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At least 19 recordsLinked to original sources
PubMed2003

During apoptosis of tumor cells HMGA1a protein undergoes methylation: identification of the modification site by mass spectrometry.

Programmed cell death is characterized by posttranslational modifications of a limited and specific set of nuclear proteins. We demonstrate that during apoptosis of different types of tumor cells there is a monomethylation of the nuclear protein HMGA1a that is associated to its previously described hyperphosphorylation/dephosphorylation process. HMGA1a methylation is strictly related to the execution of programmed cell death and is a massive event that involves large amounts of the protein. In some tumor cells, HMGA1a protein is already methylated to an extent that depends on cell type. The degree of methylation in any case definitely increases during apoptosis. In the studied cell systems (human leukaemia, human prostate tumor, and rat thyroid transformed cells) among the low-molecular-mass HMG proteins, only HMGA1a was found to be methylated. A tryptic digestion map of HPLC-purified HMGA1a protein showed that methylation occurs at arginine 25 in the consensus G(24)R(25)G(26) that belongs to one of the DNA-binding AT-hooks of the protein. An increase of HMGA1a methylation could be related to heterochromatin and chromatin remodeling of apoptotic cells.

Amino Acid Sequence↗
PubMed2001

A link between apoptosis and degree of phosphorylation of high mobility group A1a protein in leukemic cells.

Nuclear phosphoprotein HMGA1a, high mobility group A1a, (previously HMGI) has been investigated during apoptosis. A change in the degree of phosphorylation of HMGA1a has been observed during apoptosis induced in four leukemic cell lines (HL60, K562, NB4, and U937) by drugs (etoposide, camptothecin) or herpes simplex virus type-1. Both hyper-phosphorylation and de-phosphorylation of HMGA1a have been ascertained by liquid chromatography-mass spectrometry. Hyper-phosphorylation (at least five phosphate groups/HMGA1a molecule) occurs at the early apoptotic stages and is probably related to HMGA1a displacement from DNA and chromatin release from the nuclear scaffold. De-phosphorylation (one phosphate or no phosphate groups/HMGA1a molecule) accompanies the later formation of highly condensed chromatin in the apoptotic bodies. We report for the first time a direct link between the degree of phosphorylation of HMGA1a protein and apoptosis according to a process that involves the entire amount of HMGA1a present in the cells and, consequently, whole chromatin. At the same time we report that variously phosphorylated forms of HMGA1a protein are also mono-methylated.

Amino Acid Sequence↗
PubMed2001

Role of the high mobility group A proteins in human lipomas.

The HMGA family is comprised of four proteins: HMGA1a, HMGA1b, HMGA1c and HMGA2. The first three proteins are products of the same gene, HMGA1, generated through an alternative splicing mechanism. The HMGA proteins are involved in the regulation of chromatin structure and HMGA DNA-binding sites have been identified in functional regions of many gene promoters. Rearrangements of the HMGA2 gene have been frequently detected in human benign tumors of mesenchymal origin including lipomas. 12q13-15 chromosomal translocations involving the HMGA2 gene locus, account for these rearrangements. The HMGA proteins have three AT-hook domains and an acidic C-terminal tail. The HMGA2 modifications consist in the loss of the C-terminal tail and fusion with ectopic sequences. A pivotal role of the HMGA2 rearrangements in the process of lipomagenesis is suggested by experiments showing that transgenic mice carrying a truncated HMGA2 gene showed a giant phenotype together with abdominal/pelvic lipomatosis. As HMGA2 null mice showed a great reduction in fat tissue, a positive role of the HMGA2 gene in adipocytic cell proliferation is proposed. More recently, similar alterations of the HMGA1 gene have been described. As the block of the HMGA1 protein synthesis induces an increase in growth rate of the pre-adipocytic cell line 3T3-L1, we suggest a negative role of the HMGA1 proteins in adipocytic cell growth and, therefore, we propose that adipocytic cell growth derives from the balance of the HMGA1 and HMGA2 protein functions.

