Collisions of RNA polymerases behind the replication fork promote alternative RNA splicing in newly replicated chromatin.

DNA replication produces a global disorganization of chromatin structure that takes hours to be restored. However, how these chromatin rearrangements affect the regulation of gene expression and the maintenance of cell identity is not clear. Here, we use ChOR-seq and ChrRNA-seq experiments to analyze RNA polymerase II (RNAPII) activity and nascent RNA synthesis during the first hours after chromatin replication in human cells. We observe that transcription elongation is rapidly reactivated in nascent chromatin but that RNAPII abundance and distribution are altered, producing heterogeneous changes in RNA synthesis. Moreover, this first wave of transcription results in RNAPII blockages behind the replication fork, leading to changes in alternative splicing. Altogether, our results deepen our understanding of how transcriptional programs are regulated during cell division and uncover molecular mechanisms that explain why chromatin replication is an important source of gene expression variability.

Role of Chromatin Replication in Transcriptional Plasticity, Cell Differentiation and Disease.

Chromatin organization is essential to maintain a correct regulation of gene expression and establish cell identity. However, during cell division, the replication of the genetic material produces a global disorganization of chromatin structure. In this paper, we describe the new scientific breakthroughs that have revealed the nature of the post-replicative chromatin and the mechanisms that facilitate its restoration. Moreover, we highlight the implications of these chromatin alterations in gene expression control and their impact on key biological processes, such as cell differentiation, cell reprogramming or human diseases linked to cell proliferation, such as cancer.

Efficacy of Eribulin Plus Gemcitabine Combination in L-Sarcomas.

Although the overall survival of advanced soft-tissue sarcoma (STS) patients has increased in recent years, the median progression-free survival is lower than 5 months, meaning that there is an unmet need in this population. Among second-line treatments for advanced STS, eribulin is an anti-microtubule agent that has been approved for liposarcoma. Here, we tested the combination of eribulin with gemcitabine in preclinical models of L-sarcoma. The effect in cell viability was measured by MTS and clonogenic assay. Cell cycle profiling was studied by flow cytometry, while apoptosis was measured by flow cytometry and Western blotting. The activity of eribulin plus gemcitabine was evaluated in in vivo patient-derived xenograft (PDX) models. In L-sarcoma cell lines, eribulin plus gemcitabine showed to be synergistic, increasing the number of hypodiploid events (increased subG1 population) and the accumulation of DNA damage. In in vivo PDX models of L-sarcomas, eribulin combined with gemcitabine was a viable scheme, delaying tumour growth after one cycle of treatment, being more effective in leiomyosarcoma. The combination of eribulin and gemcitabine was synergistic in L-sarcoma cultures and it showed to be active in in vivo studies. This combination deserves further exploration in the clinical context.

Genome-wide and sister chromatid-resolved profiling of protein occupancy in replicated chromatin with ChOR-seq and SCAR-seq.

Elucidating the mechanisms underlying chromatin maintenance upon genome replication is critical for the understanding of how gene expression programs and cell identity are preserved across cell divisions. Here, we describe two recently developed techniques, chromatin occupancy after replication (ChOR)-seq and sister chromatids after replication (SCAR)-seq, that profile chromatin occupancy on newly replicated DNA in mammalian cells in 5 d of bench work. Both techniques share a common strategy that includes pulse labeling of newly synthesized DNA and chromatin immunoprecipitation (ChIP), followed by purification and high-throughput sequencing. Whereas ChOR-seq quantitatively profiles the post-replicative abundance of histone modifications and chromatin-associated proteins, SCAR-seq distinguishes chromatin occupancy between nascent sister chromatids. Together, these two complementary techniques have unraveled key mechanisms controlling the inheritance of modified histones during replication and revealed locus-specific dynamics of histone modifications across the cell cycle. Here, we provide the experimental protocols and bioinformatic pipelines for these methods.

Repeat RNAs associate with replication forks and post-replicative DNA.

