Biomedical subjects

Jun Ueda

Publications and source records attributed to Jun Ueda.

12 recordsLinked to original sources

PubMed2006

Zinc finger protein Wiz links G9a/GLP histone methyltransferases to the co-repressor molecule CtBP.

G9a is a SET-domain mammalian histone methyltransferase responsible for mono- and dimethylation of lysine 9 in histone H3 (H3K9) at euchromatic regions. Recently we reported that G9a forms a stoichiometric heteromeric complex with another SET-domain-containing molecule, GLP/Eu-HMTase1. Although G9a and GLP can independently methylate H3K9 in vitro, G9a/GLP heteromeric formation seems to be essential for their function as a euchromatic H3K9 methyltransferase in vivo. To further elucidate how G9a/GLP-mediated histone methylation and transcriptional regulation are controlled, we purified and characterized G9a complexes from mouse embryonic stem cells. We identified a novel G9a/GLP-associating zinc finger molecule named Wiz that can interact with G9a and GLP independently but is more stable in the G9a/GLP heteromeric complexes. Interestingly, Wiz small inhibitory RNA knocks down not only Wiz but also G9a. GLP deficiency also decreases G9a levels, suggesting that the Wiz/G9a/GLP tri-complex may protect G9a from degradation and that Wiz plays a major role in G9a/GLP heterodimer formation. Furthermore, amino acid sequence analysis of Wiz predicted two potential CtBP binding sites, and indeed CtBP binding to Wiz and association of CtBP with the Wiz/G9a/GLP complex was observed. These data indicate that Wiz not only contributes to the stability of G9a but also links the G9a/GLP heteromeric complex to the CtBP co-repressor machinery.

Amino Acid Sequence↗

PubMed2005

Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9.

Histone H3 Lys 9 (H3-K9) methylation is a crucial epigenetic mark for transcriptional silencing. G9a is the major mammalian H3-K9 methyltransferase that targets euchromatic regions and is essential for murine embryogenesis. There is a single G9a-related methyltransferase in mammals, called GLP/Eu-HMTase1. Here we show that GLP is also important for H3-K9 methylation of mouse euchromatin. GLP-deficiency led to embryonic lethality, a severe reduction of H3-K9 mono- and dimethylation, the induction of Mage-a gene expression, and HP1 relocalization in embryonic stem cells, all of which were phenotypes of G9a-deficiency. Furthermore, we show that G9a and GLP formed a stoichiometric heteromeric complex in a wide variety of cell types. Biochemical analyses revealed that formation of the G9a/GLP complex was dependent on their enzymatic SET domains. Taken together, our new findings revealed that G9a and GLP cooperatively exert H3-K9 methyltransferase function in vivo, likely through the formation of higher-order heteromeric complexes.

Animals↗

PubMed2005

Developmentally regulated expression of Sp1 in the mouse cornea.

PURPOSE: To examine the expression of transcription factor Sp1 in the cornea of the mouse eye throughout developmental stages. The environmental effect of light on Sp1 expression was also assessed. METHODS: C57BL/6 mice were set up for timed mating. Embryos on embryonic day (E)10.5, E12.5, E15.5, and E18.5 and eyes from mice on postnatal day (P)0, P7, P11, P15, P30, and P60 were collected for immunohistochemical staining and in situ hybridization. One group of mice was bred strictly in the dark between E18.5 and P15, and the eyes were collected at P0, P7, P11, and P15 time points. RESULTS: Sp1 expression was observed in the ectoderm and lens vesicle as early as E10.5. Both Sp1 protein and mRNA were abundant in the corneal basal epithelium and keratocytes until P11. Their levels were markedly reduced at P15, right after eyelid opening, and declined further between P15 and P60. In those mice bred in the dark, Sp1 was evident in the cornea at P0. The Sp1 level gradually increased until P11 and was decreased at P15. This expression pattern was nearly identical in mice bred either in a light/dark cycle or in the dark. The Sp1 level in the central lens epithelium was much lower than that in the cornea from E15.5 to late stages. CONCLUSIONS: The present study indicates that Sp1 expression is developmentally regulated, providing a basis for further investigations on the regulation of the Sp1 gene during the course of corneal development and in diseases such as keratoconus.

Animals↗

PubMed2003

Distribution of myocilin and extracellular matrix components in the corneoscleral meshwork of human eyes.

PURPOSE: To examine ultrastructurally the composition of major extracellular matrix (ECM) components and the distribution of myocilin in the trabecular lamellae of corneoscleral (CS) meshwork in normal human eyes. The codistribution of myocilin with ECM components was also investigated. METHODS: Postembedding immunoelectron microscopic studies were performed with antibodies against myocilin and other ECM components, including fibronectin, laminin, vitronectin, tenascin, elastin, fibrillin-1, microfibril-associated glycoprotein (MAGP)-1, decorin, versican, hyaluronic acid, and five types of collagen (I, III, IV, V, and VI). Double labeling of myocilin with other ECM components was performed with different sized gold particles. RESULTS: In the trabecular beams of CS meshwork, fibronectin, laminin, and collagen type IV were associated with basement membranes, whereas elastin was specifically localized to the core of elastic-like fibers. Several types of collagens, glycoproteins, proteoglycans, and hyaluronic acid were detected both in the collagen fibers and ground substances. Myocilin predominantly localized in the long-spacing collagens and sheath materials surrounding elastic-like fibers, codistributed with fibronectin, fibrillin-1, MAGP-1, decorin, and type VI collagen. CONCLUSIONS: This study illustrated the composition of ECM materials in the trabecular lamellae of CS meshwork. Myocilin was specifically localized to long-spacing collagens and the surrounding sheath of elastic-like fibers interacting with microfibril-associated elements where changes have been documented to occur in glaucomatous and aging eyes.

