Study of the chromatin binding properties of the high mobility group 1 protein (HMGB1) in living and dead cells

Scaffidi, Paola (2002). Study of the chromatin binding properties of the high mobility group 1 protein (HMGB1) in living and dead cells. PhD thesis The Open University.



HMGBl (High mobility group 1 protein) is both a nuclear factor and a secreted protein. In the cell nucleus it acts as an architectural chromatin-binding factor that bends DNA and promotes protein assembly on specific DNA targets. Outside the cell, it binds with high affinity to RAGE (receptor for advanced glycation endproducts), and is a potent mediator of inflammation.
I used a GFP-fused HMGBl in combination with photobleaching techniques to characterize the binding of the protein to chromatin. I found that the protein has differential chromatin binding properties in living, apoptotic, and necrotic cells. In living cells, HMGBl exchanges rapidly between the soluble and the chromatin-bound states. In necrotic cells, HMGBl is not associated with chromatin and is leaked into the extracellular medium. In apoptotic cells, HMGBl is retained in the nucleus because it binds stably to chromatin, in a process that can be inhibited by trichostatin A and appears to be a consequence of histone H4 deacetylation.
The differential binding of HMGBl to chromatin in necrotic and apoptotic cells is relevant with reference to the role of HMGBl in promoting inflammation: as necrosis is always accompanied by inflammation in contrast to apoptosis, we made the hypothesis that HMGBl, released by necrotic cells but retained by apoptotic ones, could be used by proinflammatory cells as a signal to distinguish necrosis from apoptosis. Indeed, necrotic cells lacking HMGBl are rather ineffective in activating proinflammatory cells, and injections of anti-HMGBl antibodies in wt mice reduce inflammatory responses induced by liver necrosis, indicating that HMGBl is necessary to promote inflammatory responses to necrosis. Moreover, if HMGBl is detached from apoptotic chromatin by TSA treatment, also apoptotic cells become proinflammatory.
Thus, HMGBl acts as a signal of unprogrammed cell death, and the retention of HMGBl by apoptotic cells appears to be a safeguard mechanism to prevent unwanted inflammatory reactions in response to programmed cell death.

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