Adipocytes↗
PubMed2003

Induced HMGA1a expression causes aberrant splicing of Presenilin-2 pre-mRNA in sporadic Alzheimer's disease.

The aberrant splicing isoform (PS2V), generated by exon 5 skipping of the Presenilin-2 (PS2) gene transcript, is a diagnostic feature of sporadic Alzheimer's disease (AD). We found PS2V is hypoxia-inducible in human neuroblastoma SK-N-SH cells. We purified a responsible trans-acting factor based on its binding to an exon 5 fragment. The factor was identified as the high mobility group A1a protein (HMGA1a; formerly HMG-I). HMGA1a bound to a specific sequence on exon 5, located upstream of the 5' splice site. HMGA1a expression was induced by hypoxia and the protein was accumulated in the nuclear speckles with the endogenous splicing factor SC35. Overexpression of HMGA1a generated PS2V, but PS2V was repressed by cotransfection with the U1 snRNP 70K protein that has a strong affinity to HMGA1a. HMGA1a could interfere with U1 snRNP binding to the 5' splice site and caused exon 5 skipping. HMGA1a levels were significantly increased in the brain tissue from sporadic AD patients. We propose a novel mechanism of sporadic AD that involves HMGA1a-induced aberrant splicing of PS2 pre-mRNA in the absence of any mutations.

Alternative Splicing↗
PubMed2001

Overexpression of proteins HMGA1 induces cell cycle deregulation and apoptosis in normal rat thyroid cells.

The high mobility group (HMG) proteins (HMGA1a, HMGA1b, and HMGA2) bind to DNA and interact with various transcriptional factors. Therefore, they play an important role in chromatin organization. HMGA protein expression is low in normal adult tissues, but abundant during embryonic development and in several experimental and human tumors. Blockage of HMGA expression inhibits the transformation of rat thyroid PC Cl 3 cells treated with oncogene-carrying retroviruses, thus implicating HMGA in rat thyroid transformation. To better understand the role of HMGA and to establish whether its up-regulated expression is sufficient to induce the transformed phenotype, we generated PC Cl 3 cells that overexpress the protein. We demonstrate that HMGA1b protein overexpression does not transform normal rat thyroid PC Cl 3 cells, but it deregulates their cell cycle: cells enter S-phase earlier and the G(2)-M transition is delayed. HMGA1-overexpressing cells undergo apoptosis through a pathway involving caspase-3 activation, probably consequent to the conflict between mitogenic pressure and the inability to proceed through the cell cycle. Using various HMGA1b gene mutations, we found that the third AT-hook domain and the acetylation site K60 are the protein regions required for induction of apoptosis in PC Cl 3 cells. In conclusion, although HMGA1 protein overexpression is associated with the malignant phenotype of rat and human thyroid cells, it does not transform normal thyroid cells in culture but leads them to programmed cell death.

Animals↗
PubMed2000

FR900482 class of anti-tumor drugs cross-links oncoprotein HMG I/Y to DNA in vivo.

