Regulation of intermediate filament gene expression.

Members of the intermediate filament protein family exhibit complex patterns of development-specific and tissue-specific expression. Studies exploring the mechanisms of gene regulation are underway and key regulatory factors are currently being described and isolated for certain genes encoding intermediate filament proteins. Selected systems from this diverse group of about 50 genes will be discussed.

Identification of a cis-acting DNA antisilencer element which modulates vimentin gene expression.

Vimentin is a tissue-specific, developmentally regulated member of the intermediate filament protein family normally expressed in cells of mesenchymal origin. Transcription factors which recognize specific cis-acting elements of the chicken gene include Sp-1 and the 95-kDa silencer protein which binds to a 40-bp silencer element at -608 (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we have identified a region upstream of the silencer element which restores gene activity. This region has been further delineated into two functional subelements of 75 and 260 bp. In transient transfection assays, the 75-bp element overrides the silencer effect of pStkCAT by 100%, while the 260-bp element is about half as active. Neither element affects gene activity when the silencer element is absent. Therefore, these elements do not function as enhancers, but they may serve only to override the silencer element and therefore can be viewed as antisilencers. In addition, the 75-bp element binds a specific 140-kDa protein, as determined by gel mobility shift assays and Southwestern (DNA-protein) blots, the binding site of which has been delineated to a 10- to 17-bp element by DNase I protection experiments. During myogenesis, a direct correlation can be made between the binding efficiency of the 140-kDa protein, the silencer protein, and gene activity in vivo. Genes known to contain a functional silencer element also contain at least one antisilencer element, as determined by sequence identity. Therefore, we have identified an antisilencer element and protein important in the developmental regulation of vimentin gene expression which may be involved in the regulation of other genes.

Multiple silencer elements are involved in regulating the chicken vimentin gene.

Vimentin, a member of the intermediate filament protein family, exhibits tissue- as well as development-specific expression. Transcription factors that are involved in expression of the chicken vimentin gene have been described and include a cis-acting silencer element (SE3) that is involved in the down-regulation of this gene (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we report the identification of two additional silencer elements (SE1 and SE2). We show by transfection analysis that all three silencer elements are functionally active and that optimal silencing occurs when multiple (at least two) silencer elements are present. In addition, the previously identified SE3 can be divided into three subregions, each of which is moderately active alone. By gel mobility shift assays, all three silencer elements plus SE3 subregions bind a protein which by Southwestern (DNA-protein) blot analysis is identical in molecular mass (approximately 95 kDa). DNase I footprinting experiments indicate that this protein binds to purine-rich sites. Therefore, multiple elements appear to be involved in the negative regulation of the chicken vimentin gene, which may be important in the regulation of other genes as well.

A negative element involved in vimentin gene expression.

Vimentin is one member of the intermediate filament multigene family which exhibits both tissue- and developmental stage-specific expression. In vivo, vimentin is expressed in cells of mesenchymal origin. Previously, we identified both enhancer and promoter elements in the chicken vimentin gene which regulate gene expression in a positive manner. In this report, we have identified a 40-base-pair region at -568 base pairs between the proximal and distal enhancer elements which represses transcriptional activity. This silencer region can also repress the heterologous herpes simplex virus thymidine kinase promoter, which is comparable to the vimentin promoter. In addition, the element is able to function in a position- and orientation-independent manner, and the amount of repression is increased by multiple copies. Here we show by gel retardation assays and DNase I footprinting that this region binds a protein in nuclear extracts from HeLa cells. Southwestern (DNA-protein) blot analysis indicates this protein is approximately 95 kilodaltons in size. Moreover, protein distribution and activity mimic the expression pattern of vimentin during myogenesis, i.e., protein binding increases as vimentin gene expression decreases. The silencer region shares strong sequence similarity with 5'-flanking sequences found in both the human and hamster vimentin genes and with other characterized silencer elements, including the human immunodeficiency virus long terminal repeat, rat growth hormone, chicken lysozyme, and rat insulin genes. Thus, a negative element appears to bind a 95-kilodalton protein involved in regulating the tissue-specific expression of the chicken vimentin gene.

A human tRNA(iMet) gene produces multiple transcripts.

A third nonallelic locus of the human methionyl-tRNA multigene family (tRNA(iMet-3) was isolated. This gene, unlike two other tRNA(iMet) loci, lacks a remarkable run of T and C residues which functions as a termination of transcription signal. Instead, three tandem termination signals, each containing no more than four thymidylate residues, function as relatively inefficient termination signals. As a result, polymerase readthrough generates at least three transcripts in vitro. The efficiency of apparent termination varies significantly at these sites. All resulting transcripts appear to be processed in vitro.

A negative regulatory factor is missing in a human metastatic breast cancer cell line.

