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M E Bianchi

Publications and source records attributed to M E Bianchi.

At least 37 records · Page 2Linked to original sources
PubMed1999

The lack of chromosomal protein Hmg1 does not disrupt cell growth but causes lethal hypoglycaemia in newborn mice.

High mobility group 1 (HMG1) protein is an abundant component of all mammalian nuclei, and related proteins exist in all eukaryotes. HMG1 binds linear DNA with moderate affinity and no sequence specificity, but bends the double helix significantly on binding through the minor groove. It binds with high affinity to DNA that is already sharply bent, such as linker DNA at the entry and exit of nucleosomes; thus, it is considered a structural protein of chromatin. HMG1 is also recruited to DNA by interactions with proteins required for basal and regulated transcriptions and V(D)J recombination. Here we generate mice harbouring deleted Hmg1. Hmg1-/- pups are born alive, but die within 24 hours due to hypoglycaemia. Hmg1-deficient mice survive for several days if given glucose parenterally, then waste away with pleiotropic defects (but no alteration in the immune repertoire). Cell lines lacking Hmg1 grow normally, but the activation of gene expression by the glucocorticoid receptor (GR, encoded by the gene Grl1) is impaired. Thus, Hmg1 is not essential for the overall organization of chromatin in the cell nucleus, but is critical for proper transcriptional control by specific transcription factors.

Animals↗
PubMed1999

Domain-domain interactions in high mobility group 1 protein (HMG1).

The high mobility group protein HMG1 is a conserved chromosomal protein with two homologous DNA-binding domains, A and B, and an acidic carboxy-terminal tail, C. The structure of isolated domains A and B has been previously determined by NMR, but the interactions of the different domains within the complete protein were unknown. By means of differential scanning calorimetry and circular dichroism we have investigated the thermal stability of HMG1, of the truncated protein A-B (HMG1 without the acidic tail C) and of the isolated domains A and B. In 3 mm sodium acetate buffer, pH 5, the thermal melting of domains A and B are identical (transition temperature tm = 43 degrees C and 41 degrees C, denaturation enthalpies DeltaH = 46 kcal.mol-1). The thermal melting of protein A-B presents two nearly identical transitions (tm = 40 degrees C and 41 degrees C, DeltaH = 44 kcal.mol-1 and 46 kcal.mol-1, respectively). We conclude that the two domains A and B within protein A-B behave as independent domains. The thermal melting of HMG1 is biphasic. The two transitions have a different value of tm (38 degrees C and 55 degrees C) and corresponding values of DeltaH around 40 kcal.mol-1. We conclude that within HMG1, the acidic tail C is interacting with one of the two domains A and B, however, the two domains A and B do not interact with each other. At 37 degrees C, one of the two domains A and B, within HMG1, is partly unfolded, whereas the other which interacts with the acidic tail C, is fully native. The interaction free energy of the acidic tail C is estimated to be in the range of 2.5 kcal.mol-1 based on simulations of the thermograms of HMG1 as a function of the interaction free energy.

Calorimetry, Differential Scanning↗
PubMed1999

The RAG1 homeodomain recruits HMG1 and HMG2 to facilitate recombination signal sequence binding and to enhance the intrinsic DNA-bending activity of RAG1-RAG2.

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.

Binding Sites↗
PubMed1998

Hmg4, a new member of the Hmg1/2 gene family.

High mobility group (HMG) proteins are abundant components of mammalian nuclei and fall into three families. The members of one such family, HMG1 and HMG2, are ubiquitously expressed and facilitate the formation of nucleoprotein complexes where the DNA is sharply bent. We have identified a mouse cDNA that codes for a novel 200-amino-acid protein of the HMG1/2 family, which we called HMG4. The mouse Hmg4 gene is highly expressed in the embryo; Hmg4 transcripts are barely detectable in adult tissues. The human HMG4 gene, which is extremely similar to its mouse homolog, has been sequenced as part of chromosome X, band q28. HMG4, HMG1, and HMG2 proteins have been highly conserved during vertebrate evolution, suggesting that each has at least some unique property. It is possible that HMG4 is required during development.

Amino Acid Sequence↗
PubMed1998

HMG box proteins bind to four-way DNA junctions in their open conformation.