BACKGROUND: Overexpression of the high-mobility group, HMG I/Y, family of chromatin oncoproteins has been implicated as a clinical diagnostic marker for both neoplastic cellular transformation and increased metastatic potential of several human cancers. These minor groove DNA-binding oncoproteins are thus an attractive target for anti-tumor chemotherapy. FR900482 represents a new class of anti-tumor agents that bind to the minor groove of DNA and exhibit greatly reduced host toxicity compared to the structurally related mitomycin C class of anti-tumor drugs. We report covalent cross-linking of DNA to HMG I/Y by FR900482 in vivo which represents the first example of a covalent DNA-drug-protein cross-link with a minor groove-binding oncoprotein and a potential novel mechanism through which these compounds exert their anti-tumor activity. RESULTS: Using a modified chromatin immunoprecipitation procedure, fragments of DNA that have been covalently cross-linked by FR900482 to HMG I/Y proteins in vivo were polymerase chain reaction-amplified, isolated and characterized. The nuclear samples from control cells were devoid of DNA fragments whereas the nuclear samples from cells treated with FR900482 contained DNA fragments which were cross-linked by the drug to the minor groove-binding HMG I/Y proteins in vivo. Additional control experiments established that the drug also cross-linked other non-oncogenic minor groove-binding proteins (HMG-1 and HMG-2) but did not cross-link major groove-binding proteins (Elf-1 and NFkappaB) in vivo. Our results are the first demonstration that FR900482 cross-links a number of minor groove-binding proteins in vivo and suggests that the cross-linking of the HMG I/Y oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent. CONCLUSIONS: We have illustrated that the FR class of anti-tumor antibiotics, represented in this study by FR900482, is able to produce covalent cross-links between the HMG I/Y oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG I/Y proteins in the minor groove of DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demonstrated that FR900482 cross-links other minor groove-binding proteins (HMG-1 and HMG-2 in the present study) in vivo; however, since HMG I/Y is the only minor groove-binding oncoprotein presently known, it is possible that these non-histone chromatin proteins are among the important in vivo targets of this family of drugs. These compounds have already been assessed as representing a compelling clinical replacement for mitomycin C due to their greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy. The capacity of FR900482 to cross-link the HMG I/Y oncoprotein with nuclear DNA in vivo potentially represents a significant elucidation of the anti-tumor efficacy of this family of anticancer agents.

Antineoplastic Agents↗
PubMed1994

The high mobility group protein HMG I(Y) can stimulate or inhibit DNA binding of distinct transcription factor ATF-2 isoforms.

The high mobility group protein HMG I(Y) stimulates the binding of a specific isoform of the activating transcription factor 2 (ATF-2(195)) to the interferon beta (IFN-beta) gene promoter. HMG I(Y) specifically interacts with the basic-leucine zipper region of ATF-2(195), and HMG I(Y) binds to two sites immediately flanking the ATF-2 binding site of the IFN-beta promoter. Here, we show that HMG I(Y) can stimulate the binding of ATF-2(195), at least in part, by promoting ATF-2 dimerization. In addition, we report the characterization of a naturally occurring isoform of ATF-2 (ATF-2(192)) that binds specifically to the IFN-beta promoter but is unable to interact with HMG I(Y). Remarkably, HMG I(Y) inhibits the binding of ATF-2(192) to the IFN-beta promoter. Thus, the ability of HMG I(Y) to specifically interact with ATF-2 correlates with its ability to stimulate ATF-2 binding to the IFN-beta promoter. Comparisons of the amino acid sequences of the basic-leucine zipper domains of ATF-2(195) and ATF-2(192) suggest that HMG I(Y) interacts with a short stretch of basic amino acids near the amino terminus of the basic-leucine zipper domain of ATF-2(195).

Activating Transcription Factor 2↗
PubMed1995

Interleukin 4-inducible phosphorylation of HMG-I(Y) is inhibited by rapamycin.

The non-histone chromosomal protein HMG-I(Y) participates in repression of transcription directed by a promoter which confers interleukin 4 (IL-4)-inducible activation in transfected B cell lines. Metabolic labeling, phosphoamino acid analyses, and in vitro phosphorylation studies demonstrate that IL-4 induces serine phosphorylation of HMG-I(Y) in B lymphocytes. Phosphopeptide mapping shows that the predominant site of phosphorylation contains a casein kinase II consensus motif. The immunosuppressive agent rapamycin has been shown preferentially to inhibit IgE production by IL-4-treated human B cells. It is shown here that rapamycin inhibits both activation of the human germ line epsilon promoter by IL-4 and IL-4-inducible phosphorylation of HMG-I(Y). These findings demonstrate a rapamycin-sensitive pathway that transduces signals from the IL-4 receptor to nuclear factors that regulate inducible transcription. The affinity of normal nuclear HMG-I(Y) for DNA is increased by dephosphorylation in vitro, whereas in vitro kinase reactions using casein kinase II decrease recombinant HMG-I(Y) binding to DNA. These data further suggest a novel mechanism in which phosphorylation triggered by IL-4 or other cytokines could regulate the effects of HMG-I(Y) on gene transcription.

