scholarly journals Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Nina Kirstein ◽  
Alexander Buschle ◽  
Xia Wu ◽  
Stefan Krebs ◽  
Helmut Blum ◽  
...  
Keyword(s):  
Replication Fork ◽  
Replication Timing ◽  
Replication Initiation ◽  
Gene Promoters ◽  
Rna Seq ◽  
Replication Time ◽  
Origin Licensing ◽  
Eukaryotic Dna ◽  
Late Origin ◽  
Active Genes

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

scholarly journals Human ORC/MCM density is low in active genes and correlates with replication time but does not solely define replication initiation zones

2020 ◽  
Author(s):  
Nina Kirstein ◽  
Alexander Buschle ◽  
Xia Wu ◽  
Stefan Krebs ◽  
Helmut Blum ◽  
...  
Keyword(s):  
Replication Fork ◽  
Replication Timing ◽  
Replication Initiation ◽  
Gene Promoters ◽  
Rna Seq ◽  
Replication Time ◽  
Origin Licensing ◽  
Eukaryotic Dna ◽  
Late Origin ◽  
Active Genes

AbstractEukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are strongly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early-than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms that specify when and where replication initiates in human cells.

scholarly journals Topologically associating domain boundaries are enriched in early firing origins and restrict replication fork progression

2020 ◽  
Author(s):  
Emilia Puig Lombardi ◽  
Madalena Tarsounas
Keyword(s):  
Binding Sites ◽  
Replication Fork ◽  
Replication Timing ◽  
Replication Initiation ◽  
Ctcf Binding ◽  
Origin Firing ◽  
Replication Fork Progression ◽  
Fork Stalling ◽  
Genomic Regions ◽  
The Impact

ABSTRACTTopologically associating domains (TADs) are units of the genome architecture defined by binding sites for the CTCF transcription factor and cohesin-mediated loop extrusion. Genomic regions containing DNA replication initiation sites have been mapped in the proximity of TAD boundaries. However, the factors that determine this positioning have not been identified. Moreover, the impact of TADs on the directionality of replication fork progression remains unknown. Here we use EdU-seq technology to map origin firing sites at 10 kb resolution and to monitor replication fork progression after restart from hydroxyurea arrest. We show that origins firing in early/mid S-phase within TAD boundaries map to two distinct peaks flanking the centre of the boundary, which is occupied by CTCF and cohesin. When transcription is inhibited chemically or deregulated by oncogene overexpression, replication origins become repositioned to the centre of the TAD. Furthermore, we demonstrate the strikingly asymmetric fork progression initiating from origins located within TAD boundaries. Divergent CTCF binding sites and neighbouring TADs with different replication timing (RT) cause fork stalling in regions external to the TAD. Thus, our work assigns for the first time a role to transcription within TAD boundaries in promoting replication origin firing and demonstrates how genomic regions adjacent to the TAD boundaries could restrict replication progression.

scholarly journals Genome-wide identification of sucrose nonfermenting-1-related protein kinase (SnRK) genes in barley and RNA-seq analyses of their expression in response to abscisic acid treatment

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhiwei Chen ◽  
Longhua Zhou ◽  
Panpan Jiang ◽  
Ruiju Lu ◽  
Nigel G. Halford ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Gene Family ◽  
Stress Responses ◽  
Phylogenetic Analyses ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Related Protein ◽  
Rna Seq ◽  
Response Elements ◽  
Genome Data

