scholarly journals Genomic Study of Replication Initiation in Human Chromosomes Reveals the Influence of Transcription Regulation and Chromatin Structure on Origin Selection

2010 ◽  
Vol 21 (3) ◽  
pp. 393-404 ◽  
Author(s):  
Neerja Karnani ◽  
Christopher M. Taylor ◽  
Ankit Malhotra ◽  
Anindya Dutta
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Open Chromatin ◽  
Lambda Exonuclease ◽  
Genomic Study ◽  
Exonuclease Digestion

DNA replication in metazoans initiates from multiple chromosomal loci called origins. Currently, there are two methods to purify origin-centered nascent strands: lambda exonuclease digestion and anti-bromodeoxyuridine immunoprecipitation. Because both methods have unique strengths and limitations, we purified nascent strands by both methods, hybridized them independently to tiling arrays (1% genome) and compared the data to have an accurate view of genome-wide origin distribution. By this criterion, we identified 150 new origins that were reproducible across the methods. Examination of a subset of these origins by chromatin immunoprecipitation against origin recognition complex (ORC) subunits 2 and 3 showed 93% of initiation peaks to localize at/within 1 kb of ORC binding sites. Correlation of origins with functional elements of the genome revealed origin activity to be significantly enriched around transcription start sites (TSSs). Consistent with proximity to TSSs, we found a third of initiation events to occur at or near the RNA polymerase II binding sites. Interestingly, ∼50% of the early origin activity was localized within 5 kb of transcription regulatory factor binding region clusters. The chromatin signatures around the origins were enriched in H3K4-(di- and tri)-methylation and H3 acetylation modifications on histones. Affinity of origins for open chromatin was also reiterated by their proximity to DNAse I-hypersensitive sites. Replication initiation peaks were AT rich, and >50% of the origins mapped to evolutionarily conserved regions of the genome. In summary, these findings indicate that replication initiation is influenced by transcription initiation and regulation as well as chromatin structure.

Where it all starts: eukaryotic origins of DNA replication

2001 ◽  
Vol 114 (4) ◽  
pp. 643-651 ◽  
Author(s):  
A.K. Bielinsky ◽  
S.A. Gerbi
Keyword(s):  
Dna Replication ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Cell Cycle Regulators ◽  
Initiation Point ◽  
The Past ◽  
Eukaryotic Genomes ◽  
Made In

Chromosomal origins of DNA replication in eukaryotic cells not only are crucial for understanding the basic process of DNA duplication but also provide a tool to analyze how cell cycle regulators are linked to the replication machinery. During the past decade much progress has been made in identifying replication origins in eukaryotic genomes. More recently, replication initiation point (RIP) mapping has allowed us to detect start sites for DNA synthesis at the nucleotide level and thus to monitor replication initiation events at the origin very precisely. Beyond giving us the precise positions of start sites, the application of RIP mapping in yeast and human cells has revealed a single, defined start point at which replication initiates, a scenario very reminiscent of transcription initiation. More importantly, studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site. Therefore, in our pursuit of identifying ORC-binding sites in higher eukaryotes, RIP mapping may lead the way.

scholarly journals Transcription shapes DNA replication initiation to preserve genome integrity

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yang Liu ◽  
Chen Ai ◽  
Tingting Gan ◽  
Jinchun Wu ◽  
Yongpeng Jiang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Open Chromatin ◽  
Dna Replication Initiation ◽  
Early Replication

Abstract Background Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage. Results We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage. Conclusion RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

scholarly journals Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

2012 ◽  
Vol 198 (4) ◽  
pp. 509-528 ◽  
Author(s):  
Peer Papior ◽  
José M. Arteaga-Salas ◽  
Thomas Günther ◽  
Adam Grundhoff ◽  
Aloys Schepers
Keyword(s):  
Cell Cycle ◽  
Internal Control ◽  
Epstein Barr Virus ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Micrococcal Nuclease ◽  
Open Chromatin ◽  
Barr Virus ◽  
A Genome ◽  
Epstein Barr

Whether or not metazoan replication initiates at random or specific but flexible sites is an unsolved question. The lack of sequence specificity in origin recognition complex (ORC) DNA binding complicates genome-scale chromatin immunoprecipitation (ChIP)-based studies. Epstein-Barr virus (EBV) persists as chromatinized minichromosomes that are replicated by the host replication machinery. We used EBV to investigate the link between zones of pre-replication complex (pre-RC) assembly, replication initiation, and micrococcal nuclease (MNase) sensitivity at different cell cycle stages in a genome-wide fashion. The dyad symmetry element (DS) of EBV’s latent origin, a well-established and very efficient pre-RC assembly region, served as an internal control. We identified 64 pre-RC zones that correlate spatially with 57 short nascent strand (SNS) zones. MNase experiments revealed that pre-RC and SNS zones were linked to regions of increased MNase sensitivity, which is a marker of origin strength. Interestingly, although spatially correlated, pre-RC and SNS zones were characterized by different features. We propose that pre-RCs are formed at flexible but distinct sites, from which only a few are activated per single genome and cell cycle.

