Fibroblast Nuclear Extracts Research Articles

Identification of Proteins Immunologically Related to Interferon Regulatory Factor–1 That Bind with Interferon Regulatory Factor Element

Interferon regulatory factor (IRF)-1 expression was surveyed in nontransformed and oncogene-transformed mouse fibroblasts, using Western immunoblot with an IRF-1-specific antiserum, to examine possible differences resulting from cellular transformation. Ten additional proteins that reacted with the IRF-1 antibody and that underwent specific competition by peptide antigen were observed in extracts of both nontransformed and oncogene-transformed cell lines. Cross-reacting proteins were also observed in mouse macrophage extracts. Protein was captured from fibroblast nuclear extracts, using oligonucleotides representing IRF-binding sequences linked to magnetic beads. Captured proteins were eluted and analyzed by immunoblot with anti-IRF-1. Along with 43-kDa IRF-1, 4 of the 7 nuclearly located cross-reacting proteins (97, 90, 66, and 33 kDa) were found to complex with the IRF binding element. These proteins, with an epitope in common with the IRF-1 C-terminal region and IRF element DNA sequence-binding capability, may represent new members of the IRF family.

Peroxisome Proliferator-activated Receptor-γ1 Is Dephosphorylated and Degraded during BAY 11-7085-induced Synovial Fibroblast Apoptosis

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a central role in whole body metabolism by regulating adipocyte differentiation and energy storage. Recently, however, PPAR-gamma has also been demonstrated to affect proliferation, differentiation, and apoptosis of different cell types. As we have previously shown that BAY 11-7085-induced synovial fibroblast apoptosis is prevented by PPAR-gamma agonist 15d-PGJ2; the expression of PPAR-gamma in these cells was studied. Both PPAR-gamma1 and PPAR-gamma2 isoforms were cloned from synovial fibroblast RNA, but only PPAR-gamma1 was detected by Western blot, showing constitutive nuclear expression. Within minutes of BAY 11-7085 treatment, a PPAR-gamma1-specific band was shifted into a form of higher mobility, suggesting dephosphorylation, as confirmed by phosphatase treatment of cell extracts. Of interest, BAY 11-7085-induced PPAR-gamma1 dephosphorylation was followed by PARP and caspase-8 cleavage as well as by PPAR-gamma1 protein degradation. PPAR-gamma1 dephosphorylation was followed by the loss of PPAR-DNA binding activity ubiquitously present in synovial fibroblast nuclear extracts. Unlike the phosphorylated form, dephosphorylated PPAR-gamma1 was found in insoluble membrane cell fraction and was not ubiquitinated before degradation. PPAR-gamma1 dephosphorylation coincided with ERK1/2 phosphorylation that accompanies BAY 11-7085-induced synovial fibroblasts apoptosis. 15d-PGJ2, PGD2, and partially UO126, down-regulated ERK1/2 phosphorylation, protected cells from BAY 11-7085-induced apoptosis, and reversed both PPAR-gamma dephosphorylation and degradation. Furthermore, PPAR-gamma antagonist BADGE induced PPAR-gamma1 degradation, ERK1/2 phosphorylation, and synovial fibroblasts apoptosis. The results presented suggest an anti-apoptotic role for PPAR-gamma1 in synovial fibroblasts. Since apoptotic marker PARP is cleaved after PPAR-gamma1 dephosphorylation but before PPAR-gamma1 degradation, dephosphorylation event might be enough to mediate BAY 11-7085-induced apoptosis in synovial fibroblasts.

Parent-of-origin-specific binding of nuclear hormone receptor complexes in the H19-Igf2 imprinting control region.

Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 gene (Igf2) and the closely linked H19 gene located on distal chromosome 7 is regulated by a 2.4-kb imprinting control region (ICR) located upstream of the H19 gene. In somatic cells, the maternally and paternally derived ICRs are hypo- and hypermethylated, respectively, with the former binding the insulator protein CCCTC-binding factor (CTCF) and acting to block access of enhancers to the Igf2 promoter. Here we report on a detailed in vivo footprinting analysis-using ligation-mediated PCR combined with in vivo dimethyl sulfate, DNase I, or UV treatment-of ICR sequences located outside of the CTCF binding domains. In mouse primary embryo fibroblasts carrying only maternal or paternal copies of distal chromosome 7, we have identified five prominent footprints specific to the maternal ICR. Each of the five footprinted areas contains at least two nuclear hormone receptor hexad binding sites arranged with irregular spacing. When combined with fibroblast nuclear extracts, these sequences interact with complexes containing retinoic X receptor alpha and estrogen receptor beta. More significantly, the footprint sequences bind nuclear hormone receptor complexes in male, but not female, germ cell extracts purified from fetuses at a developmental stage corresponding to the time of establishment of differential ICR methylation. These data are consistent with the possibility that nuclear hormone receptor complexes participate in the establishment of differential ICR methylation imprinting in the germ line.

