Lucy F. Pemberton
Associate Professor of Microbiology,
Immunology, and Cancer Biology
The Role of Nuclear Transport
in Chromatin Assembly and Transcriptional Regulation
Our lab is interested in how 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 ultimately 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. There are many proteins and protein complexes that can modify and assemble chromatin. We are interested in the role that histone chaperones and chromatin assembly proteins play in this process
Focus of the Lab. Histones are synthesized in the cytoplasm and imported into the nucleus. Our lab focuses on the early events of chromatin assembly, including how nuclear import helps coordinate the assembly of chromatin.
- We are interested in questions such as how and when are histones synthesized in the cytoplasm and imported into the nucleus? Which transport factors are important in this process?
- 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? How does this regulate replication and transcription?
- Lastly, what role do histone chaperones and the nuclear transport machinery play in the removal of histones from chromatin? Is this a mechanism for the insertion of histone variants, what is the impact on gene regulation?
Nuclear import of histones. 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 NLS within each histone overlaps with the amino termini, which are known to be post-transcriptionally modified by acetylation, methylation and phosphorylation. These modifications represent a reversible mechanism by which the karyopherin-NLS interactions are regulated, and suggests epigenetics play a role in nuclear transport.
Function of the Nucleosome Assembly Protein 1. We have shown that the histone chaperone Nucleosome Assembly Protein 1 (Nap1p) plays an important role in nuclear import of histones. Nap1p is a nucleo-cytoplasmic shuttling protein that can act as a cofactor for the import of H2A and H2B, as well as the histone variant H2A.Z. Nap1p is part of an evolutionarily conserved superfamily of proteins. Human cells have 4 Nap1 proteins, the SET protein 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 overproliferation defects in neuronal tissues. We work in the model system S. cerevisiae where there are 2 members, Nap1p, and Vps75p, a homologue of the SET protein. An understanding of the function of Nap1p and Vps75p in this simple model system will ultimately allow us to understand the role this superfamily plays in human disease.
Nap1p is recruited to sites of transcription. We have determined that Nap1p is recruited to chromatin by at least two mechanisms. In one pathway, recruitment of Nap1p is dependent on the mRNA export factor and transcription-export (TREX) component, Yra1p. We predict that Nap1p is recruited to open reading frames to disassemble and reassemble chromatin promoting passage of RNA polymerase II. We are currently analyzing the role of Nap1p at the promoter of specific genes and determining the precise mechanism of recruitment. Vps75p is also a subject of investigation as we determine how this protein functions in the nuclear import of histones and in chromatin assembly.
Using proteomics to identify interacting proteins and phosphorylation sites. We also 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 Nap1p, which will help elucidate the mechanism of their regulation.
Nap1p in mitosis. Prior studies have shown that during the cell cycle, Nap1p also has an important role in regulating entry into mitosis, and loss of Nap1p leads to characteristic morphological defects. We are currently determining whether the cell cycle and chromatin related function of Nap1p are differently regulated by phosphorylation. This analysis is being aided by use of the Amnis Image Stream in the UVA Flow Cytometry Core. This cutting edge machine simultaneously captures images of cells and measures their fluorescence intensity as they pass through the flow cell. This is one of the first reported uses of this technology to simultaneously analyze the yeast cell cycle and morphological defects. It will be important to fully understand the role that Nap1p plays in the cell cycle, as well as chromatin assembly, as this information will help us understand the how Nap1 superfamily members are involved in human cancers.
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 immunoprecipitations 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.