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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.

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.

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.

The primary structure of the Lewis lung carcinoma protein HMGY belonging to the nuclear group of proteins HMGI (high mobility group I) was determined using electrospray and fast atom bombardment mass spectrometry. It was demonstrated that the sequence of the tumor protein corresponds to the amino acid sequence derived from the cDNA from cultured cells and that the N-terminal serine residue is N-acetylated. Moreover, the two high performance liquid chromatography-purified forms Y1 and Y2 of the protein HMGY were shown to differ at the level of serine phosphorylation, since they contain three phosphate and two phosphate groups, respectively, in the C-terminal region. No other modification was detected in the remaining part of the molecule.