scholarly journals Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin

2013 ◽  
Vol 203 (1) ◽  
pp. 57-71 ◽  
Author(s):  
Nikhil Raghuram ◽  
Hilmar Strickfaden ◽  
Darin McDonald ◽  
Kylie Williams ◽  
He Fang ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Histone H1 ◽  
Dependent Manner ◽  
Prolyl Isomerase ◽  
Proline Isomerization ◽  
The Stability

Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulated the dephosphorylation of H1 in vitro and modulated the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilized H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation were associated with transcriptional activation independent of RNA polymerase II. We thus identify a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin.

scholarly journals Chromatin Profiling of Epstein-Barr Virus Latency Control Region

2007 ◽  
Vol 81 (12) ◽  
pp. 6389-6401 ◽  
Author(s):  
Latasha Day ◽  
Charles M. Chau ◽  
Michael Nebozhyn ◽  
Andrew J. Rennekamp ◽  
Michael Showe ◽  
...  
Keyword(s):  
Control Region ◽  
Histone Modifications ◽  
Transcription Initiation ◽  
Epstein Barr Virus ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Pcr Analysis ◽  
Chromatin Domain ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) escapes host immunity by the reversible and epigenetic silencing of immunogenic viral genes. We previously presented evidence that a dynamic chromatin domain, which we have referred to as the latency control region (LCR), contributes to the reversible repression of EBNA2 and LMP1 gene transcription. We now explore the protein-DNA interaction profiles for a few known regulatory factors and histone modifications that regulate LCR structure and activity. A chromatin immunoprecipitation assay combined with real-time PCR analysis was used to analyze protein-DNA interactions at ∼500-bp intervals across the first 60,000 bp of the EBV genome. We compared the binding patterns of EBNA1 with those of the origin recognition complex protein ORC2, the chromatin boundary factor CTCF, the linker histone H1, and several histone modifications. We analyzed three EBV-positive cell lines (MutuI, Raji, and LCL3459) with distinct transcription patterns reflecting different latency types. Our findings suggest that histone modification patterns within the LCR are complex but reflect differences in each latency type. The most striking finding was the identification of CTCF sites immediately upstream of the Qp, Cp, and EBER transcription initiation regions in all three cell types. In transient assays, CTCF facilitated EBNA1-dependent transcription activation of Cp, suggesting that CTCF coordinates interactions between different chromatin domains. We also found that histone H3 methyl K4 clustered with CTCF and EBNA1 at sites of active transcription or DNA replication initiation. Our findings support a model where CTCF delineates multiple domains within the LCR and regulates interactions between these domains that correlate with changes in gene expression.

scholarly journals Strong replicators associated with open chromatin are sufficient to establish an early replicating domain

2018 ◽  
Author(s):  
Caroline Brossas ◽  
Sabarinadh Chilaka ◽  
Antonin Counillon ◽  
Marc Laurent ◽  
Coralie Goncalves ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Binding Sites ◽  
Replication Origin ◽  
Molecular Mechanisms ◽  
Open Chromatin ◽  
Strongly Correlated ◽  
Topologically Associating Domains ◽  
Genome Wide ◽  
Close Physical Proximity ◽  
Temporal Program

AbstractVertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into topologically associating domains. However, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late replicating region for earlier replication. When inserted side-by-side, these modules acted in cooperation, with similar effects on two late-replicating regions. Targeted insertions of these two modules at two chromosomal sites separated by 30 kb brought these two modules into close physical proximity and induced the formation of an early-replicating domain. Thus, combinations of strong origins and cis-elements capable of opening the chromatin structure are the basic units of early-replicating domains, and are absent from late-replicated regions. These findings are consistent with those of genome-wide studies mapping strong initiation sites and open chromatin marks in vertebrate genomes.

scholarly journals Transcriptional Elements Involved in the Repression of Ribosomal Protein Synthesis

1999 ◽  
Vol 19 (8) ◽  
pp. 5393-5404 ◽  
Author(s):  
Baojie Li ◽  
Concepcion R. Nierras ◽  
Jonathan R. Warner
Keyword(s):  
Rna Polymerase Ii ◽  
Mating Type ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Ribosomal Proteins ◽  
Secretory Pathway ◽  
Rrna Genes ◽  
Mrna Levels ◽  
Growing Cell ◽  
Mating Type Genes

