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1.
Methods Mol Biol ; 2611: 39-52, 2023.
Article in English | MEDLINE | ID: mdl-36807062

ABSTRACT

Genome-wide accessible chromatin sequencing and identification has enabled deciphering the epigenetic information encoded in chromatin, revealing accessible promoters, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding. The starting biological materials are often fixed using formaldehyde crosslinking. Here, we describe accessible chromatin library preparation from low numbers of formaldehyde-crosslinked cells using a modified nick translation method, where a nicking enzyme nicks one strand of DNA and DNA polymerase incorporates biotin-conjugated dATP, dCTP, and methyl-dCTP. Once the DNA is labeled, it can be isolated for NGS library preparation. We termed this method as universal NicE-seq (nicking enzyme-assisted sequencing). We also demonstrate a single tube method that enables direct NGS library preparation from low cell numbers without DNA purification. Furthermore, we demonstrated universal NicE-seq on FFPE tissue section sample.


Subject(s)
Chromatin , DNA , DNA/genetics , Nucleosomes , Chromosome Mapping/methods , Sequence Analysis, DNA/methods , Formaldehyde , High-Throughput Nucleotide Sequencing/methods
2.
Genome Biol ; 22(1): 332, 2021 12 06.
Article in English | MEDLINE | ID: mdl-34872606

ABSTRACT

BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.


Subject(s)
Epigenesis, Genetic , Epigenomics/methods , Quality Control , 5-Methylcytosine , Algorithms , CpG Islands , DNA/genetics , DNA Methylation , Epigenome , Genome, Human , High-Throughput Nucleotide Sequencing , Humans , Sequence Alignment , Sequence Analysis, DNA/methods , Sulfites , Whole Genome Sequencing/methods
4.
Genome Res ; 31(7): 1280-1289, 2021 Jul.
Article in English | MEDLINE | ID: mdl-34140313

ABSTRACT

Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

5.
J Mol Biol ; 430(14): 2051-2065, 2018 07 06.
Article in English | MEDLINE | ID: mdl-29758262

ABSTRACT

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication. SAHH enhances DNMT1 activity in vitro, and its overexpression in mammalian cells led to hypermethylation of the genome, whereas its inhibition by adenosine periodate or siRNA-mediated knockdown resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to aberrant gene regulation. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level affects global DNA methylation levels and gene expression.


Subject(s)
Adenosylhomocysteinase/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA Methylation , Proteomics/methods , S-Adenosylhomocysteine/metabolism , Adenosylhomocysteinase/genetics , Animals , COS Cells , Chlorocebus aethiops , DNA Replication , Epigenesis, Genetic , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Metabolic Networks and Pathways , Mutation , Repetitive Sequences, Nucleic Acid , S Phase , Signal Transduction
6.
Epigenetics ; 13(3): 275-289, 2018.
Article in English | MEDLINE | ID: mdl-29498561

ABSTRACT

DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development.


Subject(s)
DNA Methylation/genetics , Epigenesis, Genetic , Myoblasts/metabolism , Transcriptional Activation/genetics , Adult , Aged , Aged, 80 and over , Cell Differentiation/genetics , Child, Preschool , Chromatin/genetics , CpG Islands/genetics , Female , Gene Expression Regulation/genetics , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Histones/genetics , Humans , Male , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/pathology , Neoplasms/genetics , Neoplasms/pathology , Organ Specificity , Promoter Regions, Genetic
7.
Genome Biol ; 18(1): 122, 2017 06 28.
Article in English | MEDLINE | ID: mdl-28655330

ABSTRACT

Open chromatin profiling integrates information across diverse regulatory elements to reveal the transcriptionally active genome. Tn5 transposase and DNase I sequencing-based methods prefer native or high cell numbers. Here, we describe NicE-seq (nicking enzyme assisted sequencing) for high-resolution open chromatin profiling on both native and formaldehyde-fixed cells. NicE-seq captures and reveals open chromatin sites (OCSs) and transcription factor occupancy at single nucleotide resolution, coincident with DNase hypersensitive and ATAC-seq sites at a low sequencing burden. OCSs correlate with RNA polymerase II occupancy and active chromatin marks, while displaying a contrasting pattern to CpG methylation. Decitabine-mediated hypomethylation of HCT116 displays higher numbers of OCSs.


