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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

MLL1 knockdown attenuated the TGFβ1-induced expression of TGFBIp and ECM-associated genes. a Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, MLL1, H3K4me3, TGFBIp, and actin protein levels were analyzed by western blot. b-d Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGFβ1 (5 ng/ml) for 8 h, and mRNA and protein levels of TGFBIp were analyzed by RT-qPCR (b) and western blot (c). mRNA levels of ECM-associated genes were analyzed by RT-qPCR (d). Gene expression was normalized to that of GAPDH (internal control), and results are expressed as fold stimulation over control shRNA-infected wild-type control (mean ± standard error (SE); **P < 0.01 vs. control, n = 3)
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Fig7: MLL1 knockdown attenuated the TGFβ1-induced expression of TGFBIp and ECM-associated genes. a Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, MLL1, H3K4me3, TGFBIp, and actin protein levels were analyzed by western blot. b-d Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGFβ1 (5 ng/ml) for 8 h, and mRNA and protein levels of TGFBIp were analyzed by RT-qPCR (b) and western blot (c). mRNA levels of ECM-associated genes were analyzed by RT-qPCR (d). Gene expression was normalized to that of GAPDH (internal control), and results are expressed as fold stimulation over control shRNA-infected wild-type control (mean ± standard error (SE); **P < 0.01 vs. control, n = 3)

Mentions: Corneal fibroblasts were first infected with lentivirus containing shRNA targeting MLL1 (shMLL1) or control shRNA (shCont), and protein levels were analyzed by western blot. As shown in Fig. 7a, MLL1 protein levels were significantly reduced in the wild-type and GCD2 cells infected with shMLL1 lentivirus compared with those in wild-type and GCD2 cells infected with shCont lentivirus. We next examined whether MLL1 shRNA can affect H3K4me3 in corneal fibroblasts, because previous studies showed that MLL1 regulates H3K4me3 in embryonic fibroblasts and immune cells [34–36]. Global H3K4me3 levels were significantly reduced in both the wild-type cells and the GCD2 cells infected with shMLL1 lentivirus (Fig. 7a). Also, TGFBIp protein levels were reduced in cells infected with shMLL1 lentivirus. Furthermore, shMLL1 significantly attenuated TGFβ1-induced TGFBIp protein and mRNA levels in wild-type corneal fibroblasts compared with shCont (Fig. 7b, c). The GCD2-homozygous cells showed very weak TGFBIp expression and inhibition induced by TGFβ1 and shMLL1, respectively (Fig. 7b, c). TGFβ1-induced COL1A2, PAI-1, and CTGF mRNA levels were inhibited by shMLL1 (Fig. 7d), whereas no significant increase and decrease in response to TGFβ1 and shMLL1 were observed in the GCD2-homozygous cells (Fig. 7d), suggesting a key role for MLL1 in modulating TGFβ1 responses in corneal fibroblasts.Fig. 7


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)

MLL1 knockdown attenuated the TGFβ1-induced expression of TGFBIp and ECM-associated genes. a Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, MLL1, H3K4me3, TGFBIp, and actin protein levels were analyzed by western blot. b-d Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGFβ1 (5 ng/ml) for 8 h, and mRNA and protein levels of TGFBIp were analyzed by RT-qPCR (b) and western blot (c). mRNA levels of ECM-associated genes were analyzed by RT-qPCR (d). Gene expression was normalized to that of GAPDH (internal control), and results are expressed as fold stimulation over control shRNA-infected wild-type control (mean ± standard error (SE); **P < 0.01 vs. control, n = 3)
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Fig7: MLL1 knockdown attenuated the TGFβ1-induced expression of TGFBIp and ECM-associated genes. a Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, MLL1, H3K4me3, TGFBIp, and actin protein levels were analyzed by western blot. b-d Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGFβ1 (5 ng/ml) for 8 h, and mRNA and protein levels of TGFBIp were analyzed by RT-qPCR (b) and western blot (c). mRNA levels of ECM-associated genes were analyzed by RT-qPCR (d). Gene expression was normalized to that of GAPDH (internal control), and results are expressed as fold stimulation over control shRNA-infected wild-type control (mean ± standard error (SE); **P < 0.01 vs. control, n = 3)
Mentions: Corneal fibroblasts were first infected with lentivirus containing shRNA targeting MLL1 (shMLL1) or control shRNA (shCont), and protein levels were analyzed by western blot. As shown in Fig. 7a, MLL1 protein levels were significantly reduced in the wild-type and GCD2 cells infected with shMLL1 lentivirus compared with those in wild-type and GCD2 cells infected with shCont lentivirus. We next examined whether MLL1 shRNA can affect H3K4me3 in corneal fibroblasts, because previous studies showed that MLL1 regulates H3K4me3 in embryonic fibroblasts and immune cells [34–36]. Global H3K4me3 levels were significantly reduced in both the wild-type cells and the GCD2 cells infected with shMLL1 lentivirus (Fig. 7a). Also, TGFBIp protein levels were reduced in cells infected with shMLL1 lentivirus. Furthermore, shMLL1 significantly attenuated TGFβ1-induced TGFBIp protein and mRNA levels in wild-type corneal fibroblasts compared with shCont (Fig. 7b, c). The GCD2-homozygous cells showed very weak TGFBIp expression and inhibition induced by TGFβ1 and shMLL1, respectively (Fig. 7b, c). TGFβ1-induced COL1A2, PAI-1, and CTGF mRNA levels were inhibited by shMLL1 (Fig. 7d), whereas no significant increase and decrease in response to TGFβ1 and shMLL1 were observed in the GCD2-homozygous cells (Fig. 7d), suggesting a key role for MLL1 in modulating TGFβ1 responses in corneal fibroblasts.Fig. 7

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