Chromosoma

Replication Timing (RT) refers to the temporal order in which the genome is replicated during S phase. Early replicating regions correlate with the transcriptionally active, accessible euchromatin (A) compartment, while late replicating regions correlate with the heterochromatin (B) compartment and repressive histone marks. Previously, widespread A/B genome compartmentalization changes were reported following Brd2 depletion. Since RT and A/B compartmentalization are two of the most highly correlated chromosome properties, we evaluated the effects of Brd2 depletion on RT. We performed E/L Repli-Seq following Brd2 depletion in the previously described Brd2 conditional degron cell line and found no significant alterations in RT after Brd2 KD. This finding prompted us to re-analyze the Micro-C data from the previous publication. We report that we were unable to detect any compartmentalization changes in Brd2 depleted cells compared to DMSO control using the same data. Taken together, our findings demonstrate that Brd2 depletion alone does not affect A/B compartmentalization or RT in mouse embryonic stem cells.

Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3 and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process - at prophase HDAC2 modulates most transcription-associated H3K9ac-marked loci and at metaphase HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity. Summary blurbCombination of immunofluorescence, western blot and ChIP-seq revealed the interplay between HDAC2, HDAC3 and SIRT1 in H3K9 deacetylation during mitosis of mammalian cells.

Chromatin is intrinsically repressive, limiting access to DNA, implying a major regulatory role. Studies with nuclei support this model. However, we have shown previously that genomic DNA is almost completely accessible in living budding yeast and human cells, except for centromeric chromatin. The fission yeast, Schizosaccharomyces pombe, possesses heterochromatin similar to mammalian heterochromatin at the pericentromeric repeats, telomeres and the silenced mating type loci. S. pombe heterochromatin is marked by histone H3K9 di- and tri-methylation (H3K9me2/3) and heterochromatin protein 1 (HP1/Swi6), potentially repressing genes by preventing access to the DNA. Here, we developed a copper-inducible DNA methyltransferase system to measure accessibility in living S. pombe cells. We find that euchromatin and heterochromatin are generally accessible, indicating that heterochromatin does not represent a significant block to DNA methyltransferases in vivo. S. pombe centromeres are much more accessible than budding yeast and human centromeres. In contrast, S. pombe chromatin is mostly inaccessible in isolated nuclei, primarily due to tight nucleosome spacing on gene bodies, with very little linker DNA. We conclude that S. pombe euchromatin and heterochromatin are both highly dynamic in vivo, suggesting that the H3K9me/HP1 system does not repress transcription by preventing access to DNA.

The architecture of mammalian mitotic chromosomes is considered to be universal across species and cell types. However, some studies suggest that features of mitotic chromosomes might be cell type or species specific. We previously reported that CTCF binding in human differentiated cell lines is lost in mitosis, whereas mouse embryonic stem cells (mESC) display prominent binding at a subset of CTCF sites in mitosis. Here, we perform parallel footprint ATAC-seq data analyses of mESCs and somatic mouse and human cells to further explore these differences. We then investigate roles of mitotically bound (bookmarked) CTCF in prometaphase chromosome organization by Hi-C. We do not find any remaining interphase structures such as TADs or CTCF loops at mitotically bookmarked CTCF sites in mESCs. This suggests that mitotic loop extruders condensin I and II are not blocked by bound CTCF, and thus that any remaining CTCF binding does not alter mitotic chromosome folding. Lastly, we compare mitotic Hi-C data generated in this study in mouse with publicly available data from human and chicken cell lines. We do not find any cell type specific differences; however, we do find a difference between species. The average genomic size of mitotic loops is much smaller in chicken (200-350 kb), compared to human (500-750 kb) and mouse (1-2 mb). Interestingly, we find that this difference in loop size is correlated with the average genomic length of the q-arm in these species, a finding we confirm by microscopy measurements of chromosome compaction. This suggests that the dimensions of mitotic chromosomes can be modulated through control of sizes of loops generated by condensins to facilitate species-appropriate shortening of chromosome arms.

