Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively splicedIghproduct made by mature B lymphocytes

Abstract

IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.IgHm | IgHd | immunoglobulin isotype |

Zfp318 Regulates IgD Expression by Abrogating Transcription Termination within the Ighm/Ighd Locus

“…Indeed, B cells lacking Mef2c still express IgD (28) suggesting that sufficient Zfp318 was present to allow for Ighd transcription. The analyses of our Zfp318 deficiency via Vav-Cre phenocopies the results described by Enders, et al, although a relative depression of T2 B cells of the spleen was not noted in their report (15). Enders, et al, further suggested that Zfp318 served to regulate IgD production by controlling the alternative splicing of the hnRNA of the Ighm/Ighd locus although no data were presented to support this model (15).…”

“…The analyses of our Zfp318 deficiency via Vav-Cre phenocopies the results described by Enders, et al, although a relative depression of T2 B cells of the spleen was not noted in their report (15). Enders, et al, further suggested that Zfp318 served to regulate IgD production by controlling the alternative splicing of the hnRNA of the Ighm/Ighd locus although no data were presented to support this model (15). Our conclusions as to the proposed function of Zfp318 differ from those presented of Enders, et al due to our RNA-Seq data which point to the regulation of transcription termination as the event which subsequently allows for the production of IgD via alternative splicing.…”

“…As our data were being prepared for submission, a report by Enders, et al detailed the results of an ethylnitrosourea (ENU) mutagenesis screen followed by whole exome sequencing that identified a single point mutation (a non-synonymous T>C transition altering the protein sequence I1347T) within Zfp318 that inhibited the production of IgD (15). This mutation mapped to the long form of the protein confirming its necessity versus that of the alternatively spliced, truncated form of the protein.…”

IgD attenuates the IgM-induced anergy response in transitional and mature B cells

“…2f,g). To exclude differences in transgene integration site between the MD4 and MM4 animals, we also prevented IgD co-expression in MD4:ML5 transgenic mice by breeding with a Zfp318 null allele22. Accumulation of anergic mature B cells was diminished in Zfp318 −/− MD4:ML5 transgenic mice where the B cells only expressed IgM HEL (Fig.…”

“…Immature B cells begin by expressing only IgM, but IgD co-expression progressively increases as they become transitional and mature B cells in the spleen due to increased expression of Zfp318 (ref. 22), which facilitates alternative mRNA splicing of the heavy chain variable (VDJ H ) exon to either IgM or IgD heavy chain constant (C)-region exons. This arrangement is evolutionarily preserved in most species of fish, amphibians, reptiles, birds and mammals1723, yet mice lacking IgD have normal B cell development and only slightly delayed antibody responses2425.…”