Regulation of heterochromatic DNA replication by histone H3 lysine 27 methyltransferases.

Multiple pathways prevent DNA replication from occurring more than once per cell cycle. These pathways block re-replication by strictly controlling the activity of pre-replication complexes, which assemble at specific sites in the genome called origins. Here we show that mutations in the homologous histone 3 lysine 27 (H3K27) monomethyltransferases, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, lead to re-replication of specific genomic locations. Most of these locations correspond to transposons and other repetitive and silent elements of the Arabidopsis genome. These sites also correspond to high levels of H3K27 monomethylation, and mutation of the catalytic SET domain is sufficient to cause the re-replication defect. Mutation of ATXR5 and ATXR6 also causes upregulation of transposon expression and has pleiotropic effects on plant development. These results uncover a novel pathway that prevents over-replication of heterochromatin in Arabidopsis.

Hepatitis B virus X protein stabilizes amplified in breast cancer 1 protein and cooperates with it to promote human hepatocellular carcinoma cell invasiveness.

UNLABELLED: Chronic infection of hepatitis B virus (HBV) is closely associated with the development of human hepatocellular carcinoma (HCC). HBV X protein (HBx) plays a key role in the progression of HCC. We recently found that amplified in breast cancer 1 (AIB1) protein is overexpressed in 68% of human HCC specimens and promotes HCC progression by enhancing cell proliferation and invasiveness. Given that both HBx and AIB1 play important oncogenic roles in HCC, we aimed to determine whether they could cooperatively promote human HCC development. Herein, we show that HBx-positive HCC tissues had a higher level of AIB1 protein, compared to HBx-negative HCC tissues. A positive correlation between HBx protein level and AIB1 protein level was established in HCC specimens. Without affecting its messenger RNA level, HBx induced a significant increase of the protein level of AIB1, which correlated with a significant extension of the half-life of AIB1 protein. Mechanistically, HBx could interact with AIB1 to prevent the interaction between envelope protein 3 ubiquitin ligase F-box and WD repeat domain containing 7 (Fbw7)α and AIB1, then inhibited the Fbw7α-mediated ubiquitination and degradation of AIB1. In addition, reporter assays and chromatin immunoprecipitation assays revealed that both HBx and AIB1 were recruited to matrix metalloproteinase-9 (MMP-9) promoter to enhance MMP-9 promoter activity cooperatively. Consistently, HBx and AIB1 cooperatively enhanced MMP-9 expression in HepG2 cells, which, in turn, increased cell-invasive ability. CONCLUSION: Our study demonstrates that HBx can stabilize AIB1 protein and cooperate with it to promote human HCC cell invasiveness, highlighting the essential role of the cross-talk between HBx and AIB1 in HBV-related HCC progression.

Impact of delayed PBMC processing on functional and genomic assays.

Peripheral blood mononuclear cells (PBMCs) are commonly isolated from whole blood samples in clinical trials. Isolated PBMCs can be cryopreserved for use in downstream assays such as flow cytometry, single-cell RNA sequencing (scRNA-seq) and enzyme-linked immunosorbent spot (ELISpot) assays to aid understanding of disease biology and treatment effects, and biomarker identification. However, due to logistical practicalities, delays from blood collection to PBMC processing may exceed 24 h, which can potentially affect PBMC function and, ultimately, downstream assay results. Whole blood samples from 20 healthy adults were collected and incubated at 20-25 °C for 2-48 h before PBMC processing. PBMC viability was measured, and flow cytometry immunophenotyping, scRNA-seq and ELISpot were performed following increasing PBMC processing delays. The RosetteSep™ granulocyte depletion kit was used to evaluate the impact of granulocyte contamination following processing delay. Processed scRNA-seq reads were used to identify cell clusters based on marker genes. scRNA-seq data was further used to determine gene expression correlation and pathway activity score in major PBMC cell types (T cells, B cells, natural killer cells, monocytes and dendritic cells) between PBMC preparations subjected to shorter (2-4 h) and longer (8-48 h) processing delays. ELISpot assays evaluated the impact of processing delays on the number of interferon-γ (IFN-γ) secreting cells from ex vivo stimulated PBMCs. PBMC viability was reduced after a 48-h processing delay. Flow cytometry showed that granulocyte contamination of PBMCs increased after 24 h. Cluster analysis of scRNA-seq data identified 23 immune cell type gene expression clusters that were not significantly changed upon granulocyte depletion. Gene expression correlations across the major PBMC cell types were < 0.8 after 24 h of delay compared with 2 or 4 h of delay. Inflammatory, proliferation and signaling pathway activities increased, whereas IFN-γ and metabolic pathway activities decreased with increasing PBMC processing delays. The number of IFN-γ secreting cells trended towards a reduction as PBMC processing delays increased. PBMC processing delays should be minimised when designing clinical trials to reduce outcome variability in downstream assays. Ideally clinical trial sites should have on-site PBMC processing capabilities or be located close to such facilities.

