Identification of the positive and negative cis-elements involved in modulating the constitutive expression of mouse thymosin β4 gene

Abstract

We previously showed that the -278 to +410 region of mouse thymosin beta4 (mT,beta4) gene supports high levels of reporter gene expression in NIH3T3 cells. This region contains part of the 5'-flanking sequences (-278 to -1), the intact first exon (+1 to +133), and portion of the first intron (+134 to +410). However, the size of this exon is much longer than those of its rat and human counterparts. To resolve the question regarding this size discrepancy, transcription start site for the mTbeta4 gene was re-examined by primer extension and bioinformatics analyses. We found that the first exon of mTbeta4 gene spans 56 bp with its cap site situated in a putative initiator highly similar to the consensus mammalian sequence. In addition, a TATA box-like motif and two consecutive downstream promoter elements were also found. To delineate the cis-elements involved in modulating the constitutive expression of mTbeta4 gene, transient transfection assay was performed. Interestingly, expression level of the reporter gene driven by the -117 to +56 region of mTbeta4 gene was approximately 8-fold higher than that directed by the SV40 promoter and significant promoter activity was found to be associated with the smaller (-56 to +56) fragment. A nuclear protein-bound silencer was located in the region between the -167 and -118 and an enhancer whose effect did not seem to be dependent on protein binding was identified in the downstream (-117 to -88) region. However, neither of these cis-elements affected reporter expression driven by a SV40 promoter. Intriguingly, mTbeta4 promoter functioned well in human colorectal (SW480) and cervical (HeLa) carcinoma cells. Taken together, our findings not only provide crucial information for further elucidation of the transcriptional regulation of mTbeta4 gene but also raise the possibility of utilizing its promoter to produce large quantity of recombinant proteins in mammalian cells.

Identification of Thymosin β4 as an effector of Hand1-mediated vascular development

“…At a biochemical level, we reveal that Tβ4 is a direct target of Hand1 and demonstrate the ability of this transcription factor to both activate and repress transcription of TB4 by binding at alternate E-box sites closely positioned within proximal sequences upstream of the functional promoter11. Our ChIP data suggest binding of the Thing1 degenerate E-box in the Tβ4 proximal regulatory region at E8.5 to cause activation of Tβ4 , and, subsequently at E11.5, a relatively stronger binding of Hand1 to the E-box than to the Thing1 box within the same Tβ4 upstream element to mediate repression of Tβ4 .…”

“…The functional Tβ4 promoter has previously been identified as residing between residues −278 and +410, relative to the ATG11. We identified two upstream putative Hand1-binding sites, a canonical E-Box site, CACATG (consensus CANNTG; nucleotide positions −1913 to −1908) for the binding of bHLH/E-factor heterodimers and a degenerate E-Box, TCTCTG (nucleotide positions −1691 to −1686), which matches the preferred binding site of HAND1/E-factor heterodimers (consensus NCTCTG12; Fig.…”

RGD-independent Cell Adhesion via a Tissue Transglutaminase-Fibronectin Matrix Promotes Fibronectin Fibril Deposition and Requires Syndecan-4/2 and α5β1 Integrin Co-signaling

“…Integrin-or ␤3 Integrinactivated Pathways-Because TG2 is also known to be a ligand for ␣4␤1 integrin (39), which has been reported to be expressed in mouse fibroblasts (40), we explored whether the RGD-independent cell adhesion in response to TG-FN involves ␣4␤1 integrins in fibroblasts (41). The cell attachment and spreading of MEF cells on FN and/or TG-FN was not significantly (p Ͼ 0.05) affected with either control mouse IgG treatment or function-blocking anti-integrin ␣4 antibody 9C10 (42) treatments at 30 g/ml (Fig.…”