P.48The role of Dnmt3b in DUX4 repression in transgenic mice

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in muscle cells. DUX4 is encoded in the D4Z4 macrosatellite repeat array, consisting of 8-100 repeat units in non-affected individuals. The most common form of FSHD, FSHD type 1, is caused by a contraction of the D4Z4 repeat to 1-10 repeat units, leading to somatic derepression of DUX4. FSHD type 2 (FSHD2) is often caused by mutations in SMCHD1, an epigenetic repressor of the D4Z4 repeat. Recently, DNMT3B mutations were identified in FSHD2 patients that lack a mutation in SMCHD1. DNMT3B is a de novo DNA methyltransferase, but the exact role of DNMT3B on repression of DUX4 remains unknown. To study the role of Dnmt3b on DUX4 repression, we crossed mice hemizygous for a FSHD-sized D4Z4 repeat (D4Z4-2.5 mice) with mice carrying a heterozygous mutation in Dnmt3b leading to exon 13 skipping (Dnmt3bMommeD14 mice). While we did not find any significant changes in DUX4 expression for several skeletal muscles and non-muscle tissues, we observed a significantly higher DUX4 expression in the spleen and lymph nodes of D4Z4-2.5/Dnmt3bMommeD14 mice compared with D4Z4-2.5 mice. In addition, we found lower DNA methylation levels at the D4Z4 repeat in D4Z4-2.5/Dnmt3bMommeD14 mice. Dnmt3b siRNA knockdown experiments in murine embryonic stem cells (mESCs) carrying either a long D4Z4 repeat (12.5 repeat units) or a short D4Z4 repeat (2.5 repeat units) confirmed that in mESCs with a short D4Z4 repeat high DUX4 expression levels were further enhanced after Dnmt3b knockdown. In contrast, DUX4 expression was not affected after Dnmt3b knockdown in mESCs with a long D4Z4 repeat that normally do not express DUX4. In short, our data suggest that Dnmt3b is involved in DUX4 repression in some somatic tissues and mESCs of transgenic FSHD mice.