Noncoding RNA has a proven ability to direct and regulate chromatin modifications by acting as scaffolds between DNA and histone-modifying complexes. However, it is unknown if ncRNA plays any role in DNA replication and epigenome maintenance, including histone eviction and reinstallment of histone modifications after genome duplication. Isolation of nascent chromatin has identified a large number of RNA-binding proteins in addition to unknown components of the replication and epigenetic maintenance machinery. Here, we isolated and characterized long and short RNAs associated with nascent chromatin at active replication forks and track RNA composition during chromatin maturation across the cell cycle. Shortly after fork passage, GA-rich-, alpha- and TElomeric Repeat-containing RNAs (TERRA) are associated with replicated DNA. These repeat containing RNAs arise from loci undergoing replication, suggesting an interaction in cis. Post-replication during chromatin maturation, and even after mitosis in G1, the repeats remain enriched on DNA. This suggests that specific types of repeat RNAs are transcribed shortly after DNA replication and stably associate with their loci of origin throughout the cell cycle. The presented method and data enable studies of RNA interactions with replication forks and post-replicative chromatin and provide insights into how repeat RNAs and their engagement with chromatin are regulated with respect to DNA replication and across the cell cycle.

Pazopanib for treatment of typical solitary fibrous tumours: a multicentre, single-arm, phase 2 trial.

BACKGROUND: Solitary fibrous tumour is an ultra-rare sarcoma, which encompasses different clinicopathological subgroups. The dedifferentiated subgroup shows an aggressive course with resistance to pazopanib, whereas in the malignant subgroup, pazopanib shows higher activity than in previous studies with chemotherapy. We designed a trial to test pazopanib activity in two different cohorts of solitary fibrous tumour: the malignant-dedifferentiated cohort, which was previously published, and the typical cohort, which is presented here. METHODS: In this single-arm, phase 2 trial, adult patients (aged ≥18 years) diagnosed with confirmed metastatic or unresectable typical solitary fibrous tumour of any location, who had progressed in the previous 6 months (by Choi criteria or Response Evaluation Criteria in Solid Tumors [RECIST]) and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 were enrolled at 11 tertiary hospitals in Italy, France, and Spain. Patients received pazopanib 800 mg once daily, taken orally, until progression, unacceptable toxicity, withdrawal of consent, non-compliance, or a delay in pazopanib administration of longer than 3 weeks. The primary endpoint was proportion of patients achieving an overall response measured by Choi criteria in patients who received at least 1 month of treatment with at least one radiological assessment. All patients who received at least one dose of the study drug were included in the safety analyses. This study is registered in ClinicalTrials.gov, NCT02066285, and with the European Clinical Trials Database, EudraCT 2013-005456-15. FINDINGS: From June 26, 2014, to Dec 13, 2018, of 40 patients who were assessed, 34 patients were enrolled and 31 patients were included in the response analysis. Median follow-up was 18 months (IQR 14-34), and 18 (58%) of 31 patients had a partial response, 12 (39%) had stable disease, and one (3%) showed progressive disease according to Choi criteria and central review. The proportion of overall response based on Choi criteria was 58% (95% CI 34-69). There were no deaths caused by toxicity, and the most frequent adverse events were diarrhoea (18 [53%] of 34 patients), fatigue (17 [50%]), and hypertension (17 [50%]). INTERPRETATION: To our knowledge, this is the first prospective trial of pazopanib for advanced typical solitary fibrous tumour. The manageable toxicity and activity shown by pazopanib in this cohort suggest that this drug could be considered as first-line treatment for advanced typical solitary fibrous tumour. FUNDING: Spanish Group for Research on Sarcomas (GEIS), Italian Sarcoma Group (ISG), French Sarcoma Group (FSG), GlaxoSmithKline, and Novartis.

Accurate Recycling of Parental Histones Reproduces the Histone Modification Landscape during DNA Replication.

