SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold attachment factor A (SAF-A), also known as Heteronuclear Ribonucleoprotein Protein U (HNRNPU), contributes to the formation of open chromatin structure. Here we demonstrate that SAF-A promotes the normal progression of DNA replication, and enables resumption of replication after inhibition. We report that cells depleted for SAF-A show reduced origin licensing in G1 phase, and consequently reduced origin activation frequency in S phase. Replication forks also progress less consistently in cells depleted for SAF-A, contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed two distinct effects of SAF-A depletion: first, the boundaries between early- and late-replicating domains become more blurred; and second, SAF-A depletion causes replication timing changes that tend to bring regions of discordant domain compartmentalisation and replication timing into concordance. Associated with these defects, SAF-A-depleted cells show elevated -H2AX formation and tend to enter quiescence. Overall we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains

Cohesin organizes the genome by forming intra-chromosomal loops and inter-sister chromatid linkages. During gamete formation by meiosis, chromosomes are reshaped to support crossover recombination and two consecutive rounds of chromosome segregation. Here we show that Eco1 acetyltransferase positions both chromatin loops and sister chromatid cohesion to organize meiotic chromosomes into functional domains in budding yeast. Eco1 acetylates the Smc3 cohesin subunit in meiotic S phase to establish chromatin boundaries, independently of DNA replication. Boundary formation by Eco1 is critical for prophase exit and for the maintenance of cohesion until meiosis II, but is independent of the ability of Eco1 to antagonize the cohesin-release factor, Wpl1. Conversely, prevention of cohesin release by Wpl1 is essential for centromeric cohesion, kinetochore monoorientation and co-segregation of sister chromatids in meiosis I. Our findings establish Eco1 as a key determinant of chromatin boundaries and cohesion positioning, revealing how local chromosome structuring directs genome transmission into gametes.

HiCEnterprise: identifying long range chromosomal contacts in Hi-C data

Motivation Computational analysis of chromosomal contact data is currently gaining popularity with the rapid advance in experimental techniques providing access to a growing body of data. An important problem in this area is the identification of long range contacts between distinct chromatin regions. Such loops were shown to exist at different scales, either mediating relatively short range interactions between enhancers and promoters or providing interactions between much larger, distant chromosome domains. A proper statistical analysis as well as availability to a wide research community are crucial in a tool for this task. Results We present HiCEnterprise, a first freely available software tool for identification of long range chromatin contacts not only between small regions, but also between chromosomal domains. It implements four different statistical tests for identification of significant contacts for user defined regions or domains as well as necessary functions for input, output and visualization of chromosome contacts. Availability The software and the corresponding documentation are available at: github.com/regulomics/HiCEnterprise. Supplementary information Supplemental data are available in the online version of the article and at the website regulomics.mimuw.edu.pl/wp/hicenterprise.

SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold attachment factor A (SAF-A), also known as Heteronuclear Ribonucleoprotein Protein U (HNRNPU), contributes to the formation of open chromatin structure. Here we demonstrate that SAF-A promotes the normal progression of DNA replication, and enables resumption of replication after inhibition. We report that cells depleted for SAF-A show reduced origin licensing in G1 phase, and consequently reduced origin activation frequency in S phase. Replication forks progress slowly in cells depleted for SAF-A, also contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed that the boundaries between early- and late- replicating domains are blurred in cells depleted for SAF-A. Associated with these defects, SAF-A-depleted cells show elevated gH2A phosphorylation and tend to enter quiescence. Overall we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

Unscheduled origin building in S-phase upon tight CDK1 inhibition suppresses CFS instability

SummaryGenome integrity requires replication to be completed before chromosome segregation. This coordination essentially relies on replication-dependent activation of a dedicated checkpoint that inhibits CDK1, delaying mitotic onset. Under-replication of Common Fragile Sites (CFSs) however escapes surveillance, which triggers chromosome breakage. Using human cells, we asked here whether such leakage results from insufficient CDK1 inhibition under modest stresses used to destabilize CFSs. We found that tight CDK1 inhibition suppresses CFS instability. Repli-Seq and molecular combing analyses consistently showed a burst of replication initiations in mid S phase across large origin-poor domains shaped by transcription, including CFSs. Strikingly, CDC6 or CDT1 depletion or CDC7-DBF4 inhibition during the S phase prevented both extra-initiations and CFS rescue, showing that CDK1 inhibition promotes targeted and mistimed building of functional extra-origins. In addition to delay mitotic onset, checkpoint activation therefore advances replication completion of chromosome domains at risk of under-replication, two complementary roles preserving genome stability.

