Expression and functional characterization of Xhmg-at-hook genes in Xenopus laevis.

High Mobility Group A proteins (HMGA1 and HMGA2) are architectural nuclear factors involved in development, cell differentiation, and cancer formation and progression. Here we report the cloning, developmental expression and functional analysis of a new multi-AT-hook factor in Xenopus laevis (XHMG-AT-hook) that exists in three different isoforms. Xhmg-at-hook1 and 3 isoforms, but not isoform 2, are expressed throughout the entire development of Xenopus, both in the maternal and zygotic phase. Localized transcripts are present in the animal pole in the early maternal phase; during the zygotic phase, mRNA can be detected in the developing central nervous system (CNS), including the eye, and in the neural crest. We show evidence that XHMG-AT-hook proteins differ from typical HMGA proteins in terms of their properties in DNA binding and in protein/protein interaction. Finally, we provide evidence that they are involved in early CNS development and in neural crest differentiation.

HMGA molecular network: From transcriptional regulation to chromatin remodeling.

Nuclear functions rely on the activity of a plethora of factors which mostly work in highly coordinated molecular networks. The HMGA proteins are chromatin architectural factors which constitute critical hubs in these networks. HMGA are referred to as oncofetal proteins since they are highly expressed and play essential functions both during embryonic development and neoplastic transformation. A particular feature of HMGA is their intrinsically disordered status, which confers on them an unusual plasticity in contacting molecular partners. Indeed these proteins are able to bind to DNA at the level of AT-rich DNA stretches and to interact with several nuclear factors. In the post-genomic era, and with the advent of proteomic tools for the identification of protein-protein interactions, the number of HMGA molecular partners has increased rapidly. This has led to the extension of our knowledge of the functional involvement of HMGA from the transcriptional regulation field to RNA processing, DNA repair, and chromatin remodeling and dynamics. This review focuses mainly on the protein-protein interaction network of HMGA and its functional outcome. HMGA molecular partners have been functionally classified and all the information collected in a freely available database (http://www.bbcm.units.it/ approximately manfiol/INDEX.HTM).

Macroscopic differences in HMGA oncoproteins post-translational modifications: C-terminal phosphorylation of HMGA2 affects its DNA binding properties.

HMGA is a family of nuclear proteins involved in a huge number of functions at the chromatin level. It consists of three members, HMGA1a, HMGA1b, and HMGA2, having high sequence homology and sharing the same structural organization (three highly conserved DNA-binding domains, an acidic C-terminal tail, and a protein-protein interaction domain). They are considered important nodes in the chromatin context, establishing a complex network of interactions with both promoter/enhancer sequences and nuclear factors. They are involved in a plethora of biological processes and their activities are finely tuned by several different post-translational modifications. We have performed an LC/MS screening on several different cell lines to investigate HMGA proteins expression and their post-translational modifications in order to detect distinctive modification patterns for each. Our analyses evidenced relevant macroscopic differences in the phosphorylation and methylation patterns of these proteins. These differences occur both within the HMGA family members and in the different cell types. Focusing on HMGA2, we have mapped its in vivo phosphorylation sites demonstrating that, similarly to the HMGA1 proteins, it is highly phosphorylated on the acidic C-terminal tail and that these modifications affect its DNA binding properties.

Interaction proteomics of the HMGA chromatin architectural factors.

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.