Chromatin organization is disrupted genome-wide during DNA replication. On newly synthesized DNA, nucleosomes are assembled from new naive histones and old modified histones. It remains unknown whether the landscape of histone post-translational modifications (PTMs) is faithfully copied during DNA replication or the epigenome is perturbed. Here we develop chromatin occupancy after replication (ChOR-seq) to determine histone PTM occupancy immediately after DNA replication and across the cell cycle. We show that H3K4me3, H3K36me3, H3K79me3, and H3K27me3 positional information is reproduced with high accuracy on newly synthesized DNA through histone recycling. Quantitative ChOR-seq reveals that de novo methylation to restore H3K4me3 and H3K27me3 levels occurs across the cell cycle with mark- and locus-specific kinetics. Collectively, this demonstrates that accurate parental histone recycling preserves positional information and allows PTM transmission to daughter cells while modification of new histones gives rise to complex epigenome fluctuations across the cell cycle that could underlie cell-to-cell heterogeneity.

BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation.

During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins.

A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks.

During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.

A genome-wide function of THSC/TREX-2 at active genes prevents transcription-replication collisions.

The THSC/TREX-2 complex of Saccharomyces cerevisiae mediates the anchoring of transcribed genes to the nuclear pore, linking transcription elongation with mRNA export and genome stability, as shown for specific reporters. However, it is still unknown whether the function of TREX-2 is global and the reason for its relevant role in genome integrity. Here, by studying two TREX-2 representative subunits, Thp1 and Sac3, we show that TREX-2 has a genome-wide role in gene expression. Both proteins show similar distributions along the genome, with a gradient disposition at active genes that increases towards the 3' end. Thp1 and Sac3 have a relevant impact on the expression of long, G+C-rich and highly transcribed genes. Interestingly, replication impairment detected by the genome-wide accumulation of the replicative Rrm3 helicase is increased preferentially at highly expressed genes in the thp1Δ and sac3Δ mutants analyzed. Therefore, our work provides evidence of a function of TREX-2 at the genome-wide level and suggests a role for TREX-2 in preventing transcription-replication conflicts, as a source of genome instability derived from a defective messenger ribonucleoprotein particle (mRNP) biogenesis.

Comparative epigenomics in distantly related teleost species identifies conserved cis-regulatory nodes active during the vertebrate phylotypic period.

The complex relationship between ontogeny and phylogeny has been the subject of attention and controversy since von Baer's formulations in the 19th century. The classic concept that embryogenesis progresses from clade general features to species-specific characters has often been revisited. It has become accepted that embryos from a clade show maximum morphological similarity at the so-called phylotypic period (i.e., during mid-embryogenesis). According to the hourglass model, body plan conservation would depend on constrained molecular mechanisms operating at this period. More recently, comparative transcriptomic analyses have provided conclusive evidence that such molecular constraints exist. Examining cis-regulatory architecture during the phylotypic period is essential to understand the evolutionary source of body plan stability. Here we compare transcriptomes and key epigenetic marks (H3K4me3 and H3K27ac) from medaka (Oryzias latipes) and zebrafish (Danio rerio), two distantly related teleosts separated by an evolutionary distance of 115-200 Myr. We show that comparison of transcriptome profiles correlates with anatomical similarities and heterochronies observed at the phylotypic stage. Through comparative epigenomics, we uncover a pool of conserved regulatory regions (≈700), which are active during the vertebrate phylotypic period in both species. Moreover, we show that their neighboring genes encode mainly transcription factors with fundamental roles in tissue specification. We postulate that these regulatory regions, active in both teleost genomes, represent key constrained nodes of the gene networks that sustain the vertebrate body plan.

Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans.

