Localization and dynamics of homeotic oncogenic protein HOXC13 in pre-initiation complex of human DNA replication origins

In metazoan cells the DNA replication origins are not well defined. Differently from what observed for bacteria cells and for budding yeast, in metazoan the origins does not show a conserved sequence and they appear to be specified by many factors. In order to better understand the mechanisms involved in the origin specification, many studies have been done to identify the proteins involved in the recognition and activation of the origins. From these kind of analysis is emerging that, beside the wellknown proteins of the pre replicative complex, also other factors might be involved. Between these, the HOX proteins seem to be able to play a role in the origin activity. One of the first studies of this involvement was done by our group and leads to the identification of three homeotic proteins able to specifically bind in vitro the human lamin B2 origin. Thus, in the study conducted during this PhD program, was investigated the involvement of one of these homeotic proteins, namely HOXC13, with human DNA replication origins and with replicative complexes. We found an interaction of HOXC13 with two crucial factors of the pre Replication Complex (pre-RC), ORC1 and Cdc6 and that HOXC13 binds a good fraction of the origins, in particular the early replicating ones, like the lamin B2 origin and other known human origins. The HOXC13 protein is bound to origin chromatin, at least for the lamin B2 origin, at a precise site within the pre-RC at specific moments of the cell cycle. Interaction with the origin occurs within the area protected by the pre-RC in G1, very close to the start sites of leading strand synthesis and to the binding sites of ORC1, ORC2, Cdc6, topoisomerase (topo) I and topo II. The protein is absent from the origin in M and appears on it at the beginning of G1, reach a peak at G1/S and as synthesis starts, the interaction of HOXC13 with the origin fades, in parallel with the transition from this large pre-RC to a smaller and differently organized post-RC. Recently also other HOX proteins have been identify as proteins involved in regulation processes of DNA replication, suggesting that the interaction of HOXC13 with the origins might occur in a multi-homeotic proteins complex. Depletion of one of these proteins however is compatible with the continuation of the cell cycle and, according with what observed for the other homeotic proteins, we found that also the depletion of HOXC13 does not alter cell cycle progression or S phase entry. This is probably due to the redundancy of homeotic proteins and indicates a relatively generic function for the HOX proteins. Among the identified elements influencing the choice and the activity of a sequence as DNA replication origin, much relevance is assumed by the chromatin structure and topology of DNA. Therefore, we analysed the effects of chromatin structure disruption using Tricostatin A, a histone deacetylase inhibitor. The alteration of chromatin caused by this treatment not only sharply reduces origin function, but also disturbs the binding of replication complex members like HOXC13 and the well known Cdc6 to the DNA replication origins, while does not affect the binding of other unrelated proteins like USF1. On the basis of this finding, we infer that an appropriate chromatin organization and DNA topology strongly influence the binding between factors of the pre Replication Complex and DNA replication origins. This influence could be a key element in origin specification. The described interactions are not restricted to a single origin nor to a single homeotic protein, leading us to conclude that HOX proteins, probably in the context of a multi-protein homeotic effectors, contribute to recruit and stabilize the replicative complexes onto early replicating origins, in presence of specific chromatin and topological configurations. The relevance of HOXC13 in DNA replication is also underlined by its involvement in oncogenesis, clearly demonstrated in acute myeloid leukaemia when HOXC13 is fused with NUP98 protein.