Subject(s)
Chromatin/genetics , DNA Methylation/genetics , Genome, Human/genetics , Regulatory Elements, Transcriptional/genetics , CpG Islands/genetics , Deoxyribonuclease I/genetics , HCT116 Cells , Humans , RNA Polymerase II/genetics , Transposases/genetics
8.
Protein Expr Purif ; 132: 143-151, 2017 04.
Article in English | MEDLINE | ID: mdl-28188826

ABSTRACT

5-Methylcytosine within CpG islands in DNA plays a crucial role in epigenetic transcriptional regulation during metazoan development. Recently, it has been established that the Ten-Eleven Translocation (TET) family, Fe(II)- and 2-oxoglutarate (2OG/αKG)-dependent oxygenases initiate 5-methylcytosine demethylation by iterative oxidation reactions. Mutations in the TET2 gene are frequently detected in patients with myeloid malignancies. Here, we describe the cloning of untagged human TET2 demethylase using Gateway technology and its efficient expression in E. coli. The untagged TET2 enzyme was purified using cation exchange and heparin sepharose chromatography. In addition, a reliable quantitative liquid chromatography-tandem mass spectrometry-based assay was utilized to analyze the activity of TET2 oxygenase. This assay was further used to analyze the activity of a number of clinical TET2 variants with mutations in the 2OG binding sites. Our results demonstrate that the activity of one TET2 mutant, TET2-R1896S, can be restored using an excess of 2OG in the reaction mixture. These studies suggest that dietary 2OG supplements, which are commonly used for several other conditions, may be used to treat some patients with myeloid malignancies harboring TET2-R1896S mutation. Results described in this paper serve as a foundation for better characterization of wild type as well as mutant TET2 demethylases.


Subject(s)
DNA-Binding Proteins , Gene Expression , Oxidoreductases , Proto-Oncogene Proteins , Chromatography, Liquid , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Dioxygenases , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Mass Spectrometry , Oxidoreductases/biosynthesis , Oxidoreductases/chemistry , Oxidoreductases/genetics , Oxidoreductases/isolation & purification , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
9.
Epigenetics ; 12(2): 123-138, 2017 02.
Article in English | MEDLINE | ID: mdl-27911668

ABSTRACT

Differentially methylated or hydroxymethylated regions (DMRs) in mammalian DNA are often associated with tissue-specific gene expression but the functional relationships are still being unraveled. To elucidate these relationships, we studied 16 human genes containing myogenic DMRs by analyzing profiles of their epigenetics and transcription and quantitatively assaying 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) at specific sites in these genes in skeletal muscle (SkM), myoblasts, heart, brain, and diverse other samples. Although most human promoters have little or no methylation regardless of expression, more than half of the genes that we chose to study-owing to their myogenic DMRs-overlapped tissue-specific alternative or cryptic promoters displaying corresponding tissue-specific differences in histone modifications. The 5mC levels in myoblast DMRs were significantly associated with 5hmC levels in SkM at the same site. Hypermethylated myogenic DMRs within CDH15, a muscle- and cerebellum-specific cell adhesion gene, and PITX3, a homeobox gene, were used for transfection in reporter gene constructs. These intragenic DMRs had bidirectional tissue-specific promoter activity that was silenced by in vivo-like methylation. The CDH15 DMR, which was previously associated with an imprinted maternal germline DMR in mice, had especially strong promoter activity in myogenic host cells. These findings are consistent with the controversial hypothesis that intragenic DNA methylation can facilitate transcription and is not just a passive consequence of it. Our results support varied roles for tissue-specific 5mC- or 5hmC-enrichment in suppressing inappropriate gene expression from cryptic or alternative promoters and in increasing the plasticity of gene expression required for development and rapid responses to tissue stress or damage.


Subject(s)
5-Methylcytosine/analogs & derivatives , DNA Methylation , 5-Methylcytosine/metabolism , Adult , Aged , Brain/metabolism , Cadherins/genetics , Cadherins/metabolism , Child, Preschool , Female , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Male , Muscle, Skeletal/metabolism , Myoblasts/metabolism , Myocardium/metabolism , Organ Specificity , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism
10.
Oncotarget ; 7(50): 83627-83640, 2016 Dec 13.
Article in English | MEDLINE | ID: mdl-27845898

