title: Functional analysis of natural mutations in two TWIST protein motifs.
abstract: The basic helix-loop-helix protein Twist, a transcriptional repressor, is essential for embryogenesis in both invertebrates and vertebrates. Haploinsufficiency of the human TWIST1 gene, which causes the craniosynostosis disorder Saethre-Chotzen syndrome (SCS), is related to failure to repress transcription of CDKN1A (which encodes p21/WAF1/CIP1), promoting osteoblast differentiation. We have examined the functional significance of natural TWIST1 variants present in craniosynostosis patients and in their healthy relatives. Both deletion and duplication variants of the glycine-rich tract Gly5AlaGly5 inhibited E2A (E12/E47)-dependent transcription of CDKN1A to a similar degree as wild-type protein, indicating that the length of this glycine tract is not critical for efficient transcriptional repression. We also evaluated a newly identified heterozygous TWIST1 variant (c.115C>G, encoding p.Arg39Gly), located within a putative nuclear localization signal (NLS), that was present in a child with mild SCS and her clinically unaffected father and grandmother. Unlike wild-type protein, this mutant required cotransfected E12 to localize to the nucleus, indicating that the NLS, including amino acid 39, is essential for nuclear localization; inhibition of E2A-dependent transcription of CDKN1A occurred normally. This analysis further dissects the structure-function relationships of TWIST and corroborates with phenotypic observations of disease expressivity.
authors.affiliation: Human Gene Sciences Center, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Funato
authors.forename: Stephen R F
authors.initials: SR
authors.lastname: Twigg
authors.forename: Norihisa
authors.initials: N
authors.lastname: Higashihori
authors.forename: Kimie
authors.initials: K
authors.lastname: Ohyama
authors.forename: Steven A
authors.initials: SA
authors.lastname: Wall
authors.forename: Andrew O M
authors.initials: AO
authors.lastname: Wilkie
authors.forename: Masataka
authors.initials: M
authors.lastname: Nakamura
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002675:Child, Preschool; D002522:Chlorocebus aethiops; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D004252:DNA Mutational Analysis; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D019913:Nuclear Localization Signals / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D051879:Twist-Related Protein 1 / Q000737:chemistry* / Q000235:genetics*; D050880:p300-CBP Transcription Factors
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1; D050880:p300-CBP Transcription Factors