Summary of research activity
Our general areas of interest are gene regulation, temporal control of DNA replication initiation, genome stability and telomere biology. At present we have projects aimed at understanding: i) how promoter nucleosome architecture is established and maintained genome-wide; ii) how transcription factors and nucleosomes interact to bring about regulated expression of genes that drive cell growth, particularly ribosomal protein genes; iii) how the telomeric protein Rif1 regulates the DNA damage response and the temporal program of DNA replication. We use the budding yeast Saccharomyces cerevisiae as our experimental system because it allows us to employ a powerful combination of genetic, molecular, biochemical and cell biological approaches to address specific mechanistic questions.
Promoter nucleosome architecture and gene regulation
The basic unit of chromatin structure, the nucleosome, is generally recognized to inhibit transcription initiation. The precise location of nucleosomes at promoter regions, where RNA polymerase must bind to initiate transcription, is thus of critical importance in understanding gene regulation. We discovered that the promoters of a large set of protein-coding genes, most of which are highly transcribed and implicated in cell growth, contain one or two dynamic ("fragile") nucleosomes, characterized by an unusual sensitivity to micrococcal nuclease digestion. Our analysis of these fragile nucleosomes uncovered two short sequence motifs whose number and distribution are strongly correlated with promoter nucleosome positions at all genes. We show that these motifs drive the action of the essential nucleosome remodeler RSC to generate a nucleosome-depleted region upstream of nearly all promoters. At highly transcribed genes one of a small set of "general regulatory factors" destabilizes the "fragile" nucleosome immediately upstream of the transcription start site. Ongoing studies are addressed at understanding the structure and dynamic properties of "fragile" nucleosomes and their relationship to transcriptional regulation.
The ribosome biogenesis transcriptional network
Ribosome biogenesis is an energy-intensive process that drives cell growth. Underlying this point is the remarkable fact that in rapidly growing yeast cells about 50% of all RNAPII initiation events occurs on a ribosomal protein (RP) gene. Consequently, ribosome biogenesis is tightly regulated by nutrient and stress signals. Current studies focus on how promoter DNA sequence determines transcription factor and nucleosome assembly at the large suite of RP gene promoters, and how the resulting promoter architecture determines transcription initiation rates at these genes. We also study a novel mechanism of regulatory crosstalk that couples RNAP II transcription of RP genes with RNAP I transcription of ribosomal RNA genes to assure a balanced production of ribosome components.
Rif1 and the control of DNA replication timing and genome stability
Telomeres, the extremities of eukaryotic chromosomes, carry out two essential functions: chromosome end protection ('capping') and end replication. The telomere capping function plays an important role in promoting genome stability, yet the mechanisms by which cells hide telomeres from DNA damage checkpoint and recombination machineries are still unclear. Current projects focus on the yeast telomere-binding protein Rif1. Though highly conserved throughout evolution, in most eukaryotes Rif1 is involved in control of the temporal program of DNA replication initiation and in mechanisms of DNA repair choice. Rif1 also controls replication timing in yeasts, and our studies demonstrated that this function is mediated through recruitment of the PP1 phosphatase (Glc7). Ongoing work is aimed at understanding how Rif1/Glc7 is directed to a specific set of replication origins and uncovering its molecular targets there. We also collaborate with the laboratory of Dr. Nicolas Thomä, which uses x-ray crystallography to study Rif1 structure and function.
Key recent publications
Nucleosome Stability Distinguishes Two Different Promoter Types at All Protein-Coding Genes in Yeast.
Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.
Rif1 controls DNA replication timing in yeast through the PP1 phosphatase Glc7.