scholarly journals Sequential Histone Modifications at Hoxd4 Regulatory Regions Distinguish Anterior from Posterior Embryonic Compartments

2004 ◽  
Vol 24 (18) ◽  
pp. 8090-8103 ◽  
Author(s):  
Mojgan Rastegar ◽  
Laila Kobrossy ◽  
Erzsebet Nagy Kovacs ◽  
Isabel Rambaldi ◽  
Mark Featherstone
Keyword(s):  
Rna Polymerase Ii ◽  
Histone Modifications ◽  
Chromatin Immunoprecipitation ◽  
Gene Activation ◽  
Open Chromatin ◽  
Rna Pol Ii ◽  
Regulatory Regions ◽  
Pol Ii ◽  
Embryonic Tissues ◽  
Intervening Sequences

ABSTRACT Hox genes are differentially expressed along the embryonic anteroposterior axis. We used chromatin immunoprecipitation to detect chromatin changes at the Hoxd4 locus during neurogenesis in P19 cells and embryonic day 8.0 (E8.0) and E10.5 mouse embryos. During Hoxd4 induction in both systems, we observed that histone modifications typical of transcriptionally active chromatin occurred first at the 3′ neural enhancer and then at the promoter. Moreover, the sequential distribution of histone modifications between E8.0 and E10.5 was consistent with a spreading of open chromatin, starting with the enhancer, followed by successively more 5′ intervening sequences, and culminating at the promoter. Neither RNA polymerase II (Pol II) nor CBP associated with the inactive gene. During Hoxd4 induction, CBP and RNA Pol II were recruited first to the enhancer and then to the promoter. Whereas the CBP association was transient, RNA Pol II remained associated with both regulatory regions. Histone modification and transcription factor recruitment occurred in posterior, Hox-expressing embryonic tissues, but never in anterior tissues, where such genes are inactive. Together, our observations demonstrate that the direction of histone modifications at Hoxd4 mirrors colinear gene activation across Hox clusters and that the establishment of anterior and posterior compartments is accompanied by the imposition of distinct chromatin states.

scholarly journals Inferring time series chromatin states for promoter-enhancer pairs based on Hi-C data

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Henriette Miko ◽  
Yunjiang Qiu ◽  
Bjoern Gaertner ◽  
Maike Sander ◽  
Uwe Ohler
Keyword(s):  
Time Series ◽  
Histone Modifications ◽  
Gene Activation ◽  
Expectation Maximization Algorithm ◽  
Chromatin State ◽  
Open Chromatin ◽  
Multiple Time ◽  
Enhancer Activity ◽  
Chromatin States ◽  
Multiple Time Points

Abstract Background Co-localized combinations of histone modifications (“chromatin states”) have been shown to correlate with promoter and enhancer activity. Changes in chromatin states over multiple time points (“chromatin state trajectories”) have previously been analyzed at promoter and enhancers separately. With the advent of time series Hi-C data it is now possible to connect promoters and enhancers and to analyze chromatin state trajectories at promoter-enhancer pairs. Results We present TimelessFlex, a framework for investigating chromatin state trajectories at promoters and enhancers and at promoter-enhancer pairs based on Hi-C information. TimelessFlex extends our previous approach Timeless, a Bayesian network for clustering multiple histone modification data sets at promoter and enhancer feature regions. We utilize time series ATAC-seq data measuring open chromatin to define promoters and enhancer candidates. We developed an expectation-maximization algorithm to assign promoters and enhancers to each other based on Hi-C interactions and jointly cluster their feature regions into paired chromatin state trajectories. We find jointly clustered promoter-enhancer pairs showing the same activation patterns on both sides but with a stronger trend at the enhancer side. While the promoter side remains accessible across the time series, the enhancer side becomes dynamically more open towards the gene activation time point. Promoter cluster patterns show strong correlations with gene expression signals, whereas Hi-C signals get only slightly stronger towards activation. The code of the framework is available at https://github.com/henriettemiko/TimelessFlex. Conclusions TimelessFlex clusters time series histone modifications at promoter-enhancer pairs based on Hi-C and it can identify distinct chromatin states at promoter and enhancer feature regions and their changes over time.

scholarly journals Interdependence between histone marks and steps in Pol II transcription

2021 ◽  
Author(s):  
Charles Danko ◽  
Zhong Wang ◽  
Alexandra Chivu ◽  
Lauren Choate ◽  
Edward Rice ◽  
...  
Keyword(s):  
Machine Learning ◽  
Histone Modifications ◽  
Dnase I ◽  
Open Chromatin ◽  
Learning Tools ◽  
Pol Ii ◽  
Histone Marks ◽  
Chromatin States ◽  
Hypersensitive Sites

