Influence of Repressive Histone and DNA Methylation upon D4Z4 Transcription in Non-Myogenic Cells.

Sunny Das,Brian P Chadwick,Michael Kyba

doi:10.1371/journal.pone.0160022

Abstract

We looked at a disease-associated macrosatellite array D4Z4 and focused on epigenetic factors influencing its chromatin state outside of the disease-context. We used the HCT116 cell line that contains the non-canonical polyadenylation (poly-A) signal required to stabilize somatic transcripts of the human double homeobox gene DUX4, encoded from D4Z4. In HCT116, D4Z4 is packaged into constitutive heterochromatin, characterized by DNA methylation and histone H3 tri-methylation at lysine 9 (H3K9me3), resulting in low basal levels of D4Z4-derived transcripts. However, a double knockout (DKO) of DNA methyltransferase genes, DNMT1 and DNMT3B, but not either alone, results in significant loss of DNA and H3K9 methylation. This is coupled with upregulation of transcript levels from the array, including DUX4 isoforms (DUX4-fl) that are abnormally expressed in somatic muscle in the disease Facioscapulohumeral muscular dystrophy (FSHD) along with DUX4 protein, as indicated indirectly by upregulation of bondafide targets of DUX4 in DKO but not HCT116 cells. Results from treatment with a chemical inhibitor of histone methylation in HCT116 suggest that in the absence of DNA hypomethylation, H3K9me3 loss alone is sufficient to facilitate DUX4-fl transcription. Additionally, characterization of a cell line from a patient with Immunodeficiency, Centromeric instability and Facial anomalies syndrome 1 (ICF1) possessing a non-canonical poly-A signal and DNA hypomethylation at D4Z4 showed DUX4 target gene upregulation in the patient when compared to controls in spite of retention of H3K9me3. Taken together, these data suggest that both DNA methylation and H3K9me3 are determinants of D4Z4 silencing. Moreover, we show that in addition to testis, there is appreciable expression of spliced and polyadenylated D4Z4 derived transcripts that contain the complete DUX4 open reading frame (ORF) along with DUX4 target gene expression in the thymus, suggesting that DUX4 may provide normal function in this somatic tissue.

Highlights

Macrosatellite repeats (MSR) are composed of near identical repeat units arranged in tandem that can span anywhere from tens to thousands of kilobases in the human genome [1,2,3].PLOS ONE | DOI:10.1371/journal.pone.0160022 July 28, 2016Chromatin and D4Z4 TranscriptionWhat biological purpose is served by the arrangement and maintenance of these large tandem repeats is unclear, given that many of these MSRs do not code for a functional gene product
D4Z4 is packaged into constitutive heterochromatin in HCT116 cells, which possess 4qA allele
double homeobox 4 (DUX4) containing D4Z4 transcripts can be stabilized by polyadenylation using the canonical polyA signal associated with exon-7, as seen in the germline [27], or for those cells that possess a 4qA haplotype on a chromosome 4, the non-canonical polyadenylation signal AUUAAA (ATTAAA in genomic DNA) found in exon-3 that is unique to 4qA variants can potentially be used [10]

Summary

Introduction

Macrosatellite repeats (MSR) are composed of near identical repeat units arranged in tandem that can span anywhere from tens to thousands of kilobases (kb) in the human genome [1,2,3].PLOS ONE | DOI:10.1371/journal.pone.0160022 July 28, 2016Chromatin and D4Z4 TranscriptionWhat biological purpose is served by the arrangement and maintenance of these large tandem repeats is unclear, given that many of these MSRs do not code for a functional gene product. While some MSRs, such as DXZ4 at Xq23 have regulatory roles such as mediating long-range interactions between regions of chromatin [4,5], others such as TAF11-Like MSR at 5p15.1 and D4Z4 at 4q35.2 have been shown to segregate with diseases such as schizophrenia [6] and onset of different types of FSHD (OMIM # 158900; 158901) [7,8,9,10,11], respectively These instances clearly demonstrate the potential impact repeat elements can have on human health. No study has investigated how D4Z4 silencing is maintained in cell types outside of the context of the disease

Methods

Results

Conclusion