Adult↗

PubMed2002

G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis.

Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes.

Acetylation↗

PubMed2002

Reporter gene construct containing 1.4-kB alpha 1-proteinase inhibitor promoter confers expression in the cornea of transgenic mice.

A 1.4-kb human alpha1-proteinase inhibitor (alpha1-PI) 5'-flanking sequence fused to the E. coli LacZ gene was used to generate transgenic mice. The 1.4-kb alpha 1-PI fragment was found to target LacZ expression preferentially in the epithelium and stroma of the mouse cornea, and moderately or weakly in white blood cells and a few other tissues, such as the skin and brain. This finding implies that the alpha 1-PI promoter may offer an option for targeting foreign genes in both the epithelial and stromal layers of the cornea in future transgenic experiments.

Animals↗

PubMed2002

Myocilin is associated with mitochondria in human trabecular meshwork cells.

The trabecular meshwork (TM) is a specialized tissue located at the chamber angle of the eye next to the cornea. This tissue is believed to be responsible for regulation of the aqueous humor outflow and control of the intraocular pressure (IOP). Alterations in functions of the TM may lead to IOP elevation and development of glaucoma, a major cause of blindness. The myocilin gene has recently been directly linked to open-angle glaucomas. The gene product was originally identified as a protein inducible in TM cells by treatment with glucocorticoids such as dexamethasone (DEX) and termed TIGR (TM inducible-glucocorticoid response). The exact nature and function of the myocilin protein so far still remain elusive. In this study, myocilin was localized to the perinuclear region of both DEX-treated and control TM cells. Its distribution overlapped considerably with that of mitochondria. Subcellular fractionation and Western blot analyses suggested a rather extensive association of myocilin with mitochondria. The DEX-treated TM cells were found to undergo apoptosis, when exposed to anti-Fas antibody, to a significantly higher degree than the untreated control cells. It appears that the TM cell integrity remains intact after DEX treatment. However, the induced myocilin or myocilin-mitochondria association seems to render the cells more susceptible to a second stress or challenge. This vulnerability may be the basis that ultimately leads to pathological consequences.

Antibodies↗

PubMed2002

Protein interactions with myocilin.

PURPOSE: To identify factors that interact in vivo with myocilin, a glaucoma gene product. METHODS: The yeast two-hybrid system with myocilin as the bait and a human skeletal muscle cDNA library as the prey was used to identify potential factors that interact with myocilin. Interactions were also examined in bovine trabecular meshwork (TM) cells through a mammalian two-hybrid system. Biochemical coimmunoprecipitation from both human TM cell lysate and in vitro translated proteins was also used to confirm results obtained from yeast analysis. RESULTS: Twenty positive clones isolated through yeast two-hybrid screening were deemed potential myocilin partners. Sequence analysis determined that two of them encoded for myocilin from amino acids 64 to 268. Myocilin was also found to interact with a component of the myosin motor protein, myosin regulatory light chain (RLC). The myocilin-myocilin and myocilin-RLC interactions revealed by the yeast system were further confirmed and demonstrated in cultured TM cells, by means of a mammalian two-hybrid system, and through biochemical coimmunoprecipitation, subcellular fractionation, immunofluorescence, and immunogold double labeling. CONCLUSIONS: These results indicate that myocilin can form homomultimers in vivo, independent of the olfactomedin-like domain. Further analysis established that the leucine zipper motif of myocilin may be necessary for the myocilin-RLC interaction. The interaction of myocilin with RLC, a component of the myosin motor protein complex, implies a role for myocilin in the actomyosin system, linking in turn this novel protein to functional status of the TM.

Animals↗

PubMed2002

Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes.