Amino Acid Sequence↗
PubMed1997

Cdc2 and mitogen-activated protein kinases modulate DNA binding properties of the putative transcriptional regulator Chironomus high mobility group protein I.

Cells of the dipteran insect Chironomus contain a high mobility group protein that is homologous to the mammalian high mobility group proteins I/Y (HMGI/Y). These proteins facilitate the assembly of higher order nucleoprotein complexes. In proliferating cells, >30% of Chironomus HMGI was found to be phosphorylated. The phosphorylation sites were mapped to Ser3, Ser22, and Ser72 and were found to be substrates for the kinases Cdc2 (and mitogen-activated protein (MAP)), MAP, and Ca2+/phospholipid-dependent protein kinase, respectively. In mitotically arrested cells, the extent of phosphorylation at Ser3 increased, whereas phosphorylation at Ser22 remained unchanged. In nondividing cells, phosphorylation at Ser3 and Ser22 was strongly reduced. The DNA binding affinity of Chironomus HMGI was not influenced by single phosphorylation at Ser3 or Ser22. In contrast, phosphorylation at both of these sites resulted in a 10-fold weakening of the binding activity and altered the mode of protein-DNA interaction. Since both human and murine HMGI/Y proteins, similarly to the insect HMGI protein, possess phosphorylation sites for Cdc2 and MAP kinases that intersperse the AT-hook DNA-binding motifs, our results may reflect a general mechanism that regulates the properties and function of this class of putative transcriptional regulators.

Amino Acid Sequence↗
PubMed1997

High mobility group I proteins interfere with the homeodomains binding to DNA.

Homeodomains (HDs) constitute the DNA binding domain of several transcription factors that control cell differentiation and development in a wide variety of organisms. Most HDs recognize sequences that contain a 5'-TAAT-3' core motif. However, the DNA binding specificity of HD-containing proteins does not solely determine their biological effects, and other molecular mechanisms should be responsible for their ultimate functional activity. Interference by other factors in the HD/DNA interaction could be one of the processes by which HD-containing proteins achieve the functional complexity required for their effects on the expression of target genes. Using gel-retardation assay, we demonstrate that two members of the high mobility group I (HMGI) family of nuclear proteins (HMGI-C and HMGY) can bind to a subset of HD target sequences and inhibit HDs from binding to the same sequences. The inhibition of the HD/DNA interaction occurs while incubating HMGI-C with DNA either before or after the addition of the HD. The reduced half-life of the HD.DNA complex in the presence of HMGI-C, and the shift observed in the CD spectra recorded upon HMGI-C binding to DNA, strongly suggest that structural modifications of the DNA are responsible for the inhibition of the HD.DNA complex formation. Moreover, by co-transfection experiments we provide evidence that this inhibition can occur also in vivo. The data reported here would suggest that HMGI proteins may be potential regulators of the function of HD-containing proteins and that they are able to interfere with the access of the HD to their target genes.

3T3 Cells↗
PubMed1997

Functional interaction between the DNA binding subunit trimerization domain of NF-Y and the high mobility group protein HMG-I(Y).

