In Drosophila melanogaster the single male chromosome X undergoes an average twofold transcriptional upregulation for balancing the transcriptional output between sexes. Previous literature hypothesised that a global change in chromosome structure may accompany this process. However, recent studies based on Hi-C failed to detect these differences. Here we show that global conformational differences are specifically present in the male chromosome X and detectable using Hi-C data on sex-sorted embryos, as well as male and female cell lines, by leveraging custom data analysis solutions. We find the male chromosome X has more mid-/long-range interactions. We also identify differences at structural domain boundaries containing BEAF-32 in conjunction with CP190 or Chromator. Weakening of these domain boundaries in male chromosome X co-localizes with the binding of the dosage compensation complex and its co-factor CLAMP, reported to enhance chromatin accessibility. Together, our data strongly indicate that chromosome X dosage compensation affects global chromosome structure.

Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in ‘regulation of RNA splicing’ and ‘mRNA catabolic process’. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.

The onset of chemo-resistant recurrence represents the principal cause of high-grade serous ovarian carcinoma (HGSOC) death. HGSOC masses are characterized by a hypoxic microenvironment, which contributes to the development of this chemo-resistant phenotype. Hypoxia regulated-miRNAs (HRMs) represent a molecular response of cancer cells to hypoxia and are involved in tumor progression. We investigated the expression of HRMs using miRNA expression data from a total of 273 advanced-stage HGSOC samples. The miRNAs associated with chemoresistance and survival were validated by RT-qPCR and target prediction, and comparative pathway analysis was conducted for target gene identification. Analysis of miRNA expression profiles indicated miR-23a-3p and miR-181c-5p over-expression as associated with chemoresistance and poor PFS. RT-qPCR data confirmed upregulation of miR-23a-3p in tumors from chemoresistant HGSOC patients and its significant association with shorter PFS. In silico miR-23a-3p target prediction and comparative pathway analysis identified platinum drug resistance as the pathway with the highest number of miR-23a-3p target genes. Among them, APAF-1 emerged as the most promising, being downregulated in platinum-resistant patients and in HGSOC chemo-resistant cells. These results highlight miR-23a-3p as a potential biomarker for HGSOC platinum response and prognosis and the miR23a-3p/APAF1 axis as a possible target to overcome platinum-resistance.

A growing amount of evidence in literature suggests that germline sequence variants and somatic mutations in non-coding distal regulatory elements may be crucial for defining disease risk and prognostic stratification of patients, in genetic disorders as well as in cancer. Their functional interpretation is challenging because genome-wide enhancer–target gene (ETG) pairing is an open problem in genomics. The solutions proposed so far do not account for the hierarchy of structural domains which define chromatin three-dimensional (3D) architecture. Here we introduce a change of perspective based on the definition of multi-scale structural chromatin domains, integrated in a statistical framework to define ETG pairs. In this work (i) we develop a computational and statistical framework to reconstruct a comprehensive map of ETG pairs leveraging functional genomics data; (ii) we demonstrate that the incorporation of chromatin 3D architecture information improves ETG pairing accuracy and (iii) we use multiple experimental datasets to extensively benchmark our method against previous solutions for the genome-wide reconstruction of ETG pairs. This solution will facilitate the annotation and interpretation of sequence variants in distal non-coding regulatory elements. We expect this to be especially helpful in clinically oriented applications of whole genome sequencing in cancer and undiagnosed genetic diseases research.

Chromatin three-dimensional (3D) organization inside the cell nucleus determines the separation of euchromatin and heterochromatin domains. Their segregation results in the definition of active and inactive chromatin compartments, whereby the local concentration of associated proteins, RNA and DNA results in the formation of distinct subnuclear structures. Thus, chromatin domains spatially confined in a specific 3D nuclear compartment are expected to share similar epigenetic features and biochemical properties, in terms of accessibility and solubility. Based on this rationale, we developed the 4f-SAMMY-seq to map euchromatin and heterochromatin based on their accessibility and solubility, starting from as little as 10 000 cells. Adopting a tailored bioinformatic data analysis approach we reconstruct also their 3D segregation in active and inactive chromatin compartments and sub-compartments, thus recapitulating the characteristic properties of distinct chromatin states. A key novelty of the new method is the capability to map both the linear segmentation of open and closed chromatin domains, as well as their compartmentalization in one single experiment.