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PubMed2001

Transcriptional regulation of human insulin receptor gene by the high-mobility group protein HMGI(Y).

We have previously identified two closely related nuclear binding proteins that specifically interact with two unique functional AT-rich sequences of the 5' regulatory region of the human insulin receptor gene. Expression of these nuclear binding proteins increases during myocyte and adipocyte differentiation, and in other tissues appears to correlate with insulin receptor content. We have hypothesized, therefore, that insulin receptor expression in the insulin target tissues is regulated at least in part by these nuclear proteins. Here we show data on purification and biochemical characterization of these DNA binding proteins. Using a conventional chromatographic purification procedure combined with electrophoresis mobility shift assay and immunoblot analyses, a unique approximately 15 kDa protein, either identical to or highly related to the architectural transcription factor HMGI(Y), has now been identified, suggesting an essential role for HMGI(Y) in regulating insulin receptor gene transcription. Direct evidence of HMGI(Y) insulin receptor promoter interactions is provided by functional analysis with the CAT reporter gene and by hormone binding studies in cells expressing HMGI(Y) antisense RNA. In these experiments, antisense HMGI(Y) specifically inhibits insulin receptor promoter function and insulin receptor protein expression, indicating that HMGI(Y) is required for proper transcription of insulin receptor gene. Moreover, our data consistently support the hypothesis that a putative defect in this nuclear binding protein may cause insulin receptor dysfunction with subsequent impairment of insulin signaling and action.

5' Untranslated Regions↗
PubMed2006

Haploinsufficiency of the Hmga1 gene causes cardiac hypertrophy and myelo-lymphoproliferative disorders in mice.

The HMGA1 protein is a major factor in chromatin architecture and gene control. It plays a critical role in neoplastic transformation. In fact, blockage of HMGA1 synthesis prevents rat thyroid cell transformation by murine transforming retroviruses, and an adenovirus carrying the HMGA1 gene in the antisense orientation induces apoptotic cell death in anaplastic human thyroid carcinoma cell lines, but not in normal thyroid cells. Moreover, both in vitro and in vivo studies have established the oncogenic role of the HMGA1 gene. In this study, to define HMGA1 function in vivo, we examined the consequences of disrupting the Hmga1 gene in mice. Both heterozygous and homozygous mice for the Hmga1-null allele show cardiac hypertrophy due to the direct role of HMGA1 on cardiomyocytic cell growth regulation. These mice also developed hematologic malignancies, including B cell lymphoma and myeloid granuloerythroblastic leukemia. The B cell expansion and the increased expression of the RAG1/2 endonuclease, observed in HMGA1-knockout spleen tissues, might be responsible for the high rate of abnormal IgH rearrangements observed in these neoplasias. Therefore, the data reported here indicate the critical role of HMGA1 in heart development and growth, and reveal an unsuspected antioncogenic potential for this gene in hematologic malignancies.

Animals↗
PubMed2000

The high mobility group I/Y protein is hypophosphorylated in endoreduplicating maize endosperm cells and is involved in alleviating histone H1-mediated transcriptional repression.

During maize endosperm development, cells shift from a mitotic cycle to endoreduplication, driving the massive synthesis of storage proteins (zeins) and starch. In this developmental context, we studied changes in expression levels of histone H1 and high mobility group I/Y (HMG-I/Y), two chromatin architectural proteins that are known to affect gene transcription. Almost no change was found in the level of histone H1 during endosperm development, despite a dramatic increase in DNA content (endoreduplication); hence, the histone H1/DNA ratio decreased substantially. Concurrently with a reduction in the Cdc2 kinase activity at the shift to endoreduplication, significant changes were found in the level and mobility of the HMG-I/Y protein; the faster migrating forms were, at least partly, hypophosphorylated. Purified maize HMG-I/Y protein was found to be phophorylated in vitro by the Cdc2 kinase and bound efficiently to the gamma-zein promoter AT-rich tract (gammaZ-AT). Using an in vitro transcription assay, we demonstrated the capability of the maize HMG-I/Y protein to relieve the inhibitory effect exerted by histone H1 on templates containing the gammaZ-AT sequence. These data suggest that during maize endosperm development transcription and perhaps replication are controlled, at least partly, by the activity of the Cdc2 kinase and the interplay between histone H1 and HMG-I/Y proteins.

