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. 2014 May;112(1):73-83.
doi: 10.1016/j.ymgme.2013.12.006. Epub 2013 Dec 16.

Transcriptional Profiling of the Human fibrillin/LTBP Gene Family, Key Regulators of Mesenchymal Cell Functions

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Free PMC article

Transcriptional Profiling of the Human fibrillin/LTBP Gene Family, Key Regulators of Mesenchymal Cell Functions

Margaret R Davis et al. Mol Genet Metab. .
Free PMC article

Abstract

The fibrillins and latent transforming growth factor binding proteins (LTBPs) form a superfamily of extracellular matrix (ECM) proteins characterized by the presence of a unique domain, the 8-cysteine transforming growth factor beta (TGFβ) binding domain. These proteins are involved in the structure of the extracellular matrix and controlling the bioavailability of TGFβ family members. Genes encoding these proteins show differential expression in mesenchymal cell types which synthesize the extracellular matrix. We have investigated the promoter regions of the seven gene family members using the FANTOM5 CAGE database for human. While the protein and nucleotide sequences show considerable sequence similarity, the promoter regions were quite diverse. Most genes had a single predominant transcription start site region but LTBP1 and LTBP4 had two regions initiating different transcripts. Most of the family members were expressed in a range of mesenchymal and other cell types, often associated with use of alternative promoters or transcription start sites within a promoter in different cell types. FBN3 was the lowest expressed gene, and was found only in embryonic and fetal tissues. The different promoters for one gene were more similar to each other in expression than to promoters of the other family members. Notably expression of all 22 LTBP2 promoters was tightly correlated and quite distinct from all other family members. We located candidate enhancer regions likely to be involved in expression of the genes. Each gene was associated with a unique subset of transcription factors across multiple promoters although several motifs including MAZ, SP1, GTF2I and KLF4 showed overrepresentation across the gene family. FBN1 and FBN2, which had similar expression patterns, were regulated by different transcription factors. This study highlights the role of alternative transcription start sites in regulating the tissue specificity of closely related genes and suggests that this important class of extracellular matrix proteins is subject to subtle regulatory variations that explain the differential roles of members of this gene family.

Keywords: Extracellular matrix; Fibrillin; Gene regulation; Latent transforming growth factor β binding protein; Promoter; Transcription start sites.

Figures

Fig. 1
Fig. 1
Expression of fibrillin/LTBP gene family members. qPCR was performed on RNA from cell lines SAOS2, MG63, H1 human embryonic stem cells and RH1 human embryonic stem cells and from primary cells ADMSC and NHDF. Results were normalized using GAPDH. FBN3 was detected solely in H1 and RH1 embryonic stem cells. See Methods for more details. (A. FBN1; C. FBN2; E. FBN3; F. LTBP1; G. LTBP2; H. LTBP3; I. LTBP4). Fluorescent immunocytochemistry was performed on Day 7 cultured human fibroblasts, NHDF. Antibody information is available in Supplementary Table 1. (B. fibrillin-1; D. fibrillin-2).
Fig. 2
Fig. 2
Expression of mesenchymal marker genes. qPCR was performed on RNA from cell lines SAOS2, MG63, H1 human embryonic stem cells and RH1 human embryonic stem cells and from primary cells ADMSC and NHDF. Results were normalized using GAPDH. See methods for more details. (A. COL1A1; B. COL1A2; D. BGN; F. ACTA2). Fluorescent immunocytochemistry was performed on Day 7 cultured human fibroblasts, NHDF. Antibody information is available in Supplementary Table 1. (C. collagen type 1; E. biglycan; G. smooth muscle actin).
Fig. 3
Fig. 3
Architecture of the human FBN1 gene. Promoters were numbered using the permissive set, based on expression, with p1@FBN1 as the highest expressing promoter. Images derived from the ZENBU browser (http://fantom.gsc.riken.jp/zenbu/) and from the USCS Genome Browser (http://genome.ucsc.edu/). Images for the other gene family members are available in the Supplementary Figure or by accessing the ZENBU browser. A. The whole of the FBN1 gene, showing the physical location, exon/intron structure (introns —horizontal lines; exons —vertical lines), robust promoters, CpG islands, and enhancers. Boxed area shows the region enlarged in C. B. Regulatory elements detected for FBN1 by the ENCODE Project , showing trimethylation of lysine 4 in Histone 3 (H3K4Me3), DNase I hypersensitivity sites and transcription factor binding sites detected by chromatin immunoprecipitation and sequencing. The FBN1 gene is aligned to the image in A. C. Enlargement of the region containing the promoters for FBN1, showing the consensus gene start site (Entrez gene), the start sites of known transcripts, the CpG island and the promoters in this region.
Fig. 4
Fig. 4
Coexpression of promoters of the fibrillin/LTBP gene family. Colored spheres (nodes) represent different promoters and the lines between them show correlated expression patterns at the threshold indicated. Spheres of the same color form a coexpression cluster of promoters with expression more similar to each other than to the other promoters in the analysis. Promoters within each of the clusters are listed in Supplementary Table 2. A. Clustering of expression pattern at low correlation coefficient threshold (0.17) using Biolayout Express3D. All 81 promoters were included. Genes whose promoters are within the different colored clusters are shown in the legend. p2@FBN3 (circled in red) clustered with p11@LTBP3, p1@LTBP4, p9@LTBP4 and p13@LTBP4. B. Clustering at moderate correlation coefficient threshold (0.50). Seventy promoters were included. Genes whose promoters are within the different colored clusters are shown on the figure. Promoters for most genes formed separate clusters, except that p6@LTBP1 (circled in red) grouped with FBN1 promoters. Two LTBP3 clusters and three LTBP4 clusters formed separate closely related groups (circled in black), although there were also other distinct clusters containing promoters for these genes. C. Clustering at high correlation coefficient threshold (0.75). Twenty-five promoters were included. Genes whose promoters are within the different colored clusters are shown on the figure. Promoters for all genes formed separate clusters with four distinct but closely related clusters (circled in black) for LTBP2.

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