High-Throughput (HTP) Dataset Index metadata provided by MGI
ID: ArrayExpress:GSE137060
Tags: WT vs. mutant, genotype
Summary: Epigenetic silencing of HLTF in human beta cells alters insulin secretion and is associated with diabetes. Here we examine the effects of Hltf-deletion on beta cell function in genetically engineered mouse models. The developmental timeline for the exclusive expression of full-length Hltf mRNA and protein overlaps with the organization of murine pancreatic islets. Reduced insulin in the pancreatic beta cells of global Hltf-deleted near-term (E18.5) fetal mice predicts postpartum hypoinsulinemia. Seventy-five percent of newborn global Hltf-deleted postprandial mice die at a ratio of 3:2 males to females with negligible serum insulin levels, and their apoptotic beta cells are devoid of insulin. They are also hypoglycemic. Newborn beta cell-specific Hltf-deleted mice succumb to the same loss of glucose homeostasis indicating the phenotype is solely attributable to loss of beta cell function. Confirmation that an intact immune system is an absolute requirement for the phenotype resulted from breeding the Hltf-deletion into the Rag2-IL2-null background. Triple null (Hltf-/-Rag2-/-IL2-/-) newborn mice that lack functional receptors for IL-2,-4,-7,-9,-15, and -21, and have severe lymphocyte developmental impairment (deficient T and B cells, no NK cells) are euglycemic and normoinsulinemic with normal survival rates. Transcriptomic profiling (RNA-seq) eliminated beta cell-specific Hltf-deletion on gene programs essential for normal development, and beta cell secretion. Significantly, Hltf-deletion induced IL33/beta cell signaling that promoted islet infiltration of fetal CD8+T cells and beta cell apoptosis. Our data suggest, Hltf-deleted newborns that are not profoundly hypoglycemic survive in the absence of memory CD8+T cells. These Hltf-deletion studies provide mechanistic insights to how Hltf-deletion in beta cells promotes their immune destruction. We previously used RNA-seq in conjunction with 3SEQ/transcriptome to quantify expression levels in brain (14), heart (15), and placenta (7). RNase levels in pancreas are 181,000-fold higher than brain (https://www.thermofisher.com/us/en/home/references/ambion-tech-support/nuclease-enzymes/tech-notes/rnase-activity-in-mouse-tissue.html). As a result, isolation of total RNA was highly variable. Total RNA (n=47 samples) was isolated, its integrity and purity were assessed (Agilent Bioanalyzer). Ultimately pancreata (n=6 samples; 3 test (Hltf rIPCre fl/fl with low (<15 mg/dL) blood sugar, and 3 Hltf +/+ euglycemic controls) perfused in situ with RNAlater (21) using a 1 CC syringe and a 26g 1/2 needle, and snap frozen were suitable (Table 2) for rRNA-depletion. cDNA was generated from rRNA-depleted samples and subjected to Illumina library preparation. Libraries were sequenced utilizing Illumina sequencing technology. Paired-end 100 nucleotide reads were aligned to genomic assembly mm10 and analyzed using the platform provided by DNAnexus, Inc. (Mountain View, CA) to generate an unbiased gene expression analysis report of RNA-seq; alternative splicing analysis of Hltf; mutation/RNA-editing analysis and parallel comparison of expression profiles between beta cell-specific Hltf-deleted pancreata and control pancreata. The power in detecting alternative splicing was dramatically increased by paired-end sequencing relative to single-end sequencing. FPKM (fragments per kilobase of transcript per million mapped reads) were mapped against mm10 with Tophat (V1.3.3) to obtain .bam mapping files that were input into Cufflinks for transcript assembly. Cuffdiff (V 1.3.0), part of the Cufflinks package, used the alignment reads for rigorous statistical comparison of two conditions (beta cell-specific Hltf-deleted and wild type control pancreata) and 3 biological replicates for each condition. The depth of sequencing was a minimum of 20 million sequencing reads per sample [90% Power, 5% significance level: 91+/- 4% of all annotated genes are sequenced at a frequency of 0.1 times/103 bases X 3 x 109 bases/sequencing read x 3 samples = 9 x104 reads/gene]. Data were imported into iPathwayGuide (Advaita Corporation) a next-generation pathway analysis tool. Standard enrichment parameters (log fold change, logFC = 0.6, p<0.05) were used.