The intermediate filament protein, vimentin, is differentially expressed in various tissues and stages of development and in metastatic versus nonmetastatic breast cancer cell lines. Previously, we have shown vimentin expression to be regulated at least in part by a silencer element which binds a M(r) 95,000 protein and an overriding, antisilencer element which binds a M(r) 140,000 protein. Southwestern blot (DNA-protein) analyses indicate that silencer protein binding activity is missing in the metastatic breast cancer cell line (MDA-MB-231), where vimentin is highly expressed, but is present in the nonmetastatic breast cancer cell line, MCF-7, where vimentin is not expressed. This suggests that the absence of a functional silencer protein may lead to expression of vimentin as well as other genes which contribute to the metastatic state.

miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis.

Identification of dysregulated microRNAs (miRNAs) in prostate cancer is critical not only for diagnosis, but also differentiation between the aggressive and indolent forms of the disease. miR-9 was identified as an oncomiR through both miRNA panel RT-qPCR as well as high-throughput sequencing analysis of the human P69 prostate cell line as compared to its highly tumorigenic and metastatic subline M12, and found to be consistently upregulated in other prostate cell lines including DU-145 and PC3. While miR-9 has been characterized as dysregulated either as an oncomiR or tumour suppressor in a variety of other cancers including breast, ovarian, and nasopharyngeal carcinomas, it has not been previously evaluated and proven as an oncomiR in prostate cancer. miR-9 was confirmed an oncomiR when found to be overexpressed in tumour tissue as compared to adjacent benign glandular epithelium through laser-capture microdissection of radical prostatectomy biopsies. Inhibition of miR-9 resulted in reduced migratory and invasive potential of the M12 cell line, and reduced tumour growth and metastases in male athymic nude mice. Analysis showed that miR-9 targets e-cadherin and suppressor of cytokine signalling 5 (SOCS5), but not NF-ĸB mRNA. Expression of these proteins was shown to be affected by modulation in expression of miR-9.

An antisilencer element is involved in the transcriptional regulation of the human vimentin gene.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. The promoter of the human vimentin gene was previously reported to contain two positive-acting regions, separated by a negative region (Rittling, S.R., Baserga, R., 1987. Functional analysis and growth factor regulation of the human vimentin promoter. Mol. Cell. Biol. 7, 3908-3915). Here, detailed studies reveal two additional regulatory elements, a new positive transcriptional element located between -717 and -757, and a new repressor element at -780 to -821. In transient transfections, the positive-acting element is able to completely override the effect of different silencer elements when fused to a heterologous promoter. However, this element does not enhance gene activity when the silencer element is absent and thus cannot be viewed as a true enhancer. Since it appears to overcome the effect of a silencer element, we refer to it as an antisilencer element. Gel mobility shift assays, UV-cross-linking experiments, and Southwestern blots reveal that a 105-kDa protein specifically binds to this region.

Nucleotide sequence of the chicken cardiac alpha actin gene: absence of strong homologies in the promoter and 3'-untranslated regions with the skeletal alpha actin sequence.

The entire nucleotide sequence of the chicken cardiac alpha-actin (CC alpha A) gene has been determined. This is the first complete sequence of a cardiac actin gene that includes the promoter region, cap site, all the introns, and the polyadenylation site. The gene contains six introns, five of which interrupt the coding region at amino acids (aa) 41, 150, 204, 267, and 327. The first intron is in the 5'-noncoding region and is 438 bp in length. The CC alpha A gene encodes an mRNA of approx. 1400 bp with 5'- and 3'-untranslated region of 59 and 184 nucleotides (nt), respectively. Like the chicken skeletal alpha-actin gene, the CC alpha A gene has the codon for the aa cysteine between the initiator ATG and the codon for the N-terminal aspartic acid residue of the mature protein. There are no strong homologies (less than 13 consecutive nt) in the promoter or 3'-untranslated regions between the CC alpha A and chicken skeletal alpha-actin genes even though both are expressed in skeletal muscle during development. However, the 3'-untranslated region of the CC alpha A gene demonstrates significant sequence homology (76% over a 200-nt region) with the same region in the partial sequence of the human cardiac gene. The conservation of these sequence homologies between identical isoforms rather than the different alpha actin genes suggests these conserved regions may have a role in regulation rather than tissue-specific expression, as previously proposed.

Flanking sequences are required for efficient transcription and stable complex formation for the human tRNAiMet3-coding gene.

An analysis of 5' and 3' deletions of the human tRNAiMet3 gene has revealed upstream regions required for efficient transcription and stable complex formation in vitro. The 5' boundary of this essential region lies between nucleotides -39 to -18 (start point = + 1), and it has been shown that 3'-flanking sequences near the first termination site are also important for stable complex formation. The transcriptional efficiency of two non-allelic loci (TMET3 and TMET2) has been compared and TMET2 is more active. An analysis of chimeric (hybrid) genes indicates that much of the difference seen is due to 5'-flanking sequences and that there may be complex interactions between 5' and 3' sequences.