C9ORF78 is a poorly characterized protein found in diverse eukaryotes. Previous work indicated overexpression of hC9ORF78 (aka HCA59) in malignant tissues indicating a possible involvement in growth regulatory pathways. Additional studies in fission yeast and humans uncover a potential function in regulating the spliceosome. In studies of GFP-tagged hC9ORF78 we observed a dramatic reduction in protein abundance in cells grown to confluence and/or deprived of serum growth factors. Serum stimulation induced synchronous re-expression of the protein in HeLa cells. This effect was also observed with the endogenous protein. Overexpressing either E2F1 or N-Myc resulted in elevated hC9ORF78 expression potentially explaining the serum-dependent upregulation of the protein. Immunofluorescence analysis indicates that hC9ORF78 localizes to nuclei in interphase but does not appear to concentrate in speckles as would be expected for a splicing protein. Surprisingly, a subpopulation of hC9ORF78 co-localizes with ACA, Mad1 and Hec1 in mitotic cells suggesting that this protein may associate with kinetochores or centromeres. Furthermore, knocking-down hC9ORF78 caused mis-alignment of chromosomes in mitosis. These studies uncover novel mitotic function and subcellular localization of cancer antigen hC9ORF78. SUMMARY STATEMENThC9ORF78 regulates chromosome segregation.

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by abnormal distribution of genetic information. Polo-like kinase 1 interacting checkpoint helicase (PICH) is a DNA translocase essential in chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by Small Ubiquitin-like modifier (SUMO). However, it is unclear how these activities are cooperating to resolve chromosome bridges. Here, we show that PICH specifically promotes the organization of SUMOylated proteins like SUMOylated TopoisomeraseIIα (TopoIIα) on mitotic chromosomes. Conditional depletion of PICH using the Auxin Inducible Degron (AID) system resulted in the retention of SUMOylated chromosomal proteins, including TopoIIα, indicating that PICH functions to control proper association of these proteins with chromosomes. Replacement of PICH with its mutants showed that PICH is required for the proper organization of SUMOylated proteins on chromosomes. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taken together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.Summary Statement Polo-like kinase interacting checkpoint helicase (PICH) interacts with SUMOylated proteins to mediate proper chromosome segregation during mitosis. The results demonstrate that PICH controls association of SUMOylated chromosomal proteins, including Topoisomerase IIα, and that function requires PICH translocase activity and SUMO binding ability.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsTopoIIαTopoisomerase IIαPICHPolo-like kinase interacting checkpoint helicaseSPRStrand passage reactionSUMOSmall ubiquitin-like modifierXEEXenopus egg extractCSFCytostatic factordnUbc9dominant negative E2 SUMO-conjugating enzymeSENPSentrin-specific proteasePIASProtein inhibitor of activated STATSIMSUMO-interacting-motifView Full Text

Centromeres are essential chromosomal components that ensure proper cell division by serving as assembly sites for kinetochores, which connect chromosomes to spindle microtubules. Centromeres are marked by the evolutionarily conserved centromere-specific histone H3 variant, CENP-A, which is deposited into centromere nucleosomes during G1 in human cells. Centromeres retain cohesin, a ring-like protein complex during mitosis, protecting sister chromatid cohesion and centromere transcription to prevent chromosome missegregation. Previous work in Drosophila has suggested that centromere transcription and centromeric RNAs are important for CENP-A deposition in chromatin. During mitosis centromeric cohesin is critical for centromere transcription. However, it is not clear how or if centromeric transcription and cohesin contribute to CENP-A deposition in G1 in human cells. To address these questions, we combined a cell synchronization strategy with the Auxin Inducible Degron technology and transcription inhibition in human cells. In contrast to Drosophila cells, our results demonstrated that neither centromeric transcription nor cohesin is required for CENP-A deposition in human cells. Our data demonstrate clear differences in the CENP-A deposition mechanism between human and Drosophila cells. These findings provide deeper insights into the plasticity underlying centromere maintenance and highlight evolutionary divergence in centromere maintenance systems across species.

Centromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of sequencing and assembling repetitive genomic regions. Using recently available PacBio long-read sequencing data from the C57BL/6 strain and chromatin profiling, we found that contrary to the previous reports of their highly homogeneous nature, centromeric and pericentromeric satellites display varied sequences and organization. We find that both centromeric minor satellites and pericentromeric major satellites exhibited sequence variations within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Additionally, we investigated CENP-A and H3K9me3 chromatin organization at centromeres and pericentromeres using Chromatin immunoprecipitation sequencing (ChIP-seq). We found that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence abundance and homogeneity at these regions. Furthermore, the transposable elements at centromeric regions are not part of functional centromeres as they lack CENP-A enrichment. Finally, we found that while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays lack phased organization. Interestingly, the homogeneous class of major satellites phase CENP-A and H3K27me3 nucleosomes as well, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites. Overall, our findings reveal that house mouse centromeres and pericentromeres, which were previously thought to be highly homogenous, display significant diversity in satellite sequence, organization, and chromatin structure.

BackgroundThe number and escape levels of genes that escape X chromosome inactivation (XCI) in female somatic cells vary among tissues and cell types, potentially contributing to specific sex differences. Here we investigate the role of CTCF, a master chromatin conformation regulator, in regulating escape from XCI. CTCF binding profiles and epigenetic features were systematically examined at constitutive and facultative escape genes using mouse allelic systems to distinguish the inactive X (Xi) and active X (Xa) chromosomes. ResultsWe found that escape genes are located inside domains flanked by convergent arrays of CTCF binding sites, consistent with the formation of loops. In addition, strong and divergent CTCF binding sites often located at the boundaries between escape genes and adjacent neighbors subject to XCI would help insulate domains. Facultative escapees show clear differences in CTCF binding dependent on their XCI status in specific cell types/tissues. Concordantly, deletion but not inversion of a CTCF binding site at the boundary between the facultative escape gene Car5b and its silent neighbor Siah1b resulted in loss of Car5b escape. Reduced CTCF binding and enrichment of a repressive mark over Car5b in cells with a boundary deletion indicated loss of looping and insulation. In mutant lines in which either the Xi-specific compact structure or its H3K27me3 enrichment was disrupted, escape genes showed an increase in gene expression and associated active marks, supporting the roles of the 3D Xi structure and heterochromatic marks in constraining levels of escape. ConclusionOur findings indicate that escape from XCI is modulated both by looping and insulation of chromatin via convergent arrays of CTCF binding sites and by compaction and epigenetic features of the surrounding heterochromatin.

Members of the conserved subfamily, JADE1S and JADE1L isoforms, are expressed in epithelial cells, fibroblasts, and epithelial cell lining in vivo. JADE1 proteins interact with histone acetyl transferase HBO1 complex. The two consecutive PHD zinc fingers of JADE1 bind chromatin. We recently reported novel effects of JADE1S on cytokinesis progression. JADE1S depletion facilitated G2/M-to-G1 transition and increased polyploidy and aneuploidy. JADE1S over-expression arrested cells in late cytokinesis, an effect reversed by AURKB inhibitor. In late cytokinesis cells JADE1S protein localized to the midbody. Results suggested a JADE1S role in final abscission delay. Here we investigated the expression of JADE1 in the central spindle, interactions with HBO1, and the role of PHD fingers in late cytokinesis arrest. The midzone begins to assemble in anaphase and forms into a midbody in cytokinesis. The midbody structure connects two daughter cells and is thought to bear factors controlling the final abscission. We questioned whether, similar to established factors, JADE1S is targeted to the central spindle structures in anaphase. Indeed, in cells transitioning from mitosis to cytokinesis, JADE1S was sequentially targeted to early midzone, midbody flanking zone, and midbody. The step-wise increase of JADE1S expression in midzone and midbody of synchronously dividing cells suggested protein recruitment. The increase of late cytokinesis arrest caused by recombinant JADE1S correlated with increased expression in midbody. Spatial analysis of the members of the chromatin passenger complex, microtubule associated proteins, and centralspindlin, revealed transient co-localization with JADE1S and mapped JADE1S within the cytokinesis bridge. Deletion of the two PHD zinc fingers inactivated JADE1S ability to arrest cells in late cytokinesis but did not affect its midbody localization. Thus, PHD zinc fingers are required for JADE1S cytokinesis delay but not for midbody targeting. Recombinant HBO1 protein decreased the proportion of late cytokinesis cells, prevented late cytokinesis arrest by JADE1S as well as its midbody localization. Enzyme inactive HBO1 mutant recapitulated the wild type phenotype. The results demonstrate antagonistic relationship and suggest HBO1-mediated midbody dislocation of JADE1S. Our study supports the role of JADE1S in cytokinesis delay and implicates protein partners.

