Chromosomal Mcm2-7 distribution is the primary driver of the genome replication program in species from yeast to humans

AbstractThe spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S-phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed identical MCM double-hexamer footprints across the species, but notable differences in their distribution: In budding yeast, complexes were present in sharp peaks comprised largely of single double-hexamers; in fission yeast, corresponding peaks typically contained 4 to 8 double-hexamers, were more disperse, and showed a striking correlation with AT content. In mouse and humans, complexes were even more disperse, with a preference for regions of high GC content. Nonetheless, most fluctuations in replication timing in all four organisms could be accounted for by differences in chromosomal MCM distribution. This analysis also identified genomic regions whose replication timing was clearly not attributable to MCM density. The most notable was the inactive X-chromosome, which replicates late in S phase despite the fact that both MCM abundance and chromosomal distribution were comparable to those on the early replicating active X-chromosome. We conclude that, although certain genomic regions, most notably the inactive X-chromosome, are subject to post-licensing regulation, most differences in replication timing along the chromosome reflect uneven chromosomal distribution of stochastically firing pre-replication complexes.

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Chromosomal Mcm2-7 distribution and the genome replication program in species from yeast to humans

PLoS Genetics ◽

10.1371/journal.pgen.1009714 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009714

Author(s):

Eric J. Foss ◽

Smitha Sripathy ◽

Tonibelle Gatbonton-Schwager ◽

Hyunchang Kwak ◽

Adam H. Thiesen ◽

...

Keyword(s):

Binding Sites ◽

Budding Yeast ◽

Replication Timing ◽

S Phase ◽

Genome Replication ◽

Sequence Specificity ◽

Chromosomal Distribution ◽

Chromosomal Replication ◽

Inactive X Chromosome ◽

Spatio Temporal

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes.

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Promyelocytic leukemia nuclear bodies associate with transcriptionally active genomic regions

The Journal of Cell Biology ◽

10.1083/jcb.200305142 ◽

2004 ◽

Vol 164 (4) ◽

pp. 515-526 ◽

Cited By ~ 151

Author(s):

Jayson Wang ◽

Carol Shiels ◽

Peter Sasieni ◽

Pei Jun Wu ◽

Suhail A. Islam ◽

...

Keyword(s):

X Chromosome ◽

Transcriptional Activity ◽

Promyelocytic Leukemia ◽

S Phase ◽

Nuclear Bodies ◽

Rna Transcripts ◽

Expression Of Genes ◽

Inactive X Chromosome ◽

Pml Bodies ◽

Genomic Regions

The promyelocytic leukemia (PML) protein is aggregated into nuclear bodies that are associated with diverse nuclear processes. Here, we report that the distance between a locus and its nearest PML body correlates with the transcriptional activity and gene density around the locus. Genes on the active X chromosome are more significantly associated with PML bodies than their silenced homologues on the inactive X chromosome. We also found that a histone-encoding gene cluster, which is transcribed only in S-phase, is more strongly associated with PML bodies in S-phase than in G0/G1 phase of the cell cycle. However, visualization of specific RNA transcripts for several genes showed that PML bodies were not themselves sites of transcription for these genes. Furthermore, knock-down of PML bodies by RNA interference did not preferentially change the expression of genes closely associated with PML bodies. We propose that PML bodies form in nuclear compartments of high transcriptional activity, but they do not directly regulate transcription of genes in these compartments.

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Cell cycle–dependent localization of macroH2A in chromatin of the inactive X chromosome

The Journal of Cell Biology ◽

10.1083/jcb.200112074 ◽

2002 ◽

Vol 157 (7) ◽

pp. 1113-1123 ◽

Cited By ~ 74

Author(s):

Brian P. Chadwick ◽

Huntington F. Willard

Keyword(s):

