Investigating crosstalk between H3K27 acetylation and H3K4 trimethylation in CRISPR/dCas-based epigenome editing and gene activation

AbstractEpigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions. While histone PTMs have been correlated with certain gene expression status, the causalities remain elusive. Histone H3 Lysine 27 acetylation (H3K27ac) and histone H3 Lysine 4 trimethylation (H3K4me3) are both associated with active genes, and located at active promoters and enhancers or around transcriptional start sites (TSSs). Although crosstalk between histone lysine acetylation and H3K4me3 has been reported, relationships between specific epigenetic marks during transcriptional activation remain largely unclear. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-based epigenome editing methods, we discovered that the ectopic introduction of H3K27ac in the promoter region lead to H3K4me3 enrichment around TSS and transcriptional activation, while H3K4me3 installation at the promoter cannot induce H3K27ac increase and failed to activate gene expression. Blocking the reading of H3K27ac by BRD proteins using inhibitor JQ1 abolished H3K27ac-induced H3K4me3 installation and downstream gene activation. Furthermore, we uncovered that BRD2, not BRD4, mediated H3K4me3 installation and gene activation upon H3K27ac writing. Our studies revealed the relationships between H3K27ac and H3K4me3 in gene activation process and demonstrated the application of CRISPR/dCas-based epigenome editing methods in elucidating the crosstalk between epigenetic mechanisms.

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Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription

eLife ◽

10.7554/elife.01632 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 54

Author(s):

Vincenzo Di Cerbo ◽

Fabio Mohn ◽

Daniel P Ryan ◽

Emilie Montellier ◽

Salim Kacem ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Lateral Surface ◽

Histone H3 ◽

Activation Function ◽

Major Mechanism ◽

Nucleosome Core ◽

Nucleosome Dynamics ◽

Transcriptional Start Sites

Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states.

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HOS15 is a transcriptional corepressor of NPR1-mediated gene activation of plant immunity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016049117 ◽

2020 ◽

Vol 117 (48) ◽

pp. 30805-30815

Author(s):

Mingzhe Shen ◽

Chae Jin Lim ◽

Junghoon Park ◽

Jeong Eun Kim ◽

Dongwon Baek ◽

...

Keyword(s):

Gene Expression ◽

Ubiquitin Ligase ◽

Transcriptional Activation ◽

Plant Immunity ◽

Gene Activation ◽

Transcriptional Corepressor ◽

Defense Gene Expression ◽

Dynamic Regulation ◽

Defense Gene ◽

Transcriptional Corepressors

Transcriptional regulation is a complex and pivotal process in living cells. HOS15 is a transcriptional corepressor. Although transcriptional repressors generally have been associated with inactive genes, increasing evidence indicates that, through poorly understood mechanisms, transcriptional corepressors also associate with actively transcribed genes. Here, we show that HOS15 is the substrate receptor for an SCF/CUL1 E3 ubiquitin ligase complex (SCFHOS15) that negatively regulates plant immunity by destabilizing transcriptional activation complexes containing NPR1 and associated transcriptional activators. In unchallenged conditions, HOS15 continuously eliminates NPR1 to prevent inappropriate defense gene expression. Upon defense activation, HOS15 preferentially associates with phosphorylated NPR1 to stimulate rapid degradation of transcriptionally active NPR1 and thus limit the extent of defense gene expression. Our findings indicate that HOS15-mediated ubiquitination and elimination of NPR1 produce effects contrary to those of CUL3-containing ubiquitin ligase that coactivate defense gene expression. Thus, HOS15 plays a key role in the dynamic regulation of pre- and postactivation host defense.

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Gene activation precedes DNA demethylation in response to infection in human dendritic cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814700116 ◽

2019 ◽

Vol 116 (14) ◽

pp. 6938-6943 ◽

Cited By ~ 39

Author(s):

Alain Pacis ◽

Florence Mailhot-Léonard ◽

Ludovic Tailleux ◽

Haley E. Randolph ◽

Vania Yotova ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dendritic Cells ◽

Transcriptional Activation ◽

Time Course ◽

Gene Activation ◽

Dna Demethylation ◽

Epigenetic Mark ◽

Distal Enhancer ◽

Human Dendritic Cells

DNA methylation is considered to be a relatively stable epigenetic mark. However, a growing body of evidence indicates that DNA methylation levels can change rapidly; for example, in innate immune cells facing an infectious agent. Nevertheless, the causal relationship between changes in DNA methylation and gene expression during infection remains to be elucidated. Here, we generated time-course data on DNA methylation, gene expression, and chromatin accessibility patterns during infection of human dendritic cells withMycobacterium tuberculosis. We found that the immune response to infection is accompanied by active demethylation of thousands of CpG sites overlapping distal enhancer elements. However, virtually all changes in gene expression in response to infection occur before detectable changes in DNA methylation, indicating that the observed losses in methylation are a downstream consequence of transcriptional activation. Footprinting analysis revealed that immune-related transcription factors (TFs), such as NF-κB/Rel, are recruited to enhancer elements before the observed losses in methylation, suggesting that DNA demethylation is mediated by TF binding to cis-acting elements. Collectively, our results show that DNA demethylation plays a limited role to the establishment of the core regulatory program engaged upon infection.

