Transcriptional activation of urokinase by the Krüppel-like factor Zf9/COPEB activates latent TGF-beta1 in vascular endothelial cells.

Understanding the regulation of genes controlling fibrinolysis and matrix homeostasis is essential for elucidating the basis of tissue repair. A recently described novel Krüppel-like factor, Zf9, is up-regulated in acute liver injury in activated hepatic stellate cells. Because Zf9 can be induced widely, its activity was examined in vascular endothelium, a key cell in vascular injury. Zf9 is induced as an immediate-early response gene in bovine aortic endothelial cells (BAECs) following treatment with serum or phorbol ester. Zf9 transcriptionally activates urokinase plasminogen activator (uPA). Recombinant Zf9-GST binds to wild-type but not mutated 'GC-box' motifs within the human uPA promoter (-63 to -32), with greatest affinity to the middle of 3 contiguous GC boxes. Transient transfection of Zf9 drives transactivation of a full-length uPA promoter- and GC box-construct, but not a uPA promoter-construct devoid of GC boxes. Transactivation of uPA by Zf9 is also supported in Drosophila S2 cells. Most importantly, transiently transfected Zf9 up-regulates endogenous uPA messenger RNA and activity in BAECs, resulting in increased bioactive transforming growth factor-beta (TGF-beta) via enhancement of proteolytic activation of the latent molecule. Furthermore, concomitant expression of Zf9 and uPA proteins was observed in arterial endothelial cells after balloon injury in rats, suggesting a potential role of Zf9 in uPA expression not only in vitro but also in vivo. These findings suggest a role of Zf9 in the injury response by enhancing uPA synthesis and subsequent activation of latent TGF-beta. (Blood. 2000;95:1309-1316)

In vivo analysis of the state of the human uPA enhancer following stimulation by TPA.

We have analysed in vivo the -2.0 kb enhancer of the human urokinase-type plasminogen activator (uPA) gene in HepG2 cells, in which gene expression can be induced by phorbol esters. The results reveal that, within the regulatory region, the enhancer, the silencer and the minimal promoter become hypersensitive to deoxyribonuclease I (DNase I) upon induction of transcription. The hypersensitivity of the enhancer can be reversed after removal of the inducer. In vivo footprinting analysis indicates that all the cis-acting elements of the enhancer, previously identified in vitro, are occupied in vivo upon 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulation of HepG2 cells. Micrococcal nuclease (MNase) cleavage of this region fails to reveal discrete nucleosomal boundaries in vivo in close proximity of the enhancer, either before or after stimulation. Furthermore, this region does not lose its nucleosomal configuration after TPA induction of transcription. An approximately 600 bp long region around the enhancer becomes more, but not fully, accessible to restriction endonucleases upon stimulation. A time-course experiment shows that this accessibility reaches a plateau after a 1 h TPA treatment suggesting the persistent presence of nucleosomes. These results indicate that TPA induces the binding of transcription factors to the uPA enhancer without chromatin remodelling of this region.

Physical interaction between retinoic acid receptor and Sp1: mechanism for induction of urokinase by retinoic acid.

Induction of urokinase plasminogen activator (uPA) by retinoic acid (RA) is the initial event preceding certain subsequent biological changes in vascular endothelial cells. We investigated the molecular mechanism by which RA stimulates the expression of uPA, which lacks a canonical RA receptor (RAR)-responsive element, in bovine and human aortic endothelial cells. Upon stimulation with RA, mRNA levels of RARalpha and beta transiently increased in parallel with the induction of uPA, and this increase was inhibited by cycloheximide. Results of transient transfection of RAR/RXR cDNAs and experiments using specific agonists and antagonists suggested that uPA induction is dependent upon RAR (initially, RARalpha) with the help of RXRalpha. Deletion analysis of the uPA promoter suggested that RAR/RXR acts on GC box region within the uPA promoter. This was further supported by inhibition of Sp1 binding to this region. Coimmunoprecipitation studies, glutathione S-transferase pull-down experiment, and mammalian two-hybrid assays suggested a physical interaction between RAR/RXR and Sp1. Furthermore, gel shift studies showed that the binding of Sp1 to the uPA GC box is significantly potentiated in the presence of RARs/RXRs. Finally, Sp1 and RAR/RXR synergistically enhanced the transactivation activity of the uPA promoter. These results suggest that (1) RA induces RARs mainly via RARalpha and that (2) RAR/RXR physically and functionally interact with Sp1, resulting in a potentiation of uPA transcription.

