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Results for “Targeting chromatin regulators”

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At least 73 records · Page 4Linked to original sources
PubMed2001

Transcriptional regulation by p53 through intrinsic DNA/chromatin binding and site-directed cofactor recruitment.

The tumor suppressor protein, p53, plays a critical role in mediating cellular response to stress signals by regulating genes involved in cell cycle arrest and apoptosis. p53 is believed to be inactive for DNA binding unless its C terminus is modified or structurally altered. We show that unmodified p53 actively binds to two sites at -1.4 and -2.3 kb within the chromatin-assembled p21 promoter and requires the C terminus and the histone acetyltransferase, p300, for transcription. Acetylation of the C terminus by p300 is not necessary for binding or promoter activation. Instead, p300 acetylates p53-bound nucleosomes in the p21 promoter with spreading to the TATA box. Thus, p53 is an active DNA and chromatin binding protein that may selectively regulate its target genes by recruitment of specific cofactors to structurally distinct binding sites.

Acetylation↗
PubMed1997

The polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes.

In Drosophila the Polycomb group genes are required for the long-term maintenance of the repressed state of many developmental regulatory genes. Their gene products are thought to function in a common multimeric complex that associates with Polycomb group response elements (PREs) in target genes and regulates higher-order chromatin structure. We show that the chromodomain of Polycomb is necessary for protein-protein interactions within a Polycomb-Polyhomeotic complex. In addition, Posterior Sex Combs protein coimmunoprecipitates Polycomb and Polyhomeotic, indicating that they are members of a common multimeric protein complex. Immunoprecipitation experiments using in vivo cross-linked chromatin indicate that these three Polycomb group proteins are associated with identical regulatory elements of the selector gene engrailed in tissue culture cells. Polycomb, Polyhomeotic, and Posterior Sex Combs are, however, differentially distributed on regulatory sequences of the engrailed-related gene invected. This suggests that there may be multiple different Polycomb group protein complexes which function at different target sites. Furthermore, Polyhomeotic and Posterior Sex Combs are also associated with expressed genes. Polyhomeotic and Posterior Sex Combs may participate in a more general transcriptional mechanism that causes modulated gene repression, whereas the inclusion of Polycomb protein in the complex at PREs leads to stable silencing.

Animals↗
PubMed2025

PATTY corrects open chromatin bias for improved bulk and single-cell CUT&Tag profiling.

Precise profiling of epigenomes is essential for better understanding chromatin biology and gene regulation. Cleavage Under Targets & Tagmentation (CUT&Tag) is an efficient epigenomic profiling technique that can be performed on a low number of cells and at the single-cell level. With its growing adoption, CUT&Tag datasets spanning diverse biological systems are rapidly accumulating in the field. CUT&Tag assays use the hyperactive transposase Tn5 for DNA tagmentation. Tn5's preference toward accessible chromatin alters CUT&Tag sequence read distributions in the genome and introduces open chromatin bias that can confound downstream analysis, an issue more substantial in sparse single-cell data. We show that open chromatin bias extensively exists in published CUT&Tag datasets, including those generated with recently optimized high-salt protocols. To address this challenge, we present PATTY (Propensity Analyzer for Tn5 Transposase Yielded bias), a comprehensive computational method that corrects open chromatin bias in CUT&Tag data by leveraging accompanying ATAC-seq. By integrating transcriptomic and epigenomic data using machine learning and integrative modeling, we demonstrate that PATTY enables accurate and robust detection of occupancy sites for both active and repressive histone modifications, including H3K27ac, H3K27me3, and H3K9me3, with experimental validation. We further develop a single-cell CUT&Tag analysis framework built on PATTY and show improved cell clustering when using bias-corrected single-cell CUT&Tag data compared to using uncorrected data. Beyond CUT&Tag, PATTY sets a foundation for further development of bias correction methods for improving data analysis for all Tn5-based high-throughput assays.

Journal Article↗
PubMed2025

PATTY corrects open-chromatin bias for improved bulk and single-cell CUT&Tag profiling.