The HMG box is an 80 amino acid domain found in a variety of eukaryotic chromosomal proteins and transcription factors. Binding to DNA is associated with recognition of structural distortion or manipulation of DNA structure. All the HMG box domains bind to four-way DNA junctions, which must therefore present some feature that is common to the binding targets of this wide variety of proteins. Since the four-way junction can itself adopt a variety of structures depending upon conditions, it is important to determine in which form it exists in complexes with HMG boxes. We find that a single HMG box domain is bound exclusively to the open square form of the junction and that conditions that stabilize the stacked X structure significantly lower affinity for the HMG box. We suggest that the HMG domain binds to one arm of the junction in the minor groove at the point of strand exchange and we present a model for the structure of the complex.

Animals↗
PubMed1998

High mobility group 1 (HMG1) protein in mouse preimplantation embryos.

High mobility group 1 protein (HMG1) has traditionally been considered a structural component of chromatin, possibly similar in function to histone H1. In fact, at the onset of Xenopus and Drosophila development, HMG1 appears to substitute for histone H1: HMG1 is abundant when histone H1 is absent after the midblastula transition histone H1 largely replaces HMG1. We show that in early mouse embryos the expression patterns of HMG1 and histone H1 are not complementary. Instead, HMG1 content increases after zygotic genome activation at the same time as histone H1. HMG1 does not remain associated to mitotic chromosomes either in embryos or somatic cells. These results argue against a shared structural role for HMG1 and histone H1 in mammalian chromatin.

Amino Acid Sequence↗
PubMed1998

High-mobility group chromatin proteins 1 and 2 functionally interact with steroid hormone receptors to enhance their DNA binding in vitro and transcriptional activity in mammalian cells.

We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by several nonsteroid nuclear receptors, including retinoid acid receptor (RAR), retinoic X receptor (RXR), and vitamin D receptor (VDR). As highly purified recombinant full-length proteins, all steroid receptors tested exhibited weak binding affinity for their optimal palindromic hormone response elements (HREs), and the addition of purified HMG-1 or -2 substantially increased their affinity for HREs. Purified RAR, RXR, and VDR also exhibited little to no detectable binding to their cognate direct repeat HREs but, in contrast to results with steroid receptors, the addition of HMG-1 or HMG-2 had no stimulatory effect. Instead, the addition of purified RXR enhanced RAR and VDR DNA binding through a heterodimerization mechanism and HMG-1 or HMG-2 had no further effect on DNA binding by RXR-RAR or RXR-VDR heterodimers. HMG-1 and HMG-2 (HMG-1/-2) themselves do not bind to progesterone response elements, but in the presence of PR they were detected as part of an HMG-PR-DNA ternary complex. HMG-1/-2 can also interact transiently in vitro with PR in the absence of DNA; however, no direct protein interaction was detected with VDR. These results, taken together with the fact that PR can bend its target DNA and that HMG-1/-2 are non-sequence-specific DNA binding proteins that recognize DNA structure, suggest that HMG-1/-2 are recruited to the PR-DNA complex by the combined effect of transient protein interaction and DNA bending. In transient-transfection assays, coexpression of HMG-1 or HMG-2 increased PR-mediated transcription in mammalian cells by as much as 7- to 10-fold without altering the basal promoter activity of target reporter genes. This increase in PR-mediated gene activation by coexpression of HMG-1/-2 was observed in different cell types and with different target promoters, suggesting a generality to the functional interaction between HMG-1/-2 and PR in vivo. Cotransfection of HMG-1 also increased reporter gene activation mediated by other steroid receptors, including glucocorticoid and androgen receptors, but it had a minimal influence on VDR-dependent transcription in vivo. These results support the conclusion that HMG-1/-2 are coregulatory proteins that increase the DNA binding and transcriptional activity of the steroid hormone class of receptors but that do not functionally interact with certain nonsteroid classes of nuclear receptors.

Animals↗
PubMed1997

Mmot1, a new helix-loop-helix transcription factor gene displaying a sharp expression boundary in the embryonic mouse brain.