A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers.

Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24 h in 1% oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1beta. A series of MMP-2 promoter-luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91 bp region from -1433 to -1342 bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between -1410 and -1362 bp that included a potential activating protein 1 (AP-1)-binding sequence, C(-1394)CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the -1410 to -1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the -1410 to -1362 bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody-supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the -1410 to -1362 bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1-JunB and FosB-JunB expression plasmids led to significant increases in transcriptional activity. These studies demonstrate that a functional AP-1 site mediates MMP-2 transcription in cardiac cells through the binding of distinctive Fra1-JunB and FosB-JunB heterodimers. The synthesis of MMP-2 is widely considered, in contrast with many members of the MMP gene family, to be independent of the AP-1 transcriptional complex. This report is the first demonstration that defined members of the Fos and Jun transcription-factor families specifically regulate this gene under conditions relevant to critical pathophysiological processes.

Rational basis for oligodeoxynucleotides to inhibit collagen synthesis in lung fibroblasts and primary fibroblasts from liver granulomas of Schistosoma mansoni-infected mice

Hepatocellular carcinoma is associated with liver fibrosis. Murine schistosomiasis infection offers a model to study hepatic fibrogenesis. Single-stranded phosphorothiate oligodeoxynucleotides containing the TGF-β regulatory element have been shown to regulate the transcription of this gene and effectively inhibit collagen synthesis in primary fibroblasts isolated from schistosomiasis-induced hepatic granulomas. While the single-stranded oligos did not decrease collagen and non-collagen protein synthesis below control levels, their double-stranded modified and unmodified counterparts did. Competitive cold oligodeoxynucleotide gel mobility shift analysis using control fibroblast nuclear extract demonstrated that the single-stranded oligos diminished binding of the TGF-β activator protein to the TGF-β regulatory element while the double-stranded oligos totally inhibited this binding. TGF-β element containing single-stranded phosphorothioate oligodeoxynucleotides and their double-stranded counterparts may be successful therapeutic agents to inhibit hepatic fibrogenesis and associated hepatocellular carcinoma.

The Y-box Binding Protein YB-1 Suppresses Collagen α1(I) Gene Transcription via an Evolutionarily Conserved Regulatory Element in the Proximal Promoter

Appropriate expression of collagen type I, a major component of connective tissue matrices, is dependent on tight transcriptional control and a number of trans-activating and repressing factors have been characterized. Here we identify the Y-box binding protein-1 (YB-1) as a novel repressor of the collagen type alpha1(I) (COL1A1) gene. Collagen type I mRNA and protein levels decreased upon overexpression of YB-1 by transfection in NRK fibroblasts. The human, rat, and mouse COL1A1 promoter -220/+115 contains three putative Y-boxes, one of these sites, designated collagen Y-box element (CYE), includes a Y-box plus an adjacent 3' inverted repeat. DNase-I footprinting and Southwestern blotting with fibroblast nuclear extract demonstrated binding of several nuclear proteins across the CYE, one of which was identified as YB-1. Recombinant YB-1 bound the CYE sequence in gel shift assays with a preference for single-stranded templates. The entire sequence (-88/-48) was required for high affinity binding. Complex formation of endogenous YB-1 with the CYE was established by supershift studies. COL1A1 promoter-reporter constructs were suppressed up to 80% by cotransfection with YB-1 in a variety of cell types. In addition, CYE conferred YB-1 responsiveness on two heterologous promoters further demonstrating the importance of this repressor region. Mung bean nuclease sensitivity analysis suggested that repression is most likely exerted through changes in DNA conformation.