Base Sequence↗
PubMed1995

The non-histone chromosomal protein HMG-I(Y) contributes to repression of the immunoglobulin heavy chain germ-line epsilon RNA promoter.

The rate of germ-line RNA transcription correlates with the rate of immunoglobulin heavy chain isotype switching. A promoter element for the transcription of RNA from the germ-line mouse immunoglobulin epsilon heavy chain constant region gene is induced by interleukin(IL)-4 and lipopolysaccharide, and is bound at its transcription initiation sites by an IL-4-inducible nuclear protein, NF-BRE. To examine the function of the binding site for this IL-4-inducible complex, substitution mutations were introduced in the promoter. These binding site mutations increased promoter activity and decreased binding of NF-BRE. To investigate the paradox of an IL-4-inducible protein binding to a repressor site in an IL-4-inducible promoter, we determined that the non-histone chromosomal protein HMG-I(Y) binds at the transcription initiation sites of the germ-line epsilon promoter. Assays with antisera against HMG-I(Y) revealed monomeric HMG-I(Y) in nuclear extracts. Cotransfection of an expression construct directing the synthesis of anti-sense HMG-I(Y) RNA also increased promoter activity, consistent with a repressor function of HMG-I(Y). Thus, the data are most consistent with a model in which HMG-I(Y) participates in repression of promoter activity. The effects of IL-4 may include derepression at this site.

Animals↗
PubMed1999

The host-cell architectural protein HMG I(Y) modulates binding of herpes simplex virus type 1 ICP4 to its cognate promoter.

The productive infection cycle of herpes simplex virus is controlled in part by the action of ICP4, an immediate-early gene product that acts as both an activator and repressor of transcription. ICP4 is autoregulatory, and IE-3, the gene that encodes it, contains a high-affinity binding site for the protein at its cap site. Previously, we had demonstrated that this site could be occupied by proteins found in nuclear extracts from uninfected cells. A HeLa cell cDNA expression library was screened with a DNA probe containing the IE-3 gene cap site, and clones expressing the architectural chromatin proteins HMG I and HMG Y were identified by this technique. HMG I is shown to augment binding of ICP4 to its cognate site in in vitro assays and to enhance the activity of this protein in short-term transient expression assays.

Animals↗
PubMed1999

High mobility group-I(Y) protein facilitates nuclear factor-kappaB binding and transactivation of the inducible nitric-oxide synthase promoter/enhancer.

Nitric oxide (NO), a free radical gas whose production is catalyzed by the enzyme NO synthase, participates in the regulation of multiple organ systems. The inducible isoform of NO synthase (iNOS) is transcriptionally up-regulated by inflammatory stimuli; a critical mediator of this process is nuclear factor (NF)-kappaB. Our objective was to determine which regulatory elements other than NF-kappaB binding sites are important for activation of the iNOS promoter/enhancer. We also wanted to identify transcription factors that may be functioning in conjunction with NF-kappaB (subunits p50 and p65) to drive iNOS transcription. Deletion analysis of the iNOS promoter/enhancer revealed that an AT-rich sequence (-61 to -54) downstream of the NF-kappaB site (-85 to -76) in the 5'-flanking sequence was important for iNOS induction by interleukin-1beta and endotoxin in vascular smooth muscle cells. This AT-rich sequence, corresponding to an octamer (Oct) binding site, bound the architectural transcription factor high mobility group (HMG)-I(Y) protein. Electrophoretic mobility shift assays showed that HMG-I(Y) and NF-kappaB subunit p50 bound to the iNOS promoter/enhancer to form a ternary complex. The formation of this complex required HMG-I(Y) binding at the Oct site. The location of an HMG-I(Y) binding site typically overlaps that of a recruited transcription factor. In the iNOS promoter/enhancer, however, HMG-I(Y) formed a complex with p50 while binding downstream of the NF-kappaB site. Furthermore, overexpression of HMG-I(Y) potentiated iNOS promoter/enhancer activity by p50 and p65 in transfection experiments, suggesting that HMG-I(Y) contributes to the transactivation of iNOS by NF-kappaB.