Precise profiling of epigenomes is essential for better understanding chromatin biology and gene regulation. Cleavage Under Targets & Tagmentation (CUT&Tag) is an efficient epigenomic profiling technique that can be performed on a low number of cells and at the single-cell level. With its growing adoption, CUT&Tag datasets spanning diverse biological systems are rapidly accumulating in the field. CUT&Tag assays use the hyperactive transposase Tn5 for DNA tagmentation. Tn5's preference toward accessible chromatin alters CUT&Tag sequence read distributions in the genome and introduces open-chromatin bias that can confound downstream analysis, an issue more substantial in sparse single-cell data. We show that open-chromatin bias extensively exists in published CUT&Tag datasets, including those generated with recently optimized high-salt protocols. To address this challenge, we present PATTY (Propensity Analyzer for Tn5 Transposase Yielded bias), a comprehensive computational method that corrects open-chromatin bias in CUT&Tag data by leveraging accompanying ATAC-seq. By integrating transcriptomic and epigenomic data using machine learning and integrative modeling, we demonstrate that PATTY enables accurate and robust detection of occupancy sites for both active and repressive histone modifications, including H3K27ac, H3K27me3, and H3K9me3, with experimental validation. We further develop a single-cell CUT&Tag analysis framework built on PATTY and show improved cell clustering when using bias-corrected single-cell CUT&Tag data compared to using uncorrected data. Beyond CUT&Tag, PATTY sets a foundation for further development of bias correction methods for improving data analysis for all Tn5-based high-throughput assays.

Journal Article↗
PubMed2002

Interferon-gamma-induced chromatin remodeling at the CIITA locus is BRG1 dependent.

SWI/SNF regulates growth control, differentiation and tumor suppression, yet few direct targets of this chromatin-remodeling complex have been identified in mammalian cells. We report that SWI/SNF is required for interferon (IFN)-gamma induction of CIITA, the master regulator of major histocompatibility complex class II expression. Despite the presence of functional STAT1, IRF-1 and USF-1, activators implicated in CIITA expression, IFN-gamma did not induce CIITA in cells lacking BRG1 and hBRM, the ATPase subunits of SWI/SNF. Reconstitution with BRG1, but not an ATPase-deficient version of this protein (K798R), rescued CIITA induction, and enhanced the rate of induction of the IFN-gamma-responsive GBP-1 gene. Not ably, BRG1 inhibited the CIITA promoter in transient transfection assays, underscoring the importance of an appropriate chromosomal environment. Chromatin immunoprecipitation revealed that BRG1 interacts directly with the endogenous CIITA promoter in an IFN-gamma-inducible fashion, while in vivo DNase I footprinting and restriction enzyme accessibility assays showed that chromatin remodeling at this locus requires functional BRG1. These data provide the first link between a cytokine pathway and SWI/SNF, and suggest a novel role for this chromatin-remodeling complex in immune surveillance.

Cell Line, Transformed↗
PubMed2005

Dynamic chromatin: concerted nucleosome remodelling and acetylation.

The flexibility of chromatin that enables translation of environmental cues into changes in genome utilisation, relies on a battery of enzymes able to modulate chromatin structure in a highly targeted and regulated manner. The most dynamic structural changes are brought about by two kinds of enzymes with different functional principles. Changes in the acetylation status of histones modulate the folding of the nucleosomal fibre. The histone-DNA interactions that define the nucleosome itself can be disrupted by ATP-dependent remodelling factors. This review focuses on recent developments that illustrate various strategies for integrating these disparate activities into complex regulatory schemes. Synergies may be brought about by consecutive or parallel action during the stepwise process of chromatin opening or closing. Tight co-ordination may be achieved by direct interaction of (de-)acetylation enzymes and remodelling ATPases or even permanent residence within the same multi-enzyme complex. The fact that remodelling ATPases can be acetylated by histone acetyltransferases themselves suggests exciting possibilities for the co-ordinate modulation of chromatin structure and remodelling enzymes.

Acetylation↗
PubMed1995

Characterization of deoxyribonucleic acid-protein interactions at a growth hormone-inducible nuclease hypersensitive site in the rat insulin-like growth factor-I gene.

Many of the growth-promoting effects of GH are mediated by insulin-like growth factor-I (IGF-I), a highly conserved, 70-residue basic peptide. Previous studies have demonstrated that GH rapidly stimulates IGF-I expression in vivo, and our laboratory has identified a GH-regulated alteration in chromatin configuration, manifested as a hormonally induced deoxyribonuclease-I (DNase-I)-hypersensitive site in the second IGF-I intron. In the current study, we have used in vivo DNase-I footprinting to map this hormonally responsive chromatin domain to an approximately 350-nucleotide region and have identified DNA-protein interactions within the hypersensitive site by in vitro gel mobility shift experiments and DNase-I footprinting studies. DNA-protein binding was localized to two adjacent segments of 32 and 48 nucleotides. In 1 of these regions, protein-DNA contacts were also detected in vivo on guanine residues by dimethylsulfate footprinting. DNA-binding activity was present in GH-deficient rats, but was not modified by hormone treatment. Our results define a rapid and reversible genomic alteration in response to GH in a GH-regulated gene and delineate a target within chromatin for GH action.