BACKGROUND: Laminopathies are diseases characterized by defects in nuclear envelope structure. A well-known example is Emery-Dreifuss muscular dystrophy, which is caused by mutations in the human lamin A/C and emerin genes. While most nuclear envelope proteins are ubiquitously expressed, laminopathies often affect only a subset of tissues. The molecular mechanisms underlying these tissue-specific manifestations remain elusive. We hypothesize that different functional subclasses of genes might be differentially affected by defects in specific nuclear envelope components. RESULTS: Here we determine genome-wide DNA association profiles of two nuclear envelope components, lamin/LMN-1 and emerin/EMR-1 in adult Caenorhabditis elegans. Although both proteins bind to transcriptionally inactive regions of the genome, EMR-1 is enriched at genes involved in muscle and neuronal function. Deletion of either EMR-1 or LEM-2, another integral envelope protein, causes local changes in nuclear architecture as evidenced by altered association between DNA and LMN-1. Transcriptome analyses reveal that EMR-1 and LEM-2 are associated with gene repression, particularly of genes implicated in muscle and nervous system function. We demonstrate that emr-1, but not lem-2, mutants are sensitive to the cholinesterase inhibitor aldicarb, indicating altered activity at neuromuscular junctions. CONCLUSIONS: We identify a class of elements that bind EMR-1 but do not associate with LMN-1, and these are enriched for muscle and neuronal genes. Our data support a redundant function of EMR-1 and LEM-2 in chromatin anchoring to the nuclear envelope and gene repression. We demonstrate a specific role of EMR-1 in neuromuscular junction activity that may contribute to Emery-Dreifuss muscular dystrophy in humans.

C. elegans epigenetic regulation in development and aging.

The precise developmental map of the Caenorhabditis elegans cell lineage, as well as a complete genome sequence and feasibility of genetic manipulation make this nematode species highly attractive to study the role of epigenetics during development. Genetic dissection of phenotypical traits, such as formation of egg-laying organs or starvation-resistant dauer larvae, has illustrated how chromatin modifiers may regulate specific cell-fate decisions and behavioral programs. Moreover, the transparent body of C. elegans facilitates non-invasive microscopy to study tissue-specific accumulation of heterochromatin at the nuclear periphery. We also review here recent findings on how small RNA molecules contribute to epigenetic control of gene expression that can be propagated for several generations and eventually determine longevity.

Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery.

The factors that sequester transcriptionally repressed heterochromatin at the nuclear periphery are currently unknown. In a genome-wide RNAi screen, we found that depletion of S-adenosylmethionine (SAM) synthetase reduces histone methylation globally and causes derepression and release of heterochromatin from the nuclear periphery in Caenorhabditis elegans embryos. Analysis of histone methyltransferases (HMTs) showed that elimination of two HMTs, MET-2 and SET-25, mimics the loss of SAM synthetase, abrogating the perinuclear attachment of heterochromatic transgenes and of native chromosomal arms rich in histone H3 lysine 9 methylation. The two HMTs target H3K9 in a consecutive fashion: MET-2, a SETDB1 homolog, mediates mono- and dimethylation, and SET-25, a previously uncharacterized HMT, deposits H3K9me3. SET-25 colocalizes with its own product in perinuclear foci, in a manner dependent on H3K9me3, but not on its catalytic domain. This colocalization suggests an autonomous, self-reinforcing mechanism for the establishment and propagation of repeat-rich heterochromatin.

Dissecting the NUP107 complex: multiple components and even more functions.

The Nuclear Pore Complex (NPC) is a fascinating structure whose functional relevance and complexity attract significant interest. Within the NPC, several different subcomplexes interact with each other to form a highly conserved and stable structure. One of these subcomplexes is the NUP107 complex, constituted by 7-9 members. A wide variety of functions have been ascribed to the NUP107 complex, ranging from NPC assembly to mRNA export to cell differentiation. Recently, genetic dissection of the NUP107 complex has provided novel insight to the assembly of the complex and has, moreover, revealed an unexpected connection with the mitotic spindle assembly checkpoint protein MAD1.

Dissection of the NUP107 nuclear pore subcomplex reveals a novel interaction with spindle assembly checkpoint protein MAD1 in Caenorhabditis elegans.

Nuclear pore complexes consist of several subcomplexes. The NUP107 complex is important for nucleocytoplasmic transport, nuclear envelope assembly, and kinetochore function. However, the underlying molecular mechanisms and the roles of individual complex members remain elusive. We report the first description of a genetic disruption of NUP107 in a metazoan. Caenorhabditis elegans NUP107/npp-5 mutants display temperature-dependent lethality. Surprisingly, NPP-5 is dispensable for incorporation of most nucleoporins into nuclear pores and for nuclear protein import. In contrast, NPP-5 is essential for proper kinetochore localization of NUP133/NPP-15, another NUP107 complex member, whereas recruitment of NUP96/NPP-10C and ELYS/MEL-28 is NPP-5 independent. We found that kinetochore protein NUF2/HIM-10 and Aurora B/AIR-2 kinase are less abundant on mitotic chromatin upon NPP-5 depletion. npp-5 mutants are hypersensitive to anoxia, suggesting that the spindle assembly checkpoint (SAC) is compromised. Indeed, NPP-5 interacts genetically and physically with SAC protein MAD1/MDF-1, whose nuclear envelope accumulation requires NPP-5. Thus our results strengthen the emerging connection between nuclear pore proteins and chromosome segregation.