During prophase I of meiosis, DNA double-strand breaks form throughout the genome, with a subset repairing as crossover events, enabling the accurate segregation of homologous chromosomes during the first meiotic division. The mechanism by which DSBs become selected to repair as crossovers is unknown, although the crossover positioning and levels in each cell indicate it is a highly regulated process. One of the proteins that localises to crossover sites is the serine/threonine cyclin-dependent kinase CDK2. Regulation of CDK2 occurs via phosphorylation at tyrosine 15 (Y15) and threonine 160 (T160) inhibiting and activating the kinase, respectively. In this study we use a combination of immunofluorescence staining on spread spermatocytes and fixed testis sections, and STA-PUT gravitational sedimentation to isolate cells at different developmental stages to further investigate the temporal phospho regulation of CDK2 during prophase I. Western blotting reveals differential levels of the two CDK2 isoforms (CDK233kDa and CDK239kDa) throughout prophase I, with inhibitory phosphorylation of CDK2 at Y15 occurring early in prophase I, localising to telomeres and diminishing as cells enter pachynema. Conversely, the activatory phosphorylation on T160 occurs later, specifically the CDK233kDa isoform, and T160 signal is detected in spermatogonia and pachytene spermatocytes, where it co-localises with the Class I crossover protein MLH3. Taken together, our data reveals intricate control of CDK2 both with regards to levels of the two CDK2 isoforms, and differential regulation via inhibitory and activatory phosphorylation.

The three-dimensional configuration of the eukaryotic genome is an emerging area of research. Chromosome conformation capture outlined genome segregation into large scale A and B compartments corresponding mainly to transcriptionally active and repressive chromatin. It remains unknown how the compartmentalization of the genome changes in growing oocytes of animals with hypertranscriptional type of oogenesis. In this type of oogenesis, highly elongated chromosomes, called lampbrush chromosomes, acquire a characteristic chromomere-loop appearance, representing one of the classical model systems for studying the structural and functional organization of chromatin domains. Here, we compared the distribution of A/B compartments in chicken somatic cells with chromatin domains in lampbrush chromosomes. We found that in lampbrush chromosomes, the extended chromatin domains, restricted by compartment boundaries in somatic cells, disintegrate into individual chromomeres. Next, we performed FISH-mapping of the genomic loci, which belong to A or B chromatin compartments as well as to A/B compartment transition regions in embryonic fibroblasts on isolated lampbrush chromosomes. We established, that in chicken lampbrush chromosomes, clusters of dense compact chromomeres bearing short lateral loops and enriched with repressive epigenetic modifications generally correspond to constitutive B compartments in somatic cells. These results suggest that gene-poor regions tend to be packed into chromomeres. Clusters of small loose chromomeres with relatively long lateral loops show no obvious correspondence with either A or B compartment identity. Some genes belonging to facultative B (sub-) compartments can be tissue-specifically transcribed during oogenesis, forming distinct lateral loops.

