Crosslinking of base modified RNAs by synthetic DYW-KP base editors implicates an enzymatic lysine as the nitrogen donor for U-to-C RNA editing.

Base editing mechanisms are being investigated as potential therapeutic tools to alleviate genetic diseases. Sequence specific C-to-U and A-to-I base editing tools are capable of altering RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyze C-to-U edits and likely use the canonical deamination mechanism. Proteins developed from consensus sequences from the DYW-KP domain family catalyze what initially appeared to be U-to-C edits leading to this investigation into mechanistic insights into U-to-C editing. The synthetic DYW-KP enzyme KP6 was found sufficient for C-to-U editing activity stimulated by the addition of carboxylic acids in vitro. Despite addition of 14 different putative amine/amide donors in vitro U-to-C editing could not be observed. C-to-U editing was found not to be concomitant with U-to-C editing thus discounting a pyrimidine transaminase mechanism. RNAs containing base modifications were covalently crosslinked to KP6, KP2, and KP3 proteins. Mass spectrometry of purified KP2 and KP6 proteins revealed an additional mass of 319 Da. A U-to-C crosslinking mechanism was projected to explain the link between crosslinking, RNA base changes, and the ∼319 Da mass. In this model an enzymatic lysine attacks C4 of uridine to form a Schiff base RNA-protein conjugate. Sequenced RT-PCR products from the fern Ceratopteris richardii indicate U-to-C base edits do not preserve proteinaceous crosslinks in planta. Hydrolysis of a protonated Schiff base conjugate releasing cytidine is hypothesized to explain the completed pathway in plants.

An allelic series of Trp63 mutations defines TAp63 as a modifier of EEC syndrome.

Human Ectrodactyly, Ectodermal dysplasia, Clefting (EEC) syndrome is an autosomal dominant developmental disorder defined by limb deformities, skin defects, and craniofacial clefting. Although associated with heterozygous missense mutations in TP63, the genetic basis underlying the variable expressivity and incomplete penetrance of EEC is unknown. Here, we show that mice heterozygous for an allele encoding the Trp63 p.Arg318His mutation, which corresponds to the human TP63 p.Arg279His mutation found in patients with EEC, have features of human EEC. Using an allelic series, we discovered that whereas clefting and skin defects are caused by loss of Trp63 function, limb anomalies are due to gain- and/or dominant-negative effects of Trp63. Furthermore, we identify TAp63 as a strong modifier of EEC-associated phenotypes with regard to both penetrance and expressivity.

Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch.

Morphogenesis of the vertebrate head relies on proper dorsal-ventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches. Endothelin-1 (Edn1)-induced signaling through the endothelin-A receptor (Ednra) is crucial for cranial NCC patterning within the mandibular portion of the first pharyngeal arch, from which the lower jaw arises. Deletion of Edn1, Ednra or endothelin-converting enzyme in mice causes perinatal lethality due to severe craniofacial birth defects. These include homeotic transformation of mandibular arch-derived structures into more maxillary-like structures, indicating a loss of NCC identity. All cranial NCCs express Ednra whereas Edn1 expression is limited to the overlying ectoderm, core paraxial mesoderm and pharyngeal pouch endoderm of the mandibular arch as well as more caudal arches. To define the developmental significance of Edn1 from each of these layers, we used Cre/loxP technology to inactivate Edn1 in a tissue-specific manner. We show that deletion of Edn1 in either the mesoderm or endoderm alone does not result in cellular or molecular changes in craniofacial development. However, ectodermal deletion of Edn1 results in craniofacial defects with concomitant changes in the expression of early mandibular arch patterning genes. Importantly, our results also both define for the first time in mice an intermediate mandibular arch domain similar to the one defined in zebrafish and show that this region is most sensitive to loss of Edn1. Together, our results illustrate an integral role for ectoderm-derived Edn1 in early arch morphogenesis, particularly in the intermediate domain.

ΔNp63α is an oncogene that targets chromatin remodeler Lsh to drive skin stem cell proliferation and tumorigenesis.

The p53 homolog p63 is essential for development, yet its role in cancer is not clear. We discovered that p63 deficiency evokes the tumor-suppressive mechanism of cellular senescence, causing a striking absence of stratified epithelia such as the skin. Here we identify the predominant p63 isoform, ΔNp63α, as a protein that bypasses oncogene-induced senescence to drive tumorigenesis in vivo. Interestingly, bypass of senescence promotes stem-like proliferation and maintains survival of the keratin 15-positive stem cell population. Furthermore, we identify the chromatin-remodeling protein Lsh as a new target of ΔNp63α that is an essential mediator of senescence bypass. These findings indicate that ΔNp63α is an oncogene that cooperates with Ras to promote tumor-initiating stem-like proliferation and suggest that Lsh-mediated chromatin-remodeling events are critical to this process.

Regulation of facial morphogenesis by endothelin signaling: insights from mice and fish.

Craniofacial morphogenesis is accomplished through a complex set of developmental events, most of which are initiated in neural crest cells within the pharyngeal arches. Local patterning cues from the surrounding environment induce gene expression within neural crest cells, leading to formation of a diverse set of skeletal elements. Endothelin-1 (Edn1) is one of the primary signals that establishes the identity of neural crest cells within the mandibular portion of the first pharyngeal arch. Signaling through its cognate receptor, the endothelin-A receptor, is critical for patterning the ventral/distal portion of the arch (lower jaw) and also participates with Hox genes in patterning more posterior arches. Edn1/Ednra signaling is highly conserved between mouse and zebrafish, and genetic analyses in these two species have provided complementary insights into the patterning cues responsible for establishing the craniofacial complex as well as the genetic basis of facial birth defect syndromes.

Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects.

The congenital malformation Split Hand-Foot Malformation (SHFM, or ectrodactyly) is characterized by a medial cleft of hands and feet, and missing central fingers. Five genetically distinct forms are known in humans; the most common (type-I) is linked to deletions of DSS1 and the distalless-related homeogenes DLX5 and DLX6. As Dlx5;Dlx6 double-knockout mice show a SHFM-like phenotype, the human orthologs are believed to be the disease genes. SHFM-IV and Ectrodactyly-Ectodermal dysplasia-Cleft lip (EEC) are caused by mutations in p63, an ectoderm-specific p53-related transcription factor. The similarity in the limb phenotype of different forms of SHFM may underlie the existence of a regulatory cascade involving the disease genes. Here, we show that p63 and Dlx proteins colocalize in the nuclei of the apical ectodermal ridge (AER). In homozygous p63- (null) and p63EEC (R279H) mutant limbs, the AER fails to stratify and the expression of four Dlx genes is strongly reduced; interestingly, the p63+/EEC and p63+/- hindlimbs, which develop normally and have a normally stratified AER, show reduced Dlx gene expression. The p63+/EEC mutation combined with an incomplete loss of Dlx5 and Dlx6 alleles leads to severe limb phenotypes, which are not observed in mice with either mutation alone. In vitro, DeltaNp63alpha induces transcription from the Dlx5 and Dlx6 promoters, an activity abolished by EEC and SHFM-IV mutations, but not by Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) mutations. ChIP analysis shows that p63 is directly associated with the Dlx5 and Dlx6 promoters. Thus, our data strongly implicate p63 and the Dlx5-Dlx6 locus in a pathway relevant in the aetio-pathogenesis of SHFM.