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Tissue-selective alternate promoters guide NLRP6 expression.

ABSTRACT: The pryin domain (PYD) domain is involved in protein interactions that lead to assembly of immune-sensing complexes such as inflammasomes. The repertoire of PYD-containing genes expressed by a cell type arms tissues with responses against a range of stimuli. The transcriptional regulation of the PYD gene family however is incompletely understood. Alternative promoter utilization was identified as a mechanism regulating the tissue distribution of human PYD gene family members, including NLRP6 that is translationally silenced outside of intestinal tissue. Results show that alternative transcriptional promoters mediate NLRP6 silencing in mice and humans, despite no upstream genomic synteny. Human NLRP6 contains an internal alternative promoter within exon 2 of the PYD, resulting in a truncated mRNA in nonintestinal tissue. In mice, a proximal promoter was used that expanded the 5' leader sequence restricting nuclear export and abolishing translational efficiency. Nlrp6 was dispensable in disease models targeting the kidney, which expresses noncanonical isoforms. Thus, alternative promoter use is a critical mechanism not just for isoform modulation but for determining expression profile and function of PYD family members.

SUBMITTER: Bracey NA

PROVIDER: S-EPMC7772780 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Tissue-selective alternate promoters guide NLRP6 expression.

Bracey Nathan A NA Platnich Jaye M JM Lau Arthur A Chung Hyunjae H Hyndman M Eric ME MacDonald Justin A JA Chun Justin J Beck Paul L PL Girardin Stephen E SE Gordon Paul Mk PM Muruve Daniel A DA

Life science alliance 20201229 3

The pryin domain (PYD) domain is involved in protein interactions that lead to assembly of immune-sensing complexes such as inflammasomes. The repertoire of PYD-containing genes expressed by a cell type arms tissues with responses against a range of stimuli. The transcriptional regulation of the PYD gene family however is incompletely understood. Alternative promoter utilization was identified as a mechanism regulating the tissue distribution of human PYD gene family members, including NLRP6 tha ...[more]

PMID: 33376129

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Project description:Alternate splicing is believed to produce the greatest diversity in transcriptional complexity and function in eukaryotic species. In this study, we present an analysis of alternative splicing events that occur in the chicken, using the recently sequenced genomic sequence and over 580,000 EST sequences mapped back to the genome. A carefully controlled EST-to-genome mapping pipeline is presented, based around the EXONERATE program using the est2genome model, which also considers several quality control steps to filter out erroneous matches. The data is then used to estimate the level of alternate splicing events with respect to Ensembl predicted transcripts. The EST-genome mappings are characterised at the exon level, in order to classify individual splicing events and provide estimates of novel transcripts not currently annotated by the Ensembl genome database. This is the first large scale analysis of this kind in an avian species, and suggests that chicken displays a similar level of alternate splicing as that found in other higher vertebrates such as human and mouse, both in terms of the number of genes that undergo alternate splicing events, and the average number of transcripts produced per gene. The EST data suggests alternate splicing may occur in some 50-60% of the chicken gene set and with an average of around 2.3 transcripts per gene which undergo this process. The EST data is also used to look at gene and transcript usage in the tissues sequenced in embryonic and adult libraries. Genes which display notable biases were analysed in more detail, including twinfilin-2 and embryonic heavy chain myosin. This also highlights several as yet functionally un-annotated genes which appear to be important in embryonic tissues and also undergo alternate splicing events. The analysis also demonstrates some of the difficulties involved in using EST-based data to annotate transcriptional activity in eukaryotic genes, where a broad spectrum of tissues and a large number of sequenced transcripts are required in order to fully characterise alternate splicing and differential expression.

Project description:INTRODUCTION:The potential of pluripotent stem cells to be used for cell therapy depends on a comprehensive understanding of the molecular mechanisms underlying their unique ability to specify cells of all germ layers while undergoing unlimited self-renewal. Alternative splicing and alternate promoter selection contribute to this mechanism by increasing the number of transcripts generated from a single gene locus and thus enabling expression of novel protein variants which may differ in their biological role. The homeodomain-containing transcription factor NANOG plays a critical role in maintaining the pluripotency of Embryonic Stem Cells (ESC). Therefore, a thorough understanding of the transcriptional regulation of the NANOG locus in ESCs is necessary. METHODS:Regulatory footprints and transcription levels were identified for NANOG in human embryonic stem cells from data obtained using high-throughput sequencing methodologies. Quantitative real-time PCR following reverse transcription of RNA extracted human ESCs was used to validate the expression of transcripts from a region that extends upstream of the annotated NANOG transcriptional start. Promoter identification and characterization were performed using promoter reporter and electrophoretic mobility shift assays. RESULTS:Transcriptionally active chromatin marking and transcription factor binding site enrichment were observed at a region upstream of the known transcriptional start site of NANOG. Expression of novel transcripts from this transcriptionally active region confirmed the existence of NANOG alternative splicing in human ESCs. We identified an alternate NANOG promoter of significant strength at this upstream region. We also discovered that NANOG autoregulates its expression by binding to its proximal downstream promoter. CONCLUSION:Our study reveals novel transcript expression from NANOG in human ESCs, indicating that alternative splicing increases the diversity of transcripts originating from the NANOG locus and that these transcripts are expressed by an alternate promoter. Alternative splicing and alternate promoter usage collaborate to regulate NANOG, enabling its function in the maintenance of ESCs.

Project description:Transcriptome diversity provides the key to cellular identity. One important contribution to expression diversity is the use of alternative promoters, which creates mRNA isoforms by expanding the choice of transcription initiation sites of a gene. The proximity of the basal promoter to the transcription initiation site enables prediction of a promoter's location based on the gene annotations. We show that annotation of alternative promoters regulating expression of transcripts with distinct first exons enables a novel methodology to quantify expression levels and tissue specificity of mRNA isoforms.The use of distinct alternative first exons in 3,296 genes was examined using exon-microarray data from 11 human tissues. Comparing two transcripts from each gene we found that the activity of alternative promoters (i.e., P1 and P2) was not correlated through tissue specificity or level of expression. Furthermore neither P1 nor P2 conferred any bias for tissue-specific or ubiquitous expression. Genes associated with specific diseases produced transcripts whose limited expression patterns were consistent with the tissue affected in disease. Notably, genes that were historically designated as tissue-specific or housekeeping had alternative isoforms that showed differential expression. Furthermore, only a small number of alternative promoters showed expression exclusive to a single tissue indicating that "tissue preference" provides a better description of promoter activity than tissue specificity. When compared to gene expression data in public databases, as few as 22% of the genes had detailed information for more than one isoform, whereas the remainder collapsed the expression patterns from individual transcripts into one profile.We describe a computational pipeline that uses microarray data to assess the level of expression and breadth of tissue profiles for transcripts with distinct first exons regulated by alternative promoters. We conclude that alternative promoters provide individualized regulation that is confirmed through expression levels, tissue preference and chromatin modifications. Although the selective use of alternative promoters often goes uncharacterized in gene expression analyses, transcripts produced in this manner make unique contributions to the cell that requires further exploration.

Dataset Information

Tissue-selective alternate promoters guide NLRP6 expression.

Publications

Tissue-selective alternate promoters guide NLRP6 expression.

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