DH82 cells: a macrophage cell line for the replication and study of equine infectious anemia virus

In vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (EIAV). Laboratory investigations of EIAV/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. In addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. This report describes an adherent canine macrophage-like cell line, DH82, that supports the replication of EIAV. This cell line was easily transfectable and supported EIAV Tat transactivation of the LTR. Electrophoretic mobility shift assays were carried out to determine which transcription factor binding sites within the LTR enhancer region were bound by DH82 nuclear extracts. It was found that five different motifs were occupied. The ets motifs that are bound by PU.1 in primary macrophage nuclear extracts specifically interacted with DH82 nuclear extracts. In addition, the PEA-2, Lvb and Oct motifs that are occupied by fibroblast nuclear extracts were also bound by DH82 nuclear extracts. Finally, the methylation-dependent binding protein (MDBP) site that is bound by all nuclear extracts investigated to date demonstrated specific interactions with DH82 nuclear extracts. The observation that both macrophage-specific and fibroblast-specific motifs were utilized by DH82 nuclear extracts suggested that both macrophage-adapted and fibroblast-adapted EIAV could replicate in DH82 cells. Indeed, infectivity studies demonstrated that strains of virus that exclusively replicate in macrophages can replicate in DH82 cells and fibroblast-adapted strains of virus can also replicate in these cells. Finally, these cells could be transfected readily with the EIAV molecular clone, pSPeiav19-2, and virus spread was detected within the culture. In conclusion, this study has identified a useful cell line that should facilitate the study of EIAV expression and replication.

Cell Specificity of the Transcription-Factor Repertoire Used by a Lentivirus: Motifs Important for Expression of Equine Infectious Anemia Virus in Nonmonocytic Cells

The equine infectious anemia virus (EIAV) long-terminal repeat (LTR) has been identified as highly variable, both in infected horses and in cell culture. This nucleotide hypervariation is localized to the LTR enhancer region. The EIAV LTR has been implicated in controlling both the cell tropism and virulence of the virus and it is postulated that the enhancer-region hypervariation may be responsible for the LTR effects. Our previous studies have demonstrated that the presence of DNA motifs bound by the ets transcription-factor family member PU.1 are critically important for EIAV expression in equine macrophages. Here we identify and characterize the EIAV LTR enhancer motifs PEA-2, Lvb, Oct, and CRE, that bind to fibroblast nuclear extracts. Three of these four motifs, PEA-2, Oct, and CRE, were determined to be important for expression of the LTR in a fibroblast cell line that supports productive infection of EIAV. These motifs that are important for expression of the LTR in fibroblasts were found to be interdigitated between the PU.1 sites. We hypothesize that the combination of motif interdigitation and cell-specific usage of these motifs may be responsible for the observed EIAV LTR enhancer-region hypervariation.

Ets transcription factors cooperate with Sp1 to activate the human tenascin-C promoter.

Tenascin-C (TN-C), an extracellular matrix glycoprotein is expressed during embryonic development, but is present only at low levels in normal adult tissues. TN-C is re-expressed during wound healing, fibrotic diseases and in cancer. To better understand the mechanisms that control TN-C gene expression, we examined the regulation of the human TN-C promoter in human fibroblasts. We demonstrate that a short segment of the TN-C promoter between bp -133 and -27 contains three evolutionarily conserved Ets binding sites (EBS). These three EBSs bind in vitro expressed Fli1 protein and mediate transactivation of the TN-C gene by Fli1. Furthermore, two proximal EBSs contribute significantly to basal activity of the TN-C promoter. GABP, which is present in human fibroblast nuclear extracts, interacts with the two proximal EBSs. In addition, several Sp1 and Sp3 binding sites have been located in close proximity to the EBSs within this promoter region. The studies performed in Drosophila cells demonstrate that either Fli1 or GABPalpha+beta1 functionally interact with Sp1 resulting in a synergistic stimulation of the TN-C promoter activity. In conclusion, this study shows for the first time that the TN-C gene is regulated by Ets proteins, which together with Sp1 act as potent activators of TN-C expression.

