Role of parp-1 enzyme in the control of quiescence [Tesi di dottorato]

Poly(ADP-ribose)polymerase-1 (PARP-1) catalyses the polymerization of ADP-ribose units from donor NAD+ molecules on target proteins, resulting in the attachment of linear or branched polymers. The negative charges of poly(ADP-ribose) change the target protein affinity for DNA. PARP-1 exerts numerous functions in cellular physiology, from maintenance of DNA stability and integrity to transcriptional regulation and cell cycle control but its role in the G0-G1 transition is not yet completely defined. The exit from quiescence is a highly regulated and conserved process started by extra cellular stimuli. These stimuli, for instance serum stimulation, trigger a signal cascade, including MAPK activation, that culminates in the transcriptional induction of Immediate Early Genes (IEGs). Our group has recently reported that PARP-1 activity promotes cell cycle re-entry through the induction of a set of IEGs, such as c-myc, c-fos, junB and Egr-1. On the basis of these previous finding we studied the mechanism by which PARP-1 modulates IEGs in fibroblast cells. We highlighted that PARP-1 affects the IEG expression at transcription level. Then analyses of chromatin status of c-myc promoter evidenced that this region is more condensed in absence of poly(ADP-ribosyl)ation upon mitogen stimulation of resting fibroblasts. Further, ChIP experiments showed a complex dynamics of PARP-1 binding and chromatin poly(ADP-ribosyl)ation at the same region during G0-G1 transition. Indeed PARP-1 is associated with silent c-myc promoter during quiescence but, following mitogen stimulation, activated PARP-1 is displaced from it in concomitance with chromatin poly(ADP-ribosyl)ation. These PARP-1 activities are associated with the switch of transcription factor occupancies on the c-myc promoter. Moreover the dynamics of PARP-1 binding at the promoter suggested a possible implication of the enzyme in the repression of c-myc gene during G0 instauration. According with this hypothesis we found that overexpression of PARP-1 accelerates c-myc shut off during G0 entry. Since many early events induced during cell-cycle re-entry are shared by different cell lineages in several physiological condition, it was investigate whether PARP activity plays a role in other cell systems undergoing G0–G1 transition. The attention was focused on skeletal myoblasts made quiescent by suspension culture. This muscle cell system mimics the function of muscle satellite reserve cells and can be activated by restoring cell adhesion to substrate. We found that in the myoblast context, the inhibition of PARP-1 activity delays the induction of proliferation and interfers with both the upregulation of IEGs and the expression of the myogenic factor MyoD that normally occurs following reserve cell activation. This kind of analysis may open new ways of investigation in the study of cell cycle exit control that characterize stem cell differentiation or quiescence.

diritti: info:eu-repo/semantics/openAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/11573/918702

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD18047614