scholarly journals H2A.Z is dispensable for both basal and activated transcription in post-mitotic mouse muscles

2019 ◽  
Author(s):  
Edwige Belotti ◽  
Nicolas Lacoste ◽  
Thomas Simonet ◽  
Christophe Papin ◽  
Kiran Padmanabhan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Start Site ◽  
Transcriptional Activity ◽  
Muscle Cells ◽  
Histone Variant ◽  
Start Site ◽  
Rna Seq ◽  
Transcription Start ◽  
Dividing Cells

ABSTRACTThe histone variant H2A.Z is enriched in nucleosomes surrounding the transcription start site of active promoters, suggesting that it might be implicated in transcription. It is also required during mitosis. However, evidences obtained so far mainly rely on correlative evidences obtained in actively dividing cells. We have defined a paradigm in which cell cycle cannot interfere with H2A.Z transcriptional studies by developing an in vivo systems to invalidate H2A.Z in terminally differentiated post-mitotic muscle cells to dissociate its role during transcription from its role during mitosis. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is rather a marker than an actor of transcriptional activity.

scholarly journals H2A.Z is dispensable for both basal and activated transcription in post-mitotic mouse muscles

2020 ◽  
Vol 48 (9) ◽  
pp. 4601-4613 ◽  
Author(s):  
Edwige Belotti ◽  
Nicolas Lacoste ◽  
Thomas Simonet ◽  
Christophe Papin ◽  
Kiran Padmanabhan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Start Site ◽  
Muscle Cells ◽  
Histone Variant ◽  
Start Site ◽  
Rna Seq ◽  
Transcription Start ◽  
Dividing Cells ◽  
Correlational Data

Abstract While the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

scholarly journals Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 5993-6004 ◽  
Author(s):  
Anne M. L. Barnard ◽  
Jeffrey Green ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Binding Site ◽  
Transcription Regulation ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Start Site ◽  
Transcription Start ◽  
Substitution Mutant

ABSTRACT FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

scholarly journals Role of the Transcription Start Site Core Region and Transcription Factor YY1 in Rous Sarcoma Virus Long Terminal Repeat Promoter Activity

1998 ◽  
Vol 72 (8) ◽  
pp. 6592-6601 ◽  
Author(s):  
Constance M. Mobley ◽  
Linda Sealy
Keyword(s):  
Transcription Factor ◽  
Transcription Start Site ◽  
Rous Sarcoma Virus ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Start Site ◽  
Transcription Start ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Transcription Factor Yy1

ABSTRACT The Rous sarcoma virus (RSV) long terminal repeat (LTR) contains a transcriptionally potent enhancer and promoter that functions in a variety of cell types. Previous studies have identified the viral sequences required for enhancer activity, and characterization of these elements has provided insight into the mechanism of RSV transcriptional activity. The objective of this study was to better define the RSV LTR promoter by examining the transcription start site core (TSSC) region. Deletion of the TSSC resulted in complete loss of transcriptional activity despite the presence of a functional TATA box, suggesting that the TSSC is required for viral expression. Homologies within the TSSC to the DNA binding motif of YY1 suggested that it might regulate promoter activity. YY1 has been shown to regulate transcription in some cellular genes and viral promoters by binding to sites overlapping the transcription start site. Gel shift assays using YY1 antibody identified YY1 as one of three complexes that bound to the TSSC. Mutation of the YY1 binding site reduced RSV transcriptional activity by more than 50%, suggesting that YY1, in addition to other TSSC-binding factors, regulates RSV transcription. Furthermore, in vitro transcription assays performed with Drosophila embryo extract (devoid of YY1 activity) showed decreased levels of RSV transcription, while transient transfection experiments overexpressing YY1 demonstrated that YY1 could transactivate the RSV LTR ∼6- to 7-fold. We propose that the TSSC plays a vital role in RSV transcription and that this function is partially carried out by the transcription factor YY1.

scholarly journals The histone variant macroH2A1.1 regulates gene expression by direct association with their transcription start site

2020 ◽  
Author(s):  
Ludmila Recoules ◽  
Alexandre Heurteau ◽  
Flavien Raynal ◽  
Fatima Moutahir ◽  
Fabienne Bejjani ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cancer Cells ◽  
Transcription Start Site ◽  
Breast Cancer Cells ◽  
Molecular Mechanisms ◽  
Histone Variants ◽  
Histone Variant ◽  
Start Site ◽  
Transcription Start

