Gv1, a Zinc Finger Gene Controlling Endogenous MLV Expression

Abstract

The genomes of inbred mice harbor around 50 endogenous murine leukemia virus (MLV) loci, although the specific complement varies greatly between strains. The Gv1 locus is known to control the transcription of endogenous MLVs and to be the dominant determinant of cell-surface presentation of MLV envelope, the GIX antigen. Here, we identify a single Krüppel-associated box zinc finger protein (ZFP) gene, Zfp998, as Gv1 and show it to be necessary and sufficient to determine the GIX+ phenotype. By long-read sequencing of bacterial artificial chromosome clones from 129 mice, the prototypic GIX+ strain, we reveal the source of sufficiency and deficiency as splice-acceptor variations and highlight the varying origins of the chromosomal region encompassing Gv1. Zfp998 becomes the second identified ZFP gene responsible for epigenetic suppression of endogenous MLVs in mice and further highlights the prominent role of this gene family in control of endogenous retroviruses.

Keywords: Gv1; Sgp3; KRAB ZFP; TRIM28; endogenous retrovirus; murine leukemia virus.

Fig. 1

The chromosomal context of Gv1.(a) Reanalysis of a large-scale backcross (fig. S2) highlighting the location of Gv1 on Chr 13 (GRCm38). The linkage peak was predicted by scaling predicted genetic distances to the flanking markers D13Mit311 and D13Mit66 to their intervening chromosomal distance. (b) Read depth ratios vs B6/J for exemplar strains (full data in fig. S4) showing CNVs within the area encompassing Gv1. C57-lineage strains are colored black and non-C57-lineage strains by phenotype: ${\mathrm{G}}_{\text{IX}}^{–}$—blue, ${\mathrm{G}}_{\text{IX}}^{+}$—red, and Sgp3⁺—green. (c)Heatmap of the median absolute error (MAE) between read depth ratio profiles for the strains in fig. S4. All non-C57-lineage strains group with ZALENDE/EiJ, whereas the C57-lineage group separately, and C57L/J with MOLF/EiJ. Coloring is according to (c), with wild-derived inbred strains in purple. (d) View of the genes within the area deleted within B6/N mice. Pseudogenes are colored gray, lincRNAs in purple, and protein coding genes in maroon. The protein coding ZFPs are annotated.

Fig. 2

Zfp998 controls endogenous MLV expression in vitro and in vivo.(a) Median fluorescence intensity of 83A25 staining for MLV SU within the indicated subsets of CD19^– thymocytes (six mice per group). (b) expression of x- and pMLV envelope measured by qRT-PCR and normalized relative to Hprt (three mice per group). Amplification of products for e- and mpMLV was negligible or absent after 40 cycles. (c)RNAseq expression analysis of endogenous MLVs in B6/N and B6/J (three mice per group). Significantly (q < 0.01, log2FC > 1) regulated MLV loci are indicated, with symbol shape denoting MLV subclass: triangle—mpMLV, circle—pMLV, and diamond—xMLV. (d) Numbers mCherry⁺ cells in otherwise untreated 293 T cells, the same cells transfected with a control plasmid, or with a Zfp998-containing expression plasmid. Symbol shape denotes the results of three independent experiments with 3/4 technical replicates. Significance values are from two-way ANOVA comparisons with no significant differences being determined between experiments or in experiment: treatment interactions. (e) Median fluorescence intensity of 83A25 staining for MLV SU within the indicated mouse genotypes (six, six, three, and six mice per group). (f)Expression of x- and pMLV envelope measured by qRT-PCR and normalized relative to Hprt (three mice per group). Amplification of products for e- and mpMLV was negligible or absent after 40 cycles. In (a), (b), (e), and (f), significance values are from Student’s t-tests with the indicated group sizes. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Fig. 3

Nanopore sequencing allows assembly of the Gv1 gene region from 129 mice.(a) Alignment of the eight contigs resulting from assembly of the BACs against Chr 13. Diagonal lines represent only those areas ≥1 kb with ≥98% identity to the GRCm38 reference and are colored according to orientation (purple—forwards, green—reverse). Sequence names detail the scaffolding order and final sizes are shown above the alignment sections. Red highlighting shows the single position of Zfp998 in C57BL/6J (horizontal) intersecting with two independent copies within the BAC assemblies (vertical). (b) Comparison of the splice acceptor sequence (black box, with arrow marking point of splicing) for exon 4 of Zfp998 in comparison to the equivalent gene regions from the two assembled scaffolds. Area shown is the reverse complement of GRCm38 13:66432223-66432240.