A Methylation-responsive MDBP/RFX Site Is in the First Exon of the Collagen α2(I) Promoter

DNA methylation inhibits transcription driven by the collagen alpha2(I) promoter and the 5' end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen alpha2(I) gene. Complex formation increased when the CpG site at +7 base pairs from the transcription start site was methylated. The identity of the protein was demonstrated by co-migration and cross-competition for a characteristic slowly migrating doublet complex formed on MDBP/RFX recognition sequences and the collagen sequences by band shift assays. A RFX1-specific antibody supershifted the collagen DNA-protein complexes. Furthermore, in vitro translated RFX1 protein formed a specific complex with the collagen sequence that was also supershifted with the RFX1 antibody. MDBP/RFX displayed a higher affinity binding to the collagen sequence if the CpG at +7 was mutated in a manner similar to TpG. This same mutation within reporter constructs inhibited transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylation-induced inactivation of collagen alpha2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to the first exon in response to methylation in this region.

Nuclear Factor-κB Mediates TNF-α Inhibitory Effect on α2(I) Collagen (COL1A2) Gene Transcription in Human Dermal Fibroblasts

Abstract Among its plethora of activities as an inflammatory mediator, TNF-α has potent regulatory control on extracellular matrix production and degradation. Earlier studies have documented that TNF-α inhibits type I collagen gene (COL1A2) expression at the transcriptional level, but the characterization of the transcription factors involved has been elusive. In the present study, using transient cell transfection of human dermal fibroblasts with a battery of 5′ end deletion/chloramphenicol acetyltransferase (CAT) reporter gene constructs, we have characterized the TNF-α response element of the COL1A2 promoter. The TNF-α response element was attributed to a specific region that comprises noncanonical activator protein-1 (AP-1) (CGAGTCA) and NF-κB (AGAGTTTCCC) binding sites. TNF-α effect was eliminated by a 2-bp substitution mutation in the NF-κB1 binding half site of the NF-κB cis element. Electrophoretic mobility shift assays (EMSA) showed that recombinant human NF-κB heterodimers as well as NF-κB1 and RelA homodimers, but not AP-1, were capable of binding this element. Further, EMSA with human fibroblast nuclear extracts demonstrated enhanced binding of a single, specific complex within 5 min of TNF-α stimulation, which reached a plateau by 1 h and was not affected by preincubation of cells with cycloheximide. Gel supershift assays identified the complex as the NF-κB (p50/p65) heterodimer, whereas Abs to nuclear factor of activated T cells (NF-AT) and Jun family members failed to recognize the complex. These data suggest that in fibroblasts TNF-α activates and initiates the nuclear translocation of NF-κB that binds a divergent NF-κB element and plays a critical role in the observed inhibition of α2(I) collagen gene transcription.

Roles of an Ets motif and a novel CACGAC direct repeat in transcription of the murine dihydrolipoamide dehydrogenase (Dld) gene

The 5'-flanking region of the murine dihydrolipoamide dehydrogenase (Dld) gene was characterized for its promoter activity. DNase I footprinting analysis of the promoter region (-545 bp to +41 bp) revealed six major protein-binding domains (termed P1 to P6) that were protected by NIH3T3 fibroblast nuclear extracts. Transient transfection assays, using a series of nested deletions of the 2.5 kb 5'-flanking region ligated to the chloramphenicol acetyltransferase reporter gene, identified that the -42-bp to +41-bp region, which harbours the P1, P2, and P3 domains, had minimal transcriptional activity. When the 5'-flanking region was extended from -42 bp to -82 bp, there was an approx. 5-fold increase in promoter activity. To identify further the cis elements involved in transcription of the Dld gene (-82 bp to +41 bp), a series of mutations were introduced into this region and evaluated for functional effects using transient transfection and electrophoretic mobility shift assays. Mutation or deletion of the CACGAC direct repeat, located from -61 bp to -46 bp, resulted in minimal promoter activity. Mutation of the Ets motif, located from -37 bp to -32 bp, reduced the minimal promoter activity by approx. 50%, whereas the deletion of this motif almost abolished the promoter activity. These results indicate that: (i) the Ets motif is required for the minimal promoter activity and (ii) the CACGAC direct repeat enhances promoter activity. Database searches failed to identify the direct repeat with the CACGAC motif and hence the CACGAC sequence may represent a novel motif. The requirement of both the Ets motif and the direct repeat element for optimal promoter activity represents a unique combination for gene transcription.

Negative modulation of ?1(I) procollagen gene expression in human skin fibroblasts: Transcriptional inhibition by interferon-?