Cell Cycle ◽

X Chromosome ◽

Degradation Pathway ◽

S Phase ◽

Histone H2a ◽

The Core ◽

Inactive X Chromosome ◽

Inactivation Center ◽

Chromatin Proteins ◽

Cycle Dependent

One of several features acquired by chromatin of the inactive X chromosome (Xi) is enrichment for the core histone H2A variant macroH2A within a distinct nuclear structure referred to as a macrochromatin body (MCB). In addition to localizing to the MCB, macroH2A accumulates at a perinuclear structure centered at the centrosome. To better understand the association of macroH2A1 with the centrosome and the formation of an MCB, we investigated the distribution of macroH2A1 throughout the somatic cell cycle. Unlike Xi-specific RNA, which associates with the Xi throughout interphase, the appearance of an MCB is predominantly a feature of S phase. Although the MCB dissipates during late S phase and G2 before reforming in late G1, macroH2A1 remains associated during mitosis with specific regions of the Xi, including at the X inactivation center. This association yields a distinct macroH2A banding pattern that overlaps with the site of histone H3 lysine-4 methylation centered at the DXZ4 locus in Xq24. The centrosomal pool of macroH2A1 accumulates in the presence of an inhibitor of the 20S proteasome. Therefore, targeting of macroH2A1 to the centrosome is likely part of a degradation pathway, a mechanism common to a variety of other chromatin proteins.

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BRCa1 participates in XIST RNa localization on inactive x chromosome

Russian Journal of Biotherapy ◽

10.17650/1726-9784-2019-18-2-21-26 ◽

2019 ◽

Vol 18 (2) ◽

pp. 21-26

Author(s):

E. A. Shestakova ◽

T. A. Bogush

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Confocal Microscopy ◽

X Chromosome ◽

Fluorescent Microscopy ◽

S Phase ◽

Immunofluorescent Staining ◽

Heterochromatin Protein ◽

Inactive X Chromosome ◽

Xist Rna

Introduction . Inactive X chromosome (Xi) is associated with noncoding XIST RNA, series of proteins and contains multiple epigenetic modifications that altogether determine a silence of the most of X-linked genes. Recently the data were obtained that tumor suppressor BRCA1 is also associated with Xi. The purpose of this study was to reveal the colocalization of BRCA1 and XIST RNA and precise spatial organization on Xi with the high resolution of confocal microscopy.Materials and methods . The object of the study is IMR90hTERT diploid immortalized fibroblast cell line. For BRCA1 and XIST RNA colocalization analysis on Xi the method of fluorescent hybridization in situ associated with immunofluorescent cell staining (immunoFISH) and confocal microscopy were used. For BRCA1 and heterochromatin protein-1 colocalization study the method of double immunofluorescent staining and common fluorescent microscopy were applied. Results . The study using confocal fluorescent microscopy with higher resolution has demonstrated at first the colocalization of BRCA1 with XIST RNA region of Xi revealed with XIST RNA probes and with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. Altogether, the data obtained suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the regulation of XIST RNA association with Xi. Moreover, according to the results of confocal microscopy, BRCA1 also colocalizes with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. This indicates a possible involvement of this protein in the replication of pericentromeric repeats in cellular chromosomes. Colocalization of BRCA1 with heterochromatin protein-1α presented in pericentromeric regions of all chromosomes supports this suggestion.Conclusions . Altogether, the data obtained in this study suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the association with noncoding inhibiting XIST RNA and in replication of heterochromatin regions.

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X chromosome choice occurs independently of asynchronous replication timing

The Journal of Cell Biology ◽

10.1083/jcb.200405117 ◽

2005 ◽

Vol 168 (3) ◽

pp. 365-373 ◽

Cited By ~ 29

Author(s):

Joost Gribnau ◽

Sandra Luikenhuis ◽

Konrad Hochedlinger ◽

Kim Monkhorst ◽

Rudolf Jaenisch

Keyword(s):

X Chromosome ◽

Molecular Mechanisms ◽

Replication Timing ◽

S Phase ◽

X Inactivation ◽

Wild Type ◽

Gene Deletions ◽

Asynchronous Replication ◽

Skewed X Inactivation

In mammals, dosage compensation is achieved by X chromosome inactivation in female cells. Xist is required and sufficient for X inactivation, and Xist gene deletions result in completely skewed X inactivation. In this work, we analyzed skewing of X inactivation in mice with an Xist deletion encompassing sequence 5 KB upstream of the promoter through exon 3. We found that this mutation results in primary nonrandom X inactivation in which the wild-type X chromosome is always chosen for inactivation. To understand the molecular mechanisms that affect choice, we analyzed the role of replication timing in X inactivation choice. We found that the two Xist alleles and all regions tested on the X chromosome replicate asynchronously before the start of X inactivation. However, analysis of replication timing in cell lines with skewed X inactivation showed no preference for one of the two Xist alleles to replicate early in S-phase before the onset of X inactivation, indicating that asynchronous replication timing does not play a role in skewing of X inactivation.

