Dynamics and interactions of an oncogenic homeotic protein within human replicative complexes

The regulation of human DNA replication operates via the time-programmed activation and deactivation of approximately 30,000 replication origins distributed along the genome. A multi-protein replicative complex recognizes and assembles onto each origin; this determines the local unwinding of the origin DNA and the start of two oppositely moving replicative forks. The mechanism that governs the selection of a specific DNA sequence as human (and, more generally, metazoan) origin, in the course of G1 phase of the cell-cycle, is still poorly understood. The lack of DNA-sequence consensus among well-characterized replication origins, together with the little bindingspecificity displayed by the Origin Recognition Complex, suggest that origin selection might rather be determined by local chromatin structures and/or accessory targeting proteins. With regard to the latter possibility, it was interesting to find out that three homeotic proteins, namely HOXC13, HOXC10, and HOXA13 display a specific affinity for a DNA fragment corresponding to the sequence covered by the Replicative Complex of the human Lamin B2 replication origin. In the study conducted during this Ph.D. program, the possible role of homeotic proteins in origin function was explored by investigating the involvement of a selected homeotic protein, namely HOXC13, within the replicative complexes in living human cells. To this purpose, recent advances in biophysical microscopy technologies were exploited to study in vivo the localization, dynamics, and interactions of HOXC13 protein in the context of DNA replication regulation. The data reported in this thesis demonstrate that HOXC13 indeed participates in origin function. The protein is a stable component of early replicating chromatin, as it displays stable chromatin binding in correspondence to the nuclear areas where replication foci of early S phase are collected. This peculiar behavior is driven by the homeodomain and relies mainly on the conserved homeodomain arginine-5 anchoring to the DNA minor groove. Furthermore, HOXC13 displays unambiguous affinity for origin sequences and for selected replicative-complex proteins. The close proximity of HOXC13 to both Cdc6 and ORC2 proteins measured in living cells proves that the homeotic protein is involved in direct protein-protein interactions within the replicative-complex; not unexpectedly, such interactions are modulated in a cell-cycle dependent fashion that is consistent with origin function. These observations are not restricted to a single origin, but rather appear to have a general significance in the nuclear architecture of DNA replication; nor are they restricted to a single homeotic protein, as the HOXC13 exerts its function via highly conserved homeodomain residues. Hence, this dissertation argues that the homeoproteins functionally contribute in a general manner, dependent on their chromatin-binding properties, to the specification of origins, likely the early replicating ones. In this view, HOX proteins, probably in the context of a multi-protein homeotic effector, contribute to recruit and stabilize the replicative complexes onto early replicating origins, in presence of specific chromatin and topological configurations. Considering that HOXC13, involved in development and differentiation, is also an oncoprotein, the data presented in this thesis, besides offering an indication for the basis of origin selection, hint at the homeotic proteins as actors in the cross-talk between development and DNA replication regulation.