HMGA1 proteins are architectural chromatin proteins known to be preferentially expressed in proliferating embryonic tissues but absent in differentiated cells [4, 19]. HMGA1 proteins have been previously implicated in the differentiation of several cell types. For example HMGA1 affects lympho-hematopoietic differentiation of mouse embryonic stem cells  and the differentiation of sperm cells . HMGA1 proteins bind to adipocyte-specific promoters and down-regulation has been shown to impair adipocytic differentiation of 3T3-L1 cells . Here we demonstrate that HMGA1 down-regulation is one of the first and essential steps to allow myogenic differentiation of C2C12 cells. In contrast, sustained expression of HMGA1a-eGFP after induction prevents myogenic differentiation. Mechanistically, the inhibition of C2C12 myogenesis is caused by a specific down-regulation of the myogenic key transcription factors MyoD and myogenin and several additional factors that are required to progress myogenesis.
Several mechanisms have been described on how HMGA proteins participate in specific gene expression, for example the formation of enhanceosomes , binding to specific promoter regions to remove inhibitory factors and to recruit chromatin remodeling complexes or to interact with other transcription factors (e.g. Smad1/4) [8, 15, 23, 24].
The genes that are specifically targeted by HMGA1a during C2C12 myogenesis remain to be examined. Certainly, the down-regulation of specific myogenic genes through HMGA1a is indirect and may represent downstream effects in myogenic gene activation cascades. For example, the decreased myogenin expression is likely caused by the down-regulation of MyoD as well as Mef2a. The latter was recently shown to be necessary for efficient expression of myogenin through the binding to its promoter . MyoD in turn might be repressed through up-regulation of its suppressor Msx1. Even though elevated Msx1 expression in C2A1a cells was just prominent until three days after induction, this initial up-regulation might be sufficient to aggravate the effects of inhibition on the myogenic program. In contrast, it is also conceivable that the differential expression of these genes observed in C2A1a cells is regulated by HMGA1 independently of each other, while affecting the differentiation program in a synergistic manner. Due to this possibility, the promoters of MyoD, myogenin as well as Msx1 are good potential candidates for being direct HMGA1a targets. Other direct candidate genes are those of the Igf-pathway which we found to be suppressed through sustained HMGA1a expression (e.g. Igf1, Igf2). Several previous reports discussed that Igf-signaling is involved in sugar metabolism  and myogenic differentiation [27–29] and Igf1 depletion impairs functional muscle development in mice [30–32]. Supporting that, Igf1 induces myogenin expression followed by cell cycle arrest and myogenic differentiation . Depletion of Igf2 in C2 cells inhibits MyoD expression and abolishes the ability of the cells to express myogenin and myosin genes . Thus, the observed deregulation of the Igf-signaling through HMGA1a over-expression may cause and/or amplify the lack of key myogenic transcription factors and is in good correlation to our observed inhibition of myogenesis.
Despite specific effects on gene promoters, sustained HMGA1a expression may also affect gene regulation through a more global regulation of chromatin architecture. For example, it has been shown that HMGA1 binds to A/T-rich scaffold attachment regions (SARs) which are thought to organize larger chromatin domains . Previous reports showed that HMGA proteins are preferentially associated with heterochromatin [6, 35]. This is supported by the preferential localization of HMGA1a in chromocenters of C2C12 cells.
HMG proteins, histone H1 and many other chromatin proteins are members of a large network of chromatin binding factors that dynamically modulate chromatin architecture through interaction and competition . The function of this network also depends on the availability of HMGA1-interactors and competitors such as histone H1 . HMGA1 proteins were found to induce transcription of previously suppressed plasmid templates by displacement of histone H1 from SAR elements . In support, it was shown that HMG proteins in general compete for chromatin binding with histone H1 in living cells . The significantly decreased levels of histone H1 in HMGA1a over-expressing C2C12 cells demonstrate a shift in the regulatory equilibrium of those two chromatin proteins, favoring HMGA1 binding to previously H1-suppressed sites. This could lead to the modulation of the structure and activity of large chromatin loops and thus affect myogenic gene expression.
The massive down-regulation of histone H1 was surprising. This raises the question how the cells could tolerate this. However, besides histone H1 additional chromatin proteins such as HMGB1, HMGN1 and MeCP2 were also misregulated. This indicates that the entire chromatin composition is altered and that the loss of histone H1 may be compensated by other chromatin proteins like HMGB1  or other differentiation specific histone H1 variants which are not detected by the H1 antibodies used. Within this context it is important that the over-expression of HMGA1a-eGFP prevented chromocenter remodeling and thus global chromatin reorganization normally accompanying differentiation. Interestingly, remodeling of chromocenters was completely recovered after knock-down of HMGA1a in C2A1a cells which was visual through regained chromocenter clustering during the restored terminal differentiation. Notably, the protein MeCP2, which stabilizes chromocenter organization in differentiated cells, was up-regulated in C2A1a cells. MeCP2 dynamically interplays with HP1 proteins, and it was suggested that this interaction in turn stabilizes chromatin organization . Consistently, premature MeCP2 expression in HMGA1a over-expressing C2A1a cells could therefore increase and stabilize the HP1 concentration on chromatin which in turn could stabilize a chromatin structure that prevents expression of genes relevant for myogenic differentiation.