Cell-type-specific promoters for C. elegans glia.
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ABSTRACT:
SUBMITTER: Fung W
PROVIDER: S-EPMC7855602 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The multicellular green alga Volvox carteri has emerged as a valuable model organism for investigating various aspects of multicellularity and cellular differentiation, photoreception and phototaxis, cell division, biogenesis of the extracellular matrix and morphogenetic movements. While a range of molecular tools and bioinformatics resources have been made available for exploring these topics, the establishment of cell type-specific promoters in V. carteri has not been achieved so far. Therefore, here, we conducted a thorough screening of transcriptome data from RNA sequencing analyses of V. carteri in order to identify potential cell type-specific promoters. Eventually, we chose two putative strong and cell type-specific promoters, with one exhibiting specific expression in reproductive cells (gonidia), the PCY1 promoter, and the other in somatic cells, the PFP promoter. After cloning both promoter regions, they were introduced upstream of a luciferase reporter gene. By using particle bombardment, the DNA constructs were stably integrated into the genome of V. carteri. The results of the expression analyses, which were conducted at both the transcript and protein levels, demonstrated that the two promoters drive cell type-specific expression in their respective target cell types. Transformants with considerably diverse expression levels of the chimeric genes were identifiable. In conclusion, the screening and analysis of transcriptome data from RNA sequencing allowed for the identification of potential cell type-specific promoters in V. carteri. Reporter gene constructs demonstrated the actual usability of two promoters. The investigated PCY1 and PFP promoters were proven to be potent molecular tools for genetic engineering in V. carteri.
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Project description:The type 1 interleukin-1 receptor (IL-1R1) mediates diverse functions of interleukin-1 (IL-1) in the nervous, immune, and neuroendocrine systems. It has been suggested previously that the versatile functions of IL-1 may in part be conferred by the multiple promoters of IL-1R1 that have been identified for the human IL-1R1 gene. Promoters for murine IL-1R1 (mIL-1R1) gene have not been studied in detail. We performed 5'-rapid amplification of cDNA ends to determine the transcription start sites (TSS) in mIL-1R1, using mRNAs derived from 24 different tissues. The results revealed three putative TSSs of mIL-1R1. Three full-length cDNAs containing these distinct TSSs were recovered in screens of cloned cDNA libraries. Translation of these cDNAs produced IL-1R1 proteins that were verified by Western blot analysis. IL-1 stimulation of the individual IL-1R1 proteins resulted in the activation of NF-kappaB. Promoter-reporter assay for genomic DNA sequences immediately upstream of the three TSSs validated that the sequences possess promoter activity in a cell type-specific manner. These promoters are termed P1, P2, and P3 of the mIL-1R1, in 5' to 3' order. Quantitative PCR analysis of P1-, P2-, and P3-specific mIL-1R1 mRNAs showed that there is tissue-specific distribution of these mRNAs in vivo, and there are distinct patterns of P1, P2, and P3 mRNA expression in different cell lines. In the brain, P3 mRNA is expressed preferentially in the dentate gyrus. Further, glucocorticoids differentially regulate these promoters in a cell type-specific manner. Together, these results suggest that the different IL-1R1 promoters contribute to the discrete and diverse actions of IL-1.
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Project description:Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions could be present in all brain cells. The effects of nuclear inclusion formation have been mainly studied in neurons, while the effect on glia has been comparatively disregarded. Astrocytes, microglia, and oligodendrocytes are glial cells that are essential for normal brain function and are implicated in several neurological diseases. Here we examined the number of nuclear mHTT inclusions in both neurons and various types of glia in the two brain areas that are the most affected in HD, frontal cortex, and striatum. We compared nuclear mHTT inclusion body formation in three HD mouse models that express either full-length HTT or an N-terminal exon1 fragment of mHTT, and we observed nuclear inclusions in neurons, astrocytes, oligodendrocytes, and microglia. When studying the frequency of cells with nuclear inclusions in mice, we found that half of the population of neurons contained nuclear inclusions at the disease end stage, whereas the proportion of GFAP-positive astrocytes and oligodendrocytes having a nuclear inclusion was much lower, while microglia hardly showed any nuclear inclusions. Nuclear inclusions were also present in neurons and all studied glial cell types in human patient material. This is the first report to compare nuclear mHTT inclusions in glia and neurons in different HD mouse models and HD patient brains. GLIA 2016;65:50-61.
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Project description:Existing studies of chromatin conformation have primarily focused on potential enhancers interacting with gene promoters. By contrast, the interactivity of promoters per se, while equally critical to understanding transcriptional control, has been largely unexplored, particularly in a cell type-specific manner for blood lineage cell types. In this study, we leverage promoter capture Hi-C data across a compendium of blood lineage cell types to identify and characterize cell type-specific super-interactive promoters (SIPs). Notably, promoter-interacting regions (PIRs) of SIPs are more likely to overlap with cell type-specific ATAC-seq peaks and GWAS variants for relevant blood cell traits than PIRs of non-SIPs. Moreover, PIRs of cell-type-specific SIPs show enriched heritability of relevant blood cell trait (s), and are more enriched with GWAS variants associated with blood cell traits compared to PIRs of non-SIPs. Further, SIP genes tend to express at a higher level in the corresponding cell type. Importantly, SIP subnetworks incorporating cell-type-specific SIPs and ATAC-seq peaks help interpret GWAS variants. Examples include GWAS variants associated with platelet count near the megakaryocyte SIP gene EPHB3 and variants associated lymphocyte count near the native CD4 T-Cell SIP gene ETS1. Interestingly, around 25.7% ~ 39.6% blood cell traits GWAS variants residing in SIP PIR regions disrupt transcription factor binding motifs. Importantly, our analysis shows the potential of using promoter-centric analyses of chromatin spatial organization data to identify biologically important genes and their regulatory regions.
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