Long noncoding RNAs (lncRNA) play an important role in gene regulation and contribute to tumorigenesis. While pan-cancer studies of lncRNA expression have been performed for adult malignancies, the ...lncRNA landscape across pediatric cancers remains largely uncharted. Here, we curated RNA sequencing data for 1,044 pediatric leukemia and extracranial solid tumors and integrated paired tumor whole genome sequencing and epigenetic data in relevant cell line models to explore lncRNA expression, regulation, and association with cancer. A total of 2,657 lncRNAs were robustly expressed across six pediatric cancers, including 1,142 exhibiting histotype-elevated expression. DNA copy number alterations contributed to lncRNA dysregulation at a proportion comparable to protein coding genes. Application of a multidimensional framework to identify and prioritize lncRNAs impacting gene networks revealed that lncRNAs dysregulated in pediatric cancer are associated with proliferation, metabolism, and DNA damage hallmarks. Analysis of upstream regulation via cell type-specific transcription factors further implicated distinct histotype-elevated and developmental lncRNAs. Integration of these analyses prioritized lncRNAs for experimental validation, and silencing of TBX2-AS1, the top-prioritized neuroblastoma-specific lncRNA, resulted in significant growth inhibition of neuroblastoma cells, confirming the computational predictions. Taken together, these data provide a comprehensive characterization of lncRNA regulation and function in pediatric cancers and pave the way for future mechanistic studies.
Comprehensive characterization of lncRNAs in pediatric cancer leads to the identification of highly expressed lncRNAs across childhood cancers, annotation of lncRNAs showing histotype-specific elevated expression, and prediction of lncRNA gene regulatory networks.
BACKGROUND. Multiple islet autoantibodies (AAbs) predict the development of type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in only approximately 15% of individuals ...who are positive for single AAbs (generally against glutamic acid decarboxylase GADA); hence, the single GADA+ state may represent an early stage of T1D. METHODS. Here, we functionally, histologically, and molecularly phenotyped human islets from nondiabetic GADA+ and T1D donors. RESULTS. Similar to the few remaining ß cells in the T1D islets, GADA+ donor islets demonstrated a preserved insulin secretory response. By contrast, a cell glucagon secretion was dysregulated in both GADA+ and T1D islets, with impaired glucose suppression of glucagon secretion. Single-cell RNA-Seq of GADA+ a cells revealed distinct abnormalities in glycolysis and oxidative phosphorylation pathways and a marked downregulation of cAMP-dependent protein kinase inhibitor ß (PKIB), providing a molecular basis for the loss of glucose suppression and the increased effect of 3-isobutyl-1-methylxanthine (IBMX) observed in GADA+ donor islets. CONCLUSION. We found that a cell dysfunction was present during the early stages of islet autoimmunity at a time when ß cell mass was still normal, raising important questions about the role of early a cell dysfunction in the progression of T1D. FUNDING. This work was supported by grants from the NIH (3UC4DK112217-01S1, U01DK123594-02, UC4DK112217, UC4DK112232, U01DK123716, and P30 DK019525) and the Vanderbilt Diabetes Research and Training Center (DK20593).
FUNDING. This work was supported by grants from the NIH (3UC4DK112217-01S1, U01DK123594-02, UC4DK112217, UC4DK112232, U01DK123716, and P30 DK019525) and the Vanderbilt Diabetes Research and Training ...Center (DK20593).
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial pneumonia of unknown etiology. The role of genetic risk factors has been the focus of numerous studies probing ...for associations of genetic variants with IPF. We aimed to determine whether single-nucleotide polymorphisms (SNPs) of four candidate genes are associated with IPF susceptibility and survival in a Portuguese population. A retrospective case–control study was performed with 64 IPF patients and 74 healthy controls. Ten single-nucleotide variants residing in the MUC5B, TOLLIP, SERPINB1, and PLAU genes were analyzed. Single- and multi-locus analyses were performed to investigate the predictive potential of specific variants in IPF susceptibility and survival. Multifactor dimensionality reduction (MDR) was employed to uncover predictive multi-locus interactions underlying IPF susceptibility. The MUC5B rs35705950 SNP was significantly associated with IPF: T allele carriers were significantly more frequent among IPF patients (75.0% vs 20.3%,
P
< 1.0 × 10
−6
). Genotypic and allelic distributions of TOLLIP, PLAU, and SERPINB1 SNPs did not differ significantly between groups. However, the MUC5B-TOLLIP T-C-T-C haplotype, defined by the rs35705950-rs111521887-rs5743894-rs5743854 block, emerged as an independent protective factor in IPF survival (HR = 0.37, 95% CI 0.17–0.78,
P
= 0.009, after adjustment for FVC). No significant multi-locus interactions correlating with disease susceptibility were detected. MUC5B rs35705950 was linked to an increased risk for IPF, as reported for other populations, but not to disease survival. A haplotype incorporating SNPs of the MUC5B-TOLLIP locus at 11p15.5 seems to predict better survival and could prove useful for prognostic purposes and IPF patient stratification.
