Germ-line transcripts of the immunoglobulin λ J–C clusters in the mouse: characterization of the initiation sites and regulatory elements

Abstract

Transcription of unrearranged immunoglobulin gene segments strongly correlates with their accessibility to the V(D)J recombination machinery. The regulatory mechanisms governing this germ-line transcription are still poorly defined. In order to identify new regulatory elements, we first carried out a detailed characterization of the transcription initiation sites for the J–C germ-line transcripts, using rapid amplification of 5′ cDNA ends, assisted by a template switching mechanism at the 5′-end of the RNA. Transcripts were observed that initiated heterogeneously, starting up to 293 (λ1), 116 bp (λ2) and 79 bp (λ3) upstream from the respective Jλ gene segment. Additional RT-PCR analysis revealed the existence of germ-line transcripts of λ and also of κ that initiate even more upstream of these transcription initiation sites, although their frequencies were low. Promoter activity was detected in vitro 5′ of Jλ2, with the minimal promoter activity mapping to the region between positions −35 and −120. In addition, computer analysis allowed the prediction of a nuclear scaffold/matrix attachment (S/MAR) region between the two J–C gene clusters at each hemi-locus. This region between the λ1/λ3 clusters binds to the nuclear matrix in vitro, and J–C λ1 germ-line transcription initiates a short distance downstream from this S/MAR element.