A phosphorylation code coordinating transcription condensate dynamics with DNA replication.

Chromatin is organized into compartments enriched with functionally-related proteins driving non-linear biochemical activities. Some compartments, e.g. transcription foci, behave as liquid condensates. While the principles governing the enrichment of proteins within condensates are being elucidated, mechanisms that coordinate condensate dynamics with other nuclear processes like DNA replication have not been identified. We show that at the G1/S cell cycle transition, large transcription condensates form at histone locus bodies (HLBs) in a cyclin-dependent kinase 1 and 2 (CDK1/2)-dependent manner. As cells progress through S phase, ataxia-telangiectasia and Rad3-related (ATR) accumulates within HLBs and dissolves the associated transcription condensates. Integration of CDK1/2 and ATR signaling creates a phosphorylation code within the intrinsically-disordered region of mediator subunit 1 (MED1) coordinating condensate dynamics with DNA replication. Disruption of this code results in imbalanced histone biosynthesis, and consequently, global DNA damage. We propose the spatiotemporal dynamics of transcription condensates are actively controlled via phosphorylation and essential for viability of proliferating cells.

A quick restart: RNA polymerase jumping onto post-replicative chromatin.

Two recent studies in Molecular Cell1 and Nature2 show that evicted RNA polymerases reassociate rapidly with post-replicative chromatin and proceed into an unusual transcription cycle, bypassing regular controls and creating a temporary window for altered gene expression.

Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency.

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned +1 and +2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.

Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control.

The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of origins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically connect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton.

ROBO3s: a novel ROBO3 short isoform promoting breast cancer aggressiveness.

Basal-like breast cancer (BLBC) is a highly aggressive breast cancer subtype frequently associated with poor prognosis. Due to the scarcity of targeted treatment options, conventional cytotoxic chemotherapies frequently remain the standard of care. Unfortunately, their efficacy is limited as BLBC malignancies rapidly develop resistant phenotypes. Using transcriptomic and proteomic approaches in human and murine BLBC cells, we aimed to elucidate the molecular mechanisms underlying the acquisition of aggressive and chemotherapy-resistant phenotypes in these mammary tumors. Specifically, we identified and characterized a novel short isoform of Roundabout Guidance Receptor 3 (ROBO3s), upregulated in BLBC in response to chemotherapy and encoding for a protein variant lacking the transmembrane domain. We established an important role for the ROBO3s isoform, mediating cancer stem cell properties by stimulating the Hippo-YAP signaling pathway, and thus driving resistance of BLBC cells to cytotoxic drugs. By uncovering the conservation of ROBO3s expression across multiple cancer types, as well as its association with reduced BLBC-patient survival, we emphasize its potential as a prognostic marker and identify a novel attractive target for anti-cancer drug development.

Decreased PRC2 activity supports the survival of basal-like breast cancer cells to cytotoxic treatments.

Breast cancer (BC) is the most common cancer occurring in women but also rarely develops in men. Recent advances in early diagnosis and development of targeted therapies have greatly improved the survival rate of BC patients. However, the basal-like BC subtype (BLBC), largely overlapping with the triple-negative BC subtype (TNBC), lacks such drug targets and conventional cytotoxic chemotherapies often remain the only treatment option. Thus, the development of resistance to cytotoxic therapies has fatal consequences. To assess the involvement of epigenetic mechanisms and their therapeutic potential increasing cytotoxic drug efficiency, we combined high-throughput RNA- and ChIP-sequencing analyses in BLBC cells. Tumor cells surviving chemotherapy upregulated transcriptional programs of epithelial-to-mesenchymal transition (EMT) and stemness. To our surprise, the same cells showed a pronounced reduction of polycomb repressive complex 2 (PRC2) activity via downregulation of its subunits Ezh2, Suz12, Rbbp7 and Mtf2. Mechanistically, loss of PRC2 activity leads to the de-repression of a set of genes through an epigenetic switch from repressive H3K27me3 to activating H3K27ac mark at regulatory regions. We identified Nfatc1 as an upregulated gene upon loss of PRC2 activity and directly implicated in the transcriptional changes happening upon survival to chemotherapy. Blocking NFATc1 activation reduced epithelial-to-mesenchymal transition, aggressiveness, and therapy resistance of BLBC cells. Our data demonstrate a previously unknown function of PRC2 maintaining low Nfatc1 expression levels and thereby repressing aggressiveness and therapy resistance in BLBC.

Consequences and Resolution of Transcription-Replication Conflicts.

