Synonymous mutagenised <i>Trp53</i> CpG+ sequence changes.

In addition to the retained wild-type CpG sites (highlighted in purple), 72 new CpG sites are created by synonymous replacement by the bases highlighted in either blue or yellow. (TIF)</p

Observed phenotypes in transgenic mice.

Intragenic CpG dinucleotides are tightly conserved in evolution yet are also vulnerable to methylation-dependent mutation, raising the question as to why these functionally critical sites have not been deselected by more stable coding sequences. We previously showed in cell lines that altered exonic CpG methylation can modify promoter start sites, and hence protein isoform expression, for the human TP53 tumor suppressor gene. Here we extend this work to the in vivo setting by testing whether synonymous germline modifications of exonic CpG sites affect murine development, fertility, longevity, or cancer incidence. We substituted the DNA-binding exons 5–8 of Trp53, the mouse ortholog of human TP53, with variant-CpG (either CpG-depleted or -enriched) sequences predicted to encode the normal p53 amino acid sequence; a control construct was also created in which all non-CpG sites were synonymously substituted. Homozygous Trp53-null mice were the only genotype to develop tumors. Mice with variant-CpG Trp53 sequences remained tumor-free, but were uniquely prone to dental anomalies causing jaw malocclusion (p </div

Exon-specific synonymous base changes in CpG- vc-<i>Trp53</i> mice.

Exon-specific synonymous base changes in CpG- vc-Trp53 mice.</p

Typical examples of jaw malocclusion.

A, Whole-mouse photograph showing the dysmorphic appearance of the incisors, as well as associated rapid malnutrition due to feeding difficulties. B, Detailed photo of incisor overgrowth defect underlying malocclusion and feeding problems.</p

Mouse exons 5–8, all non-CpG-synonymous (NCpGS) versus WT.

CpG sites highlighted in yellow remain conserved in both sequences. Green-highlighted bases in the NCpGS sequence represent all other (181) possible synonymous nucleotide changes. (TIF)</p

Synonymous mutagenised <i>Trp53</i> CpG- sequence changes.

22 CpG sites highlighted in yellow are replaced as shown by synonymous green-highlighted bases. (TIF)</p

Illustration of mutagenesis strategy based on earlier in vitro studies using human <i>TP53</i> cDNA constructs.

A, Representation of synonymous mutations introduced into cDNA constructs, exons 5–8. Open circles–ancestral CpG sites. Red symbols–additional synonymous CpG sites (CGN, NCG, NNC/GNN). The first (wild-type) cDNA lacks the three introns normally bridging exons 5–8; the second construct, with synonymous losses of wild-type CpG sites, is labeled stable; the third construct, to which synonymous CpG-containing sites have been added, is labeled missense. B, Hypothetical phenotypic effects as potential downstream somatic consequences of altered germline TP53 mutation frequencies secondary to the synonymous changes. (TIF)</p

Relationship between amino acid site-specificity of sporadic carcinogenic mutations (above) and evolutionary rate (Ka/Ks, below: red) in <i>TP53</i>.

Sequences were downloaded from NCBI Entrez Gene (http://www.ncbi.nlm.nih.gov/Entrez/Gene), and homolog data in XML format from NCBI Homolo-Gene database (ftp://ftp.ncbi.nih.gov/pub/HomoloGene/). Mutation data were downloaded from the Human Gene Mutation Database. K-estimator 6.1 (with window size of 33 codons and step size of 10 codons using Kimura 2-parameter method) and PAML 3.15 with yn00 model were used for evolutionary rate calculations. Orthologous gene pairs between human and mouse, together with their synonymous substitution (Ks), nonsynonymous substitution rate (Ka), and their ratio (Ka/Ks), were thus isolated. The Ka/Ks evolutionary rate for TP53 CpG sites in exons 5–8 was shown to approach zero, consistent with high negative selection pressure, with these same (functionally important) germline sites closely corresponding to those undergoing somatic mutation in tumors. (TIF)</p

Exon-specific synonymous base changes in CpG+ vc-<i>Trp53</i> mice.

Exon-specific synonymous base changes in CpG+ vc-Trp53 mice.</p

Differential intron retention in Jumonji chromatin modifier genes is implicated in reptile temperature-dependent sex determination

In many vertebrates, sex of offspring is determined by external environmental cues rather than by sex chromosomes. In reptiles, for instance, temperature-dependent sex determination (TSD) is common. Despite decades of work, the mechanism by which temperature is converted into a sex-determining signal remains mysterious. This is partly because it is difficult to distinguish the primary molecular events of TSD from the confounding downstream signatures of sexual differentiation. We use the Australian central bearded dragon, in which chromosomal sex determination is overridden at high temperatures to produce sex-reversed female offspring, as a unique model to identify TSD-specific features of the transcriptome. We show that an intron is retained in mature transcripts from each of two Jumonji family genes, JARID2 and JMJD3, in female dragons that have been sex-reversed by temperature but not in normal chromosomal females or males. JARID2 is a component of the master chromatin modifier Polycomb Repressive Complex 2, and the mammalian sex-determining factor SRY is directly regulated by an independent but closely related Jumonji family member. We propose that the perturbation of JARID2/JMJD3 function by intron retention alters the epigenetic landscape to override chromosomal sex-determining cues, triggering sex reversal at extreme temperatures. Sex reversal may then facilitate a transition from genetic sex determination to TSD, with JARID2/JMJD3 intron retention preserved as the decisive regulatory signal. Significantly, we also observe sex-associated differential retention of the equivalent introns in JARID2/JMJD3 transcripts expressed in embryonic gonads from TSD alligators and turtles, indicative of a reptile-wide mechanism controlling TSD.</p