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HiC-Pro is an optimized and flexible pipeline for processing Hi-C data from raw reads to normalized contact maps. HiC-Pro maps reads, detects valid ligation products, performs quality controls and generates intra- and inter-chromosomal contact maps. It includes a fast implementation of the iterative correction method and is based on a memory-efficient data format for Hi-C contact maps. In addition, HiC-Pro can use phased genotype data to build allele-specific contact maps. We applied HiC-Pro to different Hi-C datasets, demonstrating its ability to easily process large data in a reasonable time. Source code and documentation are available at http://github.com/nservant/HiC-Pro .

Concurrent chemoradiotherapy (CRT) with blockade of the PD-1 pathway may enhance immune-mediated tumor control through increased phagocytosis, cell death, and antigen presentation. The NiCOL phase 1 trial (NCT03298893) is designed to determine the safety/tolerance profile and the recommended phase-II dose of nivolumab with and following concurrent CRT in 16 women with locally advanced cervical cancer. Secondary endpoints include objective response rate (ORR), progression free survival (PFS), disease free survival, and immune correlates of response. Three patients experience grade 3 dose-limiting toxicities. The pre-specified endpoints are met, and overall response rate is 93.8% [95%CI: 69.8–99.8%] with a 2-year PFS of 75% [95% CI: 56.5–99.5%]. Compared to patients with progressive disease (PD), progression-free (PF) subjects show a brisker stromal immune infiltrate, higher proximity of tumor-infiltrating CD3 + T cells to PD-L1 + tumor cells and of FOXP3 + T cells to proliferating CD11c + myeloid cells. PF show higher baseline levels of PD-1 and ICOS-L on tumor-infiltrating EMRA CD4 + T cells and tumor-associated macrophages, respectively; PD instead, display enhanced PD-L1 expression on TAMs, higher peripheral frequencies of proliferating Tregs at baseline and higher PD-1 levels at week 6 post-treatment initiation on CD4 and CD8 T cell subsets. Concomitant nivolumab plus definitive CRT is safe and associated with encouraging PFS rates. Further validation in the subset of locally advanced cervical cancer displaying pre-existing, adaptive immune activation is warranted. A combination of chemoradiotherapy followed by brachytherapy is recommended for patients with locally-advanced cervical cancer (LACC), however there is still a high risk of disease recurrence. Here the authors report clinical outcomes and immunologic correlates of a clinical trial of the PD-1 inhibitor nivolumab in combination with chemoradiotherapy in LACC patients.

Background Sequencing technologies give access to a precise picture of the molecular mechanisms acting upon genome regulation. One of the biggest technical challenges with sequencing data is to map millions of reads to a reference genome. This problem is exacerbated when dealing with repetitive sequences such as transposable elements that occupy half of the mammalian genome mass. Sequenced reads coming from these regions introduce ambiguities in the mapping step. Therefore, applying dedicated parameters and algorithms has to be taken into consideration when transposable elements regulation is investigated with sequencing datasets. Results Here, we used simulated reads on the mouse and human genomes to define the best parameters for aligning transposable element-derived reads on a reference genome. The efficiency of the most commonly used aligners was compared and we further evaluated how transposable element representation should be estimated using available methods. The mappability of the different transposon families in the mouse and the human genomes was calculated giving an overview into their evolution. Conclusions Based on simulated data, we provided recommendations on the alignment and the quantification steps to be performed when transposon expression or regulation is studied, and identified the limits in detecting specific young transposon families of the mouse and human genomes. These principles may help the community to adopt standard procedures and raise awareness of the difficulties encountered in the study of transposable elements.

