Introducing Pitt-Hopkins syndrome-associated mutations of TCF4 to Drosophila daughterless.

Laura Tamberg,Tõnis Timmusk,Mari Sepp,Mari Palgi

doi:10.1242/bio.014696

Laura Tamberg, Tõnis Timmusk + Show 2 more

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https://doi.org/10.1242/bio.014696

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Journal: Biology open	Publication Date: Nov 30, 2015
Citations: 19	License type: CC BY 3.0

Affiliation: Tallinn University of Technology

Abstract

ABSTRACTPitt-Hopkins syndrome (PTHS) is caused by haploinsufficiency of Transcription factor 4 (TCF4), one of the three human class I basic helix-loop-helix transcription factors called E-proteins. Drosophila has a single E-protein, Daughterless (Da), homologous to all three mammalian counterparts. Here we show that human TCF4 can rescue Da deficiency during fruit fly nervous system development. Overexpression of Da or TCF4 specifically in adult flies significantly decreases their survival rates, indicating that these factors are crucial even after development has been completed. We generated da transgenic fruit fly strains with corresponding missense mutations R578H, R580W, R582P and A614V found in TCF4 of PTHS patients and studied the impact of these mutations in vivo. Overexpression of wild type Da as well as human TCF4 in progenitor tissues induced ectopic sensory bristles and the rough eye phenotype. By contrast, overexpression of DaR580W and DaR582P that disrupt DNA binding reduced the number of bristles and induced the rough eye phenotype with partial lack of pigmentation, indicating that these act dominant negatively. Compared to the wild type, DaR578H and DaA614V were less potent in induction of ectopic bristles and the rough eye phenotype, respectively, suggesting that these are hypomorphic. All studied PTHS-associated mutations that we introduced into Da led to similar effects in vivo as the same mutations in TCF4 in vitro. Consequently, our Drosophila models of PTHS are applicable for further studies aiming to unravel the molecular mechanisms of this disorder.

Highlights

Pitt-Hopkins syndrome (PTHS, OMIM #610954) is a rare human disorder characterised by severe developmental delay, autistic behaviours, absence of speech, distinct facial features, epilepsy, constipation and hyperventilation (Pitt and Hopkins, 1978; Whalen et al, 2012)
Even though the entire amino acid sequence homology between human E-proteins and Da is below 50%, the amino acid identity of basic helix-loop-helix (bHLH) domains between Da and human E-proteins reaches 75% (Fig. 1) which allows extrapolation of mutations found in bHLH of Transcription factor 4 (TCF4) of PTHS patients into Da
In this study we show that Da, the only E-protein in Drosophila with highly conserved bHLH domain, functions as human TCF4 orthologue

Summary

Introduction

Pitt-Hopkins syndrome (PTHS, OMIM #610954) is a rare human disorder characterised by severe developmental delay, autistic behaviours, absence of speech, distinct facial features, epilepsy, constipation and hyperventilation (Pitt and Hopkins, 1978; Whalen et al, 2012). Partial gene deletions, frame shift, nonsense, splice site or missense mutations in the TCF4 gene have been found in PTHS patients. These mutations are usually sporadic, but in some cases children. PTHS-associated missense mutations result in hypomorphic, non-functional or dominant-negative TCF4 alleles (Sepp et al, 2012). It is unclear whether mutations causing PTHS impair development of the nervous system or functioning of the adult central nervous system (CNS), or both. In addition to PTHS, TCF4 is associated with several other human diseases such as schizophrenia, Fuchs’ corneal endothelial dystrophy and primary sclerosing cholangitis (reviewed by Forrest et al, 2014)

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