The mammalian transcription factor, NF-Y(CBF), contains three known subunit components, NF-YA (CBF-B), NF-YB(CBF-A), and NF-YC(CBF-C), which are all required to reconstitute specific CCAAT box DNA binding activity. In this study, the high mobility group chromosomal protein, HMG-I(Y), has been shown to activate NF-Y in transient transfections in vivo using the natural murine alpha2(I) collagen promoter and a multimerized version of the proximal NF-Y(CBF) CCAAT box element. In vitro analysis of the alpha2(I) collagen promoter region inclusive of the NF-Y(CBF) binding site (-106 to -65 base pairs) failed to identify any high affinity HMG-I(Y) DNA-binding sites. However, the heterotrimeric NF-Y complex, as well as the NF-YA subunit alone, was shown to stably interact in vitro with both HMG-I(Y) and phosphorylated HMG-I, as modified by casein kinase II, using far Western and protein-protein interaction solution assays in the absence of CCAAT box DNA. Furthermore, the interaction between HMG-I(Y) and NF-Y was mapped to the highly conserved DNA binding-subunit interaction domain (DBD) of the NF-YA subunit and to a single AT-hook motif in HMG-I(Y). Recombinant HMG-I was also found to stabilize the CCAAT box DNA binding activity of recombinant NF-Y, as well as the native NF-Y complex, in vitro. Together, these results suggest a functional HMG-I(Y) protein binding site has been identified in the NF-Y complex and mapped to the conserved DBD and AT-hook regions of NF-YA and HMG-I(Y), respectively. This protein-protein interaction site may function to modulate NF-Y activity through stabilization of NF-Y binding to its CCAAT box DNA-binding site.

Animals↗
PubMed1999

The HMG-I(Y) A.T-hook peptide motif confers DNA-binding specificity to a structured chimeric protein.

Chromosomal translocations involving genes coding for members of the HMG-I(Y) family of "high mobility group" non-histone chromatin proteins (HMG-I, HMG-Y, and HMG-IC) have been observed in numerous types of human tumors. Many of these gene rearrangements result in the creation of chimeric proteins in which the DNA-binding domains of the HMG-I(Y) proteins, the so-called A.T-hook motifs, have been fused to heterologous peptide sequences. Although little is known about either the structure or biophysical properties of these naturally occurring fusion proteins, the suggestion has been made that such chimeras have probably assumed an altered in vivo DNA-binding specificity due to the presence of the A.T-hook motifs. To investigate this possibility, we performed in vitro "domain-swap" experiments using a model protein fusion system in which a single A. T-hook peptide was exchanged for a corresponding length peptide in the well characterized "B-box" DNA-binding domain of the HMG-1 non-histone chromatin protein. Here we report that chimeric A. T-hook/B-box hybrids exhibit in vitro DNA-binding characteristics resembling those of wild type HMG-I(Y) protein, rather than the HMG-1 protein. These results strongly suggest that the chimeric fusion proteins produced in human tumors as a result of HMG-I(Y) gene chromosomal translocations also retain A.T-hook-imparted DNA-binding properties in vivo.

Amino Acid Sequence↗
PubMed2000

A novel member of the BTB/POZ family, PATZ, associates with the RNF4 RING finger protein and acts as a transcriptional repressor.

We have identified a novel human gene encoding a 59-kDa POZ-AT hook-zinc finger protein (PATZ) that interacts with RNF4, a mediator of androgen receptor activity, and acts as a transcriptional repressor. PATZ cDNA was isolated through a two-hybrid interaction screening using the RING finger protein RNF4 as a bait. In vitro and in vivo interaction between RNF4 and PATZ was demonstrated by protein-protein affinity chromatography and coimmunoprecipitation experiments. Such interaction occurred through a small region of PATZ containing an AT-hook DNA binding domain. Immunofluorescence staining and confocal microscopy showed that PATZ localizes in distinct punctate nuclear regions and colocalizes with RNF4. Functional analysis was performed by cotransfection assays: PATZ acted as a transcriptional repressor, whereas its partner RNF4 behaved as a transcriptional activator. When both proteins were overexpressed a strong repression of the basal transcription was observed, indicating that the association of PATZ with RNF4 switches activation to repression. In addition, RNF4 was also found to associate with HMGI(Y), a chromatin-modeling factor containing AT-hook domains.

Amino Acid Sequence↗
PubMed2000

Distinct organization of DNA complexes of various HMGI/Y family proteins and their modulation upon mitotic phosphorylation.

