Nuclear Organization and Cell Proliferation Control

Research in my laboratory is directed at understanding the events coordinating nuclear structure. More specifically, I study how DNA replication is organized in the nucleus. I have identified and am characterizing novel sites where primary mammalian cells initiate DNA synthesis. These 5-20 perinucleolar foci contain replication proteins and support DNA synthesis through early S-phase. Later, proteins in the foci disperse and replication occurs at hundreds of sites distributed throughout the nucleus. Patterns of DNA synthesis during S-phase progression are not static as previously thought; rather, they are regulated in interesting ways. For instance, immortalized cells do not undergo perinucleolar replication. Instead they initiate DNA synthesis in a pattern distributed throughout the nucleus. This finding suggests that mutations occurring during the immortalization process disrupt normal patterns of DNA synthesis.

I seek to identify the molecular events that dictate where replication occurs with particular emphasis on the early S-phase replication foci. To accomplish this goal, my laboratory will take a multi-directional approach. This includes identifying proteins that localize to perinucleolar foci, using genetic approaches to identify the proteins that temporally regulate the organization and dispersal of the foci, and using molecular and biochemical approaches to understand the mechanisms by which these proteins function.

Already I have identified a number of interesting proteins that target early S-phase perinucleolar foci. In addition to replication proteins, I have found that members of the retinoblastoma (pRB) protein family and associated proteins localize to the perinucleolar foci. The retinoblastoma protein was the first tumor suppressor identified and its function is altered in most if not all human tumors. pRB can perform many functions, one of which is to repress transcription of genes important for S-phase entry through association with the transcription factor E2F. It remains unclear which function(s) of pRB is most important for its role as a tumor suppressor and another goal of my laboratory is to further understand how this protein acts. Interestingly, I have found that pRB regulates the dispersal of perinucleolar replication foci in mid S-phase. A major goal is to understand the mechanism by which pRB performs this function and its significance for cell cycle regulation and tumor progression.

The human nucleus contains approximately 10,000 proteins and 108 base pairs of DNA. These molecules and chromosomes have to be precisely arranged to coordinate the many events that occur in the nucleus. Yet very little is known about how this organizational process occurs. My laboratory seeks to understand the nuclear organization of specific events such as DNA synthesis and tumor suppressor function.