Chromatin accessibility underlies synthetic lethality of SWI/SNF subunits in ARID1A-mutant cancers.

ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, is frequently mutated in cancer. Deficiency in its homolog ARID1B is synthetically lethal with ARID1A mutation. However, the functional relationship between these homologs has not been explored. Here, we use ATAC-seq, genome-wide histone modification mapping, and expression analysis to examine colorectal cancer cells lacking one or both ARID proteins. We find that ARID1A has a dominant role in maintaining chromatin accessibility at enhancers, while the contribution of ARID1B is evident only in the context of ARID1A mutation. Changes in accessibility are predictive of changes in expression and correlate with loss of H3K4me and H3K27ac marks, nucleosome spacing, and transcription factor binding, particularly at growth pathway genes including MET. We find that ARID1B knockdown in ARID1A mutant ovarian cancer cells causes similar loss of enhancer architecture, suggesting that this is a conserved function underlying the synthetic lethality between ARID1A and ARID1B

The TAM receptor tyrosine kinases Axl and Mer drive the maintenance of highly phagocytic macrophages

Many apoptotic thymocytes are generated during the course of T cell selection in the thymus, yet the machinery through which these dead cells are recognized and phagocytically cleared is incompletely understood. We found that the TAM receptor tyrosine kinases Axl and Mer, which are co-expressed by a specialized set of phagocytic thymic macrophages, are essential components of this machinery. Mutant mice lacking Axl and Mer exhibited a marked accumulation of apoptotic cells during the time that autoreactive and nonreactive thymocytes normally die. Unexpectedly, these double mutants also displayed a profound deficit in the total number of highly phagocytic macrophages in the thymus, and concomitantly exhibited diminished expression of TIM-4, CD163, and other non-TAM phagocytic engulfment systems in the macrophages that remained. Importantly, these previously unrecognized deficits were not confined to the thymus, as they were also evident in the spleen and bone marrow. They had pleiotropic consequences for the double mutants, also previously unrecognized, which included dysregulation of hemoglobin turnover and iron metabolism leading to anemia

BART: bioinformatics array research tool

Abstract Background Microarray experiments comprise more than half of all series in the Gene Expression Omnibus (GEO). However, downloading and analyzing raw or semi-processed microarray data from GEO is not intuitive and requires manual error-prone analysis and a bioinformatics background. This is due to a lack of standardization in array platform fabrication as well as the lack of a simple interactive tool for clustering, plotting, differential expression testing, and testing for functional enrichment. Results We introduce the Bioinformatics Array Research Tool (BART), an R Shiny web application that automates the microarray download and analysis process across diverse microarray platforms. It provides an intuitive interface, automatically downloads and parses data from GEO, suggests groupings of samples for differential expression testing, performs batch effect correction, outputs quality control plots, converts probe IDs, generates full lists of differentially expressed genes, and performs functional enrichment analysis. We show that BART enables a more comprehensive analysis of a wider range of microarray datasets on GEO by comparing it to four leading online microarray analysis tools. Conclusions BART allows a scientist with no bioinformatics background to extract knowledge from their own microarray data or microarray experiments available from GEO. BART is functional on more microarray experiments and provides more comprehensive analyses than extant microarray analysis tools. BART is hosted on bart.salk.edu, includes a user tutorial, and is available for download from https://bitbucket.org/Luisa_amaral/bart

An RNA polymerase ribozyme that synthesizes its own ancestor

The RNA-based organisms from which modern life is thought to have descended would have depended on an RNA polymerase ribozyme to copy functional RNA molecules, including copying the polymerase itself. Such a polymerase must have been capable of copying structured RNAs with high efficiency and high fidelity to maintain genetic information across successive generations. Here the class I RNA polymerase ribozyme was evolved in vitro for the ability to synthesize functional ribozymes, resulting in the markedly improved ability to synthesize complex RNAs using nucleoside 5'-triphosphate (NTP) substrates. The polymerase is descended from the class I ligase, which contains the same catalytic core as the polymerase. The class I ligase can be synthesized by the improved polymerase as three separate RNA strands that assemble to form a functional ligase. The polymerase also can synthesize the complement of each of these three strands. Despite this remarkable level of activity, only a very small fraction of the assembled ligases retain catalytic activity due to the presence of disabling mutations. Thus, the fidelity of RNA polymerization should be considered a major impediment to the construction of a self-sustained, RNA-based evolving system. The propagation of heritable information requires both efficient and accurate synthesis of genetic molecules, a requirement relevant to both laboratory systems and the early history of life on Earth