Mitotic tethers connect partner telomeres of all segregating anaphase chromosomes in all animal cells that have been tested, as detected by laser-cutting chromosome arms during anaphase and seeing that the arm fragments move rapidly across the equator to their partner chromosome moving to the opposite pole, telomere moving towards telomere. Tethers exert anti-poleward forces on the poleward separating telomeres, but tether elasticity (that produces the backwards forces) diminishes during anaphase: as determined by the behavior of arm fragments; short tethers (early anaphase) are elastic, long tethers (late anaphase) are not elastic, and medium-length tethers transition between the two states. We developed a procedure in which the tethers still functioned after we partially-lysed anaphase crane-fly spermatocytes. The partial lysis consistently arrested chromosome movements, after which the tethers moved the chromosomes backwards, potentially allowing the elastic tethers to be studied biochemically. To ensure that tether function was not altered by the partial cell-lysis procedure, we compared backward chromosome movements in partially-lysed cells with arm fragment movements in control cells. In the partially-lysed cells the backward chromosomal movements had characteristics identical to those of arm fragments in non-lysed (control) cells. In particular, in both control and partially-lysed cells shorter tethers caused backward movements more often than did longer tethers; shorter tethers caused backward movements over greater fractional distances (of the tether) than did longer tethers; and velocities of the backwards movements were the same for tethers of different lengths. We also compared the effects of Calyculin A (an inhibitor of Protein Phosphatase1) in control versus in partially-lysed cells. Calyculin A (CalA) added to control cells in early anaphase blocks dephosphorylation, thereby maintaining tether elasticity throughout anaphase: after the chromosomes reach the poles they move backwards when the usual poleward forces are reduced. Partial lysis preserves this tether functionality: after partial lysis of CalA-treated cells the chromosomes move backward and reach the partner telomeres at even very long tether lengths. We conclude that partial cell-lysis arrests anaphase chromosome poleward movement but does not affect tether function.

We show here that treatment of HeLa cells with calyculin A, an inhibitor of Protein Phosphatases 1 and 2A, induces premature chromosome condensation (PCC) at any point in interphase of the cell cycle. Chromosomes in G1-phase PCC closely resemble metaphase chromatids in the light microscope, and measurements using FLIM-FRET show that they have the same level of chromatin compaction as metaphase chromosomes. However, histone H1 is not phosphorylated in G1- or early S-phase PCC. These results suggest that H1 phosphorylation is not required for mitotic chromosome condensation and chromatin compaction. They also confirm that Cdk1/cyclin B, which directly phosphorylates histone H1, is not active in G1 and thus is not essential for G1- PCC. We suggest that induction of G1-PCC involves protein kinases or other factors that are either held in an inactive state by protein phosphatases, or constitutively active but countered by phosphatases. The same factors may be involved in the onset of normal mitosis, becoming active when protein phosphatases are downregulated. Induction of PCC with calyculin A should provide a useful system for identifying and studying the biochemical pathways that are required for mitotic chromosome compaction, nuclear envelope breakdown, and other events of mitosis.

We explored the role of NAT10 acetyltransferase in the DNA damage response, focusing on its impact on 3D-genome architecture and DNA repair proteins. Compared to NAT10 wild-type (wt), NAT10 deficiency reduced XPC, DDB2, and p53 protein levels. In TP53 double-null (dn) cells, the NAT10 protein was undetectable, and DDB2 was significantly down-regulated. Although NAT10 depletion caused DDB2 down-regulation, it did not affect the DNA repair functions of the DDB2 protein. To this fact, protein interaction analysis revealed that UVC exposure weakens the DDB2-p53 interaction while strengthening the bond between NAT10 and DDB2. Also, AlphaFold 3 prediction tools showed a more potent interaction between DDB2 and p53 than DDB2 and NAT10 proteins implying that NAT10 rather regulates the DDB2-p53 protein complex. These proteomic NAT10-dependent changes coincided with alterations in chromatin interactions, particularly in acrocentric chromosomes, studied by the Hi-C technique. However, 3D-genome rearrangement, caused by NAT10 deficiency and UVC irradiation, did not significantly impact post-translational histone modifications. Overall, NAT10 depletion alters the pool of key DNA repair proteins and induces substantial 3D-genome reorganization. Graphical abstractThe effect of the NAT10 acetyltransferase depletion and UVC irradiation on 3D-genome nuclear architecture, histone signature, and the pool of selected DNA repair-related proteins. The figure was made using some icons adapted from BioRender software.