Interferon-gamma (IFN-gamma), a multifunctional cytokine produced by activated Th1 lymphocytes, exerts potent effects on the extracellular matrix by regulating fibroblast function. In this study, we examined the modulation of alpha1(I) procollagen gene (COL1A1) expression by recombinant IFN-gamma. The results showed that IFN-gamma stimulated the rapid accumulation of interferon regulated factor (IRF)-1 mRNA, followed by a delayed and dose-dependent inhibition of alpha1(I) procollagen mRNA expression in skin fibroblasts from several different donors. The inhibitory response was abrogated in fibroblasts stably expressing IRF-1 in the antisense orientation. A marked decrease in the amount of heterogeneous nuclear pre-mRNA preceded the inhibition of COL1A1 mRNA expression. In fibroblasts transiently transfected with COL1A1 promoter-chloramphenicol acetyltransferase reporter gene plasmids, IFN-gamma selectively inhibited promoter activity and abrogated its stimulation induced by TGF-beta. The inhibition by IFN-gamma was not due to downregulation of TGF-beta receptor mRNA expression in the fibroblasts or decreased ligand binding to the receptor. IFN-alpha and IFN-beta by themselves had little effect on promoter activity, but IFN-alpha augmented the inhibitory effect of IFN-gamma. Using a series of 5' deletion constructs, a proximal region of the COL1A1 promoter was shown to function as an IFN-gamma response element. This region of the gene harbors overlapping binding sites for transcription factors Sp1, Sp3, and NF-1 but no homologs of previously characterized IFN-gamma response elements. The putative IFN-gamma response region was sufficient to confer inhibition of reporter gene expression by treatment with IFN-gamma. Gel mobility shift analysis showed that two distinct and specific DNA-protein complexes were formed when fibroblast nuclear extracts were incubated with oligonucleotides spanning the IFN-gamma response region. IFN-gamma did not modify the ability of nuclear proteins to bind to this region. The results indicate that IFN-gamma inhibits COL1A1 expression in fibroblasts principally at the level of gene transcription. Inhibition involves IRF-1 and is mediated through a short proximal promoter segment but without an apparent change in promoter occupancy. The findings provide novel insight into the mechanism of IFN-gamma regulation of fibroblast function.

Stimulation of Type I Collagen Transcription in Human Skin Fibroblasts by TGF-β: Involvement of Smad 3

Transforming growth factor-beta (TGF-beta) stimulates the transcription of the alpha2(I) procollagen gene (COL1A2). The intracellular mediators involved in this response remain poorly understood. In this study, we demonstrate that primary human skin fibroblasts express Smads, a novel family of signaling molecules, in vitro in the absence of TGF-beta. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-beta. Transient overexpression of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans-activation of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 promoter containing putative TGF-beta response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-beta. Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-type Smad 7, which lacks the carboxy-terminal phosphorylation motif, specifically inhibited TGF-beta-induced activation of COL1A2 promoter. A CAGACA sequence shown to be a functional Smad-binding element in the plasminogen activator inhibitor-1 gene promoter was found within the TGF-beta-response region of the proximal COL1A2 promoter. Gel mobility shift assays showed protein phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-beta treatment strongly stimulated the formation of this DNA-protein complex. Smad was identified as a component of the CAGACA-binding transcription complex in TGF-beta-treated fibroblasts by antibody supershifting. These results demonstrate that (i) Smad 3 transmits TGF-beta signals from the receptor to the COL1A2 promoter in human fibroblasts, and is likely to play an important role in stimulation of COL1A2 promoter activity elicited by TGF-beta; (ii) in fibroblasts, Smads appear to function as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 promoter activity by TGF-beta.

