title: A role for repressive complexes and H3K9 di-methylation in PRDM5-associated brittle cornea syndrome.
abstract: Type 2 brittle cornea syndrome (BCS2) is an inherited connective tissue disease with a devastating ocular phenotype caused by mutations in the transcription factor PR domain containing 5 (PRDM5) hypothesized to exert epigenetic effects through histone and DNA methylation. Here we investigate clinical samples, including skin fibroblasts and retinal tissue from BCS2 patients, to elucidate the epigenetic role of PRDM5 and mechanisms of its dysregulation in disease. First we report abnormal retinal vascular morphology in the eyes of two cousins with BCS2 (PRDM5 Δ exons 9-14) using immunohistochemistry, and mine data from skin fibroblast expression microarrays from patients with PRDM5 mutations p.Arg590* and Δ exons 9-14, as well as from a PRDM5 ChIP-sequencing experiment. Gene ontology analysis of dysregulated PRDM5-target genes reveals enrichment for extracellular matrix (ECM) genes supporting vascular integrity and development. Q-PCR and ChIP-qPCR confirm upregulation of critical mediators of ECM stability in vascular structures (COL13A1, COL15A1, NTN1, CDH5) in patient fibroblasts. We identify H3K9 di-methylation (H3K9me2) at these PRDM5-target genes in fibroblasts, and demonstrate that the BCS2 mutation p.Arg83Cys diminishes interaction of PRDM5 with repressive complexes, including NuRD complex protein CHD4, and the repressive chromatin interactor HP1BP3, by co-immunoprecipitation combined with mass spectrometry. We observe reduced heterochromatin protein 1 binding protein 3 (HP1BP3) staining in the retinas of two cousins lacking exons 9-14 by immunohistochemistry, and dysregulated H3K9me2 in skin fibroblasts of three patients (p.Arg590*, p.Glu134* and Δ exons 9-14) by western blotting. These findings suggest that defective interaction of PRDM5 with repressive complexes, and dysregulation of H3K9me2, play a role in PRDM5-associated disease.
authors.affiliation: Centre for Genomic Medicine, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester Academic Health Science Centre (MAHSC), Manchester, UK, Manchester Royal Eye Hospital, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK, Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.
authors.forename: Louise F
authors.initials: LF
authors.lastname: Porter
authors.affiliation: Stem Cell Program, Boston Children's Hospital, Harvard Stem Cell and Regenerative Biology Department and Harvard Stem Cell Institute, Harvard University, Boston, MA, USA, Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Giorgio G
authors.initials: GG
authors.lastname: Galli
authors.affiliation: Centre for Genomic Medicine, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester Academic Health Science Centre (MAHSC), Manchester, UK.
authors.forename: Sally
authors.initials: S
authors.lastname: Williamson
authors.affiliation: Faculty of Life Sciences, Michael Smith Building, Manchester, UK.
authors.forename: Julian
authors.initials: J
authors.lastname: Selley
authors.affiliation: Faculty of Life Sciences, Michael Smith Building, Manchester, UK.
authors.forename: David
authors.initials: D
authors.lastname: Knight
authors.affiliation: Department of Pediatric Genetics, Marmara University Medical School, Istanbul, Turkey.
authors.forename: Nursel
authors.initials: N
authors.lastname: Elcioglu
authors.affiliation: Department of Ophthalmology, University of Medipol Medical Faculty, Istanbul, Turkey.
authors.forename: Ali
authors.initials: A
authors.lastname: Aydin
authors.affiliation: Department of Ophthalmology, Okmeydani Research and Training Hospital, Istanbul, Turkey.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Elcioglu
authors.affiliation: Centre of Molecular and Biomolecular Informatics, Radboudumc Institute for Molecular Life Sciences, Nijmegen, The Netherlands and.
authors.forename: Hanka
authors.initials: H
authors.lastname: Venselaar
authors.affiliation: Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Anders H
authors.initials: AH
authors.lastname: Lund
authors.affiliation: Manchester Royal Eye Hospital, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK, National Ophthalmic Pathology Service Laboratory, Department of Histopathology, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Bonshek
authors.affiliation: Centre for Genomic Medicine, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester Academic Health Science Centre (MAHSC), Manchester, UK, Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, MAHSC, Manchester, UK graeme.black@manchester.ac.uk.
authors.forename: Graeme C
authors.initials: GC
authors.lastname: Black
authors.affiliation: Centre for Genomic Medicine, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester Academic Health Science Centre (MAHSC), Manchester, UK.
authors.forename: Forbes D
authors.initials: FD
authors.lastname: Manson
mesh_terms: D000328:Adult; D015703:Antigens, CD / Q000235:genetics; D015820:Cadherins / Q000235:genetics; D002648:Child; D003094:Collagen / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004535:Ehlers-Danlos Syndrome / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D063990:Gene Ontology; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008297:Male; D008745:Methylation; D008875:Middle Aged; D009154:Mutation; D009414:Nerve Growth Factors / Q000235:genetics; D000075388:Netrin-1; D012171:Retinal Vessels / Q000473:pathology*; D012867:Skin / Q000166:cytology; D014157:Transcription Factors / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics; D015854:Up-Regulation; D055815:Young Adult; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D003094:Collagen; D004268:DNA-Binding Proteins; D006657:Histones; D009414:Nerve Growth Factors; D000075388:Netrin-1; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D000099152:Cadherin 5; C000617351:NTN1 protein, human; C487762:PRDM5 protein, human