Environmental Factor, January 2009, National Institute of Environmental Health Sciences
Nuclear Compartmentalization Affects Gene Activity
By Robin Arnette
According to Harinder Singh, Ph.D., the NIEHS Distinguished Lecturer for June, the mammalian immune system protects the body from a plethora of microscopic invaders, and is also an excellent model for examining fundamental questions in molecular, cell and developmental biology. Over the years Singh has focused on how transcription factors that bind specific DNA sequences regulate the development of innate and adaptive cells of the immune system. On June 9, with Laboratory of Molecular Carcinogenesis Principal Investigator Paul Wade, Ph.D., lecture host, Singh presented "Gene Regulatory Networks Orchestrating Innate and Adaptive Immune Cell Fates."
Singh divided his talk into two parts, with the first half dealing with the molecular mechanisms that underlie cell fate determination. He devoted the second portion of his seminar to a recent topic that was of interest to many in the audience - the issue of nuclear structure and the compartmentalization of the genome. "In other words," he explained, "is the positioning of genes within the three-dimensional structure of the nucleus important in terms of all of the different DNA transactions, such as transcription, recombination and replication, which have to occur?"
Singh entered this area of research after studying B and T cells, the adaptive cells of the immune system, and their ability to generate large repertoires of antigen receptors through somatic DNA recombination. The protein that performs the recombination events for both cell types is called the V(D)J recombinase, but Singh wondered why the recombinase didn't inappropriately recombine T cell receptor segments in developing B cells and vice versa. He said much of the data on the subject suggested that developmentally-regulated localized changes in chromatin structure controlled the enzyme's accessibility to its substrates. He continued, "However, there may be additional processes, such as nuclear compartmentalization, that are used to restrict access of the recombinase to one set of receptor loci in a given lineage and not the other."
Singh's group followed the positioning of these antigen receptor loci during lymphocyte development and revealed that immunoglobin genes have a default position in which both alleles are initially positioned at the nuclear lamina just underneath the inner nuclear membrane in multipotential progenitor cells. During early B cell development, these alleles move away from this compartment and undergo transcriptional activation and DNA recombination. In the T cell lineage, immunoglobulin alleles remain associated with the nuclear lamina and do not undergo recombination.
To add to the mystery, other researchers in the field had reported that genes located at the lamina or the inner nuclear membrane were inactive, while those positioned away from the compartment were active. "We therefore decided to take an active gene that resides in the nucleoplasm and attach it to the inner nuclear membrane," Singh added. "We could then examine the consequences of relocalization on its activity."
Singh engineered murine fibroblast cells to carry the hygromycin gene with a herpes virus promoter and lac operators positioned downstream. He expressed the fusion protein emerin-GFP-lacI, which was targeted to the inner nuclear membrane through emerin, an inner nuclear membrane protein. IPTG withdrawal allowed the lacI domain to bind to the operators in the hygromycin gene. The experiment worked. The hygromycin gene and several nearby chromosomal genes were repositioned into the membrane compartment and underwent repression.
Singh pointed out that this kind of radical reconfiguration of individual chromosomes occurred after the cell went through one mitotic cycle, since this was the time when the chromosomes condensed and the nuclear envelope and lamina broke down. "That's when we think you get an opportunity to decide whether a gene in a given chromosome will be attached or not to this compartment in the new nuclei that are formed," he concluded.