Key messages
The MUC5B rs35705950 minor allele is associated with IPF risk in the Portuguese.
No predictive multi-locus interactions of IPF susceptibility were identified by MDR.
A haplotype defined by MUC5B and TOLLIP SNPs is a protective factor in IPF survival.
The haplotype may be used as a prognostic tool for IPF patient stratification.
Background & Aims: Although trimethylation of histone H3 lysine 27 (H3K27me3) by polycomb repressive complex 2 is required for intestinal function, the role of the antagonistic process—H3K27me3 ...demethylation—in the intestine remains unknown. The aim of this study was to determine the contribution of H3K27me3 demethylases to intestinal homeostasis. Methods: An inducible mouse model was used to simultaneously ablate the 2 known H3K27me3 demethylases, lysine (K)-specific demethylase 6A (Kdm6a) and lysine (K)-specific demethylase 6B (Kdm6b), from the intestinal epithelium. Mice were analyzed at acute and prolonged time points after Kdm6a/b ablation. Cellular proliferation and differentiation were measured using immunohistochemistry, while RNA sequencing and chromatin immunoprecipitation followed by sequencing for H3K27me3 were used to identify gene expression and chromatin changes after Kdm6a/b loss. Intestinal epithelial renewal was evaluated using a radiation-induced injury model, while Paneth cell homeostasis was measured via immunohistochemistry, immunoblot, and transmission electron microscopy. Results: We did not detect any effect of Kdm6a/b ablation on intestinal cell proliferation or differentiation toward the secretory cell lineages. Acute and prolonged Kdm6a/b loss perturbed expression of gene signatures belonging to multiple cell lineages (adjusted P value < .05), and a set of 72 genes was identified as being down-regulated with an associated increase in H3K27me3 levels after Kdm6a/b ablation (false discovery rate, <0.05). After prolonged Kdm6a/b loss, dysregulation of the Paneth cell gene signature was associated with perturbed matrix metallopeptidase 7 localization (P < .0001) and expression. Conclusions: Although KDM6A/B does not regulate intestinal cell differentiation, both enzymes are required to support the full transcriptomic and epigenomic landscape of the intestinal epithelium and the expression of key Paneth cell genes.
Susceptibility to common human diseases such as cancer is influenced by many genetic and environmental factors that work together in a complex manner. The state of the art is to perform a genome-wide ...association study (GWAS) that measures millions of single-nucleotide polymorphisms (SNPs) throughout the genome followed by a one-SNP-at-a-time statistical analysis to detect univariate associations. This approach has identified thousands of genetic risk factors for hundreds of diseases. However, the genetic risk factors detected have very small effect sizes and collectively explain very little of the overall heritability of the disease. Nonetheless, it is assumed that the genetic component of risk is due to many independent risk factors that contribute additively. The fact that many genetic risk factors with small effects can be detected is taken as evidence to support this notion. It is our working hypothesis that the genetic architecture of common diseases is partly driven by non-additive interactions. To test this hypothesis, we developed a heuristic simulation-based method for conducting experiments about the complexity of genetic architecture. We show that a genetic architecture driven by complex interactions is highly consistent with the magnitude and distribution of univariate effects seen in real data. We compare our results with measures of univariate and interaction effects from two large-scale GWASs of sporadic breast cancer and find evidence to support our hypothesis that is consistent with the results of our computational experiment.
Pancreatic β cells, organized in the islets of Langerhans, sense glucose and secrete appropriate amounts of insulin. We have studied the roles of LKB1, a conserved kinase implicated in the control of ...cell polarity and energy metabolism, in adult β cells. LKB1-deficient β cells show a dramatic increase in insulin secretion in vivo. Histologically, LKB1-deficient β cells have striking alterations in the localization of the nucleus and cilia relative to blood vessels, suggesting a shift from hepatocyte-like to columnar polarity. Additionally, LKB1 deficiency causes a 65% increase in β cell volume. We show that distinct targets of LKB1 mediate these effects. LKB1 controls β cell size, but not polarity, via the mTOR pathway. Conversely, the precise position of the β cell nucleus, but not cell size, is controlled by the LKB1 target Par1b. Insulin secretion and content are restricted by LKB1, at least in part, via AMPK. These results expose a molecular mechanism, orchestrated by LKB1, for the coordinated maintenance of β cell size, form, and function.
