Immunology/B Cell Development

Malone Research Laboratory: William Mulvoy

CCWGG methylation in developmentally regulated B cell-specific gene silencing.

This project is aimed at defining the function of a newly discovered epigenetic DNA marker involving methylation at symmetric CCWGG (i.e., CmCWGG, W = A/T) sites, in regulating B cell gene expression in normal development. Gene silencing involves chromatin and DNA modifications that impose a higher order of repression that supercedes trans-acting transcription factor function. Silenced genes are well known to have repressive closed chromatin structure with dense CpG methylation and extensive histone deacetylation. However, the mechanisms and modifications in DNA and chromatin structure that lead to gene silencing are just now being resolved. This application on developmental B cell gene control is aimed at determining the role of DNA methylation at CCWGG sites in turning off established patterns of gene expression during key transitions in normal B cell development and during the initiation of the chromatin modifications involved in gene silencing. These studies are based on our recent finding of CCWGG methylation in silenced B29 genes in primary effusion lymphoma and in certain myeloma cells. This study also showed that CCWGG methylation significantly repressed transcription and resulted in the displacement of essential transcription factors (e.g., EBF) for B29 promoter activity.  
 
The SPECIFIC AIMS of these studies are: 
 
1) To analyze the promoters of selected B cell genes that are turned off in normal lymphocyte development for CCWGG methylation, CpG methylation, and histone deacetylation.
 
2) To establish the timing and relationship between CCWGG methylation, CpG methylation, and histone deacetylation in B cell gene silencing.
 

Studies under these aims will test the proposition that CCWGG methylation has two related regulatory functions. First, it represses transcription and represents a new mechanism for turning off gene expression in normal and abnormal development.  Second, it marks genes for subsequent chromatin modifications (histone deacetylation and CpG methylation) that culminate in gene silencing and lock genes in the extinguished state. Studies to test these important predictions are the first priorities of this research. These studies will include analyses of CCWGG methylation, CpG methylation, and chromatin changes in B cell genes (λ5, V-pre-B, mb-1, TCL1) that are turned off at key transitions in development. We will use 4 different promoters in a unique cell system to determine the kinetics of CCWGG, CpG, and histone deacetylase (HDAC) activity in gene silencing. We predict that CCWGG methylation represents a new mechanism for turning off expressed genes and gene programs that is used in all mammalian cells.  

Grant Support:

NIH R15 AREA, "CCWGG methylation in developmentally regulated B cell-specific gene silencing" (2007)