Involvement of histone H4 gene transcription factor 1 in downregulation of vimentin gene expression during skeletal muscle differentiation.

Upon in vitro myogenesis, the intermediate filament protein vimentin is replaced by desmin, the switch in gene expression occurring essentially at the transcriptional level. Trying to elucidate the molecular mechanisms of this genetic control, we show here that the vimentin promoter is specifically recognized and activated by a protein most probably identical to H4TF-1, and that this factor is present in proliferating myoblasts but disappears upon fusion of these cells into multinucleated myotubes. Our results suggest that H4TF-1 is a differentiation stage-specific factor involved in the downregulation of vimentin gene expression during myogenesis.

Regulation of the expression of the human vimentin gene: application to cellular immortalization.

Vimentin is an intermediate filament protein mainly specific of the mesoderm in vivo. In vitro vimentin synthesis is characteristic of proliferating cells, regardless of their embryonal origin, and is switched off upon differentiation of certain precursor cells. Vimentin gene expression is upregulated in some metastatic tumour cells, appearing as a marker of oncogenic progression. The vimentin network has been suggested to participate in several steps of viral infections. The promoter of the vimentin gene is comprised of multiple elements responsible for its complex transcriptional regulation. Among them, an NF-kappa B- and two AP1-binding sites mediate growth factor responsiveness. Two negative elements are present, one of which is deregulated by the HTLV-1 activator protein Tax. Transcription factor PEA3, encoded by a member of the ets oncogene family, activates the vimentin promoter in mammary tumour cells. In vitro, 878 base pairs of the vimentin 5'-regulatory region are sufficient to give high levels of transcription. These sequences were coupled to the SV40 large T antigen-encoding gene to achieve immortalization of new cell lines, either by transfection of primary cultures, or by derivation of cell explants from transgenic mice expressing the vimentin-SV40 construct. This allowed us, for instance, to immortalize endothelial, myogenic or renal epithelial cells, otherwise difficult to maintain in culture without loss of their differentiated phenotypes.

A 28-bp negative element with multiple factor-binding activity controls expression of the vimentin-encoding gene.

The promoter of the human vimentin-encoding gene (VIM) contains two enhancers separated by a negative region. The distal and proximal enhancers bind the transcription factors, AP-1 and NK-kappaB, respectively, which contribute to serum induction of Vim synthesis. We were interested in looking for particular regulatory elements that might be responsible for tissue-specific extinction and culture-dependent activation of human VIM. We have identified a 48-bp sequence in the distal enhancer which had not been reported before. This sequence includes a negative element, NE2, which confers transcriptional repression in transfection experiments and binds at least two factors in vitro. NE2 may participate in the differentiation-stage-specific control of VIM expression which involves multiple regulatory sequences and several positive and negative trans-acting factors.

Induction of a factor that binds to the polyoma virus A enhancer on differentiation of embryonal carcinoma cells.

The cell type specificity of certain enhancers, competition experiments and the interactions that occur between proteins and enhancer sequences demonstrate that enhancers are the targets of specific factors involved in transcription control. The 246-base pair BclI-PvuII restriction enzyme fragment of polyoma virus has been shown to include two distinct enhancers, Py A and Py B, composed of several subdomains which interact with nuclear proteins from mouse fibroblasts. Embryonal carcinoma (EC) cells do not permit polyoma virus infection: both viral transcription and DNA replication are blocked. Host-range mutants of polyoma virus (EC mutants) capable of overcoming the expression block in EC cells have mutations or sequence rearrangements in their enhancer region. In an attempt to understand the molecular basis of this host restriction we compared the binding patterns displayed on the viral enhancer sequences by nuclear proteins prepared from EC cells or from fibroblasts. We show that one of the fibroblast factors required for Py A enhancer function, almost undetectable in EC cells, is induced after differentiation of these cells into parietal endoderm, suggesting that this protein is crucial in the regulation of viral gene expression during cellular differentiation, and perhaps more generally in the control of gene expression during early embryonic development.

Specific interaction of cellular factors with the B enhancer of polyoma virus.

Specific interactions between proteins from mouse 3T6 cells and the enhancer sequence of polyoma virus were detected using the method of band shifting on polyacrylamide gels. Proteins eluted from 3T6 nuclei using a buffer containing 0.55 M NaCl, formed a stable complex with the B enhancer of polyoma virus. At least two different factors are involved in this interaction. The contact sites which were mapped on the DNA sequence using DNase I footprinting correspond to a GC-rich palindrome surrounded by two sequences homologous respectively to the immunoglobulin and to the immunoglobulin and SV40 enhancers. Moreover Bal31 deletion analysis confirmed that similar sequences are required for the formation of the complex. In spite of a common function and partial sequence homology among some enhancers, neither the polyoma A enhancer, the mouse immunoglobulin heavy chain gene enhancer, nor the origin-promoter-enhancer region of SV40 efficiently competed with the polyoma B enhancer for the binding of these molecules.