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. 2008 May 14;3(5):e2184.
doi: 10.1371/journal.pone.0002184.

A transcriptional enhancer from the coding region of ADAMTS5

Kristen K B Barthel  1 , Xuedong Liu
Affiliations

A transcriptional enhancer from the coding region of ADAMTS5

Kristen K B Barthel et al. PLoS One. .

Abstract

Background: The revelation that the human genome encodes only approximately 25,000 genes and thus cannot account for phenotypic complexity has been one of the biggest surprises in the post-genomic era. However, accumulating evidence suggests that transcriptional regulation may be in large part responsible for this observed mammalian complexity. Consequently, there has been a strong drive to locate cis-regulatory regions in mammalian genomes in order to understand the unifying principles governing these regions, including their genomic distribution. Although a number of systematic approaches have been developed, these all discount coding sequence.

Methodology/principal findings: Using the computational tool PRI (Pattern-defined Regulatory Islands), which does not mask coding sequence, we identified a regulatory region associated with the gene ADAMTS5 that encompasses the entirety of the essential coding exon 2. We demonstrate through a combination of chromatin immunoprecipitation and reporter gene studies that this region can not only bind the myogenic transcription factors MYOD and myogenin and the E-protein HEB but can also function as a very strong myogenic transcriptional enhancer.

Conclusions/significance: Thus, we report the identification and detailed characterization of an exonic enhancer. Ultimately, this leads to the interesting question of why evolution would be so parsimonious in the functional assignment of sequence.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ADAMTS5 PRI 1 Is Bound by Myogenic Transcription Factors in C2C12 Myoblasts and Myotubes.
(A) Schematics of PRI 1 and PRI 2 of ADAMTS5. Colored boxes represent transcription factor binding sites and are labeled. E-boxes, used to find the PRIs, are labeled in red. The portion of PRI 1 that derives from exon 2 is indicated. Numbering is with respect to the start codon. (B) Chromatin immunoprecipitation using a MYOD antibody in formaldehyde-fixed, sonicated myoblast and myotube (Day 1 of differentiation) lysate indicates that PRI 1 but not PRI 2 is bound during differentiation. Input refers to 0.1% pre-immunoprecipitation input, Ø refers to no primary antibody, MYOD refers to MYOD antibody. (C) Chromatin immunoprecipitation with MYOD, MYOG, and HEB antibodies. mMYOG PRI 5 serves as positive control, mADAMTS5 PRI 2 and mAMY2.1 serve as negative controls. Input refers to 0.1% pre-immunoprecipitation input, Ø refers to no primary antibody, MYOD refers to MYOD antibody, MYOG refers to MYOG antibody, HEB refers to HEB antibody.
Figure 2
Figure 2. PRI 1 Drives Reporter Gene Transcription during Myogenesis and Is Dependent Upon Exon-derived Sequence.
(A) Schematic of reporter gene vector construction and deletion mutants. PRIs are placed upstream of a TATA box and the firefly luciferase gene. I1 refers to sequence from Intron 1, E refers to Exon 2 sequence, I2 refers to Intron 2 sequence. (B) C2C12 cells were transfected with luciferase reporter plasmids and normalized with a renilla reporter plasmid driven by the thymidine kinase promoter. Cells were harvested as growing myoblasts (GM) and as differentiating myotubes one day after serum withdrawal (DM D1). minTATA is the background vector with a TATA box and no upstream cloned regions. Each bar represents the average of three independent experiments and error bars denote +/− SD. (C) Luciferase assay performed as in B.
Figure 3
Figure 3. Intronic and Exonic Binding Sites Contribute to Transcriptional Output of PRI 1.
(A) Luciferase results of reporter constructs of PRI 1 harboring point mutations in PRI 1 according to Table 1.
Figure 4
Figure 4. PRI Orientation Contribution to Transcriptional Output.
(A) Luciferase results of reverse reporter construct of PRI 1.
See this image and copyright information in PMC

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