection of genomic features determining transcription factor binding and enhancer function

2017 ◽  
Vol 114 (7) ◽  
pp. E1291-E1300 ◽  
Author(s):  
Sharon R. Grossman ◽  
Xiaolan Zhang ◽  
Li Wang ◽  
Jesse Engreitz ◽  
Alexandre Melnikov ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Binding Assays ◽  
Genomic Locus ◽  
Enhancer Activity ◽  
Genomic Features ◽  
Motif Site ◽  
Reporter Assays ◽  
Enhancer Function

Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.

scholarly journals Granulocyte-Macrophage Colony-Stimulating Factor Enhancer Activation Requires Cooperation between NFAT and AP-1 Elements and Is Associated with Extensive Nucleosome Reorganization

2004 ◽  
Vol 24 (18) ◽  
pp. 7914-7930 ◽  
Author(s):  
Brett V. Johnson ◽  
Andrew G. Bert ◽  
Gregory R. Ryan ◽  
Antony Condina ◽  
Peter N. Cockerill
Keyword(s):  
Regulatory Elements ◽  
Colony Stimulating Factor ◽  
Enhancer Activity ◽  
Gm Csf ◽  
Core Elements ◽  
Macrophage Colony Stimulating Factor ◽  
Enhancer Function ◽  
Macrophage Colony ◽  
Stimulating Factor

ABSTRACT The human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is activated by an NFAT-dependent enhancer forming an inducible DNase I hypersensitive (DH) site. The enhancer core comprising the DH site contains the GM330 and GM420 elements that bind NFAT and AP-1 cooperatively. Here we demonstrate that both elements are essential for enhancer activity and that Sp1 and AML1 sites in the enhancer become occupied in vivo only after activation. Chromatin structure analysis revealed that the GM-CSF enhancer core elements are divided between two adjacent nucleosomes that become destabilized and highly accessible after activation. Inducible chromatin reorganization was not restricted to the enhancer core but extended across a 3-kb domain of mobilized nucleosomes, within which the nucleosome repeat length was compressed from approximately 185 to 150 bp. The GM420 element is a high-affinity site that binds NFAT independently of AP-1 but depends on the linked AP-1 site for enhancer function. Nevertheless, just the NFAT motif from the GM420 element was sufficient to form a DH site within chromatin even in the absence of the AP-1 site. Hence, NFAT has the potential to cooperate with other transcription factors by promoting chromatin remodelling and increasing accessibility at inducible regulatory elements.

scholarly journals Integrative Epigenomic and High-Throughput Functional Enhancer Profiling Reveals Determinants of Enhancer Heterogeneity in Gastric Cancer

2021 ◽  
Author(s):  
Taotao Sheng ◽  
Shamaine Wei Ting Ho ◽  
Wen Fong Ooi ◽  
Chang Xu ◽  
Manjie Xing ◽  
...  
Keyword(s):  
Histone Modification ◽  
Cell Fate ◽  
Copy Number ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Functional Assay ◽  
Nucleotide Polymorphisms ◽  
Enhancer Activity

Background: Enhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity – however, most predicted enhancer regions remain to be functionally tested. Results: Analyzing 128 epigenomic histone modification profiles of primary GC samples, normal gastric tissues, and GC cell lines, we report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are tumor-associated in vivo (>50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. Specifically, we identified cancer-relevant genes (e.g. ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity. Conclusions: Our study indicates that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, and provides insights into the relative contribution of genetic (cis ) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.

scholarly journals Dynamic Interactions of Transcription Factors and Enhancer Reprogramming in Cancer Progression

2021 ◽  
Vol 11 ◽  
Author(s):  
Emily Zboril ◽  
Hannah Yoo ◽  
Lizhen Chen ◽  
Zhijie Liu
Keyword(s):  
Gene Expression ◽  
Cancer Progression ◽  
Expression Patterns ◽  
Therapy Resistance ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Dynamic Interactions ◽  
Genome Wide ◽  
Dna Regulatory Elements

While improved tumor treatment has significantly reduced the overall mortality rates, invasive progression including recurrence, therapy resistance and metastasis contributes to the majority of deaths caused by cancer. Enhancers are essential distal DNA regulatory elements that control temporal- or spatial-specific gene expression patterns during development and other biological processes. Genome-wide sequencing has revealed frequent alterations of enhancers in cancers and reprogramming of distal enhancers has emerged as one of the important features for tumors. In this review, we will discuss tumor progression-associated enhancer dynamics, its transcription factor (TF) drivers and how enhancer reprogramming modulates gene expression during cancer invasive progression. Additionally, we will explore recent advancements in contemporary technology including single-cell sequencing, spatial transcriptomics and CUT&RUN, which have permitted integrated studies of enhancer reprogramming in vivo. Given the essential roles of enhancer dynamics and its drivers in controlling cancer progression and treatment outcome, understanding these changes will be paramount in mitigating invasive events and discovering novel therapeutic targets.

scholarly journals Dissection of a Complex Enhancer Element: Maintenance of Keratinocyte Specificity but Loss of Differentiation Specificity

2002 ◽  
Vol 22 (12) ◽  
pp. 4293-4308 ◽  
Author(s):  
Charles K. Kaufman ◽  
Satrajit Sinha ◽  
Diana Bolotin ◽  
Jie Fan ◽  
Elaine Fuchs
Keyword(s):  
Gene Expression ◽  
Specific Activity ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Regulatory Sequence ◽  
Open Chromatin ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Enhancer Element

ABSTRACT In this report, we explored the mechanisms underlying keratinocyte-specific and differentiation-specific gene expression in the skin. We have identified five keratinocyte-specific, open chromatin regions that exist within the 6 kb of 5′ upstream regulatory sequence known to faithfully recapitulate the strong endogenous keratin 5 (K5) promoter and/or enhancer activity. One of these, DNase I-hypersensitive site (HSs) 4, was unique in that it acted independently to drive abundant and keratinocyte-specific reporter gene activity in culture and in transgenic mice, despite the fact that it was not essential for K5 enhancer activity. We have identified evolutionarily conserved regulatory elements and a number of their associated proteins that bind to this compact and complex enhancer element. The 125-bp 3′ half of this element (referred to as 4.2) is by far the smallest known strong enhancer element possessing keratinocyte-specific activity in vivo. Interestingly, its activity is restricted to a subset of progeny of K5-expressing cells located within the sebaceous gland. The other half of HSs 4 (termed 4.1) possesses activity to suppress sebocyte-specific expression and induce expression in the channel (inner root sheath) cells surrounding the hair shaft. Our findings lead us to a view of keratinocyte gene expression which is determined by multiple regulatory modules, many of which contain AP-2 and/or Sp1/Sp3 binding sites for enhancing expression in skin epithelium, but which also harbor one or more unique sites for the binding of factors which determine specificity. Through mixing and matching of these modules, additional levels of specificity are obtained, indicating that both transcriptional repressors and activators govern the specificity.

scholarly journals Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

2021 ◽  
Vol 49 (7) ◽  
pp. 3856-3875
Author(s):  
Marina Kulik ◽  
Melissa Bothe ◽  
Gözde Kibar ◽  
Alisa Fuchs ◽  
Stefanie Schöne ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factor ◽  
Dna Binding ◽  
Receptor Binding ◽  
Binding Sites ◽  
Specific Gene ◽  
Functional Diversification ◽  
Enhancer Activity ◽  
Sequence Composition

Abstract The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

A Variant Noncoding Region Regulates Prrx1 and Predisposes to Atrial Arrhythmias

2021 ◽  
Author(s):  
Fernanda M Bosada ◽  
Mathilde R Rivaud ◽  
Jae-Sun Uhm ◽  
Sander Verheule ◽  
Karel van Duijvenboden ◽  
...  
Keyword(s):  
Sodium Current ◽  
Association Studies ◽  
Noncoding Region ◽  
Specific Gene ◽  
Genome Wide Association Studies ◽  
Enhancer Activity ◽  
Atrial Cardiomyocytes ◽  
The Impact ◽  
Lineage Specific Gene

