Functional analysis of a novel ENU-induced PHD finger 11 (Phf11) mouse mutant

Abstract

Previously, human genetic studies have shown association between polymorphisms within the gene encoding plant homeodomain zinc finger protein 11 (PHF11) and asthma-related phenotypes. Initial functional studies have suggested that PHF11 may be involved in the immune response through regulation of T cell activities. In order to study further the gene's functions, we have investigated the mouse Phf11 locus. We have established and characterised a mouse line harbouring a point mutation in the PHD domain of Phf11. Full-length mouse cDNA for Phf11 was obtained by applying rapid amplification of cDNA ends (RACE). All five exons encoding the PHD domain of Phf11 were directly sequenced in 3840 mouse DNA samples from the UK MRC Harwell ENU (N-ethyl-N-nitrosourea)-mutagenised DNA archive. Mice harbouring a valine to alanine substitution, predicted to have a significant functional impact on the PHD zinc finger domain, were re-derived. These Phf11 mutant mice were outcrossed to C3H mice and then backcrossed for ten generations in order to establish a congenic line harbouring the single point mutation in Phf11. Macroscopic examination, haematology and histological examination of lung structure revealed no significant differences between mutant and wild-type mice. After administration of lipopolysaccharide, the level of expression of Il2, NF-kB and Setdb2 were significantly increased in Phf11 mutant homozygous lungs compared to control littermates. Our results provide evidence that Phf11 can operate as a Th1 cell regulator in immune responses. Moreover, our data indicate that these mice may provide a useful model for future studies on Phf11.

Fig. 1

The mouse Phf11 Locus (from the UCSF Genome Browser). The UCSC Genome Browser view of mouse Phf11 locus on Dec 2011(GRCm8/mm10) assembly. The genome structure was based on mouse chromosome 14 (chr14:59,189,998-59,464,797274,800 bp). The five paralogous copies of mouse Phf11 were showed as in Refseq genes: Phf11c, Phf11d, Phf11b, phf11a and Gm6904

Fig. 2

Phf11 expression in multiple mouse cDNA Tissues. PCRs were performed with multiple tissues mouse cDNAs. The clontech MTC multiple tissue cDNA panels are sets of first-strand cDNAs from 5 to 12 different tissues. The PCR products covered exons 2–5 for mouse Phf11. The 100 bp DNA ladder was shown in the first lane

Fig. 3

A Cross-species conservation analysis for four mutations in the PHD Domain of Phf11. Exon 4 multiple alignment (human, chimp, macaque, dog, rabbit, cow, two rat homologs and five mouse duplicates). Mutated residues coloured red and change shown in brackets with wild-type allele listed first. Each of the mutated residues is conserved in all of the aligned sequences. The mutation-screened paralog (shown as “duo2” here) is not any closer to the ancestral sequence than any of the other paralogos. B Predicted impact of identified mutations in PHD domain of Phf11 on protein function PFAM Hidden Markov Model (HMM) of the PHD domain (hmmbuild trained with PFAM seed alignment and calibrated using hmmcalibrate) was used to score the impact of each substitution. Mut mutation. Dn/Ds calculations for this exon were consistent with general selective constraint. Mut5: domain 1 of 1, from 92 to 139: score 1.0, E = 1.2e–05; Mut2: domain 1 of 1, from 92 to 139: score −3.8, E = 3.9e–05; Mut4: domain 1 of 1, from 92 to 139: score −4.1, E = 4.1e–05; Mut3: domain 1 of 1, from 92 to 139: score −7.0, E = 8.3e–05

Fig. 4

A Haemoanalysis in Mouse Blood. B Histological comparison of E18.5 Wild-type and Phf11 homozygous lungs. No difference in either lung parenchyma (a, b) or proximal airways (c, d) was observed in H&E stained sections of wild-type (a, c) and Phf11 homozygous (b, d) lungs. C Comparison of Cell-type specific markers in Wild-type and Phf11 homozygous lungs. No significant difference was observed in expression of the Type II alveolar cell marker pro SP-C (a, b) and Type I alveolar cell marker aquaporin 5 (c, d) between wild-type (a, d, e) and Phf11 homozygous (b, d) lungs. e Control lung section with primary antibody omitted

Fig. 4

A Haemoanalysis in Mouse Blood. B Histological comparison of E18.5 Wild-type and Phf11 homozygous lungs. No difference in either lung parenchyma (a, b) or proximal airways (c, d) was observed in H&E stained sections of wild-type (a, c) and Phf11 homozygous (b, d) lungs. C Comparison of Cell-type specific markers in Wild-type and Phf11 homozygous lungs. No significant difference was observed in expression of the Type II alveolar cell marker pro SP-C (a, b) and Type I alveolar cell marker aquaporin 5 (c, d) between wild-type (a, d, e) and Phf11 homozygous (b, d) lungs. e Control lung section with primary antibody omitted

Fig. 5

Quantitative PCR analysis expression levels of ten genes in phf11 mutant mouse lungs after LPS challenge. Alterations in gene expression in Phf11 homozygous lungs were expressed relative to the mean intensity in wild-type embryos, which was given a standardised value of one. P values were compared by Student’s t-test. Error bars represent SEM and significance was scored using unpaired two-tailed t-tests. *indicates statistical significance between wild-type and Phf11 homozygous mice