Down-regulation of vimentin gene expression during myogenesis is controlled by a 5'-flanking sequence.

During myogenesis, the intermediate filament proteins vimentin and desmin are differentially expressed. While desmin levels increase dramatically, vimentin mRNA levels decrease substantially. Here, we show that transfected whole- and mini-vimentin-coding genes (Vim) are expressed in fibroblasts (mouse L cells) and down-regulated during muscle cell differentiation in culture. Functional assays with 5'-end Vim::cat constructs demonstrate that this repression is controlled by a 5'-element (nt -321 to -160). This region is distinct from Vim promoter elements (nt -160 to +71) which do not contribute to vimentin's down-regulation during myogenesis.

A GC-box is required for expression of the human vimentin gene.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. The promoter of the human vimentin gene (-1416 to +73) was shown to contain two positive-acting regions, separated by a negative region, and at least eight GC-boxes as determined by sequence homology (Rittling, S.R., Baserga, R., 1987. Mol. Cell. Biol. 7, 3908-3915). We have analyzed the region -900 to +41 for protein binding by in vivo footprinting experiments using ligation-mediated PCR. For the various GC-boxes, we detect protein binding only to that GC-box (at position -64 and -55) closest to the transcriptional start site. Transient transfection assays of various vimentin 5'-end fragments and mutations thereof fused to the reporter gene cat indicate that this sequence is indispensable for promoter function regardless of the inclusion of upstream DNA sequences. In vitro binding studies confirm that this region binds protein specifically. We suggest that this GC-box and its binding factor are required for regulated expression of the human vimentin gene.

Transcriptional mapping of two yeast genes coding for glyceraldehyde 3-phosphate dehydrogenase isolated by sequence homology with the chicken gene.

Homology between the coding regions of the chicken and yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was directly demonstrated by the hybridization of a cDNA clone coding for GAPDH in the chicken with EcoRI-digested yeast DNA. A yeast EcoRI fragment library in bacteriophage lambda was screened using the chicken cDNA plasmid as probe, and two recombinant phages were isolated, each one containing a different GAPDH gene. The initiation and termination sites for the GAPDH mRNA were localized for the two different GAPDH genes and compared to those of other yeast genes. Measurements of the relative mRNA levels for the two genes show that both genes are transcribed at about the same level when yeasts are grown on glucose media.

Functional analysis of chicken vimentin distal promoter regions in cultured lens cells.

Synthesis of the cytoskeletal intermediate filament protein vimentin (Vim) in the lens is unexpected due to the mesenchymal preference of Vim-encoding gene (Vim) expression and the epithelial origin of the lens. Previous studies indicated that chicken Vim gene expression in cultured lens cells is regulated by both positive- and negative-acting sequence elements within the first -767 nucleotides (nt) of its promoter. Here, we demonstrate the existence of additional upstream chicken Vim promoter elements which function in transfected lens cells. Sequences within the nt -1360/-1156 region repressed promoter activity in transfected lens cells to levels lower than that observed for the previously defined more proximal repressor elements. The -1612/-1360 region activated promoter activity to levels similar to those observed for the strongest previously defined proximal promoter. The nt sequence analysis of the upstream promoter region revealed the presence of multiple consensus repressor and activator transcription-factor-binding sites. Several of these sites have been implicated for lens expression of enzyme-crystallin-encoding genes (cry), suggesting that Vim expression may share features with the cry genes for recruitment and high-level expression in the lens.

Perinuclear mRNA localisation by vimentin 3'-untranslated region requires a 100 nucleotide sequence and intermediate filaments.

The role of the vimentin 3'-untranslated region (3'-UTR) in mRNA localisation was studied in cells transfected with a reporter sequence linked to subregions of the 3'-UTR. In situ hybridisation showed that nucleotides 37-137, including a previously identified protein-binding domain, were sufficient to localise transcripts to perinuclear cytoplasm. Transfection of two SW13 cell lines that do and do not express vimentin showed that perinuclear localisation due to either the vimentin or c-myc 3'-UTR requires intermediate filaments. The data suggest that both a specific protein-binding region of the vimentin 3'-UTR and intermediate filaments themselves are required to determine the site of vimentin synthesis.

