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Differential regulation of the mouse and human Wnt5a alternative promoters A and B.

ABSTRACT: Wnt5a is an extracellular glycoprotein that activates Wnt signaling pathways, important in development and tissue homeostasis. Wnt5a expression is often misregulated during cancer progression. In this study, we analyzed the transcriptional regulation of two of the Wnt5a alternative promoters, termed A and B. Transient transfection of promoter A and B luciferase reporter constructs in to NIH3T3 and Caco-2 cells indicated that the separated promoters are both functional and that 300-450 base pair (bp) of upstream sequence is sufficient for activity. Promoter B constructs displayed distinct patterns of expression in the two cell types. The endogenous levels of promoter A-derived transcripts were found to be greater than the promoter B transcripts by four- to sixfold in fibroblast cells. Treatment of NIH3T3 cells with tumor necrosis factor (TNF)-alpha leads to an increase in both promoter A and B activities, but promoter B was more responsive. Using inhibitors of TNF-alpha effector proteins, we provide evidence that the transcription factor nuclear factor-kappaB and the MEK1/2 and p38 kinases have distinct roles in determining the activity levels of promoters, A and B. These results support the conclusion that Wnt5a promoters, A and B, are differentially regulated and provide a model for complex transcriptional regulation of Wnt5a.

SUBMITTER: Katula KS

PROVIDER: S-EPMC3482380 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Differential regulation of the mouse and human Wnt5a alternative promoters A and B.

Katula Karen S KS Joyner-Powell Nicole B NB Hsu Chia-Chi CC Kuk Amber A

DNA and cell biology 20121009 11

Wnt5a is an extracellular glycoprotein that activates Wnt signaling pathways, important in development and tissue homeostasis. Wnt5a expression is often misregulated during cancer progression. In this study, we analyzed the transcriptional regulation of two of the Wnt5a alternative promoters, termed A and B. Transient transfection of promoter A and B luciferase reporter constructs in to NIH3T3 and Caco-2 cells indicated that the separated promoters are both functional and that 300-450 base pair ...[more]

PMID: 23046419

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Project description:BackgroundThe genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation.Methodology/principal findingsWe have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation.SignificanceOur observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.

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

Differential regulation of the mouse and human Wnt5a alternative promoters A and B.

Publications

Differential regulation of the mouse and human Wnt5a alternative promoters A and B.

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