DNA-binding sequence specificity of DUX4.

John K. Lee,Yu Zhang,Joslynn S. Lee,Matthew Slattery,Erik A. Toso,Si Ho Choi,Hideki Aihara,Michael Kyba

doi:10.1186/s13395-016-0080-z

Abstract

BackgroundMisexpression of the double homeodomain transcription factor DUX4 results in facioscapulohumeral muscular dystrophy (FSHD). A DNA-binding consensus with two tandem TAAT motifs based on chromatin IP peaks has been discovered; however, the consensus has multiple variations (flavors) of unknown relative activity. In addition, not all peaks have this consensus, and the Pitx1 promoter, the first DUX4 target sequence mooted, has a different TAAT-rich sequence. Furthermore, it is not known whether and to what extent deviations from the consensus affect DNA-binding affinity and transcriptional activation potential.ResultsHere, we take both unbiased and consensus sequence-driven approaches to determine the DNA-binding specificity of DUX4 and its tolerance to mismatches at each site within its consensus sequence. We discover that the best binding and the greatest transcriptional activation are observed when the two TAAT motifs are separated by a C residue. The second TAAT motif in the consensus sequence is actually (T/C)AAT. We find that a T is preferred here. DUX4 has no transcriptional activity on “half-sites”, i.e., those bearing only a single TAAT motif. We further find that DUX4 does not bind to the TAATTA motif in the Pitx1 promoter, that Pitx1 sequences have no competitive band shift activity, and that the Pitx1 sequence is transcriptionally inactive, calling into question PITX1 as a DUX4 target gene. Finally, by multimerizing binding sites, we find that DUX4 transcriptional activation demonstrates tremendous synergy and that at low DNA concentrations, at least two motifs are necessary to detect a transcriptional response.ConclusionsThese studies illuminate the DNA-binding sequence preferences of DUX4.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0080-z) contains supplementary material, which is available to authorized users.

Highlights

Misexpression of the double homeodomain transcription factor DUX4 results in facioscapulohumeral muscular dystrophy (FSHD)
This analysis showed that all four variants could be recognized by DUX4, resulting in dox-dependent luciferase induction, the motif containing a central cytosine followed by a thymidine (TAATCTAATCA) had the greatest transcriptional activity in vivo (Fig. 1a)
Combined with the fact that this 30-bp sequence is not completely conserved between mouse and human, that the expected chromatin immunoprecipitation (ChIP)-seq peak was not found over the region corresponding to this 30-bp sequence in humans, or anywhere near PITX1 [11], and the fact that PITX1 was not found to be strongly upregulated by DUX4 in various human cell systems [11, 19], these data argue strongly against the model in which FSHD is caused by DUX4mediated overexpression of PITX1 [10]

Summary

Introduction

Misexpression of the double homeodomain transcription factor DUX4 results in facioscapulohumeral muscular dystrophy (FSHD). Facioscapulohumeral muscular dystrophy (FSHD) is arguably the most prevalent genetic disease of muscle [1, 2] It is caused by altered regulation of the subtelomeric chr4q macrosatellite repeat, D4Z4 [3,4,5,6]. This 3.3-kb macrosatellite sequence is typically present in ~30 tandem copies [7], while most cases of FSHD involve array contractions bringing the number of tandem repeats down to 10 or fewer [8, 9]. When this happens in the context of an allele that provides a downstream polyA signal [15], a muscle pathology ensues

Objectives

Methods

Results

Discussion

Conclusion