Several genetic factors have been proven to contribute to the specification of the metencephalic-mesencephalic territory, a process that sets the developmental foundation for prospective morphogenesis of the cerebellum and mesencephalon. However, evidence stemming from genetic and developmental studies performed in man and various model organisms suggests the contribution of many additional factors in determining the fine subdivision and differentiation of these central nervous system regions. In man, the cerebellar ataxias/aplasias represent a large and heterogeneous family of genetic disorders. Here, we describe the identification by differential screening and the characterization of Mmot1, a new gene encoding a DNA-binding protein strikingly similar to the helix-loop-helix factor Ebf/Olf1. Throughout midgestation embryogenesis, Mmot1 is expressed at high levels in the metencephalon, mesencephalon, and sensory neurons of the nasal cavity. In vitro DNA binding data suggest some functional equivalence of Mmot1 and Ebf/Olf1, possibly accounting for the reported lack of olfactory or neural defects in Ebf-/- knockout mutants. The isolation of Mmot1 and of an additional homolog in the mouse genome defines a novel, phylogenetically conserved mammalian family of transcription factor genes of potential relevance in studies of neural development and its aberrations.

Amino Acid Sequence↗
PubMed1997

High mobility group 1 protein is not stably associated with the chromosomes of somatic cells.

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.

3T3 Cells↗
PubMed1997

Recombinant HMG1 protein produced in Pichia pastoris: a nonviral gene delivery agent.

This paper describes the production of a recombinant protein from the expression system based on the methylotrophic yeast Pichia pastoris. Efficient production of rat high-mobility-group 1 (HMG1) protein was obtained using the system. Two forms of HMG1 were secreted into the culture medium: a 24.5-kDa species corresponding to the native HMG1 and a 32-kDa glycosylated derivative. Non-glycosylated recombinant HMG1 was purified easily and shown to possess the same DNA-binding properties as HMG1 purified from calf thymus. Plasmid DNA complexed to the recombinant HMG1 is taken up by a variety of mammalian cells in culture. Transient expression of a luciferase reporter gene was observed. Under selective conditions, stable expression of a neomycin gene was established as a result of integration into the genome. HMG1-mediated gene delivery was as efficient as calcium phosphate-mediated transfection but without associated cell damage. In addition, stable transfectants obtained after selection for G418 resistance usually integrated only one copy of the transfected DNA in contrast to the high unpredictable number obtained by the calcium phosphate method. HMG1 transfection complexes were not toxic to cultured cells, even at high concentrations.

Amino Acid Sequence↗
PubMed1996

HMG1 interacts with HOX proteins and enhances their DNA binding and transcriptional activation.

High mobility group protein 1 (HMG1) is a non-histone, chromatin-associated nuclear protein with a proposed role in the regulation of eukaryotic gene expression. We show that HMG1 interacts with proteins encoded by the HOX gene family by establishing protein-protein contacts between the HMG box domains and the HOX homeodomain. The functional role of these interactions was studied using the transcriptional activity of the human HOXD9 protein as a model. HMG1 enhances, in a dose-dependent fashion, the sequence-specific DNA binding activity in vitro, and the transcriptional activation in a co-transfection assay in vivo, of the HOXD9 protein. Functional interaction between HMG1 and HOXD9 is dependent on the DNA binding activity of the homeodomain, and requires the HOXD9 transcriptional activation domain. HMG1 enhances activation by HOXD9, but not by HOXD8, of the HOXD9-controlled element. Specific target recognition and functional interaction with HMG1 can be transferred to HOXD8 by homeodomain swapping. We propose that HMG1-like proteins might be general co-factors in HOX-mediated transcriptional activation, which facilitate access of HOX proteins to specific DNA targets, and/or introduce architectural constraints in the assembly of HOX-containing transcriptional complexes.

3T3 Cells↗
PubMed1996

The active gene that encodes human high mobility group 1 protein (HMG1) contains introns and maps to chromosome 13.

The human genome contains a large number of sequences related to the cDNA for High Mobility Group 1 protein (HMG1), which so far has hampered the cloning and mapping of the active HMG1 gene. We show that the human HMG1 gene contains introns, while the HMG1-related sequences do not and most likely are retrotransposed pseudogenes. We identified eight YACs from the ICI and CEPH libraries that contain the human HMG1 gene. The HMG1 gene is similar in structure to the previously characterized murine homologue and maps to human chromosome 13 band q12, as determined by in situ hybridization. The mouse Hmg1 gene maps to the telomeric region of murine Chromosome 5, which is syntenic to the human 13q12 band.

Amino Acid Sequence↗
PubMed1996

Differential binding of HMG1, HMG2, and a single HMG box to cisplatin-damaged DNA.