High mobility group (HMG) proteins HMGI, HMGY, HMGI-C, and Chironomus HMGI are DNA-binding proteins thought to modulate the assembly and the function of transcriptional complexes. Each of these proteins contains three DNA-binding domains (DBD), properties of which appear to be regulated by phosphorylation. High levels of these proteins are characteristic for rapidly dividing cells in embryonic tissues and tumors. On the basis of their occurrence, specific functions for each of these proteins have been postulated. In this study we demonstrate differences in the nature of contacts of these proteins with promoter region of the interferon-beta gene. We show that HMGI and HMGY interact with this DNA via three DBDs, whereas HMGI-C and Chironomus HMGI bind to this DNA using only two domains. Phosphorylation of HMGY protein by Cdc2 kinase leads to impairing of contacts between the N-terminally located DBD and a single promoter element. The perturbations in the architecture of the protein.DNA complexes involve changes in the degree of unbending of the intrinsically bent IFNbeta promoter. Our results provide first insights into the molecular basis of functional specificity of proteins of the HMGI/Y family and their regulation by phosphorylation.

DNA↗
PubMed2001

Nickel resistance and chromatin condensation in Saccharomyces cerevisiae expressing a maize high mobility group I/Y protein.

Expression of a maize cDNA encoding a high mobility group (HMG) I/Y protein enables growth of transformed yeast on a medium containing toxic nickel concentrations. No difference in the nickel content was measured between yeast cells expressing either the empty vector or the ZmHMG I/Y2 cDNA. The ZmHMG I/Y2 protein contains four AT hook motifs known to be involved in binding to the minor groove of AT-rich DNA regions. HMG I/Y proteins may act as architectural elements modifying chromatin structure. Indeed, a ZmHMG I/Y2-green fluorescent protein fusion protein was observed in yeast nuclei. Nickel toxicity has been suggested to occur through an epigenetic mechanism related to chromatin condensation and DNA methylation, leading to the silencing of neighboring genes. Therefore, the ZmHMG I/Y2 protein could prevent nickel toxicity by interfering with chromatin structure. Yeast cell growth in the presence of nickel and yeast cells expressing the ZmHMG I/Y2 cDNA increased telomeric URA3 gene silencing. Furthermore, ZmHMG I/Y2 restored a wild-type level of nickel sensitivity to the yeast (Delta)rpd3 mutant. Therefore, nickel resistance of yeast cells expressing the ZmHMG I/Y2 cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA.

Amino Acid Sequence↗
PubMed2002

Interactions between p53, hMSH2-hMSH6 and HMG I(Y) on Holliday junctions and bulged bases.

The ability of the tumor suppressor protein, p53, to recognize certain types of DNA lesions may represent one of the mechanisms by which this protein modulates cellular response to DNA damage. p53 DNA binding properties are regulated by several factors, such as post-translational modifications including phosphorylation and acetylation, regulation by its own C-terminal domain and interactions with other cellular proteins. Substrates resembling Holliday junctions and extra base bulges were used to study the effect of three nuclear proteins, HMG-1, HMG I(Y) and hMSH2-hMSH6, on the lesion binding properties of p53. Gel retardation assays revealed that the three proteins had varying effects on p53 binding to these substrates. HMG-1 did not influence p53 binding to Holliday junctions or 3-cytosine bulges. HMG I(Y) rapidly dissociated p53 complexes with Holliday junctions but not 3-cytosine bulges. Finally, the mismatch repair protein complex, hMSH2-hMSH6, enhanced p53 binding to both substrates by 3-4-fold. Together, these results demonstrate that p53 DNA binding activity is highly influenced by the presence of other proteins, some having a dominant effect while others have a negative effect.

Animals↗
PubMed1997

Distinct functional properties of IkappaB alpha and IkappaB beta.

The biological activity of the transcription factor NF-kappaB is controlled mainly by the IkappaB alpha and IkappaB beta proteins, which restrict NF-kappaB to the cytoplasm and inhibit its DNA binding activity. Here, we carried out experiments to determine and compare the mechanisms by which IkappaB alpha and IkappaB beta inhibit NF-kappaB-dependent transcriptional activation. First, we found that in vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Second, we showed that both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. Third, the inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes.

Animals↗