Animals↗
PubMed1993

Organization, inducible-expression and chromosome localization of the human HMG-I(Y) nonhistone protein gene.

Members of the HMG-I(Y) family of mammalian nonhistone proteins are of importance because they have been demonstrated to bind specifically to the minor groove of A.T-rich sequences both in vitro and in vivo and to function as gene transcriptional regulatory proteins in vivo. Here we report the cloning, sequencing, characterization and chromosomal localization of the human HMG-I(Y) gene. The gene has several potential promoter/enhancer regions, a number of different transcription start sites and numerous alternatively spliced exons making it one of the most complex nonhistone chromatin protein-encoding genes so far reported. The putative promoter/enhancer regions each contain a number of conserved nucleotide sequences for potential binding of inducible regulatory transcription factors. Consistent with the presence of these conserved sequences, we found that transcription of the HMG-I(Y) gene is inducible in human lymphoid cells by factors such as phorbol esters and calcium ionophores. Detailed sequence analysis confirms our earlier suggestion that alternative splicing of precursor mRNAs gives rise to the major HMG-I and HMG-Y isoform proteins found in human cells. Furthermore, the gene's exon-intron arrangement fully accounts for all of the previously cloned human HMG-I(Y) cDNAs (1,2). Also of considerable interest is the fact that each of the three different DNA-binding domain peptides present in an individual HMG-I(Y) protein is coded for by sequences present on separate exons thus potentially allowing for exon 'shuffling' of these functional domains during evolution. And, finally, we localized the gene to the short arm of chromosome 6 (6p) in a region that is known to be involved in rearrangements, translocations and other abnormalities correlated with a number of human cancers.

Alternative Splicing↗
PubMed2001

Coordination of a transcriptional switch by HMGI(Y) acetylation.

Dynamic control of interferon-beta (IFN-beta) gene expression requires the regulated assembly and disassembly of the enhanceosome, a higher-order nucleoprotein complex formed in response to virus infection. The enhanceosome activates transcription by recruiting the histone acetyltransferase proteins CREB binding protein (CBP) and p300/CBP-associated factors (PCAF)/GCN5, which, in addition to modifying histones, acetylate HMGI(Y), the architectural component required for enhanceosome assembly. We show that the accurate execution of the IFN-beta transcriptional switch depends on the ordered acetylation of the high-mobility group I protein HMGI(Y) by PCAF/GCN5 and CBP, which acetylate HMGI(Y) at distinct lysine residues on endogenous promoters. Whereas acetylation of HMGI(Y) by CBP at lysine-65 destabilizes the enhanceosome, acetylation of HMGI(Y) by PCAF/GCN5 at lysine-71 potentiates transcription by stabilizing the enhanceosome and preventing acetylation by CBP.

Acetylation↗
PubMed2000

High-mobility-group protein I can modulate binding of transcription factors to the U5 region of the human immunodeficiency virus type 1 proviral promoter.