The HMG box domain is a DNA binding domain present in the nonhistone chromosomal proteins HMG1 and HMG2 and in other proteins involved in the regulation of gene expression. Previous studies have demonstrated that HMG1 and HMG2 bind with high affinity to DNA modified with the cancer chemotherapeutic drug cisplatin (CDDP). In this report, we compare the binding of full-length HMG1 and HMG2 and the HMG boxes present in these proteins to that of CDDP-DNA. Complexes between HMG1, HMG2, or HMG Box A + B and CDDP-DNA were stable at > or = 500 mM salt, while complexes between a single HMG box and CDDP-DNA exhibited decreased stability. Analysis of a series of HMG1 Box A mutant constructs revealed different affinities for CDDP-DNA. Two constructs containing a Phe to Ala substitution at position 19 and a Tyr to Gly substitution at position 71, are noteworthy; these peptides exhibited reduced affinity for CDDP-DNA. We have generated a structure of HMG1 Box A and used it, along with the results of our binding studies, to model its interaction with CDDP-DNA. HMG1 Box A binds in the minor groove of CDDP-DNA, in agreement with earlier studies. Our model predicts that Tyr71 partially intercalates and forms an H bond with the sugar-phosphate backbone. The model also suggests that Phe 19 does not directly interact with DNA, and hence an Ala substitution at position 19 may alter protein structure. This model should provide a framework for future studies examining HMG Box-DNA interactions.

Amino Acid Sequence↗
PubMed1995

Interaction of normal and mutant SRY proteins with DNA.

In mammals, sex determination is caused by the Y-chromosome gene SRY. The DNA-binding domain of human SRY protein is similar to those of the chromatin protein HMG1. Like HMG1, SRY binds to kinked DNA structures, and bends linear DNA sharply upon binding. We analysed the biochemical properties of mutant SRY proteins from five patients with complete gonadal dysgenesis: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally, and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence, thus discriminating different target sites. We suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY in chromatin is essential for the expression of genes involved in testis differentiation.

Base Sequence↗
PubMed1995

NMR spectroscopic analysis of the DNA conformation induced by the human testis determining factor SRY.

The conformation of an eight base pair DNA oligonucleotide duplex bound to the human testis determining factor SRY and the orientation of the protein domain within the complex have been analyzed by a variety of NMR methods which permit the selective observation of protons attached to 12C nuclei in the presence of uniformly enriched 13C/15N protein. Qualitative analysis of nuclear and rotating frame Overhauser enhancement spectra at multiple mixing times indicates that the conformation of the SRY-bound DNA is distinct from that of A- and B-DNA, in agreement with the recent three-dimensional structure determination of the complex [Werner, M. W., Huth, J. R., Gronenborn, A. M., & Clore, G. M. (1995) Cell 81, 705-714]. Selective observation of intermolecular NOEs between protein and DNA indicates that partial intercalation of a protein side chain occurs between two adenine bases in the DNA octamer. The analysis of structural features by NMR for this unusual DNA conformer and the orientation of the protein domain on the DNA is discussed. The structural features of the DNA complexed to SRY are remarkably similar, but not identical, to those of DNA complexed to the TATA-binding protein (TBP).

Amino Acid Sequence↗
PubMed1995

Evolutionary conservation in the DNA-binding and -bending properties of HMG-boxes from SRY proteins of primates.

Mammalian sex determination is caused by the Y-chromosome gene SRY, which encodes a protein containing a DNA-binding domain (HMG-box) of about 70 amino acids (aa). The HMG-box is very conserved in a wide variety of mammals; conversely, the flanking non-box regions show a high degree of aa sequence divergence, even between closely related species. The HMG-box of human SRY binds sequence-specifically to linear DNA and produces a sharp bend; it also interacts with high affinity to kinked DNA structures irrespective of their sequences. Point mutations associated with sex reversal in XY human females fall within the HMG-box and either affect the affinity for DNA or modify the geometry of the DNA-protein complex. Here, we show that the DNA-binding and -bending properties of the HMG-boxes of SRY from human and seven different primates are extremely similar to each other. Together with other data, this suggests that the inability of mouse and human SRY to substitute for each other is due to differences in the conserved HMG-box, rather than the non-conserved flanking sequences.

Amino Acid Sequence↗