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Histone H3 Lysine 9 Methylation and HP1γ Are Associated with Transcription Elongation through Mammalian Chromatin.

Blood ◽

10.1182/blood.v106.11.1734.1734 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1734-1734 ◽

Cited By ~ 1

Author(s):

Christopher R. Vakoc ◽

Sean A. Mandat ◽

Ben A. Olenschock ◽

Gerd A. Blobel

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Histone Modifications ◽

Transcription Elongation ◽

Gene Activation ◽

Histone H3 ◽

H3k9 Methylation ◽

Erythroid Cells ◽

Cellular Memory ◽

Active Genes

Abstract Epigenetic regulation of gene expression plays a fundamental role during tissue specification and cellular memory. Cells that are committed to a given lineage, for example during hematopoiesis, “remember” their phenotype throughout successive rounds of cell division, reflecting alterations in chromatin structure at genes that are permanently activated or silenced. Cellular memory is anchored in specific sets of histone modifications, which together form the basis for the histone code. This is illustrated in the methylation of histone molecules: while methylation of histone H3 on lysines 4, 36, and 79 is linked with gene activation, methylation of H3 on lysines 9 and 27 and histone H4 on lysine 20 is associated with transcriptionally silent heterochromatin and repressed genes within euchromatin. Not surprisingly, dysregulation of histone methylation contributes to human diseases such as leukemias. Here we examined the methylation of histone molecules during gene activation and repression triggered by the hematopoietic transcription factor GATA-1. Surprisingly, we found that during activation by GATA-1 in erythroid cells, the levels of H3K9 di- and tri-methylation increase dramatically at all examined GATA-1-stimulated genes, including alpha- and beta-globin, AHSP, Band 3 and Glycophorin A. In contrast, at all GATA-1-repressed genes examined (GATA-2, c-kit, and c-myc) these marks are rapidly lost. Peaks of H3K9 methylation were observed in the transcribed portion of genes with lower signals at the promoter regions. Heterochromatin Protein 1γ (HP1γ), a protein containing a chromo-domain that recognizes H3K9 methylation, is also present in the transcribed region of all active genes examined. We extended these analyses to include numerous genes in diverse cell types (primary erythroid cells, primary T-lymphoid cells, epithelial cells and fibroblast) and consistently found a tight correlation between H3K9 methylation and gene activity, highlighting the general nature of our findings. Both the presence of HP1γ and H3K9 methylation at active genes are dependent upon transcription elongation by RNA polymerase II. Finally, HP1γ is in a physical complex with the elongating form of RNA polymerase II. Together, our results show that H3K9 methylation and HP1γ not only function in repressive chromatin, but play a novel and unexpected role during transcription activation. These results further elucidate new combinations of histone modifications that distinguish between repressed and actively transcribing chromatin.

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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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Drosophila Ada2b Is Required for Viability and Normal Histone H3 Acetylation

Molecular and Cellular Biology ◽

10.1128/mcb.24.18.8080-8089.2004 ◽

2004 ◽

Vol 24 (18) ◽

pp. 8080-8089 ◽

Cited By ~ 37

Author(s):

Dai Qi ◽

Jan Larsson ◽

Mattias Mannervik

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Protein Complexes ◽

P53 Protein ◽

Polytene Chromosomes ◽

Histone H3 ◽

Saga Complex ◽

Histone H3 Acetylation ◽

H3 Acetylation

ABSTRACT Regulation of chromatin through histone acetylation is an important step in gene expression. The Gcn5 histone acetyltransferase is part of protein complexes, e.g., the SAGA complex, that interact with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery. The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity. Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b. We have generated flies that lack dAda2b, which is part of a Drosophila SAGA-like complex. dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation. A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants. This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation. Although the Drosophila p53 protein was previously shown to interact with the SAGA-like complex in vitro, we find that p53 induction of reaper gene expression occurs normally in dAda2b mutants. Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation. Based on this result, we speculate that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal. This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent.

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Phosphorylated forms of GAL4 are correlated with ability to activate transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4623 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4623-4629 ◽

Cited By ~ 31

Author(s):

L M Mylin ◽

M Johnston ◽

J E Hopper

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Polyacrylamide Gel Electrophoresis ◽

Gene Activation ◽

Sodium Dodecyl ◽

Immunoblot Analysis ◽

Transcriptional Activator Protein ◽

Mel Gene ◽

High Level

GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L. Mylin, P. Bhat, and J. Hopper, Genes Dev. 3:1157-1165, 1989). Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation. We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II. To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M. Johnston and J. Dover, Proc. Natl. Acad. Sci. USA 84:2401-2405, 1987; Genetics 120;63-74, 1988). The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species. Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II. Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.

