Replacement of Iμ-Cμ intron by NeoR gene alters Iμ germ-line expression but has no effect on V(D)J recombination

Abstract

The NeoR gene has often been used to unravel the mechanisms underlying long-range interactions between promoters and enhancers during V(D)J assembly and class switch recombination (CSR) in the immunoglobulin heavy chain (IgH) locus. This approach led to the notion that CSR is regulated through competition of germ-line (GL) promoters for activities displayed by the 3′ regulatory region (3′RR). This polarized long-range effect of the 3′RR is disturbed upon insertion of NeoR gene in the IgH constant (CH) region, where only GL transcription derived from upstream GL promoters is impaired. In the context of V(D)J recombination, replacement of Eμ enhancer or Eμ core enhancer (cEμ) by NeoR gene fully blocked V(D)J recombination and μ0 GL transcription which originates 5′ of DQ52 and severely diminished Iμ GL transcription derived from Eμ/Iμ promoter, suggesting a critical role for cEμ in the regulation of V(D)J recombination and of μ0 and Iμ expression. Here we focus on the effect of NeoR gene on μ0 and Iμ GL transcription in a mouse line in which the Iμ-Cμ intron was replaced by a NeoR gene in the sense-orientation. B cell development was characterized by a marked but incomplete block at the pro-B cell stage. However, V(D)J recombination was unaffected in sorted pro-B and pre-B cells excluding an interference with the accessibility control function of Eμ. μ0 GL transcription initiation was relatively normal but the maturation step seemed to be affected most likely through premature termination at NeoR polyadenylation sites. In contrast, Iμ transcription initiation was impaired suggesting an interference of NeoR gene with the IgH enhancers that control Iμ expression. Surprisingly, in stark contrast with the NeoR effect in the CH region, LPS-induced NeoR expression restored Iμ transcript levels to normal. The data suggest that Eμ enhancer may be the master control element that counteracts the down-regulatory “Neo effect” on Iμ expression upon LPS stimulation. More importantly, they reveal a complex and developmentally regulated interplay between IgH enhancers in the control of Iμ expression.