Removal of domain D2 or D3 of the human urokinase receptor does not affect ligand affinity.

The main ligand-binding determinant of the human urokinase receptor (uPAR) is located in the amino terminal domain D1, but when isolated this domain presents a 1500 fold lower affinity than the intact three-domain uPAR (D1D2D3). uPAR mutants missing either domain 2 (D1HD3) or domain 3 (D1D2) were expressed in murine LB6 cells and showed to be properly GPI-anchored to the cell surface. Binding assays with [125I]ATF demonstrated that these mutants possessed a normal (D1D2) or slightly reduced (D1HD3) affinity, indicating that a high ligand-affinity may be achieved by a combination of D1 with domain D2 or D3.

Incorporation of chromosomal proteins HMG-14/HMG-17 into nascent nucleosomes induces an extended chromatin conformation and enhances the utilization of active transcription complexes.

The role of chromosomal proteins HMG-14 and HMG-17 in the generation of transcriptionally active chromatin was studied in a Xenopus laevis egg extract which supports complementary DNA strand synthesis and chromatin assembly. Chromosomal proteins HMG-14/HMG-17 enhanced transcription from a chromatin template carrying a 5S rRNA gene, but not from a DNA template. The transcriptional potential of chromatin was enhanced only when these proteins were incorporated into the template during, but not after, chromatin assembly. HMG-14 and HMG-17 stimulate transcription by increasing the activity, and not the number, of transcribed templates. They unfold the chromatin template without affecting the nucleosomal repeat or decreasing the content of histone B4. We suggest that HMG-14/HMG-17 enhance transcription by inducing an extended conformation in the chromatin fiber, perhaps due to interactions with histone tails in nucleosomes. By disrupting the higher order chromatin structure HMG-14/HMG-17 increase the accessibility of target sequences to components of the transcriptional apparatus.

The footprint of chromosomal proteins HMG-14 and HMG-17 on chromatin subunits.

The position of chromosomal proteins HMG-14 and HMG-17 in nucleosome cores and in chromatosomes lacking linker histones has been mapped by hydroxyl radical footprinting. Both the nucleosome core and the H1/H5 depleted chromatosome can specifically bind two molecules of HMG-14/-17. The path of HMG-14 on the surface of chromatin subunits is indistinguishable from that of HMG-17. The bound HMGs protect the DNA from hydroxyl radical cleavage 25 base-pairs from the end of the DNA in nucleosome cores and in each of the two major grooves of the DNA flanking the nucleosomal dyad axis. Thus, in both cores and H1/H5-depleted chromatosomes the proteins bridge two adjacent DNA strands on the surface of the particles. The sites occupied by HMG near the end of the chromatosome-length particles are distinct from those occupied by the H1/H5 linker histones. In the region of the dyad axis the binding sites of HMGs overlap those of the linker histones. The placement of HMG-14/-17 near the nucleosomal dyad axis raises the possibility that interactions between histone H1 and HMGs may affect the transcriptional potential of chromatin.

Deposition of chromosomal protein HMG-17 during replication affects the nucleosomal ladder and transcriptional potential of nascent chromatin.