PURPOSE: To examine ultrastructurally the composition of extracellular matrix (ECM) materials, the distribution of myocilin, and the colocalization of myocilin with ECM components in the juxtacanalicular tissue (JCT) of normal human eyes. METHODS: Postembedding immunoelectron microscopic studies were performed with antibodies specific for major ECM components, including fibronectin, laminin, vitronectin, tenascin, elastin, fibrillin-1, microfibril-associated glycoprotein (MAGP)-1, decorin, versican, and five types of collagen (I, III, IV, V, and VI). Hyaluronic acid was localized with the use of biotinylated hyaluronic acid-binding protein. Colloidal gold labeling was also performed using an anti-human myocilin polyclonal antibody. Colocalization of myocilin with ECM components was examined by double labeling, using different-sized gold particles. The possible interaction between myocilin and ECM molecules was evaluated by in vitro binding assays. RESULTS: Amorphous basement membrane-like materials in the JCT were confirmed to be made up chiefly of collagen type IV, laminin, and fibronectin. Elastin was localized to the central core of sheath-derived plaques. Fibronectin, fibrillin-1, MAGP-1, decorin, and type VI collagen were all localized to clusters of the banded material in the sheath surrounding the core, where several types of collagen, glycoproteins, and proteoglycans were also detected. Myocilin was found to associate mainly with the sheath material, overlapping extensively in distribution with fibronectin, fibrillin-1, and MAGP-1 and moderately with decorin and type VI collagen. Its localization was distinct from that of elastin. Interactions of myocilin with molecules such as fibronectin and fibrillin-1 were confirmed biochemically. CONCLUSIONS: This study illustrated ultrastructurally the composition of ECM materials in the JCT of normal human eyes. The key finding was the association of myocilin with microfibrillar architecture in sheath-derived plaques where pathologic changes have been documented to occur in eyes of patients with primary open-angle glaucoma.

Adult↗

PubMed2002

Age independent expression of myocilin in the human trabecular meshwork.

Glaucoma is a leading cause of blindness. Primary open-angle glaucoma, the most common form of glaucoma, is known to increase in prevalence with age. One of its major risk factors is an elevated intraocular pressure believed to be related to the trabecular meshwork (TM), a specialized tissue located at the chamber angle of the eye. Myocilin, a gene linked to open-angle glaucomas (OAG), has been found to be expressed in the TM and a broad range of other ocular and non-ocular tissues. The purpose of this study was to examine the distribution of myocilin protein and mRNA in the TM of normal human eyes to determine whether age-related changes exist. Studies with glaucomatous eyes were carried out concurrently. Immunoperoxidase staining experiments demonstrated positive immunoreactivity for myocilin protein in both the cells and beams in all regions of the TM. The staining intensity and the myocilin level as determined by dot blot assays were not correlated with the ages of donors ranging from 8 weeks to 93 years. Neither did myocilin mRNA, detected by in situ hybridization in cells throughout the TM and quantified by relative quantitative RT-PCR, vary with age. Myocilin mRNA and protein expression in diseased tissues was either comparable to that of normal tissues or reduced. This study represents the first in-depth investigation of myocilin expression in relation to age in the TM of human eyes. The results indicate an age independence, argue thus against a direct role of myocilin and reiterate the involvement of additional factors in the pathogenesis of glaucoma.

Adolescent↗

PubMedDate not supplied

Comparison of fornix- and limbus-based conjunctival flaps in mitomycin C trabeculectomy with laser suture lysis in Japanese glaucoma patients.

PURPOSE: To analyze the results of mitomycin C (MMC) trabeculectomy and laser suture lysis using either fornix-based (FB) or limbus-based (LB) conjunctival flaps. METHODS: Eighty-two Japanese glaucoma patients were treated by MMC trabeculectomy, with 38 eyes receiving an FB conjunctival flap and 44 eyes receiving an LB conjunctival flap. Postoperative intraocular pressure (IOP), the probability of success, and complications were compared between the FB and LB groups. RESULTS: The IOP of the FB group decreased from 21.6+/-7.90 mmHg to 9.75+/-3.23 mmHg at 12 months postoperation, and the IOP of the LB group decreased from 21.3+/-6.77 mmHg to 9.30+/-3.16 mmHg. The postoperative IOPs were similar in the two groups at all postoperative time points. A life-table analysis (Kaplan-Meier method) showed that the survival rate was similar in the two groups but tended to be lower in the early postoperative period in the FB group. Complications were similar also, with the exception of increased leakage in the FB group. CONCLUSIONS: MMC trabeculectomy results were similar between FB and LB conjunctival flaps, in agreement with previous reports. However, increased care may be warranted when an FB flap is used in MMC trabeculectomy.

Antibiotics, Antineoplastic↗

PubMedDate not supplied

The outcome of mitomycin C trabeculectomy and laser suture lysis depends on postoperative management.

PURPOSE: To verify that postoperative management affects the outcome of mitomycin C (MMC) trabeculectomy and suture lysis. METHODS: A total of 108 eyes in 108 Japanese patients were treated with MMC trabeculectomy. They were divided into two groups based on when the operation was performed: group A, 57 eyes in 1998, and group B, 51 eyes in 2001. The results, including postoperative intraocular pressure (IOP), complications, and postoperative management, were compared between groups. In addition, they were evaluated by a Kaplan-Meier life-table analysis. RESULTS: Postoperative IOP was lower and the probability of success by life-table analysis was higher in group B than in group A. Postoperative management, including laser suture lysis and subconjunctival injection of 5-fluorouracil, was started significantly earlier in group B than in group A patients. Although more postoperative complications occurred in group B, none were severe and all were adequately managed. CONCLUSIONS: Starting postoperative management earlier, particularly laser suture lysis, may be necessary to achieve lower and longer IOP control in MMC trabeculectomy.

Antibiotics, Antineoplastic↗