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Delayed DNA replication in haploid human embryonic stem cells

10.1101/2021.05.11.443666 ◽

2021 ◽

Author(s):

Matthew Micheal Edwards ◽

Michael V. Zuccaro ◽

Ido Sagi ◽

Qiliang Ding ◽

Dan Vershkov ◽

...

Keyword(s):

Stem Cells ◽

Dna Replication ◽

Embryonic Stem Cells ◽

X Chromosome ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Replication Timing ◽

Genetic System ◽

X Chromosomes ◽

Genomic Regions

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X chromosome in haploids, consistent with the lack of X chromosome inactivation. Surprisingly, we also identified 21 autosomal regions that had dramatically delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also under-replicated in polyploid placental cells. The same delays were observed in female ESCs with two active X chromosomes, suggesting that increased X chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

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Delayed DNA replication in haploid human embryonic stem cells

Genome Research ◽

10.1101/gr.275953.121 ◽

2021 ◽

Author(s):

Matthew M. Edwards ◽

Michael V. Zuccaro ◽

Ido Sagi ◽

Qiliang Ding ◽

Dan Vershkov ◽

...

Keyword(s):

Stem Cells ◽

Dna Replication ◽

Embryonic Stem Cells ◽

X Chromosome ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Replication Timing ◽

Genetic System ◽

X Chromosomes ◽

Genomic Regions

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

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Functionally homologous DNA replication genes in fission and budding yeast

Journal of Cell Science ◽

10.1242/jcs.112.14.2381 ◽

1999 ◽

Vol 112 (14) ◽

pp. 2381-2390

Author(s):

M. Sanchez ◽

A. Calzada ◽

A. Bueno

Keyword(s):

Dna Replication ◽

Fission Yeast ◽

Kinase Activity ◽

Budding Yeast ◽

S Phase ◽

Yeast Gene ◽

Cdc2 Kinase ◽

Initiation Of Dna Replication

The cdc18(+) gene of the fission yeast Schizosaccharomyces pombe is involved in the initiation of DNA replication as well as in coupling the S phase to mitosis. In this work, we show that the Saccharomyces cerevisiae CDC6 gene complements cdc18-K46 ts and cdc18 deletion mutant S. pombe strains. The budding yeast gene suppresses both the initiation and the checkpoint defects associated with the lack of cdc18(+). The Cdc6 protein interacts in vivo with Cdc2 kinase complexes. Interestingly, Cdc6 is an in vitro substrate for Cdc13/Cdc2 and Cig1/Cdc2, but not for Cig2/Cdc2-associated kinases. Overexpression of Cdc6 in fission yeast induces multiple rounds of S-phase in the absence of mitosis and cell division. This CDC6-dependent continuous DNA synthesis phenotype is independent of the presence of a functional cdc18(+) gene product and, significantly, requires only Cig2/Cdc2-associated kinase activity. Finally, these S. pombe over-replicating cells do not require any protein synthesis other than that of Cdc6. Our data strongly suggest that CDC6 and cdc18(+) are functional homologues and also support the idea that controls restricting genome duplication diverge in fission and budding yeast.

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X chromosome reactivation in mouse embryonal carcinoma cells

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2705-2712.1985 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2705-2712

Author(s):

G D Paterno ◽

C N Adra ◽

M W McBurney

Keyword(s):

X Chromosome ◽

Dna Sequences ◽

Transcriptional Repression ◽

Embryonal Carcinoma ◽

S Phase ◽

Dna Demethylation ◽

Embryonal Carcinoma Cell ◽

X Chromosomes ◽

Embryonal Carcinoma Cell Line ◽

Inactive X Chromosome

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.

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