Abstract Background Sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRKs) play important roles in regulating metabolism and stress responses in plants, providing a conduit for crosstalk between metabolic and stress signalling, in some cases involving the stress hormone, abscisic acid (ABA). The burgeoning and divergence of the plant gene family has led to the evolution of three subfamilies, SnRK1, SnRK2 and SnRK3, of which SnRK2 and SnRK3 are unique to plants. Therefore, the study of SnRKs in crops may lead to the development of strategies for breeding crop varieties that are more resilient under stress conditions. In the present study, we describe the SnRK gene family of barley (Hordeum vulgare), the widespread cultivation of which can be attributed to its good adaptation to different environments. Results The barley HvSnRK gene family was elucidated in its entirety from publicly-available genome data and found to comprise 50 genes. Phylogenetic analyses assigned six of the genes to the HvSnRK1 subfamily, 10 to HvSnRK2 and 34 to HvSnRK3. The search was validated by applying it to Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genome data, identifying 50 SnRK genes in rice (four OsSnRK1, 11 OsSnRK2 and 35 OsSnRK3) and 39 in Arabidopsis (three AtSnRK1, 10 AtSnRK2 and 26 AtSnRK3). Specific motifs were identified in the encoded barley proteins, and multiple putative regulatory elements were found in the gene promoters, with light-regulated elements (LRE), ABA response elements (ABRE) and methyl jasmonate response elements (MeJa) the most common. RNA-seq analysis showed that many of the HvSnRK genes responded to ABA, some positively, some negatively and some with complex time-dependent responses. Conclusions The barley HvSnRK gene family is large, comprising 50 members, subdivided into HvSnRK1 (6 members), HvSnRK2 (10 members) and HvSnRK3 (34 members), showing differential positive and negative responses to ABA.

scholarly journals A transcription-based mechanism for oncogenic β-catenin-induced lethality in BRCA1/2-deficient cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rebecca A. Dagg ◽  
Gijs Zonderland ◽  
Emilia Puig Lombardi ◽  
Giacomo G. Rossetti ◽  
Florian J. Groelly ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
High Throughput Sequencing ◽  
Germline Mutations ◽  
Cdk Inhibitor ◽  
Rna Seq ◽  
Gene Encoding ◽  
Origin Firing ◽  
Replication Fork Progression ◽  
Transcriptional Alterations

AbstractBRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of β-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed β-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, β-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that β-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.

scholarly journals The Rpd3-Sin3 Histone Deacetylase Regulates Replication Timing and Enables Intra-S Origin Control in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (11) ◽  
pp. 4769-4780 ◽  
Author(s):  
Jennifer G. Aparicio ◽  
Christopher J. Viggiani ◽  
Daniel G. Gibson ◽  
Oscar M. Aparicio
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Replication Timing ◽  
Histone Deacetylation ◽  
Origin Firing ◽  
Replication Checkpoint ◽  
Genome Transcription ◽  
Late Origin

ABSTRACT The replication of eukaryotic genomes follows a temporally staged program, in which late origin firing often occurs within domains of altered chromatin structure(s) and silenced genes. Histone deacetylation functions in gene silencing in some late-replicating regions, prompting an investigation of the role of histone deacetylation in replication timing control in Saccharomyces cerevisiae. Deletion of the histone deacetylase Rpd3 or its interacting partner Sin3 caused early activation of late origins at internal chromosomal loci but did not alter the initiation timing of early origins or a late-firing, telomere-proximal origin. By delaying initiation relative to the earliest origins, Rpd3 enables regulation of late origins by the intra-S replication checkpoint. RPD3 deletion suppresses the slow S phase of clb5Δ cells by enabling late origins to fire earlier, suggesting that Rpd3 modulates the initiation timing of many origins throughout the genome. Examination of factors such as Ume6 that function together with Rpd3 in transcriptional repression indicates that Rpd3 regulates origin initiation timing independently of its role in transcriptional repression. This supports growing evidence that for much of the S. cerevisiae genome transcription and replication timing are not linked.

scholarly journals Dynamic Association of the Replication Initiator and Transcription Factor DnaA with the Bacillus subtilis Chromosome during Replication Stress

2008 ◽  
Vol 191 (2) ◽  
pp. 486-493 ◽  
Author(s):  
Adam M. Breier ◽  
Alan D. Grossman
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Replication Fork ◽  
Replication Stress ◽  
Replication Initiation ◽  
Pass Through ◽  
Bacillus Subtilis Chromosome ◽  
Rapid Signaling ◽  
Replication Initiator

ABSTRACT DnaA functions as both a transcription factor and the replication initiator in bacteria. We characterized the DNA binding dynamics of DnaA on a genomic level. Based on cross-linking and chromatin immunoprecipitation data, DnaA binds at least 17 loci, 15 of which are regulated transcriptionally in response to inhibition of replication (replication stress). Six loci, each of which has a cluster of at least nine potential DnaA binding sites, had significant increases in binding by DnaA when replication was inhibited, indicating that the association of DnaA with at least some of its target sites is altered after replication stress. When replication resumed from oriC after inhibition of replication initiation, these high levels of binding decreased rapidly at origin-proximal and origin-distal regions, well before a replication fork could pass through each of the regulated regions. These findings indicate that there is rapid signaling to decrease activation of DnaA during replication and that interaction between DnaA bound at each site and the replication machinery is not required for regulation of DnaA activity in response to replication stress.

