The Affymetrix technology is nowadays a well-established method for the analysis of gene expression profiles in cancer research studies. However, changes in gene expression levels are not the only ...way to link genes and disease. The existence of gene isoforms specifically linked with cancer or apoptosis is increasingly found in literature. Hence it is of great interest to associate the results of a gene expression study with updated evidences on the transcript structure and its possible variants.
We present here a web-based software tool, Splicy, whose primary task is to retrieve data on the mapping of Affymetrix probes to single exons of gene transcripts and displaying graphically this information projected on the gene physical structure. Starting from a list of Affymetrix probesets the program produces a series of graphical displays, each relative to a transcript associated with the gene targeted by a given probe. The information on the transcript-by-transcript and exon-by-exon mapping of probe pairs can be retrieved both graphically and in the form of tab-separated files. The mapping of single probes to NCBI RefSeq or EMBL cDNAs is handled by the ISREC mapping tables used in the CleanEx Expression Reference Database Project. We currently maintain these mappings for most popular human and mouse Affymetrix chips, and Splicy can be queried for matches with human and mouse NCBI RefSeq or EMBL cDNAs.
Splicy generates probeset annotations and images describing the relation between the single probes and intron/exon structure of the target transcript in all its known variants. We think that Splicy will be useful for giving to the researcher a clearer picture of the possible transcript variants linked with a given gene and an additional view on the interpretation of microarray experiment data. Splicy is publicly available and has been realized in the framework of a bioinformatics grant from the Italian Cancer Research Association.
Immune escape represents a major driver of acute myeloid leukemia (AML) reemergence after allogeneic hematopoietic cell transplantation (allo-HCT), with up to 40% of relapses prompted by nongenomic ...loss of HLA class II expression in leukemia cells. By integrative analysis of gene expression, DNA methylation, and chromatin accessibility in paired diagnosis/relapse primary samples and in the respective patient-derived xenografts (PDX), we identify the polycomb repressive complex 2 (PRC2) as a key epigenetic driver of this immune escape modality. We report that loss of expression of HLA class II molecules is accompanied by a PRC2-dependent reduction in chromatin accessibility. Pharmacologic inhibition of PRC2 subunits rescues HLA class II expression in AML relapses in vitro and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML posttransplantation relapse.
Loss of HLA class II expression represents a frequent mechanism of leukemia posttransplantation relapse. Here we identify PRC2 as the main epigenetic driver of this immune escape modality and show that its chemical inhibition can reinstate a proficient graft-versus-leukemia effect, providing an innovative rationale for personalized epigenetic immunotherapies. See related commentary by Köhler and Zeiser, p. 1410. This article is highlighted in the In This Issue feature, p. 1397.
Genetic mutations in TBC1D24 have been associated with multiple phenotypes, with epilepsy being the main clinical manifestation. The TBC1D24 protein consists of the unique association of a ...Tre2/Bub2/Cdc16 (TBC) domain and a TBC/lysin motif domain/catalytic (TLDc) domain. More than 50 missense and loss-of-function mutations have been described and are spread over the entire protein. Through whole genome/exome sequencing we identified compound heterozygous mutations, R360H and G501R, within the TLDc domain, in an index family with a Rolandic epilepsy exercise-induced dystonia phenotype (http://omim.org/entry/608105). A 20-year long clinical follow-up revealed that epilepsy was self-limited in all three affected patients, but exercise-induced dystonia persisted into adulthood in two. Furthermore, we identified three additional sporadic paediatric patients with a remarkably similar phenotype, two of whom had compound heterozygous mutations consisting of an in-frame deletion I81_K84 and an A500V mutation, and the third carried T182M and G511R missense mutations, overall revealing that all six patients harbour a missense mutation in the subdomain of TLDc between residues 500 and 511. We solved the crystal structure of the conserved Drosophila TLDc domain. This allowed us to predict destabilizing effects of the G501R and G511R mutations and, to a lesser degree, of R360H and potentially A500V. Next, we characterized the functional consequences of a strong and a weak TLDc mutation (TBC1D24G501R and TBC1D24R360H) using Drosophila, where TBC1D24/Skywalker regulates synaptic vesicle trafficking. In a Drosophila model neuronally expressing human TBC1D24, we demonstrated that the TBC1D24G501R TLDc mutation causes activity-induced locomotion and synaptic vesicle trafficking defects, while TBC1D24R360H is benign. The neuronal phenotypes of the TBC1D24G501R mutation are consistent with exacerbated oxidative stress sensitivity, which is rescued by treating TBC1D24G501R mutant animals with antioxidants N-acetylcysteine amide or α-tocopherol as indicated by restored synaptic vesicle trafficking levels and sustained behavioural activity. Our data thus show that mutations in the TLDc domain of TBC1D24 cause Rolandic-type focal motor epilepsy and exercise-induced dystonia. The humanized TBC1D24G501R fly model exhibits sustained activity and vesicle transport defects. We propose that the TBC1D24/Sky TLDc domain is a reactive oxygen species sensor mediating synaptic vesicle trafficking rates that, when dysfunctional, causes a movement disorder in patients and flies. The TLDc and TBC domain mutations' response to antioxidant treatment we observed in the animal model suggests a potential for combining antioxidant-based therapeutic approaches to TBC1D24-associated disorders with previously described lipid-altering strategies for TBC domain mutations.