HMG I/Y appears to be a multifunctional protein that relies on in its ability to interact with DNA in a structure-specific manner and with DNA, binding transcriptional activators via distinct protein-protein interaction surfaces. To investigate the hypothesis that HMG I/Y may have a role in human immunodeficiency virus type 1 (HIV-1) expression, we have analyzed whether HMG I/Y interacts with the 5' long terminal repeat and whether this interaction can modulate transcription factor binding. Using purified recombinant HMG I, we have identified several high-affinity binding sites which overlap important transcription factor binding sites. One of these HMG I binding sites coincides with an important binding site for AP-1 located downstream of the transcriptional start site, in the 5' untranslated region at the boundary of a positioned nucleosome. HMG I binding to this composite site inhibits the binding of recombinant AP-1. Consistent with this observation, using nuclear extracts prepared from Jurkat T cells, we show that HMG I (but not HMG Y) is strongly induced upon phorbol myristate acetate stimulation and this induced HMG I appears to both selectively inhibit the binding of basal DNA-binding proteins and enhance the binding of an inducible AP-1 transcription factor to this AP-1 binding site. We also report the novel finding that a component present in this inducible AP-1 complex is ATF-3. Taken together, these results argue that HMG I may play a fundamental role in HIV-1 expression by determining the nature of transcription factor-promoter interactions.

5' Untranslated Regions↗
PubMed1995

Regulation of cell-type-specific interleukin-2 receptor alpha-chain gene expression: potential role of physical interactions between Elf-1, HMG-I(Y), and NF-kappa B family proteins.

The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is rapidly and potently induced in T cells in response to mitogenic stimuli. Previously, an inducible enhancer between nucleotides -299 and -228 that contains NF-kappa B and CArG motifs was identified. We now report the characterization of a second essential positive regulatory element located between nucleotides -137 and -64 that binds Elf-1 and HMG-I(Y). This element had maximal activity in lymphoid cells, paralleling the cell type specificity of Elf-1 expression. Transcription from the IL-2R alpha promoter was inhibited when either the Elf-1 or the HMG-I(Y) binding site was mutated. Coexpression of both proteins activated transcription of the -137 to -64 element in COS-7 cells. Elf-1 physically associated with HMG-I and with NF-kappa B p50 and c-Rel in vitro, suggesting that protein-protein interactions might functionally coordinate the actions of the upstream and downstream positive regulatory elements. This is the first report of a physical interaction between an Ets family member and NF-kappa B family proteins. These findings provide significant new insights into the protein-protein and protein-DNA interactions that regulate cell-type-specific and inducible IL-2R alpha gene expression and also have implications for other genes regulated by Elf-1 and NF-kappa B family proteins.

Animals↗
PubMed2000

HMG-I/Y, a new c-Myc target gene and potential oncogene.

The HMG-I/Y gene encodes the HMG-I and HMG-Y proteins, which function as architectural chromatin binding proteins important in the transcriptional regulation of several genes. Although increased expression of the HMG-I/Y proteins is associated with cellular proliferation, neoplastic transformation, and several human cancers, the role of these proteins in the pathogenesis of malignancy remains unclear. To better understand the role of these proteins in cell growth and transformation, we have been studying the regulation and function of HMG-I/Y. The HMG-I/Y promoter was cloned, sequenced, and subjected to mutagenesis analysis. A c-Myc-Max consensus DNA binding site was identified as an element important in the serum stimulation of HMG-I/Y. The oncoprotein c-Myc and its protein partner Max bind to this site in vitro and activate transcription in transfection experiments. HMG-I/Y expression is stimulated by c-Myc in a Myc-estradiol receptor cell line in the presence of the protein synthesis inhibitor cycloheximide, indicating that HMG-I/Y is a direct c-Myc target gene. HMG-I/Y induction is decreased in Myc-deficient fibroblasts. HMG-I/Y protein expression is also increased in Burkitt's lymphoma cell lines, which are known to have increased c-Myc protein. Like Myc, increased expression of HMG-I protein leads to the neoplastic transformation of both Rat 1a fibroblasts and CB33 cells. In addition, Rat 1a cells overexpressing HMG-I protein form tumors in nude mice. Decreasing HMG-I/Y proteins using an antisense construct abrogates transformation in Burkitt's lymphoma cells. These findings indicate that HMG-I/Y is a c-Myc target gene involved in neoplastic transformation and a member of a new class of potential oncogenes.