A cell-free system from Xenopus eggs was used to study the role of chromosomal protein HMG-17 in the generation of the chromatin structure of transcriptionally active genes. Addition of HMG-17 protein to the extracts, which do not contain structural homologs of the HMG-14/-17 protein family, indicates the protein is incorporated into the nascent template during replication, prior to completion of chromatin assembly. The protein binds to and stabilizes the structure of the nucleosomal core thereby improving the apparent periodicity of the nucleosomal spacing of nascent chromatin. Assembly of HMG-17 into the nascent chromatin structure significantly increased the transcription potential of the 5S RNA gene and satellite I chromatin. Kinetic studies indicate that the increase in transcriptional potential is observed only when HMG-17 is incorporated into nucleosomes during chromatin assembly.

Nucleosome core binding region of chromosomal protein HMG-17 acts as an independent functional domain.

Chromosomal proteins HMG-14 and HMG-17 have a modular structure. Here we examine whether the putative nucleosome-binding domain in these proteins can function as an independent module. Mobility shift assays with recombinant HMG-17 indicate that synthetic molecules can be used to analyze the interaction of this protein with the nucleosome core. Peptides corresponding to various regions of the protein have been synthesized and their interaction with nucleosome cores analyzed by mobility shift, thermal denaturation and DNase I digestion. A 30 amino acid long peptide, corresponding to the putative nucleosome-binding domain of HMG-17, specifically shifts the mobility of cores as compared to free DNA, elevates the tm of both the premelt and main melt of the cores and protects from DNase I digestion the same nucleosomal DNA sites as the intact protein. The binding of both the peptide and the intact protein is lost upon digestion of the histone tails by trypsin. The nucleosomal binding sites of the peptide appear identical to those of the intact protein. Thus, a region of the protein can acts as an independent functional domain. This supports the notion that HMG-14 and HMG-17 are modular proteins. This finding is relevant to the understanding of the function and evolution of HMG-14/-17, the only nucleosome core particle binding proteins known to date.

Recombinant human chromosomal proteins HMG-14 and HMG-17.

Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed. The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site. Expression of the recombinant proteins does not significantly affect bacterial growth. The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time. After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction. Methods for rapid extraction and purification of the recombinant proteins are described. The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays. The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins. A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes.

Developmental changes in the expression of high mobility group chromosomal proteins.

The high mobility group (HMG) chromosomal proteins may modulate the structure of distinct regions in chromatin, thereby affecting processes such as development and differentiation. Here we report that the levels of the HMG chromosomal proteins and their mRNAs change significantly during erythropoiesis. Erythroid cells from 5-day chicken embryos contain 2.5-10 times more HMG mRNAs than cells from 14-day embryos, whereas circulating cells from adult animals are devoid of HMG and most other mRNAs. Nuclear run-off experiments and Northern analysis of RNA from various developmental stages and from Percoll-fractionated cells indicate that the genes are transcribed in early cells of either the primitive or definitive erythroid lineage. The rate of synthesis of the various HMGs changes during erythropoiesis; in erythroid cells from 7-day embryos the ratio of HMG-14b or HMG-17 to HMG-14a is, respectively, 8 and 10 times lower than in 9-day erythroids. HMG-14a, the major chicken HMG-14 species, is synthesized mainly in primitive cells, while HMG-14b is preferentially synthesized in definitive cells. Thus, the change from primitive to definitive erythroid lineage during embryogenesis is accompanied by a change in the expression of HMG chromosomal proteins. Conceivably, these changes may affect the structure of certain regions in chromatin; however, it is not presently clear whether the switch in HMG protein gene expression is a consequence or a prerequisite for proper differentiation.

Differentiation-dependent alteration in the chromatin structure of chromosomal protein HMG-17 gene during erythropoiesis.

