RESEARCH ABSTRACT The genomic DNA in all eukaryotic cells is wrapped around basic proteins known as histones to form nucleoprotein filaments called chromatin. Histones are essential as they not only package the DNA, but also regulate access to the genetic information contained in the DNA. Yeast cells lacking histones are inviable, whereas histone gene repression during S-phase triggers spontaneous DNA damage and cell cycle arrest in human cells. Due to their positive charge, histones can also bind non-specifically to the negatively charged DNA and adversely affect processes that require access to DNA. Not surprisingly, elevated histone protein levels lead to genomic instability in yeast, which is a hallmark of human cancer cells. Hence, a major challenge for the cell is to strictly coordinate histone synthesis with their orderly deposition onto the DNA. Cells have evolved a variety of mechanisms to achieve this very delicate balance between histone and DNA synthesis. Histone synthesis is tightly coupled to DNA replication in proliferating cells. Further, DNA damage during S- phase leads to a rapid decrease in the rate of DNA synthesis that is accompanied by dramatic reduction of histone mRNA levels. However, the molecular mechanisms responsible for this downregulation of histone mRNA levels upon replication inhibition are unknown. We have discovered that the essential DNA damage checkpoint kinase Rad53 (the budding yeast homolog of the human tumor suppressor Chk2) is necessary for histone gene regulation following DNA damage. Furthermore, we find that the multi-subunit ATP-dependent chromatin remodeling complex RSC (Remodel the Structure of Chromatin) is recruited to the histone gene promoters in a Rad53 dependent manner in cells where DNA replication has been inhibited. Our long term goal is to understand the molecular mechanisms by which cells ensure a delicate balance between histone and DNA synthesis. To address this issue, we will dissect the pathway by which Rad53 regulates histone transcript levels following DNA damage and replication arrest. Using the budding yeast as a model system, we plan to focus on the following questions: (1) How is the latent potential of Rad53 to downregulate histone mRNA levels regulated during the normal progression of S-phase? (2) What is the relevance of RSC localization at the histone gene promoters and how is this recruitment regulated? (3.) What are the functional interactions between factors such as Rad53, RSC and the histone regulator Hir complex (which are known to be involved in histone gene regulation), in the downregulation of histone transcript levels upon replication arrest? Histones and chromatin structure regulate all aspects of DNA metabolism and mounting evidence suggests that they play a major role in regulating genomic stability and possibly cancer. As such, a thorough understanding of the regulation of essential chromatin components such as the histones is essential for the understanding the processes that lead to genomic instability and ultimately cancer. Our objective is to understand the molecular mechanisms by which cells ensure a delicate balance between histone synthesis and their incorporation into DNA during replication, and apply this knowledge to get a better understanding of the processes that may be involved in cancer prevention in humans. ? ? ?
|Morillo-Huesca, Macarena; Maya, Douglas; Muñoz-Centeno, Mari Cruz et al. (2010) FACT prevents the accumulation of free histones evicted from transcribed chromatin and a subsequent cell cycle delay in G1. PLoS Genet 6:e1000964|
|Singh, Rakesh Kumar; Kabbaj, Marie-Helene Miquel; Paik, Johanna et al. (2009) Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis. Nat Cell Biol 11:925-33|