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Regulation of macrophage cyclooxygenase-2 gene expression by modifications of histone H3

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00338.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. L956-L962 ◽

Cited By ~ 32

Author(s):

Gye Young Park ◽

Myungsoo Joo ◽

Tetyana Pedchenko ◽

Timothy S. Blackwell ◽

John W. Christman

Keyword(s):

Gene Expression ◽

Chromatin Structure ◽

Gene Activation ◽

Histone H3 ◽

Major Effect ◽

Cyclooxygenase 2 ◽

Raw 264.7 Cells ◽

Transcriptional Level ◽

Sequence Motif ◽

Cox 2

Some transcription factors involved in the regulation of cyclooxygenase 2 (COX-2) expression in macrophage, including NF-κB, interact with p300, which contains histone acetyltransferase (HAT) enzyme complex. Chromatin structure is regulated by modifying enzymes, including HAT, and plays an important role in eukaryotic gene regulation through histone modification. We hypothesized that changes in chromatin structure related to phosphorylation and acetylation of histone H3 adjacent to key DNA binding sequence motif in the COX-2 promoter contribute to COX-2 gene activation in macrophages. Sodium butyrate (NaBT) is a short-chain fatty acid that possesses histone deacetyltransferase-inhibiting activity. Our data show that NaBT accentuates LPS-induced COX-2 gene expression at a transcriptional level, even though NaBT alone does not induce the COX-2 gene expression. Using a chromatin immunoprecipitation assay, we showed that costimulation of RAW 264.7 cells with NaBT and LPS synergistically increases COX-2 gene expression through both acetylation and phosphorylation of histone H3 at the promoter site. Our data show that NaBT accentuates LPS-induced COX-2 gene expression through MAP kinase-dependent increase of phosphorylation and acetylation of histone H3 at the COX-2 promoter site. These data indicate that posttranslational modification of histone H3 has a major effect on COX-2 gene expression by macrophages.

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PfGCN5-Mediated Histone H3 Acetylation Plays a Key Role in Gene Expression in Plasmodium falciparum

Eukaryotic Cell ◽

10.1128/ec.00062-07 ◽

2007 ◽

Vol 6 (7) ◽

pp. 1219-1227 ◽

Cited By ~ 80

Author(s):

Long Cui ◽

Jun Miao ◽

Tetsuya Furuya ◽

Xinyi Li ◽

Xin-zhuan Su ◽

...

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Histone Acetylation ◽

Transcriptional Activation ◽

Histone H3 ◽

Epigenetic Mechanism ◽

Promoter Regions ◽

Specific Expression ◽

Transcriptional Initiation ◽

Translational Start

ABSTRACT Histone acetylation, regulated by the opposing actions of histone acetyltransferases (HATs) and deacetylases, is an important epigenetic mechanism in eukaryotic transcription. Although an acetyltransferase (PfGCN5) has been shown to preferentially acetylate histone H3 at K9 and K14 in Plasmodium falciparum, the scale of histone acetylation in the parasite genome and its role in transcriptional activation are essentially unknown. Using chromatin immunoprecipitation (ChIP) and DNA microarray, we mapped the global distribution of PfGCN5, histone H3K9 acetylation (H3K9ac) and trimethylation (H3K9m3) in the P. falciparum genome. While the chromosomal distributions of H3K9ac and PfGCN5 were similar, they are radically different from that of H3K9m3. In addition, there was a positive, though weak correlation between relative occupancy of H3K9ac on individual genes and the levels of gene expression, which was inversely proportional to the distance of array elements from the putative translational start codons. In contrast, H3K9m3 was negatively correlated with gene expression. Furthermore, detailed mapping of H3K9ac for selected genes using ChIP and real-time PCR in three erythrocytic stages detected stage-specific peak H3K9ac enrichment at the putative transcriptional initiation sites, corresponding to stage-specific expression of these genes. These data are consistent with H3K9ac and H3K9m3 as epigenetic markers of active and silent genes, respectively. We also showed that treatment with a PfGCN5 inhibitor led to reduced promoter H3K9ac and gene expression. Collectively, these results suggest that PfGCN5 is recruited to the promoter regions of genes to mediate histone acetylation and activate gene expression in P. falciparum.

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A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.

10.1101/2021.09.07.459151 ◽

2021 ◽

Author(s):

Elena Garcia-Perez ◽

Borja Diego-Martin ◽

Alfredo Quijano-Rubio ◽

Elena Moreno Gimenez ◽

Diego Orzaez ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Nicotiana Benthamiana ◽

Gene Networks ◽

Rna Binding ◽

Gene Activation ◽

Dependent Manner ◽

Copper Binding ◽

Control Of Gene Expression ◽

Guide Rna

CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations in place, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold for the endogenous N. benthamiana DFR gene, with negligible expression in the absence of the trigger. The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

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