The Ig heavy chain protein but not its message controls early B cell development.

Abstract

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged <i>Igh</i> allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive <i>Igh</i> allele is expressed, a phenomenon known as <i>Igh</i> allelic exclusion. In contrast to a productively rearranged <i>Igh</i> allele, the <i>Igh</i> messenger RNA (mRNA) (<i>IgHR</i>) from a nonproductively rearranged <i>Igh</i> allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable <i>IgHR</i> to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the <i>Igh</i>^{<i>Ter5H∆TM</i>} mouse model from <i>Igh</i>^<i>Ter5H</i> mice having a premature termination codon at position +5 in leader exon of <i>Igh</i>^<i>Ter5H</i> allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of <i>Igh</i>^<i>Ter5H</i> message, indicating that previous conclusions regarding a role of <i>IgHR</i> in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the <i>Igh</i>^{<i>Ter5H∆TM</i>} knock-in allele, which generated stable <i>IgHR</i> but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not <i>IgHR</i> expression.