Animals↗
PubMed2003

In vivo footprinting and DNA affinity chromatography for analysis of p53 DNA binding ability.

p53 is a sequence-specific DNA binding protein. The p53 consensus is two copies of 5'- RRRC(A/T)(T/A)GYYY-3'. The interaction of p53 with specific DNA binding sites (DBS) has been analyzed extensively using electrophoretic mobility shift analysis (EMSA). These studies do not address the interaction of p53 with nuclear chromatin or the stability of p53-DBS interactions. In vivo footprinting examines the dynamic interactions of p53 protein in nuclear chromatin. p53 DBS affinity chromatography compares the stability of p53 from different cellular extracts with different DBS. Isogenic strains expressing high p53 levels, and deleted for p53, are required for controlled experiments using both methods. Different systems can be used to generate sufficient p53 protein (including DNA damage), and this results in the analysis of different forms of p53. A comparison of different cellular sources of high levels of p53 (in the presence and absence of DNA damage) vs different p53 DBS is required to appreciate the complexity of the regulation. Methods for comparing p53 from three different cellular sources with different DBS are presented here. The p53 research community needs to expand this analysis to complete the picture of how p53 differentially regulates transcription of target genes in nuclear chromatin.

Animals↗
PubMed2001

Characterization of nucleophosmin (B23) as a Myc target by scanning chromatin immunoprecipitation.

The genetic program through which a specific transcription factor regulates a biological response is fundamental to our understanding how instructions in the genome are implemented. The emergence of DNA microarray technology for gene expression analysis has generated vast numbers of target genes resulting from specific transcription factor activity. We use the oncogenic transcription factor c-Myc as proof-of-principle that human genome sequence analysis and scanning of a specific gene by chromatin immunoprecipitation can be coupled to identify target transcription factor binding sequences. We focused on nucleophosmin, also known as B23, which was identified as a candidate Myc-responsive gene from a subtractive hybridization screen, and we found that sequences in intron 1, and not 5' sequences in the proximal promoter, are bound by c-Myc in vivo. Hence, a scanning chromatin immunoprecipitation (SChIP) strategy is useful in analyzing functional transcription factor-binding sites.

3T3 Cells↗
PubMed2017

Rapid and reversible epigenome editing by endogenous chromatin regulators.

Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.

CRISPR-Cas Systems↗
PubMed2001

Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A.

We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.

Cell Line, Transformed↗
PubMed2004

Identifying estrogen receptor alpha target genes using integrated computational genomics and chromatin immunoprecipitation microarray.

The estrogen receptor alpha (ERalpha) regulates gene expression by either direct binding to estrogen response elements or indirect tethering to other transcription factors on promoter targets. To identify these promoter sequences, we conducted a genome-wide screening with a novel microarray technique called ChIP-on-chip. A set of 70 candidate ERalpha loci were identified and the corresponding promoter sequences were analyzed by statistical pattern recognition and comparative genomics approaches. We found mouse counterparts for 63 of these loci and classified 42 (67%) as direct ERalpha targets using classification and regression tree (CART) statistical model, which involves position weight matrix and human-mouse sequence similarity scores as model parameters. The remaining genes were considered to be indirect targets. To validate this computational prediction, we conducted an additional ChIP-on-chip assay that identified acetylated chromatin components in active ERalpha promoters. Of the 27 loci upregulated in an ERalpha-positive breast cancer cell line, 20 having mouse counterparts were correctly predicted by CART. This integrated approach, therefore, sets a paradigm in which the iterative process of model refinement and experimental verification will continue until an accurate prediction of promoter target sequences is derived.

Animals↗
PubMed1998

Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling.

Lymphocyte activation is accompanied by visible changes in chromatin structure. We find that antigen receptor signaling induces the rapid association of the BAF complex with chromatin. PIP2, which is regulated by activation stimuli, is sufficient in vitro to target the BAF complex to chromatin, but it has no effect on related chromatin remodeling complexes containing SNF2L or hISWI. Purification and peptide sequencing of the subunits of the complex revealed beta-actin as well as a novel actin-related protein, BAF53. beta-actin and BAF53 are required for maximal ATPase activity of BRG1 and are also required with BRG1 for association of the complex with chromatin/matrix. This work indicates that membrane signals control the activity of the mammalian SWI/SNF or BAF complex and demonstrates a direct interface between signaling and chromatin regulation.