Background: Analysis of scATAC-seq data has been recently scaled to thousands of cells. While processing of other types of single cell data was boosted by the implementation of alignment-free ...techniques, pipelines available to process scATAC-seq data still require large computational resources. We propose here an approach based on pseudoalignment, which reduces the execution times and hardware needs at little cost for precision.
Methods: Public data for 10k PBMC were downloaded from 10x Genomics web site. Reads were aligned to various references derived from DNase I Hypersensitive Sites (DHS) using
kallisto and quantified with
bustools. We compared our results with the ones publicly available derived by
cellranger-atac.
Results: We found that
kallisto does not introduce biases in quantification of known peaks and cells groups are identified in a consistent way. We also found that cell identification is robust when analysis is performed using DHS-derived reference in place of
de novo identification of ATAC peaks. Lastly, we found that our approach is suitable for reliable quantification of gene activity based on scATAC-seq signal, thus allows for efficient labelling of cell groups based on marker genes.
Conclusions: Analysis of scATAC-seq data by means of
kallisto produces results in line with standard pipelines while being considerably faster; using a set of known DHS sites as reference does not affect the ability to characterize the cell populations
Focal deletions occur frequently in the cancer genome. However, the putative tumor‐suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, ...we implemented a computational approach based on non‐negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1‐dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5′‐capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC. These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation.
Synopsis
Computational analysis of frequent cancer genome deletions reveals that PNRC1‐dependent nucleolar recruitment of the cytoplasmic mRNA decapping complex blocks ribosomal maturation and ablates oncogene‐induced cell proliferation.
Nuclear receptor co‐activator PNRC1 is frequently deleted in cancer cells.
PNRC1 interacts with the DCP1α/DCP2 decapping complex and stimulates its translocation into the nucleolus.
PNRC1 expression blocks ribosomal RNA processing in cancer cells.
The nucleolar PNRC1‐DCP1α/DCP2 complex targets the U3 and U8 snoRNAs for decapping.
PNRC1 expression ablates oncogene‐induced proliferation, suggesting a tumor suppressive role.
Computational analysis of frequent cancer genome deletions reveals that mRNA decapping activity blocks ribosomal maturation and ablates oncogene‐induced proliferation.
Abstract
Background
Biological networks are often used to describe the relationships between relevant entities, particularly genes and proteins, and are a powerful tool for functional genomics. Many ...important biological problems can be investigated by comparing biological networks between different conditions or networks obtained with different techniques.
Findings
We show that contrast subgraphs, a recently introduced technique to identify the most important structural differences between 2 networks, provide a versatile tool for comparing gene and protein networks of diverse origin. We demonstrate the use of contrast subgraphs in the comparison of coexpression networks derived from different subtypes of breast cancer, coexpression networks derived from transcriptomic and proteomic data, and protein–protein interaction networks assayed in different cell lines.
Conclusions
These examples demonstrate how contrast subgraphs can provide new insight in functional genomics by extracting the gene/protein modules whose connectivity is most altered between 2 conditions or experimental techniques.
DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the ...chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3.
Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers ...from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks. ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs' position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome. Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final evaluation in the therapeutically relevant setting of interest.
Invariant natural killer T cells (iNKT) cells are T lymphocytes displaying innate effector functions, acquired through a distinct thymic developmental program regulated by microRNAs (miRNAs). ...Deleting miRNAs by Dicer ablation (Dicer KO) in thymocytes selectively impairs iNKT cell survival and functional differentiation. To unravel this miRNA-dependent program, we systemically identified transcripts that were differentially expressed between WT and Dicer KO iNKT cells at different differentiation stages and predicted to be targeted by the iNKT cell-specific miRNAs. TGF-β receptor II (TGF- βRII), critically implicated in iNKT cell differentiation, was found up-regulated in iNKT Dicer KO cells together with enhanced TGF-β signaling. miRNA members of the miR-17∼92 family clusters were predicted to target Tgfbr2 mRNA upon iNKT cell development. iNKT cells lacking all three miR-17∼92 family clusters (miR- 17∼92, miR-106a∼363, miR-106b∼25) phenocopied both increased TGF-βRII expression and signaling, and defective effector differentiation, displayed by iNKT Dicer KO cells. Consistently, genetic ablation of TGF-β signaling in the absence of miRNAs rescued iNKT cell differentiation. These results elucidate the global impact of miRNAs on the iNKT cell developmental program and uncover the targeting of a lineage-specific cytokine signaling by miRNAs as a mechanism regulating innate-like T-cell development and effector differentiation.
Multiple myeloma is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins, which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The ...genomic landscape of multiple myeloma is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of multiple myeloma cells. One of these is
that is lost in more than 10% of patients with multiple myeloma. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A, which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitution of FAM46C in multiple myeloma cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased translation of IgG mRNA, but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER bound protein FNDC3A to reside in the cytoplasmic side of the ER. FNDC3A was lost in some multiple myeloma cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that multiple myeloma cells remodel their trafficking machinery to cope with ER stress. SIGNIFICANCE: This study identifies a new multiple myeloma-specific tumor suppressor complex that regulates autophagy and unconventional secretion, highlighting the sensitivity of multiple myeloma cells to the accumulation of protein aggregates.