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The architectural factors HMGA are highly connected hubs in the chromatin network and affect key cellular functions. HMGA have a causal involvement in cancer development; in fact, truncated or chimeric HMGA forms, resulting from chromosomal rearrangements, lack the constitutively phosphorylated acidic C-terminal tail and display increased oncogenic potential, suggesting a functional role for this domain. HMGA belong to the intrinsically disordered protein category, and this prevents the use of classical approaches to obtain structural data. Therefore, we combined limited proteolysis, ion mobility separation-mass spectrometry (IMS-MS), and electrospray ionization-mass spectrometry (ESI-MS) to obtain structural information regarding full length and C-terminal truncated HMGA forms. Limited proteolysis indicates that HMGA acidic tail shields the inner portions of the protein. IMS-MS and ESI-MS show that HMGA proteins can assume a compact form and that the degree of compactness is dependent upon the presence of the acidic tail and its constitutive phosphorylations. Moreover, we demonstrate that C-terminal truncated forms and wild type proteins are post-translationally modified in a different manner. Therefore, we propose that the acidic tail and its phosphorylation could affect HMGA post-translational modification status and likely their activity. Finally, the mass spectrometry-based approach adopted here proves to be a valuable new tool to obtain structural data regarding intrinsically disordered proteins.

The high mobility group A (HMGA) chromatin architectural transcription factors are a group of proteins involved in development and neoplastic transformation. They take part in an articulated interaction network, both with DNA and other nuclear proteins, organizing multimolecular complexes at chromatin level. Here, we report the development of a novel in vitro strategy for the identification of HMGA molecular partners based on the combination of an RP-HPLC prefractionation procedure, 2-DE gels, blot-overlay and MS. To demonstrate that our approach could be a reliable screening method we confirmed a representative number of interactions in vitro by GST pull-down and far-Western and in vivo by co-affinity purification. This approach allowed us to enlarge the HMGA molecular network confirming their involvement also in non-transcriptional-related processes such as RNA processing and DNA repair.

The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg(57) and Arg(59), two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization.

DNA-based activities rely on an extremely coordinated sequence of events performed by several chromatin-associated proteins which act in concert. High Mobility Group A (HMGA) proteins are non-histone architectural nuclear factors that participate in the regulation of specific genes but they are also believed to have a more general role in chromatin dynamics. The peculiarity of these proteins is their flexibility, both in terms of DNA-binding and in protein-protein interactions. Since these proteins act as core elements in the assembly of multiprotein complexes called enhanceosomes, and have already displayed the ability to interact with several different proteins, we started a proteomic approach for the systematic identification of their molecular partners. By a combination of affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry we have identified about twenty putative HMGA interactors which could be roughly assigned to three different classes: mRNA processing proteins, chromatin remodelling related factors and structural proteins. Direct HMGA interaction with some of these proteins was confirmed by glutathione-S-transferase pull-down assays and the HMGA domain involved was mapped. Blot-overlay experiments reveal that members of the HMGA family share most of their molecular partners but, interestingly, it seems that there are some cell-type specific partners. Taken together, these experimental data indicate that HMGA proteins are highly connected nodes in the chromatin protein network. Since these proteins are strongly implicated with cancer development, the identification of molecules able to perturb the HMGA molecular network could be a possible tool to interfere with their oncogenic activity.

The structural characteristics of the three nuclear phosphoproteins of the high mobility group A family are outlined and related to their participation in chromatin structure alteration in many biological processes such as gene expression, neoplastic transformation, differentiation, and apoptosis. The elevated expression of these proteins in tumor cells and their post-translational modifications, such as phosphorylation, acetylation and methylation, are discussed and suggested as suitable targets for cancer chemotherapy.

Digestion of sea urchin sperm nuclei with micrococcal nuclease yields nucleosomal monomer fragments of 151 and 164 base pairs. Prior trypsin treatment of the sperm chromatin does not alter the size of these monomer DNA fragments despite the fact that the H1 histone is reduced to a limit globular peptide of about 83 residues. Heterologous reconstitution experiments show that this peptide is capable of protecting an extra 22 base pairs beyond the core particle in a chromatosome. Nuclease digestion of reconstitutes from DNA and sperm core histones yields a core monomer of about 141 base pairs. It is concluded that this sperm chromatin contains a chromatosome of 164 bp essentially similar to that observed in the more usual chromatins. Edman degradation of the H1 limit peptide shows its sequence to be closely analogous to the corresponding peptide of calf H1 and chicken H5. Circular dichroism studies of histone H1 from the sperm of three sea urchin species demonstrate the presence of trypsin-sensitive helical regions outside the globular domain that are absent in calf H1 and chicken H5.

Nuclei isolated from the liver of rats undergoing an acute inflammatory reaction induced by turpentine treatment show increased RNA synthesis. This increase is essentially determined by a faster polyribonucleotide-elongation rate while the number of transcribing polymerase molecules is unchanged. The sensitivity of chromatin to micrococcal-nuclease digestion and the composition of chromosomal proteins are not affected by the acute-phase process. Therefore the increased RNA synthesis by liver nuclei from acutely inflamed rats does not seem to correlate with major changes in chromatin structure.

The nuclear magnetic resonance (NMR) spectrum of chromatin at ionic strengths below about 0.5 M may be attributed solely to its histone H1 component. The effect of various ions and urea on the complex has been investigated using NMR and confirm that the contraction of the complex on increase of ionic strength is largely due to electrostatic interactions. A detailed study of the H1 - DNA complex has also been undertaken. The behaviour of H1 in the two cases is virtually identical, implying that in chromatin the H1 is complexed with the DNA rather than with the other histones. Microcalorimetric measurements reveal that the binding of H1 to DNA is athermic or involves a heat of reaction which is very small indeed.

1. Histones H1 and H5 in chromatin and in free solution can be cross-linked to higher multimers. Is this due to a specific protein/protein interaction? If so, this interaction might be the structural basis of the condensation of the chromosomal nucleofilament, known to be mediated by histones H1 and H5. 2. Since only the central domain of H1 and H5 exhibits tertiary folding and globular structure, this is the most likely site of specific interaction. 3. Formaldehyde has been used to test whether the central domains of histone H1 from calf thymus or from sea urchin sperm or histone H5 from chicken erythrocytes self-interact. 4. The cross-linking shown by each globular peptide was compared with that of its parent histone. 5. In all three cases the peptide cross-linked to a much lower extent than its intact parent histone and the observed cross-linked rates were roughly in proportion to the relative number of lysine residues parent histone and peptide. 6. It is concluded that there is no specific self-interaction between the globular domains of either H1 or H5 molecules in free solution. 7. This result suggests that specific H1/H1 protein/protein interactions are not the basic cause of chromatin condensation.