Transcription-replication conflicts occur when the two critical cellular machineries responsible for gene expression and genome duplication collide with each other on the same genomic location. Although both prokaryotic and eukaryotic cells have evolved multiple mechanisms to coordinate these processes on individual chromosomes, it is now clear that conflicts can arise due to aberrant transcription regulation and premature proliferation, leading to DNA replication stress and genomic instability. As both are considered hallmarks of aging and human diseases such as cancer, understanding the cellular consequences of conflicts is of paramount importance. In this article, we summarize our current knowledge on where and when collisions occur and how these encounters affect the genome and chromatin landscape of cells. Finally, we conclude with the different cellular pathways and multiple mechanisms that cells have put in place at conflict sites to ensure the resolution of conflicts and accurate genome duplication.

New insights into re-entrant melting of microgel particles by polymer-induced aggregation experiments.

While microgels are in general described as soft particles, polystyrene (PS) microgels can be synthesized in a way that cross-link density has only a minor influence on their physical properties. Even though the particles swell in a good solvent, the imparted slight softness still allows a mapping on hard sphere behaviour for a large range of cross-link densities [Schneider et al., Soft Matter, 2017, 13, 445]. Nevertheless, the hard sphere analogy breaks down as soon as polymer chains are added to these systems. Quantitative differences between PS microgels and true hard sphere systems appear and the differences between stronger and weaker cross-linked PS microgels can be observed. To gain deeper insights into the origin of these deviations from true hard sphere behaviour, this work is addressed to a systematic study of the colloid-polymer interactions in PS microgel-polymer mixtures. We investigated the aggregation behaviour (namely aggregation concentration and cluster structure) as a function of colloid size, cross-link density and colloid-polymer size ratio in very dilute colloidal suspensions. Our results show that the interplay of cross-link density and polymer size is a key parameter for the strength of the colloid-polymer interactions. Furthermore, the centre-to-centre distance of the colloidal particles in the formed clusters decreases if the cross-link density is decreased, allowing for a higher packing density. This may also explain the unusually high fluid packing fractions observed in the re-entry region of the phase diagram of PS microgel-free PS polymer mixtures.

Branched conformational properties of macromolecules in close relation to chemical synthesis. II. Influence of excluded volume interactions.

The description of perturbed particle conformations needs as a prerequisite the algorithm of unperturbed chains which is outlined in Paper I [J. Chem. Phys. 143, 114906 (2015)]. The mean square segment length ⟨r(2)(n)⟩=b(2)n(2ν) with ν = 0.588 for linear chains in a good solvent is used as an approximation also for branched samples. The mean square radius of gyration is easily derived, but for the hydrodynamic, the segment distribution by Domb et al. [Proc. Phys. Soc., London 85, 624 (1965)] is required. Both radii can analytically be expressed by Gamma functions. For the angular dependence of scattered light, the Fourier transform of the Domb distribution for self-avoiding random walk is needed, which cannot be obtained as an analytical function and was derived by numerical integration. The summation over all segment length in the particle was performed with an analytic fit-curve for the Fourier transform and was carried out numerically. Results were derived (i) for uniform and polydisperse linear chains, (ii) or f-functional randomly branched polymers and their monodisperse fractions, (iii) for random A3B2 co-polymers, and (iv) for AB2 hyper-branched samples. The deviation of the Gaussian approximation with the variance of ⟨r(2)(n)⟩=b(2)n(2ν) slightly overestimates the excluded volume interaction but still remains a fairly good approximation for region of qR(g) < 10.

Trends and forecasts of hospital admissions for acute and chronic pancreatitis in the Netherlands.

BACKGROUND: The incidence and prevalence of acute and chronic pancreatitis have increased in Western countries. It is likely, the number of hospital admissions has increased correspondingly. AIMS: To analyze the trends in hospital admissions in the Netherlands for acute and chronic pancreatitis from 1992 to 2004 and to forecast the number of admissions up to 2010. METHODS: Analysis of hospital admissions for acute and chronic pancreatitis accumulated in a nationwide database. Curve fitting regression models were used to explore future trends. RESULTS: The number of acute pancreatitis admissions rose in 1992-2004 from 1,785 to 3,120 (74.8% increase). The overall 'annual number' of acute pancreatitis admissions increased from 11.8 to 19.2 per 100,000 person-years. The linear regression model predicted 3,205 [95% confidence intervals (CI), 3,111-3,299] and 3,537 (95% CI, 3,429-3,645) admissions for 2007 and 2010, respectively, a further increase of at least 9.9% in 2010 compared with 2004. In the 12-year time period, chronic pancreatitis admissions showed an increase of 75.4% (from 790 to 1,386). The overall 'annual number' of chronic pancreatitis admissions increased from 5.2 to 8.5 per 100,000 person-years. The cubic regression model predicted 1868 (95% CI, 1,619-2,117) and 3,173 (95% CI, 2,456-3,890) admissions for 2007 and 2010, respectively, an additional increase of 77.2% in 2010 compared with 2004. CONCLUSION: Hospital admissions for acute and chronic pancreatitis have increased substantially from 1992-2004. This trend will most likely continue for the near future and the burden and costs to the Dutch health care system will increase accordingly.