|Coordinate||46,409,692 bp (GRCm38)|
|Base Change||T ⇒ C (forward strand)|
|Gene Name||zinc finger protein 318|
|Synonym(s)||2610034E08Rik, D530032D06Rik, TZF|
|Chromosomal Location||46,383,731-46,420,920 bp (+)|
|MGI Phenotype||PHENOTYPE: Mice homozygous for an ENU-induced allele exhibit reduced male fertility and altered IgM and IgD levels. Null mutants displayed normal level of circulating B cells with decreased IgD and increased IgM levels. [provided by MGI curators]|
|Amino Acid Change||Tyrosine changed to Histidine|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000109109] [ENSMUSP00000116544] [ENSMUSP00000116132]|
AA Change: Y1119H
|Predicted Effect||possibly damaging
PolyPhen 2 Score 0.892 (Sensitivity: 0.82; Specificity: 0.94)
|Predicted Effect||probably benign|
|Predicted Effect||probably benign|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Last Updated||2018-12-06 3:13 PM by Anne Murray|
|Record Created||2014-10-12 7:58 PM by Ming Zeng|
The Wonton phenotype was identified among N-nitroso-N-ethylurea (ENU)-mutagenized G3 mice of the pedigree R1166, some of which showed a reduction in IgD mean fluorescence intensity (MFI) on B cells in the peripheral blood (Figure 1). A CRISPR-generated Zfp318 knockout mouse exhibited reduction in IgD MFI on B cells in the peripheral blood (Figure 2; P = 6.16 x 10-9; recessive model of inheritance), confirming that the Zfp318 mutation in the Wonton mice leads to the IgD MFI phenotype.
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 37 mutations. The reduced IgD MFI was linked by continuous variable mapping to a mutation in Zfp318: a T to C transition at base pair 46,409,692 (v38) on chromosome 17, or base pair 26,191 in the GenBank genomic region NC_000083 encoding Zfp318. Linkage was found with an additive model of inheritance to the normalized IgD MFI (P value of 5.698 x 10-10), wherein 5 variant homozygotes and 16 heterozygous mice departed phenotypically from 17 homozygous reference mice (Figure 3). The mutation corresponds to residue 3,428 in the mRNA sequence NM_207671 within exon 8 of 10 total exons.
The mutated nucleotide is indicated in red. The mutation results in a tyrosine (Y) to histidine (H) substitution at position 1119 (Y1119H) in the long isoform of the ZFP318 protein (see Protein Prediction section for more details on the isoforms of ZFP318), and is strongly predicted by Polyphen-2 to cause loss of function (score = 0.892).
Zfp318 encodes zinc finger protein 318 [ZFP318; alternatively, testicular zing-finger protein (TZF); Figure 4]. Zinc finger proteins are nucleic acid-binding proteins that function in the activation or repression of downstream target genes. ZFP318 has two C2H2 zinc fingers (amino acids 1085-1119 and amino acids 1155-1189, SMART) (1). C2H2 zinc fingers are found in several transcription factors including Wilms’ tumor WT1, the RNA-binding nuclear protein (NP220), and RIN zinc finger (RIN ZF) (2;3). The C2H2 zinc fingers in ZFP318 are U1-type zinc fingers that are similar to the RNA-binding zinc fingers in the spliceosome U1C protein (4;5). U1C functions in the initiation and regulation of pre-mRNA splicing as part of the U1 snRNP. ZFP318 also has a myosin II-homology region (amino acids 687-799), a serine-rich region (amino acids 799-853), four basic amino acid-rich regions [RRKR (amino acids 143-145), KKSILKKR (amino acids 200-207), KRRR (amino acids 341-344), and RKKRR (amino acids 739-743)] that are putative nuclear localization sequences, a poly-glutamate region, and a poly-proline region (1;6). The myosin II-homology domain is 30% identical and 55% homologous to the coiled-coil domain of the heavy chain of myosin II (1). Amino acids 512-663 are not within a predicted domain, but are essential for ZFP318-mediated repression of androgen receptor expression (see the Background section for more information) (7).
Zfp318 encodes two isoforms that share the first 1,114 N-terminal amino acids (6). The 2,237-amino acid long isoform has two C2H2/U1 ribonucleoprotein type zinc finger domains, while the shorter 1,154-amino acid isoform lacks the second zinc finger and poly-proline domain. The two isoforms of ZFP318 can form both homo- and heterodimers (8). Formation of a heterodimer between the two isoforms inhibited ZFP318 homodimer formation (8).
The Wonton mutation affects amino acid Tyr1119 within the N-terminal zinc finger domain of the long ZFP318 isoform only.
