Profiling lnc-RNA in CHO cells using NGS technologies

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The BAF and PRC2 Complex Subunits Dpf2 and Eed Antagonistically Converge on Tbx3 to Control ESC Differentiation.

BAF complexes are composed of different subunits with varying functional and developmental roles, although many subunits have not been examined in depth. Here we show that the Baf45 subunit Dpf2 maintains pluripotency and ESC differentiation potential. Dpf2 co-occupies enhancers with Oct4, Sox2, p300, and the BAF subunit Brg1, and deleting Dpf2 perturbs ESC self-renewal, induces repression of Tbx3, and impairs mesendodermal differentiation without dramatically altering Brg1 localization. Mesendodermal differentiation can be rescued by restoring Tbx3 expression, whose distal enhancer is positively regulated by Dpf2-dependent H3K27ac maintenance and recruitment of pluripotency TFs and Brg1. In contrast, the PRC2 subunit Eed binds an intragenic Tbx3 enhancer to oppose Dpf2-dependent Tbx3 expression and mesendodermal differentiation. The PRC2 subunit Ezh2 likewise opposes Dpf2-dependent differentiation through a distinct mechanism involving Nanog repression. Together, these findings delineate distinct mechanistic roles for specific BAF and PRC2 subunits during ESC differentiation

A Histone Map of Human Chromosome 20q13.12

We present a systematic search for regulatory elements in a 3.5 Mb region on human chromosome 20q13.12, a region associated with a number of medical conditions such as type II diabetes and obesity.We profiled six histone modifications alongside RNA polymerase II (PolII) and CTCF in two cell lines, HeLa S3 and NTERA-2 clone D1 (NT2/D1), by chromatin immunoprecipitation using an in-house spotted DNA array, constructed with 1.8 kb overlapping plasmid clones. In both cells, more than 90% of transcription start sites (TSSs) of expressed genes showed enrichments with PolII, di-methylated lysine 4 of histone H3 (H3K4me2), tri-methylated lysine 4 of histone H3 (H3K4me3) or acetylated H3 (H3Ac), whereas mono-methylated lysine 4 of histone H3 (H3K4me1) signals did not correlate with expression. No TSSs were enriched with tri-methylated lysine 27 of histone H3 (H3K27me3) in HeLa S3, while eight TSSs (4 expressed) showed enrichments in NT2/D1. We have also located several CTCF binding sites that are potential insulator elements.In summary, we annotated a number of putative regulatory elements in 20q13.12 and went on to verify experimentally a subset of them using dual luciferase reporter assays. Correlating this data to sequence variation can aid identification of disease causing variants

Comparison of Variant Calls from Whole Genome and Whole Exome Sequencing Data Using Matched Samples

Whole exome sequencing (WES) has been extensively used in genomic research. As sequencing costs decline it is being replaced by whole genome sequencing (WGS) in large-scale genomic studies, but more comparative information on WES and WGS datasets would be valuable. Thus, we have extensively compared variant calls obtained from WGS and WES of matched germline DNA samples from 96 lung cancer patients. WGS provided more homogeneous coverage with higher genotyping quality, and identified more variants, than WES, regardless of exome coverage depth. It also called more reference variants, reflecting its power to call rare variants, and more heterozygous variants that met applied quality criteria, indicating that WGS is less prone to allelic drop outs. However, increasing WES coverage reduced the discrepancy between the WES and WGS results. We believe that as sequencing costs further decline WGS will become the method of choice even for research confined to the exome.QC 20201118</p

A Multi-Omics Approach to Liver Diseases : Integration of Single Nuclei Transcriptomics with Proteomics and HiCap Bulk Data in Human Liver