Animals↗
PubMed2002

Dominant-negative HMGA1 blocks mu enhancer activation through a novel mechanism.

The immunoglobulin mu intronic enhancer is a potent B cell-specific transcriptional activator. The enhancer is activated by the appropriate combination of transcription factors, amongst which are ets and bHLH proteins. HMGA1 (formerly HMG-I(Y)) is a demonstrated co-activator of the mu enhancer. HMGA1 functions through direct interaction with PU.1, one of the ets proteins critical for enhancer activation. New data demonstrates dominant negative HMGA1 dramatically decreases enhancer activity in B cells. EMSA analysis demonstrated that DN HMGA1 disrupts established PU.1/mu enhancer binding. Similarly, DN HMGA1 blocks mu enhancer binding by Ets-1. In sharp contrast, DN HMGA1 had no effect on binding activity of the ETS DNA binding domains of either PU.1 or Ets-1, or the bHLH-zip protein TFE3, suggesting specificity. Taken together, the data suggest that DN HMGA1 utilizes a novel mechanism to specifically block interaction between ets proteins and mu enhancer DNA, suggesting DN HMGA1 represents a new, highly specific means of regulating mu enhancer activity.

Animals↗
PubMed2001

HMG-1 enhances HMG-I/Y binding to an A/T-rich enhancer element from the pea plastocyanin gene.

High-mobility-group proteins HMG-1 and HMG-I/Y bind at overlapping sites within the A/T-rich enhancer element of the pea plastocyanin gene. Competition binding experiments revealed that HMG-1 enhanced the binding of HMG-I/Y to a 31-bp region (P31) of the enhancer. Circularization assays showed that HMG-1, but not HMG-I/Y, was able to bend a linear 100-bp DNA containing P31 so that the ends could be ligated. HMG-1, but not HMG-I/Y, showed preferential binding to the circular 100-bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG-1 was not responsible for enhanced binding of HMG-I/Y. Direct interaction of HMG-I/Y and HMG-1 in the absence of DNA was demonstrated by binding of 35S-labeled proteins to immobilized histidine-tagged proteins, and this was due to an interaction of the N-terminal HMG-box-containing region of HMG-1 and the C-terminal AT-hook region of HMG-I/Y. Kinetic analysis using the IAsys biosensor revealed that HMG-1 had an affinity for immobilized HMG-I/Y (Kd = 28 nM) similar to that for immobilized P31 DNA. HMG-1-enhanced binding of HMG-I/Y to the enhancer element appears to be mediated by the formation of an HMG-1-HMG-I/Y complex, which binds to DNA with the rapid loss of HMG-1.

Carrier Proteins↗
PubMed1999

Nuclear factor of activated T cells contributes to the function of the CD28 response region of the granulocyte macrophage-colony stimulating factor promoter.

The granulocyte macrophage colony stimulating factor (GM-CSF) promoter contains a 10 bp element known as CK-1 or CD28RE that specifically responds to the co-stimulatory signal delivered to T cells via the CD28 surface receptor. This element is a variant NFkappaB site that does not function alone but requires an adjacent promoter region that includes a classical NFkappaB element, an Sp-1 site and a putative activator protein-1 (AP-1)-like binding site. The entire region is referred to as the CD28 response region (CD28RR). The GM-CSF CK-1 element has been shown to bind NFkappaB proteins, in particular c-Rel, whose binding and function is dependent on the architectural transcription factor HMGI(Y). It has been previously suggested that the nuclear factor of activated T cells (NFAT) family of proteins also plays a role in the activity of this region. We show here that recombinant NFATp but not AP-1 can bind to the GM-CSF CD28RR. NFATp present in activated Jurkat T cell extracts can also interact with the CD28RR. The binding of NFATp and Rel proteins requires the same core CK-1 sequences, and appears to be mutually exclusive. We investigated the functional significance of NFATp binding to CK-1 by overexpressing the protein in Jurkat T cells and found that NFATp cannot activate the CD28RR alone but can cooperate with signals generated by phorbol 12-myristate 13-acetate/calcium ionophore. The CD28RR is therefore a complex region that can bind and respond to a combination of transcription factors and signals.