In Drosophila , a complex consisting of Calypso and ASX catalyzes H2A deubiquitination and has been reported to act as part of the Polycomb machinery in transcriptional silencing. The mammalian homologs of these proteins (BAP1 and ASXL1/2/3, respectively), are frequently mutated in various cancer types, yet their precise functions remain unclear. Using an integrative approach based on isogenic cell lines generated with CRISPR/Cas9, we uncover an unanticipated role for BAP1 in gene activation. This function requires the assembly of an enzymatically active BAP1-associated core complex (BAP1.com) containing one of the redundant ASXL proteins. We investigate the mechanism underlying BAP1.com-mediated transcriptional regulation and show that it does not participate in Polycomb-mediated silencing. Instead, our results establish that the function of BAP1.com is to safeguard transcriptionally active genes against silencing by the Polycomb Repressive Complex 1. In Drosophila , the Calypso–ASX complex catalyzes H2A deubiquitination and aids Polycomb in transcriptional silencing. Here the authors show that the orthologous complex, BAP1.com, promotes gene activation by counteracting PRC1-mediated gene silencing.

The Polycomb group of proteins is required for the proper orchestration of gene expression due to its role in maintaining transcriptional silencing. It is composed of several chromatin modifying complexes, including Polycomb Repressive Complex 2 (PRC2), which deposits H3K27me2/3. Here, we report the identification of a cofactor of PRC2, EZHIP (EZH1/2 Inhibitory Protein), expressed predominantly in the gonads. EZHIP limits the enzymatic activity of PRC2 and lessens the interaction between the core complex and its accessory subunits, but does not interfere with PRC2 recruitment to chromatin. Deletion of Ezhip in mice leads to a global increase in H3K27me2/3 deposition both during spermatogenesis and at late stages of oocyte maturation. This does not affect the initial number of follicles but is associated with a reduction of follicles in aging. Our results suggest that mature oocytes Ezhip−/− might not be fully functional and indicate that fertility is strongly impaired in Ezhip−/− females. Altogether, our study uncovers EZHIP as a regulator of chromatin landscape in gametes. Polycomb Repressive Complex 2 (PRC2) plays critical roles in transcriptional silencing during development. Here the authors identify EZHIP as a cofactor of PRC2 expressed predominantly in the gonads, finding that EZHIP limits the enzymatic activity of PRC2 in germ cells in mice.

Chromatin modifications orchestrate the dynamic regulation of gene expression during development and in disease. Bulk approaches have characterized the wide repertoire of histone modifications across cell types, detailing their role in shaping cell identity. However, these population-based methods do not capture cell-to-cell heterogeneity of chromatin landscapes, limiting our appreciation of the role of chromatin in dynamic biological processes. Recent technological developments enable the mapping of histone marks at single-cell resolution, opening up perspectives to characterize the heterogeneity of chromatin marks in complex biological systems over time. Yet, existing tools used to analyze bulk histone modifications profiles are not fit for the low coverage and sparsity of single-cell epigenomic datasets. Here, we present ChromSCape, a user-friendly interactive Shiny/R application distributed as a Bioconductor package, that processes single-cell epigenomic data to assist the biological interpretation of chromatin landscapes within cell populations. ChromSCape analyses the distribution of repressive and active histone modifications as well as chromatin accessibility landscapes from single-cell datasets. Using ChromSCape, we deconvolve chromatin landscapes within the tumor micro-environment, identifying distinct H3K27me3 landscapes associated with cell identity and breast tumor subtype. Bulk approaches fail to capture the cell-to-cell heterogeneity of chromatin landscapes, while single-cell approaches provide low coverage datasets. Here, the authors present ChromSCape, a user-friendly interactive application that processes single-cell epigenomic data to assist the biological interpretation of chromatin landscapes within cell populations, as demonstrated in the context of cancer.