AbstractThe histone variant macroH2A1 (mH2A1) is involved in cellular growth, differentiation and reprogramming, but the underlying molecular mechanisms are a matter of debate. Different roles of mH2A1 in gene expression may relate to functional differences of its two splicing isoforms, mH2A1.1 and mH2A1.2. Here, we map for the first time genome-wide localization of endogenous mH2A1.1 and link the distribution of mH2A1.1 to control of gene expression in human breast cancer cells. In addition to localization shared with mH2A1.2 to facultative heterochromatin, mH2A1.1 specifically associates with regulatory elements required for gene activation, super-enhancers and promoters of highly expressed genes. Depending on the recruitment profile of mH2A1.1 to these elements, selective depletion of mH2A1.1 up- or downregulates its target genes. mH2A1.1 represses transcription when its binding is spread over the entire gene and promoter, and activates transcription when its binding is strictly confined to the transcription start site (TSS). Notably, RNA Polymerase II was frequently in pause at mH2A1.1-activated genes. Functionally, mH2A1.1-dependent regulation of a subset of paused genes impedes mammary tumor cell migration. Molecular mechanisms of mH2A1.1 function at the TSS uncovered by our study define an intriguing new mode of transcription regulation in cancer cells.Author SummaryControl of gene expression driving cellular functions from differentiation to epistasis and causing, when dysfunctional, uncountable diseases, relies on modifications of chromatin structure. One key element enabling chromatin plasticity is the replacement of canonical histones by histone variants. Among histone variants macroH2A1 (mH2A) is an extraordinary H2A variant possessing a large non-histone domain placed outside of the nucleosome. Two splicing isoforms, mH2A1.1 and mH2A1.2, are produced, but these are rarely studied separately because they only differ in a 30 amino acid region and are difficult to distinguish experimentally, which likely explains contradictory functions reported in the literature. Here, we take advantage of a mH2A1.1 specific antibody to generate the first genome-wide chromatin-associated map of this histone variant in the invasive breast cancer cells line MDA-MB231. We confirm that mH2A1.1, like mH2A1.2, is enriched at facultative heterochromatin in agreement with its reported role as a repressor. However, we discovered that unlike its splicing isoform, mH2A1.1 specifically binds to super-enhancers and the transcription start site of highly transcribed genes. mH2A1.1 is necessary for regulating transcription of these genes. At the cellular level, we demonstrate that mH2A1.1 inhibits migration capacity of highly metastatic breast cancer cells. Our study characterizes for the first time binding profiles of mH2A1.1 that are linked to regulation of gene expression, thereby providing a new molecular mechanisms which govern the plasticity of human tumor cells.

Drosophila OVO regulates ovarian tumor transcription by binding unusually near the transcription start site

Development ◽  
2001 ◽  
Vol 128 (9) ◽  
pp. 1671-1686 ◽  
Author(s):  
J. Lu ◽  
B. Oliver
Keyword(s):  
Transcription Start Site ◽  
Ovarian Tumor ◽  
Core Promoter ◽  
Regulatory Region ◽  
Start Site ◽  
Loss Of Function ◽  
Transcription Start ◽  
Germline Expression ◽  
Promoter Swapping

Evolutionarily conserved ovo loci encode developmentally regulated, sequence-specific, DNA-binding, C(2)H(2)-zinc-finger proteins required in the germline and epidermal cells of flies and mice. The direct targets of OVO activity are not known. Genetic experiments suggest that ovo acts in the same regulatory network as ovarian tumor (otu), but the relative position of these genes in the pathway is controversial. Three OVO-binding sites exist in a compact regulatory region that controls germline expression of the otu gene. Interestingly, the strongest OVO-binding site is very near the otu transcription start, where basal transcriptional complexes must function. Loss-of-function, gain-of-function and promoter swapping constructs demonstrate that OVO binding near the transcription start site is required for OVO-dependent otu transcription in vivo. These data unambiguously identify otu as a direct OVO target gene and raise the tantalizing possibility that an OVO site, at the location normally occupied by basal components, functions as part of a specialized core promoter.

