title: Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts.
abstract: BACKGROUND
  TGFβ1-induced expression of transforming growth factor β-induced protein (TGFBIp) and extracellular matrix (ECM) genes plays a major role in the development of granular corneal dystrophy type 2 (GCD2: also called Avellino corneal dystrophy). Although some key transcription factors are known, the epigenetic mechanisms modulating TGFBIp and ECM expression remain unclear. We examined the role of chromatin markers such as histone H3 lysine methylation (H3Kme) in TGFβ1-induced TGFBIp and ECM gene expression in normal and GCD2-derived human corneal fibroblasts.
  
  
  METHODS
  Wild-type (n = 3), GCD2-heterozygous (n = 1), and GCD2-homozygous (n = 3) primary human corneal fibroblasts were harvested from human donors and patients prepared. Microarray and gene-expression profiling, Chromatin immunoprecipitation microarray analysis, and Methylated DNA isolation assay-assisted CpG microarrays was performed in Wild-type and GCD2-homozygous human cells.
  
  
  RESULTS
  Transcription and extracellular-secretion levels of TGFBIp were high in normal cells compared with those in GCD2-derived cells and were related to H3K4me3 levels but not to DNA methylation over the TGFBI locus. TGFβ1 increased the expression of TGFBIp and the ECM-associated genes connective tissue growth factor, collagen-α2[Ι], and plasminogen activator inhibitor-1 in normal corneal fibroblasts. Increased levels of gene-activating markers (H3K4me1/3) and decreased levels of repressive markers (H3K27me3) at the promoters of those gene accompanied the changes in expression. TGFβ1 also increased recruitment of the H3K4 methyltransferase MLL1 and of SET7/9 and also the binding of Smad3 to the promoters. Knockdown of both MLL1 and SET7/9 significantly blocked the TGFβ1-induced gene expression and inhibited TGFβ1-induced changes in promoter H3K4me1/3 levels. Those effects were very weak, however, in GCD2-derived corneal fibroblasts.
  
  
  CONCLUSIONS
  Taken together, the results show the functional role of H3K4me in TGFβ1-mediated TGFBIp and ECM gene expression in corneal fibroblasts. Pharmacologic and other therapies that regulate these modifications could have potential cornea-protective effects for granular corneal dystrophy.
authors.affiliation: Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul, 120-752, South Korea.
authors.forename: Yong-Sun
authors.initials: YS
authors.lastname: Maeng
authors.affiliation: Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul, 120-752, South Korea.
authors.forename: Ga-Hyun
authors.initials: GH
authors.lastname: Lee
authors.affiliation: Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul, 120-752, South Korea.
authors.forename: Seung-Il
authors.initials: SI
authors.lastname: Choi
authors.affiliation: Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Kyu Seo
authors.initials: KS
authors.lastname: Kim
authors.affiliation: Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul, 120-752, South Korea. mysdm70@gmail.com.|Institute of Vision Research, Severance Biomedical Science Institute, Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea. mysdm70@gmail.com.
authors.forename: Eung Kweon
authors.initials: EK
authors.lastname: Kim
mesh_terms: D003315:Cornea / Q000166:cytology / Q000473:pathology; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000473:pathology*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006720:Homozygote; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D051788:Myeloid-Lymphoid Leukemia Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D051900:Smad3 Protein / Q000378:metabolism; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D000099222:betaIG-H3 Protein
chemical_list: D016326:Extracellular Matrix Proteins; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones; D008239:Lysine; D051788:Myeloid-Lymphoid Leukemia Protein; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D000099222:betaIG-H3 Protein; C497422:KMT2A protein, human; C540312:SETD7 protein, human