DNA-PK activation by ionizing radiation-induced DNA single-strand breaks

Purpose : To assess the ability of 60Co gamma-radiation-induced plasmid DNA single-strand breaks (gamma-ssb) to activate the DNA-dependent protein kinase (DNA-PK) in vitro. Materials and methods : Plasmid DNA was gamma-irradiated under aerobic conditions to yield 0-6 gamma -ssb and 0.1 double-strand breaks (dsb) per plasmid molecule. The irradiated DNA was used to stimulate DNA-PK in crude HF19 fibroblast nuclear extracts and/or purified HeLa cell DNA-PK protein, and the activation compared with that obtained with a single enzymatically generated plasmid DNA ssb (GpII endonuclease) or dsb (Eco RI endonuclease). Results: gamma-Irradiated plasmid DNA activates DNA-PK in both crude and purified preparations and the kinase activity increases linearly with dose. As significant DNA-PK activation was detectable using irradiated plasmids which contain 0.1dsb/molecule, it was concluded that this activation is due to gamma-ssb. However, using purified DNA-PK, this activation is relatively weak as 3 gamma -ssb is equivalent to one GpII-generated DNA ssb or one end of an Eco RI-generated dsb in DNA-PK assays. Conclusions: As gamma-ssb are in a 20-fold excess of gamma-dsb in vivo for low LET radiation, gamma-ssb may contribute significantly to DNAPK signalling of gamma-radiation-induced DNA damage in vivo.

Modulation of human alpha1(I) procollagen gene activity by interaction with Sp1 and Sp3 transcription factors in vitro.

In previous work, we delineated a proximal region of the human alpha1(I) collagen gene (COL1A1) promoter necessary to direct its basal transcription in fibroblasts. This region has potential recognition sites for a variety of DNA-binding proteins. Here, we show that the -129/-107-bp sequence in this region of the promoter, which harbors an inverted CCAAT motif closely linked to a GC-rich direct repeat and is perfectly conserved between mouse and human, specifically bound the transcription factors Sp1, Sp3, and CTF/NF-1 in nuclear extracts from human skin and lung fibroblasts. Drosophila Schneider L2 cells lacking endogenous Sp activity were used to investigate the effect of expression of Sp or CTF/NF-1 transcription factors on COLlAl promoter activity. Expression of Sp1 caused potent trans-activation of a COL1A1 promoter (-174 to +42bp). In contrast, expression of Sp3, which binds to the same recognition sites as Sp1, and CTF/NF-1 stimulated COL1A1 promoter activity only at higher concentrations, and Sp2 did not transactivate. Expression of a 10-fold excess of Sp3, but not CTF/NF-1 or Sp2, abrogated the stimulation of COL1A1 promoter activity induced by Sp1. TGF-beta at concentrations previously shown to increase COL1A1 transcription caused a decrease in the relative amount of Sp3 in fibroblast nuclear extracts. These results suggest that both Sp1 and Sp3 bind to the proximal COL1A1 promoter and stimulate its activity; however, their interaction with each other may result in repression of Sp1-induced COL1A1 transcription. Alterations in the relative amounts or DNA-binding activities of these transcription factors in a cell- or signal-specific manner may contribute to the control of transcription from the COL1A1 promoter. The present findings, and recent observations implicating Sp1 and its homologs in regulating the expression of several collagen genes, suggest that the family of Sp1 transcription factors play a role in physiological and pathological modulation of connective tissue accumulation.

Identification of novel stress-induced genes downstream of chop.

CHOP (GADD153) is a small nuclear protein that dimerizes avidly with members of the C/EBP family of transcription factors. Normally undetectable, it is expressed at high levels in cells exposed to conditions that perturb protein folding in the endoplasmic reticulum and induce an endoplasmic reticulum stress response. CHOP expression in stressed cells is linked to the development of programmed cell death and, in some instances, cellular regeneration. In this study, representational difference analysis was used to compare the complement of genes expressed in stressed wild-type mouse embryonic fibroblasts with those expressed in cells nullizygous for chop. CHOP expression, in concert with a second signal, was found to be absolutely required for the activation by stress of a set of previously undescribed genes referred to as DOCs (for downstream of CHOP). DOC4 is a mammalian ortholog of a Drosophila gene, Tenm/Odz, implicated in patterning of the early fly embryo, whereas DOC6 encodes a newly recognized homolog of the actin-binding proteins villin and gelsolin. These results reveal the existence of a novel CHOP-dependent signaling pathway, distinct from the known endoplasmic reticulum unfolded protein response, which may mediate changes in cell phenotype in response to stress.

Transcriptional regulation of MHC class I gene expression in rat oligodendrocytes.