Zfp318 is expressed at high levels in spermatogenic cells of the mouse testis and is upregulated at the pachytene spermatocyte stage of mouse spermatogenesis (1;6). Zfp318 was not detected in spermatogonia, in spermatogenic cells at early stages, or in Sertoli cells (6). Zfp318 was also expressed at moderate levels in the adrenal gland, uterus, prostate gland, muscle, and kidney (6;9). Zfp318 expression increases during B cell maturation from immature B cells into follicular B cells (10-12). Expression of Zfp318 is low in pro-B cells in the bone marrow, with moderate expression in immature (CD93+, CD62L−) B cells in the spleen, and high amounts in mature follicular (CD93−, CD62L+) B cells (13). Both ZFP318 isoforms localize to the nucleus (6). The short isoform localized to subnuclear speckles that contained histone deacetylase 2 (HDAC2). In addition ZFP318 was adjacent to nuclear speckles that contained serine/arginine-rich splicing factor (SRSF2) (6;7;9).
During B cell differentiation from immature to mature follicular or marginal zone cells, antibody isotypes serve as cell surface markers of B cell maturation, as distinct receptors for B cell activation, and as secreted mediators of antibody effector functions (14). During B cell maturation, immature B cells in the bone marrow only express the IgM isotype on their cell surface (15). The IgM isotype is comprised of heavy (H) chains with an N-terminal variable domain, C-terminal constant region domains, a transmembrane segment, and a cytoplasmic tail. Upon maturation into circulating follicular B cells, the B cells coexpress a second isotype, IgD. Mature follicular B cells display cell surface B cell receptors comprised of the same variable domain joined to either IgD or IgM constant regions (16;17). Upon B cell activation by antigens and helper T cells, the B cells undergo isotype switching and lose IgM and IgD, switching to express the same variable domain linked to IgG, IgA, or IgE constant region domains. Isotype switching involves DNA recombination of the Ig heavy chain locus, Igh, and subsequent deletion of Ighm and Ighd constant region exons and the reorganization of the Ighg, Ighe, or Igha constant region exons immediately 3’ to the VDJH variable exon. The VDJH exon is then spliced to IgG, IgE, or IgA constant region exons in the mRNA (18;19).
ZFP318 functions as a transcription factor to regulate the expression of Ighm and Ighd during B cell maturation (13;20). B cell expression of IgD occurs developmentally through alternative splicing of the VDJH exon to the Ighm and Ighd exons (16;21;22). ZFP318 is required for balancing IgD and IgM output from Igh, but is not necessary for B cell maturation (13). Enders et al. proposed that ZFP318 regulates the alternative RNA splicing of Ighm and Ighd constant region exons by binding to HDAC2 (Figure 5) (13;23). Pioli et al. proposed that ZFP318 regulates hRNA transcription of IgD by blocking the recognition of an IgM-specific transcriptional stop site within the final Ighm exon subsequently synthesizing the full Ighm/Ighd hnRNA that is required for IgD production (20).
ZFP318 also functions as a corepressor of the androgen receptor (AR), a member of the nuclear receptor subfamily, in the testis (8;9). ZFP318 forms specific foci located in the proximity to the splicing factor compartment in nuclei and is recruited into the AR foci in the presence of 5α-dihydrotestosterone (DHT) (9). In the presence of DHT, ZFP318 recruited HDAC2 and formed a complex with the AR (7). Furthermore, the HDAC inhibitor TSA prevented ZFP318 transcriptional repression (7). Taken together, the recruitment of HDAC2 into the ZFP318/AR complex mediates ZFP318-mediated repression of the AR (7).
Mice with B cell-specific deletion of ZFP318 (B-Zfp318-/-) exhibited normal B cell development (20). However, T1, T2, follicular, and marginal zone B cells from the B-Zfp318-/- mice expressed diminished IgD mRNA and protein (20). In addition, T2, follicular, and marginal zone B cells from the B-Zfp318-/- mice expressed elevated levels of IgM. An ENU-induced mutation, Zfp318m1Anu, resulted in an isolueucine to threonine substitution at amino acid 1347 in the long ZFP318 isoform. The Zfp318m1Anu mice exhibited diminished IgD and increased IgM on mature B cells in the peripheral blood as well as on splenic B cells (13). In the bone marrow, the percentage of B cells as well as the subset distribution of developing B cells was not significantly different in the Zfp318m1Anu mice compared to wild-type mice. However, there was a small increase in the proportion of recirculating B cells. The percentage of circulating B cells in the Zfp318m1Anu mice was normal. The long isoform functions in normal IgD expression, and any residual IgD-promoting activity was proposed to be due to the action of the short isoform (13). Alternatively, the long isoform may be the sole isoform to promote IgD expression, but mutations may only result in partial loss of activity. The Wonton mutation is predicted to only alter the long isoform of ZFP318. The reduced IgD phenotype of the Wonton mice indicates that the mutant ZFP318 protein has loss of function.
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7. Tao, R. H., Kawate, H., Wu, Y., Ohnaka, K., Ishizuka, M., Inoue, A., Hagiwara, H., and Takayanagi, R. (2006) Testicular Zinc Finger Protein Recruits Histone Deacetylase 2 and Suppresses the Transactivation Function and Intranuclear Foci Formation of Agonist-Bound Androgen Receptor Competitively with TIF2. Mol Cell Endocrinol. 247, 150-165.
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|Science Writers||Anne Murray|
|Authors||Ming Zeng, Bruce Beutler|