Abstract The role of histone modifications in transcription remains incompletely understood. Here we used experimental perturbations combined with sensitive machine learning tools that infer the distribution of histone marks using maps of nascent transcription. Transcription predicted the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. Blocking transcription rapidly removed two punctate marks, H3K4me3 and H3K27ac, from chromatin indicating that transcription is required for active histone modifications. Transcription was also required for maintenance of H3K27me3 consistent with a role for RNA in recruiting PRC2. A subset of DNase-I hypersensitive sites were refractory to prediction, precluding models where transcription initiates pervasively at any open chromatin. Our results, in combination with past literature, support a model in which active histone modifications serve a supportive, rather than a regulatory, role in transcription.

scholarly journals Mechanism of RNA polymerase II stalling by DNA alkylation

2017 ◽  
Vol 114 (46) ◽  
pp. 12172-12177 ◽  
Author(s):  
Stefano Malvezzi ◽  
Lucas Farnung ◽  
Claudia M. N. Aloisi ◽  
Todor Angelov ◽  
Patrick Cramer ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Anticancer Agents ◽  
Excision Repair ◽  
Minor Groove ◽  
Dna Alkylation ◽  
Drug Induced ◽  
Rna Pol Ii ◽  
Pol Ii

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

scholarly journals CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

2007 ◽  
Vol 27 (5) ◽  
pp. 1631-1648 ◽  
Author(s):  
Igor Chernukhin ◽  
Shaharum Shamsuddin ◽  
Sung Yun Kang ◽  
Rosita Bergström ◽  
Yoo-Wook Kwon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Activation ◽  
Ctcf Binding ◽  
Pol Ii ◽  
Genome Wide ◽  
Target Sites ◽  
On Chip

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

scholarly journals HNF4A Regulates the Formation of Hepatic Progenitor Cells from Human iPSC-Derived Endoderm by Facilitating Efficient Recruitment of RNA Pol II

Genes ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 21 ◽  
Author(s):  
Ann DeLaForest ◽  
Francesca Di Furio ◽  
Ran Jing ◽  
Amy Ludwig-Kubinski ◽  
Kirk Twaroski ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Chromatin Immunoprecipitation ◽  
Molecular Mechanisms ◽  
Cell Formation ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
High Throughput Dna Sequencing ◽  
Hepatocyte Nuclear Factor 4 ◽  
Induced Pluripotent ◽  
Human Ipsc

Elucidating the molecular basis of cell differentiation will advance our understanding of organ development and disease. We have previously established a protocol that efficiently produces cells with hepatocyte characteristics from human induced pluripotent stem cells. We previously used this cell differentiation model to identify the transcription factor hepatocyte nuclear factor 4 α (HNF4A) as being essential during the transition of the endoderm to a hepatic fate. Here, we sought to define the molecular mechanisms through which HNF4A controls this process. By combining HNF4A chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) analyses at the onset of hepatic progenitor cell formation with transcriptome data collected during early stages of differentiation, we identified genes whose expression is directly dependent upon HNF4A. By examining the dynamic changes that occur at the promoters of these HNF4A targets we reveal that HNF4A is essential for recruitment of RNA polymerase (RNA pol) II to genes that are characteristically expressed as the hepatic progenitors differentiate from the endoderm.

scholarly journals Widespread Backtracking by RNA Pol II Is a Major Effector of Gene Activation, 5′ Pause Release, Termination, and Transcription Elongation Rate

2019 ◽  
Vol 73 (1) ◽  
pp. 107-118.e4 ◽  
Author(s):  
Ryan M. Sheridan ◽  
Nova Fong ◽  
Angelo D’Alessandro ◽  
David L. Bentley
Keyword(s):  
Transcription Elongation ◽  
Gene Activation ◽  
Elongation Rate ◽  
Rna Pol Ii ◽  
Pol Ii

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

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1734-1734 ◽  
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.

AT7519, a Potent Multi-Targeted CDK Inhibitor, Is Active in CLL Patient Samples Independent of Stage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3161-3161
Author(s):  
Vicky Lock ◽  
Laurence Cooke ◽  
Murray Yule ◽  
Neil T Thompson ◽  
K. Della Croce ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Rna Polymerase Ii ◽  
Parp Cleavage ◽  
Regulation Of Transcription ◽  
Cytotoxic Effects ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Cyclin Dependent Kinases

