Limits...
Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts.

Maeng YS, Lee GH, Choi SI, Kim KS, Kim EK - BMC Med Genomics (2015)

Bottom Line: 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.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.Knockdown of both MLL1 and SET7/9 significantly blocked the TGFβ1-induced gene expression and inhibited TGFβ1-induced changes in promoter H3K4me1/3 levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul, 120-752, South Korea.

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.

No MeSH data available.


Related in: MedlinePlus

TGFβ1 enhanced Smad3 recruitment to the TGFBIp and ECM-associated gene promoters. Smad3 recruitment at the indicated gene promoters in wild-type and GCD2 homozygous corneal fibroblasts was stimulated by TGFβ1 (5 ng/ml) for 8 h. Smad3 recruitment was determined by ChIP assays with Smad3 antibody. Results were normalized to the input, and Smad3 occupancy was expressed as the fold enrichment over respective wild-type control samples (mean ± standard error (SE); **P < 0.01 vs. wild type, n = 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4638082&req=5

Fig4: TGFβ1 enhanced Smad3 recruitment to the TGFBIp and ECM-associated gene promoters. Smad3 recruitment at the indicated gene promoters in wild-type and GCD2 homozygous corneal fibroblasts was stimulated by TGFβ1 (5 ng/ml) for 8 h. Smad3 recruitment was determined by ChIP assays with Smad3 antibody. Results were normalized to the input, and Smad3 occupancy was expressed as the fold enrichment over respective wild-type control samples (mean ± standard error (SE); **P < 0.01 vs. wild type, n = 3)

Mentions: Next, we examined whether TGFβ1 altered the Smad3 occupancy on the promoters of the TGFBIp, COL1A2, PAI-1, and CTGF genes using ChIP assays with Smad3 antibodies. As shown in Fig. 4, Smad3 recruitment was significantly increased on the TGFBIp, COL1A2, PAI-1, and CTGF promoters after TGFβ1 stimulation in the GCD2-homozygous cells relative to that in the wild-type cells (Fig. 4). The GCD2-homozygous cells showed very small increases in Smad3 recruitment to the TGFBIp and ECM-associated gene promoters, however (Fig. 4), suggesting that TGFβ1 increases the expression of TGFBIp and ECM-associated genes by increasing Smad3 recruitment to the TGFBIp and ECM-associated gene promoters in corneal fibroblasts.Fig. 4


Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts.

Maeng YS, Lee GH, Choi SI, Kim KS, Kim EK - BMC Med Genomics (2015)

TGFβ1 enhanced Smad3 recruitment to the TGFBIp and ECM-associated gene promoters. Smad3 recruitment at the indicated gene promoters in wild-type and GCD2 homozygous corneal fibroblasts was stimulated by TGFβ1 (5 ng/ml) for 8 h. Smad3 recruitment was determined by ChIP assays with Smad3 antibody. Results were normalized to the input, and Smad3 occupancy was expressed as the fold enrichment over respective wild-type control samples (mean ± standard error (SE); **P < 0.01 vs. wild type, n = 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4638082&req=5

Fig4: TGFβ1 enhanced Smad3 recruitment to the TGFBIp and ECM-associated gene promoters. Smad3 recruitment at the indicated gene promoters in wild-type and GCD2 homozygous corneal fibroblasts was stimulated by TGFβ1 (5 ng/ml) for 8 h. Smad3 recruitment was determined by ChIP assays with Smad3 antibody. Results were normalized to the input, and Smad3 occupancy was expressed as the fold enrichment over respective wild-type control samples (mean ± standard error (SE); **P < 0.01 vs. wild type, n = 3)
Mentions: Next, we examined whether TGFβ1 altered the Smad3 occupancy on the promoters of the TGFBIp, COL1A2, PAI-1, and CTGF genes using ChIP assays with Smad3 antibodies. As shown in Fig. 4, Smad3 recruitment was significantly increased on the TGFBIp, COL1A2, PAI-1, and CTGF promoters after TGFβ1 stimulation in the GCD2-homozygous cells relative to that in the wild-type cells (Fig. 4). The GCD2-homozygous cells showed very small increases in Smad3 recruitment to the TGFBIp and ECM-associated gene promoters, however (Fig. 4), suggesting that TGFβ1 increases the expression of TGFBIp and ECM-associated genes by increasing Smad3 recruitment to the TGFBIp and ECM-associated gene promoters in corneal fibroblasts.Fig. 4

Bottom Line: 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.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.Knockdown of both MLL1 and SET7/9 significantly blocked the TGFβ1-induced gene expression and inhibited TGFβ1-induced changes in promoter H3K4me1/3 levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul, 120-752, South Korea.

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.

No MeSH data available.


Related in: MedlinePlus