The fetal antibody repertoire is vastly overrepresented with V genes from the DH-proximal VH gene families, especially the most 3' functional member of the VH7183 family, 81X. In contrast, the IgH rearrangements in the adult bone marrow are skewed much more to the DH-distal VHJ558 genes. The induction of accessibility for rearrangement of the VHJ558 genes in the adult has been shown to be due (3 IL-7 signaling, but the factor(s) which control the induction of histone acetylation and accessibility of the DH-proximal genes in either fetal or adult pro-B cells is not known. We hypothesize that thymic stromal lymphopoietin (TSLP) and/or Flt3 ligand may induce histone acetylation of the DH proxima1 gene families such as VH7183 which are used so predominantly in fetal life. Predominance of the influence of these cytokines, and less so of IL-7, in fetal life therefore would bias the repertoire in favor of proximal genes, whereas the action of all three cytokines in the adult bone marrow would generate a diverse repertoire of VH gene usage. This will be tested by culturing purified pro-B cells from fetal or adult RAG-deficient mice with TSLP, Flt3 ligand, or IL-7, either alone or in combination. Cells will then be assayed by chromatin immunoprecipitation (ChIP) for induction of acetylation of histones associated with Vh genes of different families. We will also perform a detailed analysis of the histone modifications of the region flanking 81X in order to gain insight into the extremely high frequency of rearrangement of this V gene in fetal life, and its preferential rearrangement even in adult pro-B cells. We predict that there will be regions of histone acetylation, methylation, or phosphorylation which may mark either boundary regions or regions of unusual accessibility which could result in the high rearrangement of this one gene. We further predict that these modifications would be most pronounced in fetal pro-B cells. Finally, we will determine by gene targeting it the apparent gradient of accessibility across the VH locus from proximal to distal VH families is real, or if it is a reflection of the fact that murine VH genes are clustered by family in the genome, which nay mask gene-specific control of rearrangement. Together, these studies should give insight into the epigenetic mechanisms and cis-elements which control VH gene rearrangement in fetal and adult pro-B cells.
Showing the most recent 10 out of 36 publications
