Missense mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, cause clinically similar but distinctive disorders, LEOPARD (LS) and Noonan (NS) syndromes. The LS is an autosomal dominant disorder with pleomorphic developmental abnormalities including lentigines, cardiac defects, short stature and deafness. Biochemical analyses indicated that LS alleles engender loss-of-function (LOF) effects, while NS mutations result in gain-of-function (GOF). These biochemical findings lead to an enigma that how PTPN11 mutations with opposite effects on function result in disorders that are so similar. To study the developmental effects of the commonest LS PTPN11 alleles (Y279C and T468M), we generated LS transgenic fruitflies using corkscrew (csw), the Drosophila orthologue of PTPN11. Ubiquitous expression of the LS csw mutant alleles resulted in ectopic wing veins and, for the Y279C allele, rough eyes with increased R7 photoreceptor numbers. These were GOF phenotypes mediated by increased RAS/MAPK signaling and requiring the LS mutant's residual phosphatase activity. Our findings provide the first evidence that LS mutant alleles have GOF developmental effects despite reduced phosphatase activity, providing a rationale for how PTPN11 mutations with GOF and LOF produce similar but distinctive syndromes.

TBX1 encodes a DNA binding transcription factor that is commonly deleted in human DiGeorge syndrome and plays an important role in heart development. Mechanisms of Tbx1 function, such as Tbx1 interacting regulatory proteins and transcriptional target specificity, are largely unknown. Ash2l is the mammalian homolog of Drosophila Ash2 (absent small homeotic 2) and is a core component of a multimeric histone methyltransferase complex that epigenetically regulates transcription via methylation of histone lysine residues. We undertook an unbiased yeast two-hybrid screen to look for functionally relevant Tbx1-interacting proteins and report a physical and functional interaction between Tbx1 and Ash2l. Tbx1 interacts with Ash2l in both yeast and mammalian cells and Ash2l acts as a transcriptional co-activator in luciferase reporter assays. Expression analysis shows that Tbx1 and Ash2l have overlapping mRNA and protein expression patterns during development. By generating an Ash2l knockout mouse utilizing gene-trap technology, we show that although Ash2l heterozygous mice are normal, Ash2l-null embryos die early during gestation. Thus, Ash2l is required for the earliest stages of embryogenesis. Furthermore, our finding of a physical interaction between Tbx1 and Ash2l suggest that at least some functions of Tbx1 may be mediated by direct interactions with a histone methyltransferase complex.

The family of Ap2 transcription factors comprises five members with highly conserved DNA-binding domains. Among the family members, Ap2␦ is the most divergent, because it lacks highly conserved residues within the transactivation domain (TAD) and has weak affinity for known Ap2 binding sites. To identify specific Ap2␦ coactivators/regulators during development, we performed a yeast two-hybrid screen, using Ap2␦'s TAD. We identified the trithorax superfamily member, Ash2l, as a binding partner that interacts exclusively with Ap2␦. We showed that Ash2l positively mediates Ap2␦ transactivation in a dose-dependent manner. Given the known role of Ash2l in histone modification, we determined whether Ap2␦ was able to form a complex with that activity. Our results showed that Ap2␦ associates with endogenous ASH2L and a member of the MLL family of histone lysine methyltransferases (HKMTs), MLL2 (ALR), forming a complex that methylates lysine 4 of histone H3 (H3K4). Additionally, we showed that Ap2␦ is necessary for recruitment of Ash2l and Alr to the Hoxc8 locus and that recruitment of this complex leads to H3K4 trimethylation (H3K4me3) and subsequent gene activation. Altogether, we provide evidence of an association between a highly restricted genespecific transcription factor and a Su(var), Enhancer of Zeste, Trithorax (SET)1/trithorax-like complex with H3K4 methyltransferase activity. Our studies also document a functional role for Ap2␦ in recruiting histone methyltransferases (HMTs) to specific gene targets, such as Hoxc8. This role provides a mechanism through which these transcription factors can have diverse effects despite nearly identical DNA-binding motifs.