Binding Sites↗
PubMed1994

A novel AT-rich DNA binding protein that combines an HMG I-like DNA binding domain with a putative transcription domain.

There is growing evidence that AT-rich promoter elements play a role in transcription of plant genes. For the promoter of the nuclear gene for chloroplast glutamine synthetase from pea (GS2), the deletion of a 33-bp AT-rich sequence (box 1 native) from the 5' end of a GS2 promoter-beta-glucuronidase (GUS) fusion resulted in a 10-fold reduction in GUS activity. The box 1 native element was used in gel shift analysis and two distinct complexes were detected. One complex is related to the low-mobility complex reported previously for AT-rich elements from several other plant promoters. A multimer of the box 1 sequence was used to isolate a cDNA encoding an AT-rich DNA binding protein (ATBP-1). ATBP-1 is not a high-mobility group protein, but it is a novel protein that combines a high-mobility group I/Y-like DNA binding domain with a glutamine-rich putative transcriptional domain.

Amino Acid Sequence↗
PubMed1998

Modulation of activity of Moloney murine leukemia virus preintegration complexes by host factors in vitro.

We have explored the requirements for host proteins in the integration of Moloney murine leukemia virus (MoMuLV) cDNA in vitro. Following infection, it is possible to lyse cells and obtain preintegration complexes (PICs) capable of integrating the MoMuLV cDNA into an added target DNA in vitro (intermolecular integration). PICs can be stripped of required proteins by gel filtration in high-salt buffers (600 mM KCI), allowing the nature of the removed factors to be investigated by in vitro reconstitution. In a previous study of human immunodeficiency virus type 1 (HIV-1) PICs, the host protein HMG I(Y) was found to be able to restore activity to salt-stripped PICs. In contrast, salt stripping and reconstitution of MoMuLV PICs led to the proposal that a host factor is important for a different activity, blocking integration into the cDNA itself (autointegration). In this report, we investigated reconstitution of salt-stripped MoMuLV PICs and found that addition of cellular extract from uninfected NIH 3T3 cells could block autointegration and also restore intermolecular integration. Isolation of the intermolecular integration-complementing activity yielded HMG I(Y), as in the HIV-1 case. However, HMG I(Y) could not block autointegration, implicating a different host factor in this process. Additionally, when MoMuLV PICs were partially purified but not salt stripped, the intermolecular integration activity was reduced but could be stimulated by the addition of any of several purified DNA binding proteins. In summary, three activities were detected: (i) the intermolecular integration cofactor HMG I(Y), (ii) an autointegration barrier protein, and (iii) stimulatory DNA binding proteins.

3T3 Cells↗
PubMed2003

The barrier-to-autointegration factor is a component of functional human immunodeficiency virus type 1 preintegration complexes.

Retroviral integration in vivo is mediated by preintegration complexes (PICs) derived from infectious virions. In addition to the integrase enzyme and cDNA substrate, PICs contain a variety of viral and host cell proteins. Whereas two different cell proteins, high-mobility group protein A1 (HMGA1) and the barrier-to-autointegration factor (BAF), were identified as integration cofactors based on activities in in vitro PIC assays, only HMGA1 was previously identified as a PIC component. By using antibodies against known viral and cellular PIC components, we demonstrate here functional coimmunoprecipitation of endogenous BAF protein with human immunodeficiency virus type 1 (HIV-1) PICs. Since integrase protein and integration activity were also coimmunoprecipitated by anti-BAF antibodies, we conclude that BAF is a component of HIV-1 PICs. These data are consistent with the model that BAF functions as an integration cofactor in vivo.

Animals↗