RNA-seq RNAaccess identified as the preferred method for gene expression analysis of low quality FFPE samples.

Clinical tumor tissues that are preserved as formalin-fixed paraffin-embedded (FFPE) samples result in extensive cross-linking, fragmentation, and chemical modification of RNA, posing significant challenges for RNA-seq-based gene expression profiling. This study sought to define an optimal RNA-seq protocol for FFPE samples. We employed a common RNA extraction method and then compared RNA-seq library preparation protocols including RNAaccess, RiboZero and PolyA in terms of sequencing quality and concordance of gene expression using FFPE and case-matched fresh-frozen (FF) triple-negative breast cancer (TNBC) tissues. We found that RNAaccess, a method based on exome capture, produced the most concordant results. Applying RNAaccess to FFPE gastric cancer tissues, we established a minimum RNA DV200 requirement of 10% and a RNA input amount of 10ng that generated highly reproducible gene expression data. Lastly, we demonstrated that RNAaccess and NanoString platforms produced highly concordant expression profiles from FFPE samples for shared genes; however, RNA-seq may be preferred for clinical biomarker discovery work because of the broader coverage of the transcriptome. Taken together, these results support the selection of RNA-seq RNAaccess method for gene expression profiling of FFPE samples. The minimum requirements for RNA quality and input established here may allow for inclusion of clinical FFPE samples of sub-optimal quality in gene expression analyses and ultimately increasing the statistical power of such analyses.

Effects of JAK1-Preferential Inhibitor Filgotinib on Circulating Biomarkers and Whole Blood Genes/Pathways of Patients With Moderately to Severely Active Crohn's Disease.

BACKGROUND: Pro-inflammatory cytokines are dysregulated in Crohn's disease (CD) and could serve as surrogate markers to improve diagnostic and therapeutic approaches, potentially addressing an unmet need. We profiled circulating biomarkers and whole blood transcriptional pathway activity to identify those associated with CD using data from the phase 2 FITZROY study with filgotinib, an oral preferential janus kinase-1 inhibitor. METHODS: Patients with serum and whole blood samples taken from the induction period were included. Serum cytokines were measured (ELISA), whole blood RNA sequenced, and stool samples taken to measure fecal calprotectin (FC). Spearman's Rank correlations were assessed between biomarkers and baseline disease activity; post-treatment endoscopic improvement was measured by the Simplified Endoscopy Score for CD (SES-CD), FC and the Crohn's Disease Activity Index. Effect of filgotinib on circulating biomarkers was also evaluated. RESULTS: Serum biomarkers (n = 168) and whole blood RNA sequencing (n = 104) were assessed. Moderate correlation between serum analytes with SES-CD and FC was noted; most highly correlated were acute phase proteins CRP (rho = 0.35 [SES-CD] and 0.47 [FC]), serum amyloid A (rho = 0.40 and 0.39, respectively) and pro-inflammatory cytokines interleukin (IL)-6 (rho = 0.31 and 0.30, respectively), IL-22 (rho = 0.36 and 0.35, respectively), and oncostatin M (rho = 0.35 and 0.33, respectively). Filgotinib treatment was associated with reduction of many candidate biomarkers, particularly in patients with treatment response. Early changes in IL-6 and IL-10 may be prognostic for endoscopic response. CONCLUSIONS: Several circulating factors with potential as CD activity biomarkers were identified. Larger studies are necessary to investigate the best utility of these markers for CD.

AGOUTI: improving genome assembly and annotation using transcriptome data.

BACKGROUND: Genomes sequenced using short-read, next-generation sequencing technologies can have many errors and may be fragmented into thousands of small contigs. These incomplete and fragmented assemblies lead to errors in gene identification, such that single genes spread across multiple contigs are annotated as separate gene models. Such biases can confound inferences about the number and identity of genes within species, as well as gene gain and loss between species. RESULTS: We present AGOUTI (Annotated Genome Optimization Using Transcriptome Information), a tool that uses RNA sequencing data to simultaneously combine contigs into scaffolds and fragmented gene models into single models. We show that AGOUTI improves both the contiguity of genome assemblies and the accuracy of gene annotation, providing updated versions of each as output. Running AGOUTI on both simulated and real datasets, we show that it is highly accurate and that it achieves greater accuracy and contiguity when compared with other existing methods. CONCLUSION: AGOUTI is a powerful and effective scaffolder and, unlike most scaffolders, is expected to be more effective in larger genomes because of the commensurate increase in intron length. AGOUTI is able to scaffold thousands of contigs while simultaneously reducing the number of gene models by hundreds or thousands. The software is available free of charge under the MIT license.