Actins↗
PubMed2026

The Jumonji C domain-containing proteins GmJMJ19 and GmJMJ20 link florigen signaling with epigenetic regulation of photoperiodic flowering and post-flowering plant height in soybean.

Soybean (Glycine max) is a photoperiod-sensitive legume whose latitudinal adaptation depends on the precise control of flowering time and plant height. Histone demethylases of the JmjC domain-containing (JMJ) protein family have been implicated in these processes across plant species, but their specific roles in soybean remain largely unexplored. Here, we identify soybean GmJMJ19 and GmJMJ20, two closely related JMJD5/KDM8 orthologs, as master epigenetic regulators that coordinately control both photoperiodic flowering and post-flowering plant height. Both genes exhibit intrinsic, rhythmic expression peaking at ZT12, and their encoded proteins physically interact with the florigen proteins FT2a and FT5a. Loss-of-function mutants display delayed flowering under long days (LDs) and increased plant height under both LDs and short days (SDs), whereas overexpression phenocopies the mutant flowering phenotype, indicating revealing a critical dosage requirement for proper function. Mechanistically, GmJMJ19 and GmJMJ20 are recruited by the FT/FD transcriptional complex to directly activate AP1a and AP1c expression through chromatin modulation. Population genomic analyses reveal distinct selection signatures: GmJMJ19 underwent sustained directional selection during cultivation, whereas GmJMJ20 experienced an early domestication sweep with limited subsequent change. Haplotype analysis identifies coordinated latitudinal clines, with the JMJ19H1/JMJ20H1 combination predominating at high latitudes to promote early flowering and limit height, while JMJ19H2/JMJ20H2 and wild JMJ19H3/JMJ20H3 alleles prevail at low latitudes, conferring later flowering and increased height. Collectively, our findings establish GmJMJ19 and GmJMJ20 as central chromatin regulators linking florigen signaling to downstream target expression and provide valuable allelic resources for breeding regionally adapted soybean varieties across a wide range of latitudinal environments.

Histone modulation↗
PubMed2006

Evidence for activator and repressor functions of the response regulator MtrA from Corynebacterium glutamicum.

Previous analysis of a Corynebacterium glutamicum Delta mtrAB mutant showed that the MtrAB two-component signal transduction system influences the expression of genes involved in cell wall metabolism or osmoregulation, but it remained unknown whether this influence is direct or indirect. In order to identify the direct target genes of the response regulator MtrA, chromatin immunoprecipitation as a genome-wide approach and DNA affinity chromatography as a gene-specific approach were used. The results indicate that mepA and nlpC, both encoding putative cell wall peptidases, are directly repressed by MtrA, whereas proP and betP, both encoding carriers for compatible solutes, are directly activated by MtrA.

ATP-Binding Cassette Transporters↗
PubMed2006

Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.

ATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease associated with defects in such proteins. To better understand the role of ATRX in development and the associated abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked) syndrome, we conditionally inactivated the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues. Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the murine trophoblast and present an example of escape from imprinted X chromosome inactivation.

Alleles↗
PubMed2000

Direct examination of histone acetylation on Myc target genes using chromatin immunoprecipitation.

Overexpression of c-Myc can lead to altered transcriptional regulation of cellular genes and to neoplastic transformation. Although DNA binding is clearly required, the mechanism by which recruitment of c-Myc to target promoters results in transcriptional activation is highly debated. Much of this controversy comes from the difficulty in clearly defining a true Myc target gene. We have previously determined that cad is a bona fide Myc target gene and thus now use the cad promoter as a model to study Myc function. Others have shown that Myc can interact indirectly with histone acetylases and have suggested that Myc mediates transcriptional activation by causing an increase in the levels of acetylated histones on target promoters. To directly test this model, we employed a chromatin immunoprecipitation assay to examine the levels of acetylated histones on the cad promoter. Although Myc was bound to the cad promoter in S phase but not in G(0) phase, we found high levels of acetylated histones on the promoter in both stages. We also examined acetylated histones on the cad promoter before and after differentiation of U937 cells. Although the levels of c-Myc bound to the cad promoter were greatly reduced after differentiation, we saw high levels of acetylated histones on the cad promoter both before and after differentiation. Finally, we found that a 30-fold change in binding of N-Myc to the telomerase promoter did not result in a concomitant change in histone acetylation. Thus, recruitment of a Myc family member to a target promoter does not necessarily influence the amount of acetylated histones at that promoter. Further investigations are in progress to define the role of Myc in transcriptional activation.

3T3 Cells↗