MHC class I molecules are normally expressed at very low levels in the brain and their up-regulation in response to cytokines and viral infections has been associated with a number of neurological disorders. Here we demonstrate that the down-regulation of surface class I molecules in differentiated primary rat oligodendrocytes was accompanied by reduced steady-state levels of class I heavy-chain mRNA. Transient expression assays were performed in oligodendrocytes and fibroblasts, using a mouse H-2Kb class I promoter chloramphenicol acetyltransferase plasmid termed pH2KCAT (which contained 5'-flanking sequences from -2033 to +5 bp of the H-2Kb gene relative to the transcriptional start site at +1 bp). These assays showed that H-2Kb promoter activity was reduced in oligodendrocytes but not in class I-expressing fibroblasts. H-2Kb promoter activity was up-regulated in oligodendrocytes co-transfected with a plasmid expression vector encoding the transcriptional activator tax of human T-cell leukaemia virus type I, showing that down-regulation of promoter activity was reversible. Deletion mutant analysis of the H-2Kb promoter revealed the presence of negative regulatory elements that were functional in oligodendrocytes at -1.61 to -1.07 kb and -242 to -190 bp. Deletion of sequences in pH2KCAT encompassing the downstream element totally abolished promoter activity in both oligodendrocytes and fibroblasts, whereas a deletion within the upstream negative regulatory element increased promoter activity specifically in oligodendrocytes. The upstream negative regulatory element also down-regulated a linked heterologous herpes simplex virus thymidine kinase promoter in oligodendrocytes, but not in fibroblasts. Gel retardation assays using overlapping DNA probes that spanned the entire -1.61 to -1.07 kb region revealed the presence of a number of DNA-binding activities that were present in oligodendrocyte, but not in fibroblast nuclear extracts.

Y-box proteins interact with the S1 nuclease-sensitive site in the chicken alpha 2(I) collagen gene promoter.

The sequence of the chicken alpha 2(I) collagen promoter from -712 to -85, relative to exon 1, has been shown to be important for transcriptional activity. Within this region a pyrimidine/purine asymmetrical element at -200 bp forms an in vitro S1 nuclease-sensitive site. The pyrimidine-rich strand of this element interacts specifically with single-stranded DNA-binding proteins present in fibroblast nuclear extracts [Bayarsaihan and Lukens (1996) Biochem. J. 314, 293-296]. To identify these proteins we performed expression screening of a chick embryo fibroblast cDNA library using a single-stranded polypyrimidine sequence derived from this element. One of the isolated clones was found to encode a member of the cold-shock gene family, either chicken YB-1 or a highly homologous protein. This protein and a known chicken Y-box protein were both found to bind sequence-specifically to the pyrimidine-rich strand of the pyrimidine/purine asymmetrical element in the chicken alpha 2(I) collagen promoter. The binding mechanism of these proteins could be based on the formation of a non-canonical triplex DNA structure (H-DNA). Although members of this widespread and conserved protein family have been reported to modulate the expression of a number of genes, the findings reported here provide the first evidence for a possible role of cold-shock proteins in the regulation of type I collagen genes.

A Novel Site in the Muscle Creatine Kinase Enhancer Is Required for Expression in Skeletal but Not Cardiac Muscle

Expression of the muscle creatine kinase (MCK) gene in skeletal and heart muscle is controlled in part by a 5' tissue-specific enhancer. In order to identify new regulatory elements, we designed mutations in a previously untested conserved portion of this enhancer. Transfection analysis of these mutations delineated a new control element, named Trex (Transcriptional regulatory element x), which is required for full transcriptional activity of the MCK enhancer in skeletal but not cardiac muscle cells. Gel mobility shift assays demonstrate that myocyte, myoblast, and fibroblast nuclear extracts but not primary cardiomyocyte nuclear extracts contain a trans-acting factor that binds specifically to Trex. The Trex sequence is similar (7/8 bases) to the TEF-1 consensus DNA-binding site involved in regulating other muscle genes. To determine if TEF-1 interacts with Trex, selected TEF-1 binding sites such as GTIIc and M-CAT and two anti-TEF-1 antisera were used in gel shift assays. These experiments strongly suggest that a factor distinct from TEF-1 binds specifically to Trex. Thus it appears that MCK transcription is regulated in skeletal muscles through a Trex-dependent pathway while Trex is not required for MCK expression in heart. This distinction could account partially for the difference in levels of muscle creatine kinase in these tissues.