Epigenetic Upregulation of the O-Linked Beta-N-Acetylglucosamine Transferase (OGT) in Response to Dronabinol Results in Antileukemic Efficacy In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2437-2437 ◽  
Author(s):  
Kerstin Maria Kampa-Schittenhelm ◽  
Hans-Joerg Buehring ◽  
Michael Bonin ◽  
Wichard Vogel ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Protein Expression ◽  
Transcription Start Site ◽  
Cannabinoid Receptors ◽  
Leukemia Cells ◽  
Start Site ◽  
Transcription Start ◽  
Potent Inducer ◽  
Induction Of Apoptosis

Abstract The O-linked β-N-acetyl glucosamine (O-GlcNAc) transferase (OGT) is a master regulator enzyme adding O-GlcNAc to serine or threonine residues in a multitude of target proteins. Aberrant O-GlcNAc modification is implicated in pathologies of metabolic and neurodegenerative diseases as well as cancers and autoimmunity. We herein provide evidence that dronabinol (FDA approval as Marinol), the natural (−)-Δ9-Tetrahydrocannabinol, is a potent inducer of OGT via epigenetic hypomethylation of the transcription start site -thereby exerting antileukemic activity in acute leukemia in vivo. We have recently shown, that dronabinol, the natural (−)-Δ9-Tetrahydrocannabinol, has growth-inhibiting antitumor efficacy - including acute leukemia. We now reveal a novel mechanism-of-action via epigenetic modulation of OGT, an enzyme linked to genes involved in leukemogenesis such as AKT, MLL5, TET2 or ASXL1, releasing leukemia blasts from differentiation blockage in vivo and sensitizing cells towards induction of apoptosis. gDNA methylation gene arrays using Jurkat leukemia cells revealed global modulation of methylation patterns upon dronabinol treatment. OGT was identified as the highest altered gene (-42%, pval 3,68E-38) - correlating with an increase of OGT protein expression in Western immunoblots. Consistently, hypomethylation of the transcription start site of OGT and induction of OGT protein expression upon dronabinol were confirmed in an independent array using native patient samples. To study whether epigenetic activity is driven via the cannabinoid receptors, Jurkat cells were pretreated with CB1 (LY320135) and/or CB2 (JTE-907) antagonists, and exposed to dronabinol. Importantly, upregulation of OGT protein expression upon dronabinol was suppressed by inhibition of either receptor. Even more, inhibition of CB1 and/or CB2 reduced induction of apoptosis - and was most profound when inhibiting both receptors simultaneously. Similarly, retroviral knockdown of OGT in Jurkat and native leukemia blasts rendered cells less susceptible towards induction of apoptosis. Furthermore, we have evidence, that OGT has lead to release of the differentiation block in leukemia cells in vivo. Supportive treatment with dronabinol of an unfit patient with secondary acute myeloid leukemia resulted in direct disease control: Tantalizingly, besides a proapoptotic effect, the leukemic clone was maturing - with loss of CD34 and upregulation of CD11c, CD14 and CD15. Remarkably, immunophenotypic and genotypic (using NGS) profiling of the predominant monocyte population present two months after start of treatment, revealed that these mature monocytes derived from the leukemia clone (presenting mutations in EZH2 and ASXL1 among others - both known candidate genes of OGT). Mimicking this observation, we treated cells of this and other patients as well as defined leukemia models such as MOLM14 with dronabinol ex vivo and revealed upregulation of differentiation markers, such as CD11c, CD15 or CEBPA by flow cytometry and immunoblots - which was abrogated by lentiviral OGT-interference. Our findings provide a strong rationale for further exploring dronabinol as an agent with remarkable antileukemic efficacy achievable in vivo. In specific, overriding the differentiation blockage in leukemia cells may open up alternative therapeutic approaches similar to promyelocytic leukemia. Disclosures Off Label Use: Marinol: FDA approved for chemotherapy related nausea... here we demonstrate significant anti-leukemic effects.

scholarly journals Cooperation of E2F-p130 and Sp1-pRb Complexes in Repression of the Chinese Hamster dhfr Gene

2001 ◽  
Vol 21 (4) ◽  
pp. 1121-1131 ◽  
Author(s):  
Young-Chae Chang ◽  
Sharon Illenye ◽  
Nicholas H. Heintz
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Transcription Start Site ◽  
Trichostatin A ◽  
Chinese Hamster ◽  
Serum Starvation ◽  
Start Site ◽  
Transcription Start ◽  
Serum Stimulation ◽  
Cell Extracts