The liver is the largest solid organ and a primary metabolic hub. In recent years, intact cell nuclei were used to perform single-nuclei RNA-seq (snRNA-seq) for tissues difficult to dissociate and for flash-frozen archived tissue samples to discover unknown and rare cell subpopulations. In this study, we performed snRNA-seq of a liver sample to identify subpopulations of cells based on nuclear transcriptomics. In 4282 single nuclei, we detected, on average, 1377 active genes and we identified seven major cell types. We integrated data from 94,286 distal interactions (p &lt; 0.05) for 7682 promoters from a targeted chromosome conformation capture technique (HiCap) and mass spectrometry proteomics for the same liver sample. We observed a reasonable correlation between proteomics and in silico bulk snRNA-seq (r = 0.47) using tissue-independent gene-specific protein abundancy estimation factors. We specifically looked at genes of medical importance. The DPYD gene is involved in the pharmacogenetics of fluoropyrimidine toxicity and some of its variants are analyzed for clinical purposes. We identified a new putative polymorphic regulatory element, which may contribute to variation in toxicity. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and we investigated all known risk genes. We identified a complex regulatory landscape for the SLC2A2 gene with 16 candidate enhancers. Three of them harbor somatic motif breaking and other mutations in HCC in the Pan Cancer Analysis of Whole Genomes dataset and are candidates to contribute to malignancy. Our results highlight the potential of a multi-omics approach in the study of human diseases.De två första författarna delar förstaförfattarskapet</p

Whole-genome sequencing and gene network modules predict gemcitabine/carboplatin-induced myelosuppression in non-small cell lung cancer patients

Gemcitabine/carboplatin chemotherapy commonly induces myelosuppression, including neutropenia, leukopenia, and thrombocytopenia. Predicting patients at risk of these adverse drug reactions (ADRs) and adjusting treatments accordingly is a long-term goal of personalized medicine. This study used whole-genome sequencing (WGS) of blood samples from 96 gemcitabine/carboplatin-treated non-small cell lung cancer (NSCLC) patients and gene network modules for predicting myelosuppression. Association of genetic variants in PLINK found 4594, 5019, and 5066 autosomal SNVs/INDELs with p ≤ 1 × 10−3 for neutropenia, leukopenia, and thrombocytopenia, respectively. Based on the SNVs/INDELs we identified the toxicity module, consisting of 215 unique overlapping genes inferred from MCODE-generated gene network modules of 350, 345, and 313 genes, respectively. These module genes showed enrichment for differentially expressed genes in rat bone marrow, human bone marrow, and human cell lines exposed to carboplatin and gemcitabine (p &lt; 0.05). Then using 80% of the patients as training data, random LASSO reduced the number of SNVs/INDELs in the toxicity module into a feasible prediction model consisting of 62 SNVs/INDELs that accurately predict both the training and the test (remaining 20%) data with high (CTCAE 3–4) and low (CTCAE 0–1) maximal myelosuppressive toxicity completely, with the receiver-operating characteristic (ROC) area under the curve (AUC) of 100%. The present study shows how WGS, gene network modules, and random LASSO can be used to develop a feasible and tested model for predicting myelosuppressive toxicity. Although the proposed model predicts myelosuppression in this study, further evaluation in other studies is required to determine its reproducibility, usability, and clinical effect.Funding agencies: Swedish Cancer Society, the Swedish Research Council, Linköping University, ALF grants Region Östergötland, the Funds of Radiumhemmet, Marcus Borgströms stiftelse, Stiftelsen Assar Gabrielssons Fond</p

Current challenges in understanding the role of enhancers in disease

Enhancers play a central role in the spatiotemporal control of gene expression and tend to work in a cell-type-specific manner. In addition, they are suggested to be major contributors to phenotypic variation, evolution and disease. There is growing evidence that enhancer dysfunction due to genetic, structural or epigenetic mechanisms contributes to a broad range of human diseases referred to as enhanceropathies. Such mechanisms often underlie the susceptibility to common diseases, but can also play a direct causal role in cancer or Mendelian diseases. Despite the recent gain of insights into enhancer biology and function, we still have a limited ability to predict how enhancer dysfunction impacts gene expression. Here we discuss the major challenges that need to be overcome when studying the role of enhancers in disease etiology and highlight opportunities and directions for future studies, aiming to disentangle the molecular basis of enhanceropathies

Visualization and analysis of gene expression in tissue sections by spatial transcriptomics

Analysis of the pattern of proteins or messenger RNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics.This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics