Role of HMGA1a and ORC interaction in origin definition and heterochromatin organization
Final Report Abstract
Key player for origin recognition is the origin recognition complex (ORC). This protein complex is not only essential for DNA replication, but also important for the eukaryotic chromosome cycle, i.e. the organization of chromatin through binding to heterochromatin protein 1 (HP1). Currently it is not known how ORC discriminates between its role at origins of DNA replication and its function as chromosome-organization element. Additional components supporting either function have been proposed. We initially observed that the high mobility group protein A (HMGA) also co-localizes with ORC. No role in origin definition or DNA replication has been ascribed to HMGA proteins previously. In a set of biochemical and imaging experiments we showed that HMGA1a can target ORC to DNA supporting the idea that chromatin constituents can target ORC to DNA replication in metazoan cells. We also found that HMGA1 proteins bind to ORC and HP1α in heterochromatin to build HMGA1a/ORC/HP1α heterochromatin. Our data indicate that HMGA1 stabilizes the binding of at least Orc1 and HP1α. Thus, in cells were HMGA proteins are present, heterochromatin may be stabilized and maintained through interplay with ORC and HP1α. In this scenario the HMGA1a/ORC/HP1α get-together may nucleate the assembly of additional regulatory factors. Such additional factors could be HMGN proteins which display a cell-cycle regulated distribution and which we found to interact with HMGA1- and HP1- proteins. Interestingly, we also found that HMGA1 proteins not only modulate chromatin directly through interaction with other chromatin components but also modulate chromatin composition through influencing the expression of other architectural chromatin proteins such as histone H1 and MeCP2. Super resolution imaging suggested that HMGA1 is able to cluster DNA. So far, binding of HMGA1 proteins to their substrates has been considered only in a 1D way, meaning that one HMGA1 molecule binds to one DNA molecule containing multiple appropriately spaced A/T-tracts in close proximity using its three DNA binding AT-hooks. To investigate DNA cross-linking activities, we developed a novel DNA-capture assay. The results demonstrated that HMGA1 proteins indeed are able to cross-link individual DNA fibers. Experiments with mutated HMGA1 proteins show that AT-hooks together with conserved basic amino acids located between AT-hooks II and III are essential to maintain this activity. Electron microscopy studies revealed that HMGA1 proteins are able to create DNA networks, loops and coils in linearized DNA. It is conceivable that the spatial organization of a DNA scaffold through the cross-linking capacity of HMGA1 proteins plays a significant role in these processes. Moreover, besides HMGA1 proteins additional chromatin proteins possess a similar intrinsic HMGA1 like DNA cross-linking activity indicating that DNA-crosslinking might be a generally used mechanism to structurally and functionally arrange DNA in the cell nucleus.
Publications
- (2008) Cell cycle-dependent binding of HMGN proteins to chromatin. Mol Biol Cell.,19 (5):1816-24
Cherukuri S., Hock R., Ueda T., Catez F., Rochman M., Bustin M.
- (2008) Interaction between HMGA1a and the origin recognition complex creates site-specific replication origins. Proc. Natl. Acad. Sci. USA,105
Thomae A.W., Pich D., Brocher J., Spindler M.P., Berens C., Hock R., Hammerschmidt W., Schepers A.
- (2009) The dynamics of HMG protein-chromatin interactions in living cells. Biochem Cell Biol., 87(1):127-137
Gerlitz G., Hock R., Ueda T., Bustin M.
- (2010) Binding and interplay of HMG proteins on chromatin: lessons from live cell imaging. Biochim Biophys Acta., 1799 (1-2):15-27
Catez F. and Hock R.
- (2010) HMGA1 down-regulation is crucial for chromatin composition and a gene expression profile permitting myogenic differentiation. BMC Cell Biol., Aug 11: 64
Brocher J., Vogel B., Hock R.
- (2011). Cross-linking of DNA through HMGA1 suggests as DNA scaffold. Nucleic Acids Res. 39
Vogel, B, Löschberger, A, Sauer, M., Hock, R.
(See online at https://doi.org/10.1093/nar/gkr396)
