GSE137060
Helicase-like transcription factor (Hltf)-deletion activates Hmgb1-Rage axis and granzyme A-mediated killing of pancreatic ß cells resulting in neonatal lethality
Epigenetic mechanisms are integral to pancreatic ß cell function. Promoter hypermethylation of the helicase like-transcription factor (HLTF) gene—a component of the cellular DNA damage response that contributes to genome stability—has been implicated in age-associated changes in ß cells.  To study HLTF, we generated global and ß cell-specific (ß) Hltf knockout (KO) immune competent (IC) and immune deficient (ID) Rag2-/IL2- mice. IC global and ß Hltf KO mice were neonatal lethal whereas ID global and ß Hltf KO newborn mice had normal survival. This focused our investigation on the effects of Rag2 interruption with common gamma chain interruption on ß cell function/survival. Three-way transcriptomic (RNAseq) analyses of whole pancreata from IC and ID newborn ß Hltf KO and wild type (Hltf +/+) controls combined with spatially resolved transcriptomic analysis of formalin fixed paraffin embedded tissue, immunohistochemistry and laser scanning confocal microscopy showed DNA damage caused by ß Hltf KO in IC mice upregulated the Hmgb1-Rage axis and a gene signature for innate immune cells. Perforin-delivered granzyme A (GzmA) activation of DNase, Nme1, showed damaged nuclear single-stranded DNA (?H2AX immunostaining). This caspase-independent method of cell death was supported by transcriptional downregulation of Serpinc1 gene that encodes a serine protease inhibitor of GzmA. Increased transcriptional availability of complement receptors C3ar1 and C5ar1 likely invited crosstalk with Hmgb1 to amplify inflammation. This study explores the complex dialog between ß cells and immune cells during development. It has implications for the initiation of type I diabetes in utero when altered gene expression that compromises genome stability invokes a localized inflammatory response. Overall design: Global and ß cell-specific (ß) Hltf knockout (KO) immune competent (IC) and immune deficient (ID) Rag2-/IL2- mice were generated for this study. Three-way transcriptomic (RNAseq) data of whole pancreata from IC and ID newborn ß Hltf KO and wild type (Hltf +/+) controls are presented here. Five basic steps were necessary to implement spatial transcriptomics technology. Step 1, placement of FFPE tissue (abdominal segments) on capture areas of a Visium gene expression (GEX) slide. Step 2, H&E staining followed by brightfield microscopic imaging with ZEISS Axioscan 7 high-performance slide scanner (White Plains, NY). Step 3, permeabilization of tissue and construction of barcoded libraries with a final sample index PCR all according to the manufacturer?s instructions. Step 4, NGS short-read sequencing (Illumina NovaSeq) of barcoded libraries by Genewiz (Azenta US, Inc, South Plainfield, NJ). Step 5, data analysis of tissue images and sequencing files in FASTQ format with Space Ranger run on Ubuntu 22.04 LTS ?Thelio Mira-b3 by System76, Inc. (Denver, CO). The space ranger aggr pipeline was used to aggregate data from replicate samples and from samples from the different biological conditions (IC, ID). Loupe browser visualization software was accessed in a desktop application via Windows (Dell Optiplex 990). CPRIT Grant # RP190524 (TTUHSC Cancer Animal Facility Core, PI Dr. Scott L. Trasti): This infrastructure grant provided funding for the ZEISS Axioscan 7 high-performance slide scanner.