Pemberton, Lucy F.
Associate Professor, Microbiology, Immunology, and Cancer Biology
- PhD, Imperial Cancer Research Fund, London, UK
PO Box 800577
MSB (Hospital West), Rm 7201
Nuclear Transport in Chromatin Assembly and Transcriptional Regulation
Our lab is interested in how the assembly and disassembly of chromatin regulates gene expression, replication and DNA repair. Correct regulation is fundamental to cellular processes such as cell division, differentiation and development, and misregulation can lead to genomic instability and cancer.
Background. Chromatin consists of histone proteins and DNA, assembled into individual repeating units called nucleosomes. New nucleosomes must be assembled every cell division following replication, and nucleosomes must be rapidly disassembled and assembled to allow the transcription and DNA repair machineries access to the DNA. The control of nucleosome architecture and chromatin structure is a key mechanism in the regulation of gene expression that is shared among all eukaryotes. There are many proteins and protein complexes that can modify and assemble chromatin: Histone modifiers such as HATs, HDACs, and HMTs, the ATP-dependent chromatin remodelers, such as SWI/SNF and RSC, and the histone chaperones. We are interested in the role that histone chaperones and chromatin assembly proteins play in this process and how they interact with other chromatin modifiers.
Function of Nucleosome Assembly Protein 1 family of proteins.
Nap1 is a nucleo-cytoplasmic shuttling protein and is part of an evolutionarily conserved superfamily of proteins. Vps75 is structurally similar to Nap1. The best-characterized function of Vps75 is as an interaction partner and import factor for the HAT, Rtt109. Vps75 is required for Rtt109 mediated acetylation of H3. Human cells have five Nap1 proteins, the SET protein (similar to Vps75) and the TSPY and TSPYL protein families. Different members of this superfamily have been shown to be upregulated or mutated in various cancers such as leukemias and gonadoblastoma. These proteins likely have a developmental role, as in mice loss of a neuronal-specific member of the family is embryonic lethal, and mutant embryos show over proliferation defects in neuronal tissues.
We work in the model system S. cerevisiae. An understanding of the function of Nap1 and Vps75 in this simple model system will ultimately allow us to understand the role this superfamily plays in human disease.
Focus of the Lab. Histones are synthesized in the cytoplasm and imported into the nucleus. Our lab focuses on early and late events of chromatin assembly and the role of histone chaperones.
We are interested in questions such as how and when are histones synthesized in the cytoplasm and how do histone chaperones cooperate with transport factors in the nuclear transport process?
What is the sequence of events once these proteins are in the nucleus? How do the histone chaperones coordinate the assembly of histones onto DNA?
What is the function of histone chaperones in chromatin dynamics, and what is their role in transcription elongation? Is this function regulated by the coordinated activities of histone modifiers?
Are histone chaperones direct players in the nucleosome eviction and reassembly cycle around the elongating polymerase, are ATP dependent chromatin remodelers assisting in this function?
Histone Chaperones and Transcription Nap1 can assemble chromatin in vitro and is recruited to sites of transcription. Nap1 genetically interacts with many factors involved in transcription. We propose that Nap1 is recruited to open reading frames to disassemble and reassemble chromatin-promoting passage of RNA polymerase II. We have shown Nap1 and Vps75 regulate both transcription and acetylation. We are currently determining how these histone chaperones interact with the transcription and acetylation machinery, and whether they modulate chromatin structure through interactions with ATP dependent chromatin remodelers.
Nuclear import of histones and histone chaperones. Nuclear transport is intimately involved in the response of cells to external signals and is important in the development of cancer, and infection by viruses such as HIV. Nuclear import and export occurs through the nuclear pore complex (NPC), a large structure embedded in the nuclear membrane, and is mediated by an evolutionarily conserved group of transport factors called karyopherins or importins. We have identified the specific karyopherins and determined the nuclear localization sequences (NLS) for each of the core histones. The NLSs within each histone are post-transcriptionally modified by acetylation, methylation and phosphorylation. We propose these modifications play a role in nuclear transport. We are characterizing how histone modifications also regulate the interactions of histones with histone chaperones and so regulate the histone trafficking and chromatin assembly.
Using proteomics to identify interacting proteins and phosphorylation sites. We have shown that Nap1 is a phosphorylated, nucleocytoplasmic shuttling protein. We have an ongoing collaboration with the lab of Don Hunt in the UVA Chemistry Dept. Using Mass Spectrometry we have carried out proteomic screens to search for interacting proteins of the histones, chromatin assembly factors and nuclear import factors. The proteins identified will help us understand how the above proteins function. We have also used Mass Spectrometry analysis to identify the phosphorylation sites on different proteins such as Nap1, and acetylation sites on histones, which will help elucidate the mechanism of their regulation.
Techniques used. The lab is located in the Center for Cell Signaling and is part of the following three BIMS programs; the Microbiology, Infectious Disease and Immunology program, the Biochemistry, Molecular Biology and Genetics program, as well as the Molecular Cell and Developmental Biology program. Our investigation into the above research areas is ongoing. The techniques that are routinely employed in the laboratory are a combination of:
- cell biology including fluorescence microscopy
- biochemistry including protein production, in vitro binding analysis and the purification and analysis of protein complexes from yeast cells
- molecular biology approaches including chromatin immunoprecipitation and real-time PCR, microarrays and chromatin assembly analysis
- genetics involving the generation and use of yeast mutants as well as various genome-wide screens
In summary, the correct assembly and remodeling of chromatin is necessary for the maintenance of genomic stability in eukaryotic cells, highlighting the importance of understanding this process.