ABSTRACT

The transcription factor LSF is highly expressed in hepatocellular carcinoma (HCC) and promotes oncogenesis. Factor quinolinone inhibitor 1 (FQI1), inhibits LSF DNA-binding activity and exerts anti-proliferative activity. Here, we show that LSF binds directly to the maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1) and its accessory protein UHRF1 both in vivo and in vitro. Binding of LSF to DNMT1 stimulated DNMT1 activity and FQI1 negated the methyltransferase activation. Addition of FQI1 to the cell culture disrupted LSF bound DNMT1 and UHRF1 complexes, resulting in global aberrant CpG methylation. Differentially methylated regions (DMR) containing at least 3 CpGs, were significantly altered by FQI1 compared to control cells. The DMRs were mostly concentrated in CpG islands, proximal to transcription start sites, and in introns and known genes. These DMRs represented both hypo and hypermethylation, correlating with altered gene expression. FQI1 treatment elicits a cascade of effects promoting altered cell cycle progression. These findings demonstrate a novel mechanism of FQI1 mediated alteration of the epigenome by DNMT1-LSF complex disruption, leading to aberrant DNA methylation and gene expression.


Subject(s)
Antineoplastic Agents/pharmacology , Benzodioxoles/pharmacology , Carcinoma, Hepatocellular/drug therapy , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA Methylation/drug effects , DNA-Binding Proteins/genetics , Epigenesis, Genetic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Liver Neoplasms/drug therapy , Quinolones/pharmacology , Transcription Factors/genetics , Animals , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , COS Cells , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Cycle/drug effects , Cell Proliferation/drug effects , Chlorocebus aethiops , CpG Islands , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA-Binding Proteins/metabolism , HEK293 Cells , Humans , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Protein Binding , Transcription Factors/metabolism , Transfection , Ubiquitin-Protein Ligases
11.
Nucleic Acids Res ; 44(4): 1642-56, 2016 Feb 29.
Article in English | MEDLINE | ID: mdl-26553800

ABSTRACT

Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity resulting in hypomethylation of the genome that was rescued by transfection of DNMT1. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, overexpression of 14-3-3 also resulted in enhanced cell invasion. Analysis of TCGA breast cancer patient data showed significant correlation for DNA hypomethylation and reduced patient survival with increased 14-3-3 expressions. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNA methylation and altered gene expression that contributes to cell invasion.


Subject(s)
14-3-3 Proteins/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation/genetics , Gene Expression Regulation , 14-3-3 Proteins/metabolism , Animals , Cell Movement/genetics , Cell Proliferation/genetics , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/biosynthesis , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Replication/genetics , Mice , NIH 3T3 Cells , Phosphorylation
12.
PLoS One ; 9(11): e113717, 2014.
Article in English | MEDLINE | ID: mdl-25422886

ABSTRACT

Pathological activation of the hypoxia-inducible-factor (HIF) pathway leading to expression of pro-angiogenic genes, such as vascular endothelial growth factor (VEGF), is the fundamental cause of neovascularization in ocular ischemic diseases and cancers. We have shown that pure honokiol inhibits the HIF pathway and hypoxia-mediated expression of pro-angiogenic genes in a number of cancer and retinal pigment epithelial (RPE) cell lines. The crude extracts, containing honokiol, from Magnolia plants have been used for thousands of years in the traditional oriental medicine for a number of health benefits. We have recently demonstrated that daily intraperitoneal injection of honokiol starting at postnatal day (P) 12 in an oxygen induced retinopathy mouse model significantly reduced retinal neovascularization at P17. Here, we evaluate the mechanism of HIF inhibition by honokiol in RPE cells. Using chromatin immunoprecipitation experiments, we demonstrate that honokiol inhibits binding of HIF to hypoxia-response elements present on VEGF promoter. We further show using a number of in vitro angiogenesis assays that, in addition to anti-HIF effect, honokiol manifests potent anti-angiogenic effect on human retinal micro vascular endothelial cells. Our results suggest that honokiol possesses potent anti-HIF and anti-angiogenic properties. These properties of honokiol make it an ideal therapeutic agent for the treatment of ocular neovascular diseases and solid tumors.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Biphenyl Compounds/therapeutic use , Eye Diseases/drug therapy , Hypoxia-Inducible Factor 1/antagonists & inhibitors , Lignans/therapeutic use , Neovascularization, Pathologic/drug therapy , Angiogenesis Inhibitors/pharmacology , Biphenyl Compounds/pharmacology , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelium, Vascular/drug effects , HeLa Cells , Humans , Lignans/pharmacology , Luciferases/antagonists & inhibitors , Retinal Pigment Epithelium/cytology
13.
Toxicol Rep ; 1: 1152-1161, 2014.
Article in English | MEDLINE | ID: mdl-28962325