Abstract Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II (RNA pol II). The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 on CLL cells isolated from patients. Primary cell samples were isolated from a total of 15 patients with CLL with various stages of disease (8 Stage 0, 0/I or II and 7 Stage IV) and who were either treatment naïve or had received a variety of prior therapies. Patient samples were characterised for cytogenetic abnormalities (11q, 17p and 13q deletion or trisomy 12) as well IgVH mutation and ZAP70 expression. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–700nM. AT7519 appears equally effective at inhibiting the survival of CLL cells harbouring a variety of mutations including those representative of patients that fall within poorer prognosis treatment groups. The amount of AT7519 required to induce cell death in 50% of the CLL cell population increased as exposure time was decreased but significant cell death was obtained at doses approximating to 1uM following 4–6h of treatment. These doses are equivalent to exposures achieved in ongoing AT7519 clinical studies indicating that cytotoxic doses can be achieved in patients on well tolerated schedules. The mechanism of AT7519 cytotoxic effects was investigated by western blotting for a variety of cell cycle and apoptotic markers following incubation with compound. Short term treatments (4–6h) resulted in inhibition of phosphorylation of the transcriptional marker RNA pol II and the downregulation of the anti-apoptotic protein Mcl-1. Additional antiapoptotic proteins including XIAP and Bcl-2 remained unchanged. The reduction in Mcl-1 protein levels was associated with an increase in the apoptotic marker cleaved PARP. No inhibition of cell cycle markers such as phospho-retinoblastoma protein was observed in the same samples suggesting that the cytotoxic effects of AT7519 in CLL patient samples is due to its transcriptional activity alone. Together the data suggest AT7519 offers a promising treatment strategy for patients with advanced B-cell leukemia and lymphoma.

scholarly journals Increased processing of SINE B2 non coding RNAs unveils a novel type of transcriptome de-regulation underlying amyloid beta neuro-pathology

2020 ◽  
Author(s):  
Yubo Cheng ◽  
Babita Gollen ◽  
Luke Saville ◽  
Christopher Isaac ◽  
Jogender Mehla ◽  
...  
Keyword(s):  
Stress Response ◽  
Rna Polymerase Ii ◽  
Amyloid Beta ◽  
Transcriptional Activation ◽  
Rna Processing ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Amyloid Toxicity ◽  
B2 Rna ◽  
Non Coding Rnas

ABSTRACTMore than 97% of the mammalian genome is non-protein coding, and repetitive elements account for more than 50% of noncoding space. However, the functional importance of many non-coding RNAs generated by these elements and their connection with pathologic processes remains elusive. We have previously shown that B2 RNAs, a class of non-coding RNAs that belong to the B2 family of SINE repeats, mediate the transcriptional activation of stress response genes (SRGs) upon application of a stimulus. Notably, B2 RNAs bind RNA Polymerase II (RNA Pol II) and suppress SRG transcription during pro-stimulation state. Upon application of a stimulus, B2 RNAs are processed into fragments and degraded, which in turn releases RNA Pol II from suppression and upregulates SRGs. Here, we demonstrate a novel role for B2 RNAs in transcriptome response to amyloid beta toxicity and pathology in the mouse hippocampus. In healthy hippocampi, activation of SRGs is followed by a transient upregulation of pro-apoptotic factors, such as p53 and miRNA-34c, which target SRGs creating a negative feedback loop that facilitates transition to the pro-stimulation state. Using an integrative RNA genomics approach, we show that in mouse hippocampi of an amyloid precursor protein knock-in mouse model and in an in vitro cell culture model of amyloid beta toxicity, this regulatory loop is dysfunctional due to increased levels of B2 RNA processing, constitutively elevated SRG expression and high p53 levels. Evidence indicates that Hsf1, a master regulator of stress response, mediates B2 RNA processing in cells, and is upregulated during amyloid toxicity accelerating the processing of SINE RNAs and SRG hyper-activation. Our study reveals that in mouse, SINE RNAs constitute a novel pathway deregulated in amyloid beta pathology, with potential implications for similar cases in the human brain, such as Alzheimer’s disease (AD). This data attributes a role to SINE RNA processing in a pathological process as well as a new function to Hsf1 that is independent of its transcription factor activity.

scholarly journals Cohesin removal reprograms gene expression upon mitotic entry

2019 ◽  
Author(s):  
Carlos Perea-Resa ◽  
Leah Bury ◽  
Iain Cheeseman ◽  
Michael D. Blower
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Rna Polymerase Ii ◽  
Chromosome Segregation ◽  
List Type ◽  
Mitotic Chromosomes ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Mitotic Entry ◽  
Nascent Transcripts

SummaryEntering mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA Polymerase II (RNAPII) in mitosis. Here, we demonstrate that chromatin-bound cohesin is sufficient to retain RNAPII at centromeres while WAPL-mediated removal of cohesin during prophase is required for RNAPII dissociation from chromosome arms. Failure to remove cohesin from chromosome arms results in a failure to release elongating RNAPII and nascent transcripts from mitotic chromosomes and dramatically alters gene expression. We propose that prophase cohesin removal is the key step in reprogramming gene expression as cells transition from G2 to mitosis, and is temporally coupled with chromosome condensation to coordinate chromosome segregation with changes in gene expression.HighlightsMitotic centromere transcription requires cohesinCohesin removal releases elongating RNA Pol II and nascent RNA from chromatinThe prophase pathway reprograms gene expression during mitosis

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