Developmental Biology Section
The Developmental Biology Section investigates the transcriptional regulation of cell fate determination during metazoan development. Using the C. elegans system, we exploit forward and reverse genetic approaches to identify and characterize transcription factor function required for proper development of specific cell types, at single-cell resolution. Historically, our interest has primarily been directed at understanding muscle cell specification and differentiation as a model for both embryonic and postembryonic development. Our goal is to fully describe the transcriptional cascade that orchestrates the formation of this tissue from just after fertilization, throughout embryogenesis, and into adulthood.
Genetic Mechanisms Section
The Genetic Mechanisms Section investigates the mechanics of cellular processes that impact the genomic structure and the heritance of the genomic material. We study mechanisms of reactions that impact the stability of the linear organization of the genome, as well as the 3-dimensional dynamics involved in the heritance of bacterial chromosomes.
The Mechanism of DNA Repair, Replication, and Recombination Section is interested in studying DNA recombination, repair, and replication. In particular, we are interested in V(D)J recombination, mismatch repair, nucleotide excision repair, and translesion DNA synthesis. We use X-ray crystallography, molecular biology, and various biochemical and biophysical approaches to find out the molecular mechanisms in these biological processes.
Molecular Genetics Section
The Molecular Genetics Section studies the rearrangement of immunoglobulin and T-cell receptor genes (known as V(D)J recombination). This process is essential for the development of lymphoid cells and is unique in sharing some properties with site-specific recombination and with the repair of radiation damage to DNA. Our aim is to understand V(D)J recombination in detail and to apply this knowledge to the immune system. Our research has shown that recombination begins with site-specific DNA breaks, which can be made by the isolated RAG1 and RAG2 proteins, and that a DNA hairpin is produced on one side of each break. This reaction shares many properties with mobile genetic elements (transposons), and we are interested in the potential role of transposition in causing chromosomal translocations of the types found in leukemias and lymphomas. Researchers in this section are also investigating the separate ubiquitin ligase activity of RAG1 and its covalent modification by auto-ubiquitylation.
Molecular Virology Section
The Molecular Virology Section focuses on mechanistic aspects of retroviral DNA integration. After entering the host cell, a DNA copy of the viral genome is made by reverse transcription. Integration of this viral DNA into a chromosome of the host cell is an essential step in the retroviral replication cycle. The key player in the retroviral DNA integration process is the virally encoded integrase protein. Integrase processes the ends of the viral DNA and covalently inserts these processed ends into host DNA. We study the molecular mechanism of these reactions using biochemical, biophysical, and structural techniques. Researchers in this section collaborate closely with NIDDK colleagues who use X-ray crystallography and NMR. Our work also investigates cellular proteins that play important accessory roles in the integration process. Of particular interest is the mechanism that prevents integrase using the viral DNA as a target for integration. Such autointegration would result in destruction of the viral DNA. We have identified a cellular protein, which we called barrier-to-autointegration factor (BAF) that prevents integration of the viral DNA into itself. Our studies suggest that compaction of the viral DNA by BAF makes it inaccessible as a target for integration.
Physical Chemistry Section
The Physical Chemistry Section studies the structure and function of several types of proteins. Specific projects investigate the relationship between chromatin structure and gene expression in eukaryotes. Researchers focus on the epigenetic mechanisms and the structure of both the individual nucleosomes (the fundamental chromatin subunits) and the folded polynucleosome fiber. Recent investigations on the role of histone variants in regulation of chromatin structure and gene expression suggest that unstable nucleosome core particles (NCPs) play a role in making active promoters more accessible for binding by regulatory factors. Other work examines long-range chromatin organization and the boundaries between independently regulated domains, which play a role in regulation of gene expression. We have focused on the properties of insulator elements that help to establish such boundaries. Our research has identified proteins that bind to the insulator sites, as well as the co-factors that those proteins recruit, and the results suggest how the enhancer blocking or barrier activity arises. Present work is examining these mechanisms in detail and includes biochemical and functional analysis of the complexes. Other studies examine the effects of protein knockdown on the local and long-range chromatin structure and histone modification patterns. In particular, we are studying long-range interactions within the nucleus in human pancreatic beta cells and between the insulin gene and other genes that may be co-regulated. This is part of a larger effort to study chromatin structure of the insulin locus and its relationship to insulin gene expression and secretion.
Protein Stability and Quality Control Section
The Protein Stability and Quality Control Section (1) performs research using in vitro reconstitution and cell-based assays to elucidate the cellular mechanisms underlying protein quality control at the endoplasmic reticulum (ER); (2) develops reagents and tools to disrupt ER protein homeostasis and evaluates their activities in anti-cancer therapy; (3) studies the catalysis mechanisms for various enzymes in the ubiquitin proteasome system; (4) extends our studies to address fundamental questions in other protein quality control systems; and (5) supports the career development of research trainees in scientific or biomedical pursuits.
The Structural Biochemistry Section studies the molecular mechanisms underlying protein activity modulation. To function properly, cells must coordinate and choreograph a large number of simultaneous events and processes. Proteins carry out these essential processes. We primarily use X-ray crystallography to study how cells regulate the activity and function of protein-protein and protein-DNA complexes. X-ray crystallography produces high-resolution "snapshots" to visualize subtle changes in protein structure that often accompany functional regulation. With these snapshots in hand, we use a variety of biochemical, biophysical and simulation approaches to relate their structures and biological functions. Specific projects investigate how proteins control the movement of mobile genetic elements, such as transposons or viruses. One of our current areas of emphasis is the Rep protein of adeno-associated virus (AAV). This protein catalyzes the integration of the AAV genome into a specific locus in human chromosome 19, making it an extremely useful tool for gene therapy studies. In addition, we are studying how a ubiquitous group of chaperone proteins known as 14-3-3s are able to direct when and where in a cell to deliver proteins that regulate gene expression.
Susan K. Buchanan, Ph.D.
The Structural Biology of Membrane Biology Section focuses on the structure determination of integral membrane proteins by x-ray crystallography and functional analysis of these proteins using biophysical, biochemical, and cell biological techniques. We study transporters embedded in the outer membranes of Gram-negative bacteria, which are surface accessible and therefore have the potential to be good vaccine and/or drug targets against infectious diseases. We also study the membrane-associated, or soluble protein, partners that interact with outer membrane transporters to better understand how these systems function in vivo. Current topics in the lab include (1) small molecule and protein import across the bacterial outer membrane, (2) protein secretion by pathogenic bacteria, and (3) protein import across mitochondrial outer membranes. Our lab currently studies several distinct proteins. Some are common to many different kinds of bacteria and are required for their survival, while others are uniquely involved in the development of E. coli or Yersinia pestis (the bacteria that causes the plague) infections. Recently, we have also started to study mammalian proteins, which may play a role in the progression of neurodegenerative states like Alzheimer’s and Parkinson’s diseases.