A Stat1alpha factor regulates the expression of the human vimentin gene by IFN-gamma.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. Here, we report an increase in vimentin gene transcription induced by the cytokine interferon-y (IFN-gamma). Northern blot analysis and reporter gene assays reveal that IFN-gamma induces vimentin gene transcription in HeLa cells. However, no increase in vimentin mRNA synthesis was observed de novo in MCF-7 cells, which do not already express vimentin. Band shift analysis shows that the Stat1alpha protein mediates vimentin induction by IFN-gamma. A human mutant fibroblast cell line (U3A), which lacks Stat1alpha but expresses vimentin mRNA, yields no increase in vimentin mRNA levels on the addition of IFN-gamma. These results suggest that the induction of vimentin gene expression might be an important part of a complex cellular response to IFN-gamma.

The chicken vimentin gene: aspects of organization and transcription during myogenesis.

The intermediate filament gene vimentin exists in a single copy in the chick haploid genome. However, it exhibits the curious property of producing at least three functional vimentin mRNA transcripts in vivo through the differential utilization of multiple polyadenylylation sites. According to one group in erythroid cells there may be a tissue-specific utilization of one of these poly A addition sites. The chicken and hamster vimentin genes exhibit remarkable nucleotide sequence homology both within coding and 3'-noncoding regions (82%). This nucleotide homology extends both to the size and juxtaposition of exons. With the noted exception of valine, even the frequency of codons utilized is strongly conserved across the widely different species. Of course, this strong homology at the DNA level extends to an amino acid homology of 92% between vimentins and 65% between related proteins in the same species.

Vimentin gene expression during myogenesis: two functional transcripts from a single copy gene.

We have previously reported that a single vimentin gene is present in the chicken genome (22). In vivo transcription of this gene yields two distinct classes of mRNA's (approximatley 2200 and-2500 nts) generated through the possible differential utilization of polyadenylylation sites. In this report we demonstrate that 1) both transcripts are functional in vitro and direct the cell-free synthesis of the vimentin polypeptide, as judged by two dimensional gel analysis; 2) three of the four possible adenylylation signals indicated in the sequence of the gene are utilized in vivo as determined by S1 analysis; 3) furthermore, these adenylylation sites are utilized identically in all tissues of the chicken examined; 4) the adenylylation site closest to the body of the message is apparently not used as judged by the S1 conditions employed in the assay. Levels of vimentin mRNA decrease in different tissues of the chicken during the embryonic to adult transition. There is no evidence for a tissue specific or developmentally regulated pattern of expression for either of the two vimentin transcripts. The same pattern of vimentin mRNA expression is seen in all tissues examined, only the level of expression is altered.

Regulation of chicken vimentin gene expression by serum, phorbol ester, and growth factors: identification of a novel fibroblast growth factor-inducible element.

Vimentin is a cytoskeletal protein that belongs to the intermediate filament protein family. It is normally expressed in cells of mesenchymal origin and is developmentally as well as cell cycle regulated. Multiple silencer elements as well as unique antisilencer element are responsible for regulating the chicken vimentin gene. The silencer elements bind a protein of M(r) 90,000 (the silencer protein), whereas the antisilencer element binds a protein of M(r) 110,000-120,000 (the antisilencer protein). In this study, we examined the effect of serum, phorbol ester, transforming growth factor beta, and fibroblast growth factor of gene expression and identify the regions in the 5'-end of the chicken vimentin gene responsible for induction. The binding activity of both the silencer and the antisilencer proteins are affected by 12-O-tetradecanoylphorbol-13-acetate treatment, whereas the antisilencer element is inducible by fibroblast growth factor.

Characterization of the chicken vimentin gene: single copy gene producing multiple mRNAs.

Genomic clones and cDNA plasmids were isolated for the intermediate filament protein vimentin from chicken. The identity of the various clones was determined both by mRNA selection [Paterson, B. M. & Roberts, B. E. (1981) in Gene Amplification and Analysis, Structural Analysis or Nucleic Acids, eds. Chirikjian, J. G. & Papas, T. S. (Elsevier, North Holland), Vol. 2, pp. 418-435] and nucleotide sequence analysis. Restriction analysis, hybridization data, and heteroduplex studies confirmed that all of the genomic isolates contained overlapping fragments of an identical vimentin gene. No evidence for the existence of a second vimentin gene could be found by a Southern analysis either by using coding fragments from the purified vimentin gene or by using cDNA plasmids as probe. Likewise, copy-number experiments verified that the vimentin gene was present only once in the haploid chicken genome. However, in a RNA blot analysis, at least two equally abundant vimentin mRNA species of approximately 2,200 and 2,500 nucleotides in length were detected in all RNAs tested. Sequence analysis revealed that the vimentin gene contained two sets of tandem polyadenylylation sites, 249 and 532 nucleotides downstream from the stop codon for protein synthesis. It is proposed that the larger mRNA species arise because of complete transcription of the 3'-end of the vimentin gene (560 nucleotides of 3' nontranslated sequence), whereas the smaller mRNA species terminate after the first set of polyadenylylation sites.