Glioblastoma Model Using Human Cerebral Organoids

Summary: We have developed a cancer model of gliomas in human cerebral organoids that allows direct observation of tumor initiation as well as continuous microscopic observations. We used CRISPR/Cas9 technology to target an HRasG12V-IRES-tdTomato construct by homologous recombination into the TP53 locus. Results show that transformed cells rapidly become invasive and destroy surrounding organoid structures, overwhelming the entire organoid. Tumor cells in the organoids can be orthotopically xenografted into immunodeficient NOD/SCID IL2RG−/− animals, exhibiting an invasive phenotype. Organoid-generated putative tumor cells show gene expression profiles consistent with mesenchymal subtype human glioblastoma. We further demonstrate that human-organoid-derived tumor cell lines or primary human-patient-derived glioblastoma cell lines can be transplanted into human cerebral organoids to establish invasive tumor-like structures. Our results show potential for the use of organoids as a platform to test human cancer phenotypes that recapitulate key aspects of malignancy. : Ogawa et al. show that human cerebral organoids can be used as a platform for tumor formation. CRISPR/Cas9 manipulation of oncogenes/tumor suppressors initiates tumorigenesis in cerebral organoids, allowing microscopic observation of tumor development. Additionally, human cerebral organoids can be used as a platform for tumor cell transplantation. Keywords: cerebral organoid, glioblastoma, glioma, CRISPR/Cas

The Aging Astrocyte Transcriptome from Multiple Regions of the Mouse Brain

Summary: Aging brains undergo cognitive decline, associated with decreased neuronal synapse number and function and altered metabolism. Astrocytes regulate neuronal synapse formation and function in development and adulthood, but whether these properties change during aging, contributing to neuronal dysfunction, is unknown. We addressed this by generating aged and adult astrocyte transcriptomes from multiple mouse brain regions. These data provide a comprehensive RNA-seq database of adult and aged astrocyte gene expression, available online as a resource. We identify astrocyte genes altered by aging across brain regions and regionally unique aging changes. Aging astrocytes show minimal alteration of homeostatic and neurotransmission-regulating genes. However, aging astrocytes upregulate genes that eliminate synapses and partially resemble reactive astrocytes. We further identified heterogeneous expression of synapse-regulating genes between astrocytes from different cortical regions. We find that alterations to astrocytes in aging create an environment permissive to synapse elimination and neuronal damage, potentially contributing to aging-associated cognitive decline. : The aging brain has reduced synapse number and decreased neuronal activity, functions regulated by neighboring astrocytes. Boisvert et al. investigated if aging astrocytes are contributing to these changes and found that aged astrocytes show increased expression of genes for inflammatory and synapse elimination pathways and decreased cholesterol synthesis enzymes. Keywords: astrocyte, synapse, aging, neurodegeneratio

Accurate annotation of human protein-coding small open reading frames.

Functional protein-coding small open reading frames (smORFs) are emerging as an important class of genes. However, the number of translated smORFs in the human genome is unclear because proteogenomic methods are not sensitive enough, and, as we show, Ribo-seq strategies require additional measures to ensure comprehensive and accurate smORF annotation. Here, we integrate de novo transcriptome assembly and Ribo-seq into an improved workflow that overcomes obstacles with previous methods, to more confidently annotate thousands of smORFs. Evolutionary conservation analyses suggest that hundreds of smORF-encoded microproteins are likely functional. Additionally, many smORFs are regulated during fundamental biological processes, such as cell stress. Peptides derived from smORFs are also detectable on human leukocyte antigen complexes, revealing smORFs as a source of antigens. Thus, by including additional validation into our smORF annotation workflow, we accurately identify thousands of unannotated translated smORFs that will provide a rich pool of unexplored, functional human genes

Accurate annotation of human protein-coding small open reading frames.

Functional protein-coding small open reading frames (smORFs) are emerging as an important class of genes. However, the number of translated smORFs in the human genome is unclear because proteogenomic methods are not sensitive enough, and, as we show, Ribo-seq strategies require additional measures to ensure comprehensive and accurate smORF annotation. Here, we integrate de novo transcriptome assembly and Ribo-seq into an improved workflow that overcomes obstacles with previous methods, to more confidently annotate thousands of smORFs. Evolutionary conservation analyses suggest that hundreds of smORF-encoded microproteins are likely functional. Additionally, many smORFs are regulated during fundamental biological processes, such as cell stress. Peptides derived from smORFs are also detectable on human leukocyte antigen complexes, revealing smORFs as a source of antigens. Thus, by including additional validation into our smORF annotation workflow, we accurately identify thousands of unannotated translated smORFs that will provide a rich pool of unexplored, functional human genes

Heterozygous Mutations in SMARCA2 Reprogram the Enhancer Landscape by Global Retargeting of SMARCA4

Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4