The expression of the gene coding for chromosomal protein HMG-17 is down regulated during chicken erythrocyte maturation. The transcriptional down regulation is associated with major alterations in the chromatin structure of this gene. The 5' region of the gene contains both constitutive and developmental stage-specific deoxyribonuclease I (DNase I) hypersensitive sites. The constitutive sites bracket the "CpG island" present in the gene, which remains hypomethylated throughout the various developmental stages. During erythropoiesis, the gene acquires a distinct structure that, upon digestion with micrococcal nuclease (MNase) yields an unusual repeat. Two nucleosomes, with a 200 base-pair repeat, are positioned immediately downstream from the start of transcription. Immediately downstream and upstream from these nucleosomes, the boundaries between MNase sites change to a 75 base-pair repeat, which indicates an unusual chromatin structure. The differentiation related changes in the DNase I and MNase digestion pattern in the 5' region of the gene suggest that sequences present in the first intron may be involved in gene regulation. The results may be relevant to the regulation of the entire HMG-14/-17 gene family.

Immunochemical analysis of the exposure of high mobility group protein 14 and 17 surfaces in chromatin.

Antisera were elicited against synthetic peptides corresponding either to regions common to all members of the high mobility group 14 and 17 protein family protein or to distinct domains of the HMG-14 or HMG-17 subgroup. The antisera were used to probe the accessibility of various HMG domains in chromatin. Competitive enzyme-linked immunosorbent assays indicate that the central region of the proteins, which contains their DNA binding domain and is positively charged, is exposed to a smaller degree than the C-terminal region of the proteins, which has a net negative charge. The C-terminal regions of the HMG-14 and HMG-17 proteins are exposed and available to interact with other proteins.

Expression of chromosomal proteins HMG-14 and HMG-17 in transformed human cells.

The relation between cellular phenotype and expression of chromosomal high mobility group proteins 14 and 17 (HMG-14 and HMG-17) has been examined in human cell lineages. Quantitation of HMG-14 and HMG-17 mRNA in several human cell lines revealed differences in both the steady state mRNA level and in the ratio of HMG-14 to HMG-17 mRNA. Analysis of phenotypically distinct derivatives of human bronchial epithelial cells revealed small differences between both the steady state mRNA levels and the relative amount of these proteins among the clonal variants. The effect of myeloid differentiation on the mRNA level of HMG-14 and HMG-17 was examined in the human promyelocytic leukemia cell line HL-60 following treatment with several granulocytic and monocytic differentiating agents. The ratio of HMG-17 mRNA to either HMG-14 or histone H4 mRNA varied among the cell phenotypes suggesting that phenotype switching may result in detectable alterations in the expression of the HMG-14 and HMG-17 genes. The data suggest that, although the ratio of HMG-14 to HMG-17 mRNA varies among human cell lines, these variations are relatively small.

Chromosomal protein HMG-14. Identification, characterization, and chromosome localization of a functional gene from the large human multigene family.

The human HMG-14 multigene family is one of the largest retropseudogene families known. To identify and isolate a functional human HMG-14 gene, genomic clones, selected with the cDNA, were screened with a set of 6 oligonucleotides. A single genomic clone was isolated suggesting that the human genome contains few, and perhaps only one, functional genes. An 8882-base pair (bp) genomic clone containing the complete, 6804-bp-long human gene together with 850 bp 5' to the start of transcription and 1228 bp 3' to the end of transcription was sequenced. The gene is comprised of 6 exons ranging in size from 30 to 839 bp, two of which code for the entire DNA binding site of the protein, and has several features typical of "housekeeping" genes. Using human-rodent somatic cell hybrids, the HMG-14 gene was localized to human chromosome 21. A restriction fragment length polymorphism, useful for further analysis and mapping, has been detected. The present article, which describes the first isolation and characterization of a gene coding for chromosomal protein HMG-14, indicates that genes coding for HMG-14 and HMG-17 may share several distinctive characteristics. Comparison with the human and chicken HMG-17 genes reveals that all contain 6 exons, that all have exons of similar size, that all have 5' regions highly enriched in GC residues and that all have features typical of housekeeping genes.