Interphase Cytogenetic Analysis of G0 Lymphocytes Exposed to α-Particles, C-Ions, and Protons Reveals their Enhanced Effectiveness for Localized Chromosome Shattering—A Critical Risk for Chromothripsis

For precision cancer radiotherapy, high linear energy transfer (LET) particle irradiation offers a substantial advantage over photon-based irradiation. In contrast to the sparse deposition of low-density energy by χ- or γ-rays, particle irradiation causes focal DNA damage through high-density energy deposition along the particle tracks. This is characterized by the formation of multiple damage sites, comprising localized clustered patterns of DNA single- and double-strand breaks as well as base damage. These clustered DNA lesions are key determinants of the enhanced relative biological effectiveness (RBE) of energetic nuclei. However, the search for a fingerprint of particle exposure remains open, while the mechanisms underlying the induction of chromothripsis-like chromosomal rearrangements by high-LET radiation (resembling chromothripsis in tumors) await to be elucidated. In this work, we investigate the transformation of clustered DNA lesions into chromosome fragmentation, as indicated by the induction and post-irradiation repair of chromosomal damage under the dynamics of premature chromosome condensation in G0 human lymphocytes. Specifically, this study provides, for the first time, experimental evidence that particle irradiation induces localized shattering of targeted chromosome domains. Yields of chromosome fragments and shattered domains are compared with those generated by γ-rays; and the RBE values obtained are up to 28.6 for α-particles (92 keV/μm), 10.5 for C-ions (295 keV/μm), and 4.9 for protons (28.5 keV/μm). Furthermore, we test the hypothesis that particle radiation-induced persistent clustered DNA lesions and chromatin decompaction at damage sites evolve into localized chromosome shattering by subsequent chromatin condensation in a single catastrophic event—posing a critical risk for random rejoining, chromothripsis, and carcinogenesis. Consistent with this hypothesis, our results highlight the potential use of shattered chromosome domains as a fingerprint of high-LET exposure, while conforming to the new model we propose for the mechanistic origin of chromothripsis-like rearrangements.

Replication timing alterations in leukemia affect clinically relevant chromosome domains

Key Points DNA replication timing of >100 pediatric leukemic samples identified BCP-ALL subtype-specific genome alteration signatures. Comparative analyses identified features of specific stages of B-cell differentiation and potential associations with clinical outcome.

Nuclear Disposition of Alien Chromosome Introgressions into Wheat and Rye Using 3D-FISH

During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl’s configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl’s orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery.

HiCEnterprise: Identifying long range chromosomal contacts in HiC data

Computational analysis of chromosomal capture data is currently gaining popularity with the rapid advance in experimental techniques providing access to growing body of data. An important problem in this area is the identification of long-range contacts be- tween distinct chromatin regions. Such loops were shown to exist at different scales, either mediating interactions between enhancers and promoters or providing much longer interactions between functionally interacting distant chromosome domains. A proper statistical analysis is crucial for accurate identification of such interactions from experi- mental data. We present HiCEnterprise, a software tool for identification of long-range chromatin contacts. It implements three different sta- tistical tests for identification of significant contacts at different scales as well as necessary functions for input, output and visualization of chromosome contact matrices.

HiCEnterprise: Identifying long range chromosomal contacts in HiC data

Computational analysis of chromosomal capture data is currently gaining popularity with the rapid advance in experimental techniques providing access to growing body of data. An important problem in this area is the identification of long-range contacts be- tween distinct chromatin regions. Such loops were shown to exist at different scales, either mediating interactions between enhancers and promoters or providing much longer interactions between functionally interacting distant chromosome domains. A proper statistical analysis is crucial for accurate identification of such interactions from experi- mental data. We present HiCEnterprise, a software tool for identification of long-range chromatin contacts. It implements three different sta- tistical tests for identification of significant contacts at different scales as well as necessary functions for input, output and visualization of chromosome contact matrices.