Rationale: Atrial Fibrillation (AF) is the most common cardiac arrhythmia diagnosed in clinical practice. Genome-wide association studies have identified AF-associated common variants across 100+ genomic loci, but the mechanism underlying the impact of these variant loci on AF susceptibility in vivo has remained largely undefined. One such variant region, highly associated with AF, is found at 1q24, close to PRRX1, encoding the Paired Related Homeobox 1 transcription factor. Objective: To identify the mechanistic link between the variant region at 1q24 and AF predisposition. Methods and Results: The mouse orthologue of the noncoding variant genomic region (R1A) at 1q24 was deleted using CRISPR genome editing. Among the genes sharing the topologically associated domain with the deleted R1A region (Kifap3, Prrx1, Fmo2, Prrc2c), only the broadly expressed gene Prrx1 was downregulated in mutants, and only in cardiomyocytes. Expression and epigenetic profiling revealed that a cardiomyocyte lineage-specific gene program (Mhrt, Myh6, Rbm20, Tnnt2, Ttn, Ckm) was upregulated in R1A-/- atrial cardiomyocytes, and that Mef2 binding motifs were significantly enriched at differentially accessible chromatin sites. Consistently, Prrx1 suppressed Mef2-activated enhancer activity in HL-1 cells. Mice heterozygous or homozygous for the R1A deletion were susceptible to atrial arrhythmia induction, had atrial conduction slowing and more irregular RR intervals. Isolated R1A-/- mouse left atrial cardiomyocytes showed lower action potential upstroke velocities and sodium current, as well as increased systolic and diastolic calcium concentrations compared to controls. Conclusions: The noncoding AF variant region at 1q24 modulates Prrx1 expression in cardiomyocytes. Cardiomyocyte-specific reduction of Prrx1 expression upon deletion of the noncoding region leads to a profound induction of a cardiac lineage-specific gene program and to propensity for AF. These data indicate that AF-associated variants in humans may exert AF predisposition through reduced PRRX1 expression in cardiomyocytes.

scholarly journals Genetic influences on cell type specific gene expression and splicing during neurogenesis elucidate regulatory mechanisms of brain traits

2020 ◽  
Author(s):  
Nil Aygün ◽  
Angela L. Elwell ◽  
Dan Liang ◽  
Michael J. Lafferty ◽  
Kerry E. Cheek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Elements ◽  
Regulatory Mechanisms ◽  
Specific Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Risk Variants ◽  
Allele Specific ◽  
Cell Type Specific ◽  
Regulatory Effects

SummaryInterpretation of the function of non-coding risk loci for neuropsychiatric disorders and brain-relevant traits via gene expression and alternative splicing is mainly performed in bulk post-mortem adult tissue. However, genetic risk loci are enriched in regulatory elements of cells present during neocortical differentiation, and regulatory effects of risk variants may be masked by heterogeneity in bulk tissue. Here, we map e/sQTLs and allele specific expression in primary human neural progenitors (n=85) and their sorted neuronal progeny (n=74). Using colocalization and TWAS, we uncover cell-type specific regulatory mechanisms underlying risk for these traits.

Protein signaling and regulation of gene transcription in leukemia: role of the Casein Kinase II-Ikaros axis

2016 ◽  
Vol 64 (3) ◽  
pp. 735-739 ◽  
Author(s):  
Chandrika S Gowda ◽  
Chunhua Song ◽  
Yali Ding ◽  
Malika Kapadia ◽  
Sinisa Dovat
Keyword(s):  
Gene Transcription ◽  
Target Genes ◽  
Cellular Proliferation ◽  
Lymphoblastic Leukemia ◽  
Regulation Of Gene Expression ◽  
Regulatory Elements ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Protein Signaling

Protein signaling and regulation of gene expression are the two major mechanisms that regulate cellular proliferation in leukemia. Discerning the function of these processes is essential for understanding the pathogenesis of leukemia and for developing the targeted therapies. Here, we provide an overview of one of the mechanisms that regulates gene transcription in leukemia. This mechanism involves the direct interaction between Casein Kinase II (CK2) and the Ikaros transcription factor. Ikaros (IKZF1) functions as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Impaired Ikaros function results in the development of high-risk leukemia. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. In vivo, Ikaros is a target for CK2, a pro-oncogenic kinase. CK2 directly phosphorylates Ikaros at multiple amino acids. Functional experiments showed that CK2-mediated phosphorylation of Ikaros, regulates Ikaros’ DNA binding affinity, subcellular localization and protein stability. Recent studies revealed that phosphorylation of Ikaros by CK2 regulates Ikaros binding and repression of the terminal deoxytransferase (TdT) gene in normal thymocytes and in T-cell ALL. Available data suggest that the oncogenic activity of CK2 in leukemia involves functional inactivation of Ikaros and provide a rationale for CK2 inhibitors as a potential treatment for ALL.

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