Antisense Intergenic Transcription Precedes Igh D-to-J Recombination and Is Controlled by the Intronic Enhancer E_μ

Abstract

V(D)J recombination is believed to be regulated by alterations in chromatin accessibility to the recombinase machinery, but the mechanisms responsible remain unclear. We previously proposed that antisense intergenic transcription, activated throughout the mouse Igh V_H region in pro-B cells, remodels chromatin for V_H-to-DJ_H recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh D_HJ_H region before D-to-J recombination, indicating that this is a widespread process in V(D)J recombination. Transcription initiates near the Igh intronic enhancer E_μ and is abrogated in mice lacking this enhancer, indicating that E_μ regulates D_H antisense transcription. E_μ was recently demonstrated to regulate D_H-to-J_H recombination of the Igh locus. Together, these data suggest that E_μ controls D_H-to-J_H recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which E_μ can regulate chromatin accessibility throughout the D_H region. In contrast, E_μ deletion has no effect on V_H antisense intergenic transcription, which is rarely associated with D_H antisense transcription, suggesting differential regulation and separate roles for these processes at sequential stages of V(D)J recombination. These results support a directive role for antisense intergenic transcription in enabling access to the recombination machinery.

SUPPLEMENTAL MATERIAL

We thank Peter Fraser and Lyubomira Chakalova for critical review of the manuscript and Geoff Morgan for help with FACS.

D.B., A.W., and A.E.C. were supported by the Biotechnology and Biological Sciences Research Council including grant number BB/C508769/1 (D.B.), and R.A. and E.M.O. were supported by grants from the National Institutes of Health (P01 HL68744 and CA100905, E.M.O.; T32 CA09385, R.A.) and a Cancer Center Support grant (P30 CA68485, Vanderbilt-Ingram Cancer Center).

We declare that we have no financial interests that pose a conflict of interest with regard to this article.