Discovering high mobility group A molecular partners in tumour cells

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.     

Cross-linking of DNA through HMGA1 suggests a DNA scaffold

Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.     

HMGA1a is involved in specific splice site regulation of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) utilizes a highly complex splice site regulation system, taking advantage of host proteins, to express its own viral protein in an orderly way. We show here that one of the host proteins, high mobility group A protein 1a (HMGA1a), is involved in splice site regulation of 3′ splice site 2 (A2) and 5′splice site 3 (D3) of HIV-1 genomic RNA. shRNA knockdown of HMGA1 in HeLa cells resulting in a decrease of HMGA1 showed a significant decrease of Vpr mRNA. RNA electophoretic mobility shift assays showed HMGA1a specifically binds to a sequence adjacently upstream D3. In vitro splicing using heterologous pre-mRNA with A2 and D3, showed HMGA1a induced a splicing intermediate which decreased when an RNA decoy of the HMGA1a binding site was added. RT-PCR of in vitro splicing products revealed that HMGA1a induced an incomplete splicing product resulting from usage of A2 but inhibition of D3, which is reminiscent of the splicing pattern necessary for Vpr mRNA formation. HMGA1a interacted with hnRNPA1 shown by coimmunoprecipitation and supershifted U1 snRNP in an RNA electophoretic mobility shift assay. We conclude that HMGA1a anchors U1 snRNP to inhibit D3 function, and that HMGA1a inhibits hnRNPA1 function on exon splicing silencer of Vpr (ESSV) to activate A2 function. We show here for the first time that HMGA1a is involved in specific splice site regulation of HIV-1.     

High-level expression of DNA architectural factor HMGA2 and its association with nucleosomes in human embryonic stem cells

The state of chromatin in human embryonic stem (hES) cells is a key factor determining stem cell identity. The non-histone chromatin-associated factor HMGA2 has been studied mostly in the mouse where its function seems critical for embryonic cell growth and adipocytic cell differentiation. Here we show that HMGA2 is highly expressed in two undifferentiated human embryonic stem cell lines at a level of at least 10(5) copies per individual stem cell. Interestingly, expression is further upregulated by a factor of three at day 7 of embryoid body formation, before it quickly drops to or below the level found in undifferentiated cells. We also show that HMGA2 is stably associated with inter- and metaphase hES cell chromatin, and that up to 12 HMGA2 protomers stably associate in vitro with a single nucleosome core particle of known atomic structure. Our data lend support to the possibility that HMGA2 interacts with nucleosomes in a way that imposes a global effect on the state of ES cell chromatin, which may contribute to the establishment of both ES cell identity and the initiation of specific differentiation programs.