Background Many transcript isoforms generated by intronic polyadenylation (IPA) encode isoforms of canonical proteins. Microproteins are an emerging class of small proteins translated from small open reading frames (sORFs) in noncoding RNAs and mRNAs, but their production by IPA isoforms is unknown. Results Here, by crossing 3′-seq, Ribo-Seq, and mass-spectrometry data, we identify 297 genes with a microprotein-coding IPA isoform terminating in a 5′UTR intron (coined miP-5′UTR-IPA isoform). By 3′-seq and long-read RNA-seq analyses in lung cancer cells treated with cisplatin, a DNA-cross-linking anticancer drug, we find that cisplatin globally favors the expression of (miP-5′UTR-)IPA isoforms relative to full-length mRNAs, mainly by decreasing the latter through an inhibition of transcription processivity in a FANCD2 and senataxin-dependent manner. The cisplatin-regulated miP-5′UTR-IPA isoform in the PRKAR1B gene is translated, as it is associated with light polysome fractions and contains Ribo-Seq-supported sORFs in its alternative last exon, and the microprotein (PRKAR1B-IPA-miP2) encoded by its sORF#2 is detected by Western blot and immunofluorescence. CRISPR editing of either the IPA site or the sORF#2 initiation site leads to decreased cell growth inhibition by cisplatin and camptothecin, another genotoxic drug. Mechanistically, PRKAR1B-IPA-miP2 promotes p53 protein induction by cisplatin. Finally, 70 miP-5′UTR-IPA isoforms are detected in normal cells, and 143 are upregulated by cisplatin. Conclusions Here, we show that IPA isoforms are a novel source of microproteins, and we reveal the novel paradigm of miP-5′UTR-IPA genes that produce both a canonical full-length mRNA and a microprotein-coding IPA isoform.

Background Normalization is essential to ensure accurate analysis and proper interpretation of sequencing data, and chromosome conformation capture data such as Hi-C have particular challenges. Although several methods have been proposed, the most widely used type of normalization of Hi-C data usually casts estimation of unwanted effects as a matrix balancing problem, relying on the assumption that all genomic regions interact equally with each other. Results In order to explore the effect of copy-number variations on Hi-C data normalization, we first propose a simulation model that predict the effects of large copy-number changes on a diploid Hi-C contact map. We then show that the standard approaches relying on equal visibility fail to correct for unwanted effects in the presence of copy-number variations. We thus propose a simple extension to matrix balancing methods that model these effects. Our approach can either retain the copy-number variation effects (LOIC) or remove them (CAIC). We show that this leads to better downstream analysis of the three-dimensional organization of rearranged genomes. Conclusions Taken together, our results highlight the importance of using dedicated methods for the analysis of Hi-C cancer data. Both CAIC and LOIC methods perform well on simulated and real Hi-C data sets, each fulfilling different needs.

Glioblastoma (GBM) is a highly lethal type of cancer. GBM recurrence following chemoradiation is typically attributed to the regrowth of invasive and resistant cells. Therefore, there is a pressing need to gain a deeper understanding of the mechanisms underlying GBM resistance to chemoradiation and its ability to infiltrate. Using a combination of transcriptomic, proteomic, and phosphoproteomic analyses, longitudinal imaging, organotypic cultures, functional assays, animal studies, and clinical data analyses, we demonstrate that chemoradiation and brain vasculature induce cell transition to a functional state named VC-Resist (vessel co-opting and resistant cell state). This cell state is midway along the transcriptomic axis between proneural and mesenchymal GBM cells and is closer to the AC/MES1-like state. VC-Resist GBM cells are highly vessel co-opting, allowing significant infiltration into the surrounding brain tissue and homing to the perivascular niche, which in turn induces even more VC-Resist transition. The molecular and functional characteristics of this FGFR1-YAP1-dependent GBM cell state, including resistance to DNA damage, enrichment in the G2M phase, and induction of senescence/stemness pathways, contribute to its enhanced resistance to chemoradiation. These findings demonstrate how vessel co-option, perivascular niche, and GBM cell plasticity jointly drive resistance to therapy during GBM recurrence. In patient with glioblastoma, a major cause of resistance to chemotherapy and radiotherapy is the high degree to intratumoral heterogeneity and cell plasticity. Here, the authors demonstrate that chemoradiation induces the reprograming of glioblastoma cells into an invasive and vessel co-opting state, termed VC-Resist, capable of promoting resistance to therapy.

The mouse X-inactivation center ( Xic ) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis -regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other’s TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis -regulatory landscapes can affect the regulation of mammalian developmental processes. Swapping the Xist/Tsix transcriptional units and placing their promoters in each other’s topologically associating domain shows that the topological partitioning of the X-inactivation center is critical to ensure proper X inactivation during development.