Chromosome congression and alignment on the metaphase plate involves lateral and microtubule plus-end interactions with the kinetochore. Here we take advantage of our ability to efficiently generate a GFP-marked acentric X chromosome fragment in Drosophila neuroblasts to identify forces acting on chromosome arms that drive congression and alignment. We find acentrics efficiently align on the metaphase plate, often more rapidly than kinetochore-bearing chromosomes. Unlike intact chromosomes, the paired sister acentrics oscillate as they move to and reside on the metaphase plate in a plane distinct and significantly further from the main mass of intact chromosomes. Consequently, at anaphase onset acentrics are oriented either parallel or perpendicular to the spindle. Parallel-oriented sisters separate by sliding while those oriented perpendicularly separate via unzipping. This oscillation, together with the fact that in monopolar spindles acentrics are rapidly shunted away from the poles, indicates that distributed plus-end directed forces are primarily responsible for acentric migration. This conclusion is supported by the observation that reduction of EB1 preferentially disrupts acentric alignment. In addition, reduction of Klp3a activity, a gene required for the establishment of pole-to-pole microtubules, preferentially disrupts acentric alignment. Taken together these studies suggest that plus-end forces mediated by the outer pole-to-pole microtubules are primarily responsible for acentric metaphase alignment. Surprisingly, we find that a small fraction of sister acentrics are anti-parallel aligned indicating that the kinetochore is required to ensure parallel alignment of sister chromatids. Finally, we find induction of acentric chromosome fragments results in a global reorganization of the congressed chromosomes into a torus configuration. Article SummaryThe kinetochore serves as a site for attaching microtubules and allows for successful alignment, separation, and segregation of replicated sister chromosomes during cell division. However, previous studies have revealed that sister chromosomes without kinetochores (acentrics) often align to the metaphase plate, undergo separation and segregation, and are properly transmitted to daughter cells. In this study, we discuss the forces acting on chromosomes, independent of the kinetochore, underlying their successful alignment in early mitosis.

BackgroundOrganization of the eukaryotic genome is essential for proper function, including gene expression. In metazoans, chromatin loops and Topologically Associated Domains (TADs) organize genes into transcription factories, while chromosomes occupy nuclear territories in which silent heterochromatin is compartmentalized at the nuclear periphery and active euchromatin localizes to the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation typified by heterochromatic centromeres and telomeres independently clustering at the nuclear membrane, while interspersed heterochromatic loci aggregate across Megabases of linear genomic distance to loop chromatin in TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. ResultsWe examined the genome organization of a Neurospora strain harboring a [~]47.4 kilobase deletion within a temporarily silent, facultative heterochromatic region, as well as the genome organization of a strain deleted of a 110.6 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion minimally effects local chromatin structure or telomere clustering, the constitutive heterochromatin deletion alters local chromatin structure, the predicted three-dimensional chromosome conformation, and the expression of some genes, which are qualitatively repositioned into the nucleus center, while increasing Hi-C variability. ConclusionsOur work elucidates how an individual constitutive heterochromatic region impacts genome organization and function. Specifically, one silent region indirectly assists in the hierarchical folding of the entire Neurospora genome by aggregating into the "typical" heterochromatin bundle normally observed in wild type nuclei, which may promote normal gene expression by positioning euchromatin in the nucleus center.