ABSTRACT The ribosomal proteins (RPs) of Saccharomyces cerevisiae are encoded by 137 genes that are among the most transcriptionally active in the genome. These genes are coordinately regulated: a shift up in temperature leads to a rapid, but temporary, decline in RP mRNA levels. A defect in any part of the secretory pathway leads to greatly reduced ribosome synthesis, including the rapid loss of RP mRNA. Here we demonstrate that the loss of RP mRNA is due to the rapid transcriptional silencing of the RP genes, coupled to the naturally short lifetime of their transcripts. The data suggest further that a global inhibition of polymerase II transcription leads to overestimates of the stability of individual mRNAs. The transcription of most RP genes is activated by two Rap1p binding sites, 250 to 400 bp upstream from the initiation of transcription. Rap1p is both an activator and a silencer of transcription. The swapping of promoters between RPL30 and ACT1 orGAL1 demonstrated that the Rap1p binding sites ofRPL30 are sufficient to silence the transcription ofACT1 in response to a defect in the secretory pathway. Sir3p and Sir4p, implicated in the Rap1p-mediated repression of silent mating type genes and of telomere-proximal genes, do not influence such silencing of RP genes. Sir2p, implicated in the silencing both of the silent mating type genes and of genes within the ribosomal DNA locus, does not influence the repression of either RP or rRNA genes. Surprisingly, the 180-bp sequence of RPL30 that lies between the Rap1p sites and the transcription initiation site is also sufficient to silence the Gal4p-driven transcription in response to a defect in the secretory pathway, by a mechanism that requires the silencing region of Rap1p. We conclude that for Rap1p to activate the transcription of an RP gene it must bind to upstream sequences; yet for Rap1p to repress the transcription of an RP gene it need not bind to the gene directly. Thus, the cell has evolved a two-pronged approach to effect the rapid extinction of RP synthesis in response to the stress imposed by a heat shock or by a failure of the secretory pathway. Calculations based on recent transcriptome data and on the half-life of the RP mRNAs suggest that in a rapidly growing cell the transcription of RP mRNAs accounts for nearly 50% of the total transcriptional events initiated by RNA polymerase II. Thus, the sudden silencing of the RP genes must have a dramatic effect on the overall transcriptional economy of the cell.

scholarly journals Mutational bias in spermatogonia impacts the anatomy of regulatory sites in the human genome

2021 ◽  
Author(s):  
Vera B Kaiser ◽  
Lana Talmane ◽  
Yatendra Kumar ◽  
Fiona Semple ◽  
Marie MacLennan ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Meiotic Recombination ◽  
Binding Sites ◽  
Evolutionary Process ◽  
Mutational Bias ◽  
Open Chromatin ◽  
Structural Variant ◽  
Regulatory Architecture ◽  
Mutational Processes ◽  
Regulatory Sites

Mutation in the germline is the ultimate source of genetic variation, but little is known about the influence of germline chromatin structure on mutational processes. Using ATAC-seq, we profile the open chromatin landscape of human spermatogonia, the most proliferative cell-type of the germline, identifying transcription factor binding sites (TFBSs) and PRDM9-binding sites, a subset of which will initiate meiotic recombination. We observe an increase in rare structural variant (SV) breakpoints at PRDM9-bound sites, implicating meiotic recombination in the generation of structural variation. Many germline TFBSs, such as NRF, are also associated with increased rates of SV breakpoints, apparently independent of recombination. Singleton short insertions (>=5 bp) are highly enriched at TFBSs, particularly at sites bound by testis active TFs, and their rates correlate with those of structural variant breakpoints. Short insertions often duplicate the TFBS motif, leading to clustering of motif sites near regulatory regions in this male-driven evolutionary process. Increased mutation loads at germline TFBSs disproportionately affect neural enhancers with activity in spermatogonia, potentially altering neurodevelopmental regulatory architecture. Local chromatin structure in spermatogonia is thus pervasive in shaping both evolution and disease.

scholarly journals Mutational bias in spermatogonia impacts the anatomy of regulatory sites in the human genome

2021 ◽  
Author(s):  
Vera B. Kaiser ◽  
Lana Talmane ◽  
Yatendra Kumar ◽  
Fiona Semple ◽  
Marie MacLennan ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Meiotic Recombination ◽  
Binding Sites ◽  
Evolutionary Process ◽  
Mutational Bias ◽  
Open Chromatin ◽  
Structural Variant ◽  
Regulatory Architecture ◽  
Mutational Processes ◽  
Regulatory Sites

Mutation in the germline is the ultimate source of genetic variation, but little is known about the influence of germline chromatin structure on mutational processes. Using ATAC-seq, we profile the open chromatin landscape of human spermatogonia, the most proliferative cell type of the germline, identifying transcription factor binding sites (TFBSs) and PRDM9 binding sites, a subset of which will initiate meiotic recombination. We observe an increase in rare structural variant (SV) breakpoints at PRDM9-bound sites, implicating meiotic recombination in the generation of structural variation. Many germline TFBSs, such as NRF1, are also associated with increased rates of SV breakpoints, apparently independent of recombination. Singleton short insertions (≥5 bp) are highly enriched at TFBSs, particularly at sites bound by testis active TFs, and their rates correlate with those of structural variant breakpoints. Short insertions often duplicate the TFBS motif, leading to clustering of motif sites near regulatory regions in this male-driven evolutionary process. Increased mutation loads at germline TFBSs disproportionately affect neural enhancers with activity in spermatogonia, potentially altering neurodevelopmental regulatory architecture. Local chromatin structure in spermatogonia is thus pervasive in shaping both evolution and disease.

RNA Polymerase II Pausing and Chromatin Structure Control piRNA Biogenesis Across Nematode Evolution

2018 ◽  
Author(s):  
T. Beltran ◽  
C. Barroso ◽  
T. Y. Birkle ◽  
L. Stevens ◽  
H. T. Schwartz ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Structure Control
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