Chromosome Banding and Heterochromatin in Vicia Faba

<p>This study documents the distribution of bands in Vicia faba root-tip chromosomes as shown by acid treatment, quinacrine mustard fluorescence, various forms of Giemsa banding and orcein banding methods, and demonstrates the coincidence of these bands with the position of heterochromatin as shown by cold treatment and late replication. Heterochromatin in the large metacentric M chromosome is located in two areas: (a) around the centromere and (b) adjacent to the secondary constriction. The latter is not late-replicating but is judged to represent classical nucleolus-associated heterochromatin. Heterochromatin in the smaller sub-telocentric S chromosomes is located in the intercalary and proximal areas of their long arms and in the short arm of two chromosomes. The variable expression of particular chromosome segments with different banding techniques testifies to certain differences between heterochromatic regions and emphasizes the existence of several classes of heterochromatin. In situ molecular hybridization of labelled complementary RNA to chromosomal DNA indicates the presence of repetitive DNA in both euchromatin and heterochromatin of the V. faba genome.</p>

Chromosome Banding and Heterochromatin in Vicia Faba

<p>This study documents the distribution of bands in Vicia faba root-tip chromosomes as shown by acid treatment, quinacrine mustard fluorescence, various forms of Giemsa banding and orcein banding methods, and demonstrates the coincidence of these bands with the position of heterochromatin as shown by cold treatment and late replication. Heterochromatin in the large metacentric M chromosome is located in two areas: (a) around the centromere and (b) adjacent to the secondary constriction. The latter is not late-replicating but is judged to represent classical nucleolus-associated heterochromatin. Heterochromatin in the smaller sub-telocentric S chromosomes is located in the intercalary and proximal areas of their long arms and in the short arm of two chromosomes. The variable expression of particular chromosome segments with different banding techniques testifies to certain differences between heterochromatic regions and emphasizes the existence of several classes of heterochromatin. In situ molecular hybridization of labelled complementary RNA to chromosomal DNA indicates the presence of repetitive DNA in both euchromatin and heterochromatin of the V. faba genome.</p>

Regulation of Mus81-Eme1 structure-specific endonuclease by Eme1 SUMO-binding and Rad3(ATR) kinase is essential in the absence of Rqh1(BLM) helicase

The Mus81-Eme1 structure-specific endonuclease is crucial for the processing of DNA recombination and late replication intermediates. In fission yeast, stimulation of Mus81-Eme1 in response to DNA damage at the G2/M transition relies on Cdc2(CDK1) and DNA damage checkpoint-dependent phosphorylation of Eme1 and is critical for chromosome stability in absence of the Rqh1(BLM) helicase. Here we identify Rad3(ATR) checkpoint kinase consensus phosphorylation sites and two SUMO interacting motifs (SIM) within a short N-terminal domain of Eme1 that is required for cell survival in absence of Rqh1(BLM). We show that catalytic stimulation of Mus81-Eme1 depends entirely on direct phosphorylation of Eme1 by Rad3(ATR) and that while Eme1 also undergoes Chk1-mediated phosphorylation, this is not essential for catalytic modulation. Both Rad3(ATR)- and Chk1-mediated phosphorylation of Eme1 as well as the SIMs are independently critical for cell fitness in absence of Rqh1(BLM) and abrogating bimodal phosphorylation of Eme1 along with mutating the SIMs is incompatible with rqh1∆ cell viability. Our findings unravel an elaborate regulatory network that is essential for Mus81-Eme1 to fulfill functions that are essential in absence of Rqh1(BLM).

Mutational signature SBS8 predominantly arises due to late replication errors in cancer

Although a majority of somatic mutations in cancer are passengers, their mutational signatures provide mechanistic insights into mutagenesis and DNA repair processes. Mutational signature SBS8 is common in most cancers, but its etiology is debated. Incorporating genomic, epigenomic, and cellular process features for multiple cell-types we develop genome-wide composite epigenomic context-maps relevant for mutagenesis and DNA repair. Analyzing somatic mutation data from multiple cancer types in their epigenomic contexts, we show that SBS8 preferentially occurs in gene-poor, lamina-proximal, late replicating heterochromatin domains. While SBS8 is uncommon among mutations in non-malignant tissues, in tumor genomes its proportions increase with replication timing and speed, and checkpoint defects further promote this signature - suggesting that SBS8 probably arises due to uncorrected late replication errors during cancer progression. Our observations offer a potential reconciliation among different perspectives in the debate about the etiology of SBS8 and its relationship with other mutational signatures.

Pervasive transcription-dependent chromatin remodeling influences the replication initiation program

ABSTRACTIn Eukaryotic organisms, replication initiation follows a temporal program. Among the parameters that regulate this program in Saccharomyces cerevisiae, chromatin structure has been at the center of attention without considering the contribution of transcription. Here, we revisit the replication initiation program in the light of pervasive transcription. We find that noncoding RNA transcription termination in the vicinity of replication origins or ARS (Autonomously Replicating Sequences) maximizes replication initiation by restricting transcriptional readthrough into ARS. Consistently, high natural nascent transcription correlates with low ARS efficiency and late replication timing. High readthrough transcription is also linked to chromatin features such as high levels of H3K36me3 and deacetylated nucleosomes. Moreover, forcing ARS readthrough transcription promotes these histone modifications. Finally, replication initiation defects induced by increased transcriptional readthrough are partially rescued in the absence of H3K36 methylation. Altogether, these observations indicate that natural pervasive transcription into ARS influences replication initiation through chromatin remodeling.

Replication timing shapes the cancer epigenome and the nature of chromosomal rearrangements

HighlightsReplication timing alterations are conserved in cancers of different cell originsLong-range epigenetic deregulation in cancer involves altered replication timingCancer late-replicating loci are hypomethylated and acquire facultative heterochromatinReplication timing status potentiates cis and trans chromosomal rearrangementsSummaryReplication timing is known to facilitate the establishment of epigenome, however, the intimate connection between DNA replication timing and changes to the genome and epigenome in cancer remain uncharted. Here, we perform Repli-Seq and integrated epigenome analysis and show that early-replicating loci are predisposed to hypermethylation and late-replicating loci to hypomethylation, enrichment of H3K27me3 and concomitant loss of H3K9me3. We find that altered replication timing domains correspond to long-range epigenetically deregulated regions in prostate cancer, and a subset of these domains are remarkably conserved across cancers from different tissue origins. Analyses of 214 prostate and 35 breast cancer genomes reveal that late-replicating DNA is prone to cis and early-replicating DNA to trans chromosomal rearrangements. We propose that differences in epigenetic deregulation related to spatial and temporal positioning between early and late replication potentiate the landscape of chromosomal rearrangements in cancer.