Nab2 functions in the metabolism of RNA driven by polymerases II and III.

Gene expression in eukaryotes is an essential process that includes transcription, RNA processing, and export. One important player in this interface is the poly(A)(+)-RNA-binding protein Nab2, which regulates the mRNA poly(A)(+)-tail length and export. Here we show that Nab2 has additional roles during mRNA transcription, tRNA metabolism, and ribosomal subunit export. Nab2 is associated with the entire open reading frame of actively transcribed RNA polymerase (RNAP) II and III genes. As a consequence, nab2 mutations confer translation defects that are detected by polysome profiling. Genome-wide analysis of expression of a conditional degron nab2 mutant shows that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is direct. Taken together, our results identify novel functions for Nab2 in transcription and metabolism of most types of RNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, and that it is a central player in the general and coordinated control of gene expression from transcription to translation.

A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors.

To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G-less cassettes separated by either a long and GC-rich or a short and GC-poor DNA sequence (G-less-based run-on (GLRO) assay). We demonstrate that PAF, THSC/TREX-2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre-mRNA processing and nuclear export with transcription elongation.

New suppressors of THO mutations identify Thp3 (Ypr045c)-Csn12 as a protein complex involved in transcription elongation.

Formation of a ribonucleoprotein particle (mRNP) competent for export requires the coupling of transcription with mRNA processing and RNA export. A key link between these processes is provided by the THO complex. To progress in our understanding of this coupling, we have performed a search for suppressors of the transcription defect caused by the hpr1Δ mutation. This has permitted us to identify mutations in the genes for the RNA polymerase II mediator component Med10, the Sch9 protein kinase, and the Ypr045c protein. We report a role in transcription elongation for Ypr045c (Thp3) and the Csn12 component of the COP9 signalosome. Thp3 and Csn12 form a complex that is recruited to transcribed genes. Their mutations suppress the gene expression defects of THO complex mutants involved in mRNP biogenesis and export and show defects in mRNA accumulation. Transcription elongation impairment of thp3Δ mutants is shown by in vivo transcript run-on analysis performed in G-less systems. Thp3-Csn12 establishes a novel link between transcription and mRNA processing that opens new perspectives on our understanding of gene expression and reveals novel functions for a component of the COP9 signalosome. Thp3-Csn12 also copurifies with ribosomal proteins, which opens the possibility that it has other functions in addition to transcription.

Sem1 is a functional component of the nuclear pore complex-associated messenger RNA export machinery.

The evolutionarily conserved protein Sem1/Dss1 is a subunit of the regulatory particle (RP) of the proteasome, and, in mammalian cells, binds the tumor suppressor protein BRCA2. Here, we describe a new function for yeast Sem1. We show that sem1 mutants are impaired in messenger RNA (mRNA) export and transcription elongation, and induce strong transcription-associated hyper-recombination phenotypes. Importantly, Sem1, independent of the RP, is functionally linked to the mRNA export pathway. Biochemical analyses revealed that, in addition to the RP, Sem1 coenriches with components of two other multisubunit complexes: the nuclear pore complex (NPC)-associated TREX-2 complex that is required for transcription-coupled mRNA export, and the COP9 signalosome, which is involved in deneddylation. Notably, targeting of Thp1, a TREX-2 component, to the NPC is perturbed in a sem1 mutant. These findings reveal an unexpected nonproteasomal function of Sem1 in mRNA export and in prevention of transcription-associated genome instability. Thus, Sem1 is a versatile protein that might stabilize multiple protein complexes involved in diverse pathways.