ABSTRACT

BACKGROUND: Epigenetic modifications, particularly DNA methylation and posttranslational histone modifications regulate heritable changes in transcription without changes in the DNA sequence. Despite a number of studies showing clear links between environmental factors and DNA methylation, little is known about the effect of environmental factors on the recently identified histone lysine methylation. Since their identification numerous studies have establish critical role played by these enzymes in mammalian development. OBJECTIVES: Identification of the Jumonji (Jmj) domain containing histone lysine demethylase have added a new dimension to epigenetic control of gene expression by dynamic regulation of histone methylation marks. The objective of our study was to evaluate the effect of prohexadione and trinexapac, widely used plant growth regulators of the acylcyclohexanediones class, on the enzymatic activity of histone lysine demethylases and histone modifications during the neural stem/progenitor cell differentiation. METHODS: Here we show that prohexadione, but not trinexapac, directly inhibits non-heme iron (II), 2-oxoglutarate-dependent histone lysine demethylase such as Jmjd2a. We used molecular modeling to show binding of prohexadione to Jmjd2a. We also performed in vitro demethylation assays to show the inhibitory effect of prohexadione on Jmjd2a. Further we tested this molecule in cell culture model of mouse hippocampal neural stem/progenitor cells to demonstrate its effect toward neuronal proliferation and differentiation. RESULTS: Molecular modeling studies suggest that prohexadione binds to the 2-oxoglutarate binding site of Jmjd2a demethylase. Treatment of primary neural stem/progenitor cells with prohexadione showed a concentration dependent reduction in their proliferation. Further, the prohexadione treated neurospheres were induced toward neurogenic lineage upon differentiation. CONCLUSIONS: Our results describe an important chemico-biological interaction of prohexadione, in light of critical roles played by histone lysine demethylases in human health and diseases.

14.
Biochem Biophys Res Commun ; 438(4): 697-702, 2013 Sep 06.
Article in English | MEDLINE | ID: mdl-23921228

ABSTRACT

Aberrant activation of the hypoxia inducible factor (HIF) pathway is the underlying cause of retinal neovascularization, one of the most common causes of blindness worldwide. The HIF pathway also plays critical roles during tumor angiogenesis and cancer stem cell transformation. We have recently shown that honokiol is a potent inhibitor of the HIF pathway in a number of cancer and retinal pigment epithelial cell lines. Here we evaluate the safety and efficacy of honokiol, digoxin, and doxorubicin, three recently identified HIF inhibitors from natural sources. Our studies show that honokiol has a better safety to efficacy profile as a HIF inhibitor than digoxin and doxorubicin. Further, we show for the first time that daily intraperitoneal injection of honokiol starting at postnatal day (P) 12 in an oxygen-induced retinopathy (OIR) mouse model significantly reduced retinal neovascularization at P17. Administration of honokiol also prevents the oxygen-induced central retinal vaso-obliteration, characteristic feature of the OIR model. Additionally, honokiol enhanced physiological revascularization of the retinal vascular plexuses. Since honokiol suppresses multiple pathways activated by HIF, in addition to the VEGF signaling, it may provide advantages over current treatments utilizing specific VEGF antagonists for ocular neovascular diseases and cancers.


Subject(s)
Biphenyl Compounds/therapeutic use , Drugs, Chinese Herbal/therapeutic use , Lignans/therapeutic use , Retina/drug effects , Retina/pathology , Retinal Neovascularization/drug therapy , Retinal Neovascularization/pathology , Animals , Antibiotics, Antineoplastic/therapeutic use , Cell Line , Digoxin/therapeutic use , Doxorubicin/therapeutic use , Enzyme Inhibitors/therapeutic use , Humans , Hypoxia-Inducible Factor 1/antagonists & inhibitors , Hypoxia-Inducible Factor 1/metabolism , Mice , Mice, Inbred C57BL , Oxygen , Retina/metabolism , Retinal Neovascularization/chemically induced , Retinal Neovascularization/genetics , Transcriptional Activation/drug effects
15.
Int J Pharm ; 454(1): 444-52, 2013 Sep 15.
Article in English | MEDLINE | ID: mdl-23827654