scholarly journals FOXO1 Mitigation of FOXL2C143W/SMAD3 Transcriptomic Landscape in a Model of Granulosa Cell Tumor

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A1018-A1019
Author(s):  
Christian Secchi ◽  
Paola Benaglio ◽  
Francesca Mulas ◽  
Martina Belli ◽  
Dwayne Stupack ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cell Tumor ◽  
Direct Consequence ◽  
Transcript Abundance ◽  
Cell Tumor ◽  
Gene Promoters ◽  
Rna Seq ◽  
Illumina Hiseq ◽  
Rare Type ◽  
Exact Test

Abstract Background: Adult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary. Postmenopausal genital bleeding is the main aGCT clinical sign which is attributed to estrogen excess driven by CYP19 upregulation. Typically, aGCTs that are diagnosed at an initial stage can be treated with surgery. However, recurrences are mostly fatal1. Current studies are focused on finding new molecular markers and targets that aim to treat the aGCTs recurrence. Between 95-97% of aGCTs harbor a somatic mutation in the FOXL2 gene, Cys134Trp (c.402C<G)2. A TGF-β pathway protein, SMAD3, was identified as an essential partner in FOXL2C134W transcriptional activity driving CYP19 upregulation3. Recently, the antitumoral FOXO1 gene has been recognized as a potential target for suppressing the FOXL2C134W pathogenic action4. Aim: The objective of this study was to examine whether FOXO1 upregulation affects the FOXL2C143W/SMAD3 transcriptomic landscape. Methods: RNA-seq analysis was performed comparing the effect of FOXL2WT/SMAD3 and FOXL2C143W/SMAD3 overexpression in presence of FOXO1 by transfection of an established human GC line (HGrC1). RNA-seq libraries were prepared using the illumina TrueSeq and sequenced using an illumina HiSeq Platform4000. To quantify transcript abundance for each sample we used salmon (1.1.0) with default parameters, using indexes from hg38. Data was subsequently imported in R using the tximport package and processed with the DESeq2 package. Results: RNA-seq data show that FOXL2C143W/SMAD3 significantly drives 717 genes compared with the WT and enabled us to identify targets (TGFB2, SMARCA4, HSPG2, MKI67, NFKBIA) and neoplastic pathways directly associated with the mutant. To provide evidence that the differences in gene expression were attributed to a direct consequence of FOXL2 binding, we annotated gene promoters with previously published FOXL2 ChIP-seq analysis. The majority (73-40%) of the differential expressed genes (DEGs) between FOXL2C134W and FOXL2WT had a FOXL2 binding site at their promoters, which was a significantly higher proportion than in non-DEGs (Fisher’s exact test, murine: p= 7.9x10-157; human, p= 9.9x10-39). Surprisingly, the number of DEGs between FOXL2C134W + FOXO1 and FOXL2WT was much lower (230) with respect to the number of DEGs between FOXL2C134W and FOXL2WT (717, of which 130 in common; linear regression slope ß = 0 .58), suggesting that the effect of FOXL2C134W compared with FOXL2WT is moderated by the addition of FOXO1. Conclusions: Our transcriptomic study provides the first evidence that FOXO1 can efficiently mitigate 40% of the altered genome-wide effect specifically related to FOXL2C134W in a model of human aGCT.1 Farkkila, A. et al. Ann Med (2017). 2 Jamieson, S. & Fuller, P. J. Endocr Rev (2012). 3 Belli, M. et al. Endocrinology (2018). 4 Belli, M et al. J Endocr Soc (2019).

scholarly journals High-throughput analysis of DNA replication in single human cells reveals constrained variability in the location and timing of replication initiation

2021 ◽  
Author(s):  
Dashiell J Massey ◽  
Amnon Koren
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Replication Timing ◽  
S Phase ◽  
Human Cells ◽  
Replication Initiation ◽  
High Throughput Analysis ◽  
Timing Variability ◽  
Fixed Set ◽  
Fixed Order

DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

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