GWAS has revealed many loci for type 2 diabetes (T2D). However, GWAS just reports genomic signals and not necessarily the precise localization of effector genes, with eQTLs making inferences to only ...a subset of such loci. Chromatin conformation capture-based techniques that detect contacts between distant regions of the genome offer an opportunity to understand GWAS signals that principally reside in non-coding regions, thus likely influencing regulatory elements. To move beyond analyzing one locus at a time and to improve on the low resolution of available Hi-C data, we developed a high resolution Capture-C based method to simultaneously characterize the genome-wide interactions of all human promoters in any cell type. We applied this to the immortalized human β-cell line, EndoC-βH1, a model relevant to T2D. We designed a custom Agilent SureSelect library targeting both ends of DpnII restriction fragments that overlap promoters of protein-coding, plus noncoding, transcripts, totaling 36,691 RNA baited fragments. Following sequencing, we investigated significant interactions at varying resolutions depending on how the fragments were leveraged and/or collapsed. In parallel, we generated ATAC-seq open chromatin maps to filter for informative proxy SNPs (r2>0.8) to each of the 104 T2D independent sentinels reported to date, yielding overall 150 (harbored in 94 DpnII fragments) for 50 of these loci. By filtering our promoter ’interactome’ results at 4 DpnII fragment resolution (median distance between interacting regions ∼115kb, median region size =1,440bp) based on these proxies and openness of the regions they interact with, we observed contacts relevant to 17 of the original loci. Some ’nearest’ genes were supported e.g., GIPR and ZFAND3, while at other loci more distant genes were implicated e.g., OGFOD2 at ’MPHOSPH9’ and MESP2 at ’AP3S2’.
In conclusion, we observed informative contacts at putative effector genes for 16% of T2D GWAS loci in this particular cellular context.
Disclosure
E. Manduchi: None. M. Johnson: None. M. Leonard: None. S. Lu: None. K.M. Hodge: None. A. Chesi: None. A.D. Wells: None. S.F.A. Grant: None.
Abstract
Genome-Wide Association Studies (GWAS) have implicated >60 loci in the susceptibility to systemic lupus erythematosus (SLE). However, GWAS reports signals in non-coding genomic regions, not ...the precise location of culprit genes. Chromatin conformation capture (3C) technologies that detect physical contacts between regions of the genome offer a powerful opportunity to map disease variants to target genes. We developed a massively parallel, high-resolution method to characterize the genome-wide interactomes of 36,691 promoters of protein-coding, noncoding, antisense, snRNA, miRNA, snoRNA and lincRNA genes in any cell type. Using this method, we generated promoter interactomes of primary human T follicular helper (TFH) cells from healthy tonsil, a cell type relevant to SLE as TFH operate upstream of pathogenic autoantibody-producing B cells. These sub-1kb TFH promoter interactome datasets were intersected with maps of TFH open chromatin generated by ATAC-seq and SLE SNPs from the 63 candidate loci, resulting in detection of consistent interactions between genes and accessible SNPs at 48 loci. We find that ~25% of accessible SLE SNPs interact with the nearest gene, e.g. STAT4 and IKZF3, while ~75% of accessible SNPs ‘skip’ the nearest gene to interact with distant genes, e.g. LCLAT1 at the ‘LBH’ locus, and the master TFH transcription factor BCL6 at the ‘LPP-TPRG1’ locus. Gene ontology analysis confirms that genes directly implicated by SNP interactions reside in SLE-relevant networks while ‘nearest to SNP’ genes do not. In conclusion, high-resolution, 3-dimensional promoter interactions with accessible, disease-associated SNPs in disease-relevant tissue connect variants to relevant genes with high apparent accuracy.
Although trimethylation of histone H3 lysine 27 (H3K27me3) by polycomb repressive complex 2 is required for intestinal function, the role of the antagonistic process—H3K27me3 demethylation—in the ...intestine remains unknown. The aim of this study was to determine the contribution of H3K27me3 demethylases to intestinal homeostasis.
An inducible mouse model was used to simultaneously ablate the 2 known H3K27me3 demethylases, lysine (K)-specific demethylase 6A (Kdm6a) and lysine (K)-specific demethylase 6B (Kdm6b), from the intestinal epithelium. Mice were analyzed at acute and prolonged time points after Kdm6a/b ablation. Cellular proliferation and differentiation were measured using immunohistochemistry, while RNA sequencing and chromatin immunoprecipitation followed by sequencing for H3K27me3 were used to identify gene expression and chromatin changes after Kdm6a/b loss. Intestinal epithelial renewal was evaluated using a radiation-induced injury model, while Paneth cell homeostasis was measured via immunohistochemistry, immunoblot, and transmission electron microscopy.
We did not detect any effect of Kdm6a/b ablation on intestinal cell proliferation or differentiation toward the secretory cell lineages. Acute and prolonged Kdm6a/b loss perturbed expression of gene signatures belonging to multiple cell lineages (adjusted P value < .05), and a set of 72 genes was identified as being down-regulated with an associated increase in H3K27me3 levels after Kdm6a/b ablation (false discovery rate, <0.05). After prolonged Kdm6a/b loss, dysregulation of the Paneth cell gene signature was associated with perturbed matrix metallopeptidase 7 localization (P < .0001) and expression.
Although KDM6A/B does not regulate intestinal cell differentiation, both enzymes are required to support the full transcriptomic and epigenomic landscape of the intestinal epithelium and the expression of key Paneth cell genes.
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