Role of LSH in the establishment of epigenetic gene silencing
Torrea Muguerza2018.pdf (8.923Mb)
Torrea Muguerza, Natalia Isabel
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DNA methylation is essential for mammalian development and transcriptional repression of genes and retrotransposons during embryo development and in somatic cells. The patterns of DNA methylation are established by de novo DNA methyltransferases, which are regulated by developmental signalling and require access to chromatin. Besides DNA methyltransferases, other proteins have recently been implicated in DNA methylation, such as the ATP-dependent chromatin remodeler LSH. The absence of LSH in mouse embryos leads to defects in DNA methylation and development. In relation to this, mutations in LSH have been found to cause Immunodeficiency–Centromeric instability–Facial anomalies (ICF) syndrome. This syndrome is characterized by centromeric instability and CpG hypomethylation of centromeric satellite repeats, and is most often caused by mutations in the catalytic domain of the DNA methyltransferase DNMT3B. LSH is essential for developmentally programmed de novo DNA methylation of large chromosomal domains including promoters of protein coding genes and repetitive sequences. Importantly, fibroblasts derived from chromatin remodeling ATPase LSH-null mouse embryos, which lack DNA methylation at transposons and specific gene promoters, are capable of re-establishing normal patterns of DNA methylation and transcriptional silencing of misregulated genes upon re-expression of LSH. The ATP hydrolysis by LSH is essential for its function in gene silencing and de novo DNA methylation. However, the molecular mechanisms of LSH-dependent gene silencing and de novo DNA methylation are yet unclear. Here we use an inducible system that enables controlled expression of LSH in Lsh-null mouse embryonic fibroblasts (MEFs) to follow chromatin dynamics, transcriptional silencing and establishment of de novo DNA methylation. This conditionally reversible Lsh knockout cellular system allowed us to study the order of events occurring immediately after LSH restoration in MEF cell lines in order to elucidate the molecular mechanism of LSH-dependent gene silencing. We have demonstrated that LSH upon its restoration localises to the promoters of LSH-dependent loci leading to a mild decrease in the occupancy of H3, which reinforces the previously shown role of LSH as a chromatin remodeler. Simultaneously, there is removal of acetyl groups from H3 tails when LSH is bound to these target regions, which might be facilitated by the interaction of HDACs with LSH. The removal of H3Ac marks is followed by deposition of H3K9me2 by G9a/GLP histone methylases at the same time point when misregulated genes are silenced. This suggests that LSH creates a suitable substrate for G9a/GLP promoting gene silencing. Surprisingly, transcriptional repression occurs without acquisition of DNA methylation at the promoters of these loci. This order of events implies that LSH plays a role as a chromatin remodeler leading to changes in chromatin structure and modifications that facilitate epigenetic gene silencing without DNA methylation in the initial period when LSH is restored in MEF cell lines. Furthermore, deposition of H3K9me2 by the G9a/GLP complex is critical for silencing of specific genes, but not for repetitive elements such as IAPs. The histone modification H3K27me3 seems to play a transitory role in the silencing of IAP retrotransposons in the absence of G9a/GLP activity. In conclusion, this work has demonstrated that changes in chromatin modifications leading to a transcriptionally repressive chromatin state can be established in somatic cells by the chromatin remodeler LSH without acquisition of DNA methylation. This suggests that the primary role of LSH is to promote changes in chromatin structure and modifications that lead to gene silencing and not DNA methylation, which most likely occurs as a consequence of transcriptional silencing.