ABSTRACT In mammalian cells reiterated binding sites for Sp1 and two overlapping and inverted E2F sites at the transcription start site regulate the dhfr promoter during the cell growth cycle. Here we have examined the contributions of the dhfr Sp1 and E2F sites in the repression of dhfr gene expression. In serum-starved cells or during serum stimulation, the Chinese hamsterdhfr gene was not derepressed by trichostatin A (TSA), an inhibitor of histone deacetylases (HDAC). Immunoprecipitation experiments showed that HDAC1 and hypophosphorylated retinoblastoma protein (pRb) are associated with Sp1 in serum-starved CHOC400 cells. In transfection experiments, reporter plasmids containing the reiterated dhfr Sp1 sites were stimulated 10-fold by TSA, while a promoter containing four dhfr E2F sites and a TATA box was responsive to E2F but was completely unaffected by TSA. HDAC1 did not coprecipitate with p130-E2F DNA binding complexes, the predominant E2F binding activity in cell extracts after serum starvation, suggesting that p130 imposes a TSA-insensitive state on thedhfr promoter. In support of this notion, recruitment of GAL4-p130 to a dihydrofolate reductase-GAL4 reporter rendered the promoter insensitive to TSA, while repression by GAL4-pRb was sensitive to TSA. Upon phosphorylation of pRb and p130 after serum stimulation, the Sp1-pRb and p130-E2F interactions were lost while the Sp1-HDAC1 interaction persisted into S phase. Together these studies suggest a dynamic model for the cooperation of pRb and p130 in repression ofdhfr gene expression during withdrawal from the cell cycle. We propose that, during initial phases of cell cycle withdrawal, the binding of dephosphorylated pRb to Sp1-HDAC1 complexes and complexes of E2F-1 -to -3 with DP results in transient, HDAC-dependent suppression of dhfr transcription. Upon withdrawal of cells into G0, recruitment of p130 to E2F-4–DP-1 complexes at the transcription start site results in a TSA-insensitive complex that cooperates with Sp1-HDAC-pRb complexes to stably repressdhfr promoter activity in quiescent cells.

scholarly journals Analyses of Subgenomic Promoters of Hibiscus Chlorotic Ringspot Virus and Demonstration of 5′ Untranslated Region and 3′-Terminal Sequences Functioning as Subgenomic Promoters

2006 ◽  
Vol 80 (7) ◽  
pp. 3395-3405 ◽  
Author(s):  
Weimin Li ◽  
Sek-Man Wong
Keyword(s):  
Transcription Start Site ◽  
Untranslated Region ◽  
Mutational Analysis ◽  
Ringspot Virus ◽  
Base Sequence ◽  
Minus Strand ◽  
Start Site ◽  
Transcription Start ◽  
Subgenomic Rnas

ABSTRACT Hibiscus chlorotic ringspot virus (HCRSV), which belongs to the genus Carmovirus, generates two 3′-coterminal subgenomic RNAs (sgRNAs) of 1.4 kb and 1.7 kb. Transcription start sites of the two sgRNAs were identified at nucleotides (nt) 2178 and 2438, respectively. The full promoter of sgRNA1, a 118-base sequence, is localized between positions +6 and −112 relative to its transcription start site (+1). Similarly, a 132-base sequence, from +6 to −126, defines the sgRNA2 promoter. Computer analysis revealed that both sgRNA promoters share a similar two-stem-loop (SL1 + SL2) structure, immediately upstream of the transcription start site. Mutational analysis of the primary sequence and secondary structures showed further similarities between the two subgenomic promoters. The basal portion of SL2, encompassing the transcription start site, was essential for transcription activity in each promoter, while SL1 and the upper portion of SL2 played a role in transcription enhancement. Both the 5′ untranslated region (UTR) and the last 87 nt at the 3′ UTR of HCRSV genomic RNA are likely to be the putative genomic plus-strand and minus-strand promoters, respectively. They function well as individual sgRNA promoters to produce ectopic subgenomic RNAs in vivo but not to the same levels of the actual sgRNA promoters. This suggests that HCRSV sgRNA promoters share common features with the promoters for genomic plus-strand and minus-strand RNA synthesis. To our knowledge, this is the first demonstration that both the 5′ UTR and part of the 3′ UTR can be duplicated and function as sgRNA promoters within a single viral genome.

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