We describe the anatomy of replication forks by comparing the lengths of synthesized BrdU-labelled DNA in wild type, mrc1{Delta} and cds1{Delta} Schizoasaccharomyces pombe. We correlated Rad51 and Cdc45 proteins relative to their positions on the fork, replicated tract, or unreplicated regions. We did this by using chromatin fiber images. These fibers track pixel intensity data, which is analyzed using our program: R-ODD-BLOBS. We compared the lengths of BrdU tracts and proteins, as well as the percentage of Rad51 and Cdc45 colocalization, and compared our results with literature findings. We measured average BrdU lengths consistent with current literature; cds1{Delta} was the longest at [~]2.9 kb (8.6 pixels, px), wild type was [~] 2.5 kb (7.5 px), and mrc1{Delta} was the shortest at [~]1.7 kb (5.1 px). Intriguingly, Rad51 was found at 22% more replicated areas in mrc1{Delta} than in wild type. This suggests that homologous recombination repair may be more common at mrc1{Delta} forks. In this study, we summarize the usefulness of a computational modeling tool to assess large datasets of chromatin spread data. In turn, we find patterns of DNA replication length and protein components at replication forks, to describe the anatomy of a fork and how structures change with checkpoint loss. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/621594v2_figa1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1fd59forg.highwire.dtl.DTLVardef@1f234org.highwire.dtl.DTLVardef@1c46b51org.highwire.dtl.DTLVardef@61ce37_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACT:C_FLOATNO R-ODD-BLOBS uses chromatin fiber data to rigorously model replication fork structures. DNA replication forks are multi-subunit structures that must pair and regulate DNA copying activity of the polymerases with unwinding activity of helicase. Chromatin fiber data retains proteins, and can be used to detect DNA synthesis (blue) and associated DNA replication fork proteins such as MCM4 helicase (MCM4) and replication protein A (RPA). In our work, we have used homologous recombination protein Rad51 and helicase factor Cdc45 to understand how DNA replication fork structures are destabilized during hydroxyurea treatment, and how they fail to recover because of Cdc45/helicase mis-localization. C_FIG

Mammalian genomes are partitioned into sub-megabase to megabase-sized units of preferential interactions called topologically associating domains or TADs, which are likely important for the proper implementation of gene regulatory processes. These domains provide structural scaffolds for distant cis regulatory elements to interact with their target genes within the three-dimensional nuclear space and architectural proteins such as CTCF as well as the cohesin complex participate in the formation of the boundaries between them. However, the importance of the genomic context in providing a given DNA sequence the capacity to act as a boundary element remains to be fully investigated. To address this question, we randomly relocated a topological boundary functionally associated with the mouse HoxD gene cluster and show that it can indeed act similarly outside its initial genomic context. In particular, the relocated DNA segment recruited the required architectural proteins and induced a significant depletion of contacts between genomic regions located across the integration site. The host chromatin landscape was re-organized, with the splitting of the TAD wherein the boundary had integrated. These results provide evidence that topological boundaries can function independently of their site of origin, under physiological conditions during mouse development. AUTHOR SUMMARYDuring development, enhancer sequences tightly regulate the spatio-temporal expression of target genes often located hundreds of kilobases away. This complex process is made possible by the folding of chromatin into domains, which are separated from one another by specific genomic regions referred to as boundaries. In order to understand whether such boundary sequences require their particular genomic contexts to achieve their isolating effect, we analyzed the impact of introducing one such boundary, taken from the HoxD gene cluster, into a distinct topological domain. We show that this ectopic boundary splits the host domain into two sub-domains and affects the expression levels of a neighboring gene. We conclude that this sequence can work independently from its genomic context and thus carries all the information necessary to act as a boundary element.

The centromeric protein-A (CENP-A) is an evolutionary conserved histone H3 variant that marks the identity of the centromeres. Several mechanisms regulate the centromeric deposition of CENP-A as its mislocalization causes erroneous chromosome segregation, leading to aneuploidy-based diseases, including cancers. The most crucial deposition factor is a CENP-A specific chaperone, HJURP (Scm3 in budding yeast), which specifically binds to CENP-A. However, the discovery of HJURP as a DDR (DNA damage repair) protein and evidence of its binding to Holliday junctions in vitro indicate a CENP-A-deposition-independent role of these chaperones. In this study, using budding yeast, we demonstrate that Scm3 is crucial for the DDR pathway as scm3 cells are sensitive to DNA damage. We further observe that the scm3 mutant interacts with the rad52 DDR mutant and is compromised in activating DDR-mediated arrest. We demonstrate that Scm3 associates with the DNA damage sites and undergoes posttranslational modifications upon DNA damage. Overall, from this report and earlier studies on HJURP, we conclude that DDR functions of CENP-A chaperones are conserved across eukaryotes. Thus, the revelation that these chaperones confer genome stability in more than one pathway has clinical significance.