ABSTRACT

A decrease in tissue oxygen levels (aka hypoxia) mediates a number of vascular retinal diseases. Despite introduction of novel therapeutics, treatment of retinal disorders remains challenging, possibly due to complex nature of hypoxia signaling. To date, the differential effect of hypoxia on expression of efflux and influx transporters in retinal cells has not been studied. Therefore, the objective of this study was to delineate molecular and functional expression of membrane transporters in human retinal pigment epithelial (RPE) cells cultured under normoxic and hypoxic conditions. Quantitative real time polymerase chain reaction (qPCR), ELISA and immunoblot analysis were performed to examine the RNA and protein expression levels of transporters. Further, functional activity was evaluated by performing the uptake of various substrates in both normoxic and hypoxic conditions. qPCR analysis showed elevated expression of efflux transporters (P-glycoprotein, multidrug resistant protein 2, breast cancer resistant protein) and influx transporters (folate receptor-α, cationic and neutral amino acid transporter, sodium dependent multivitamin transporter) in a time dependent manner. Immunoblot analysis further confirmed elevated expression of breast cancer resistant protein and sodium dependent multivitamin transporter. A decrease in the uptake of efflux transporter substrates (digoxin, lopinavir and abacavir) and enhanced uptake of influx transporter substrates (arginine, folic acid and biotin) in hypoxia relative to normoxia further confirmed elevated expression of transporters, respectively. This study demonstrates for the first time that hypoxic conditions may alter expression of efflux and influx transporters in RPE cells. These findings suggest that hypoxia may further alter disposition of ophthalmic drugs.


Subject(s)
Epithelial Cells/metabolism , Membrane Transport Proteins/metabolism , Pharmaceutical Preparations/metabolism , Retinal Pigment Epithelium/metabolism , Administration, Ophthalmic , Biological Transport , Blotting, Western , Cell Hypoxia , Cell Line , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation , Humans , Membrane Transport Proteins/genetics , Pharmaceutical Preparations/administration & dosage , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
16.
Biochem Biophys Res Commun ; 438(1): 71-7, 2013 Aug 16.
Article in English | MEDLINE | ID: mdl-23872148

ABSTRACT

Hemangioblastomas of the retina, central nervous system, and kidney are observed in patients with mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. Mutations in the VHL lead to constitutive activation of hypoxia-inducible-factor (HIF) pathway. HIF-mediated expression of pro-angiogenic genes causes extensive pathological neovascularization in hemangioblastomas. A number of studies have shown coexistence of pro-angiogenic and stem cell markers in 'tumorlet-like stromal cells' in the retinal and optic nerve hemangioblastomas, leading to suggestions that hemangioblastomas originate from developmentally arrested stem cells or embryonic progenitors. Since recent studies have shown that the HIF pathway also plays a role in the maintenance/de-differentiation of normal and cancerous stem cells, we evaluated the role of the HIF pathway in the expression of stem cell markers in VHL-/- renal cell carcinoma cells under normoxia or VHL+/+ retinal pigment epithelial cells under hypoxia. Here we show that the expression of stem cell markers in hemangioblastomas is due to activation of the HIF pathway. Further, we show that honokiol, digoxin, and doxorubicin, three recently identified HIF inhibitors from natural sources, blocks the expression of stem cell markers. Our results show the mechanism for the cytological origin of neoplastic stromal cells in hemangioblastomas, and suggest that inhibition of the HIF pathway is an attractive strategy for the treatment of hemangioblastomas.


Subject(s)
Hemangioblastoma/metabolism , Hemangioblastoma/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Cell Hypoxia , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans
17.
Biochem Biophys Res Commun ; 436(2): 115-20, 2013 Jun 28.
Article in English | MEDLINE | ID: mdl-23727577

ABSTRACT

Methylation of DNA at the carbon-5 position of cytosine plays crucial roles in the epigenetic transcriptional silencing during metazoan development. Recent identification of Ten-Eleven Translocation (TET)-family demethylases have added a new dimension to dynamic regulation of 5-methylcytosine (5mC), and thus, inheritable and somatic gene silencing. The interest in hematology was particularly stimulated by the recent discovery of TET2 mutations in myeloid malignancies which were proven to be leukemogenic in murine knockout models. The TET-family enzymes are Fe(II), 2-oxoglutarate-dependent oxygenases and catalyze demethylation of 5mC by iterative oxidation reactions. In the last decade results from numerous studies have established a key role for these enzymes in epigenetic transcriptional regulation in eukaryotes primarily by hydroxylation reactions. The TET catalyzed hydroxylation and dehydration reactions in the mammalian system exemplify the diversity of oxidation reactions catalyzed by Fe(II), 2-oxoglutarate-dependent oxygenases, and suggest an existence of other types of oxidation reactions catalyzed by these enzymes in the eukaryotes, which are so far only documented in prokaryotes. Here, we review the TET-mediated 5mC oxidation in light of the putative reaction mechanism of Fe(II), 2-oxoglutarate-dependent oxygenases.


Subject(s)
5-Methylcytosine/metabolism , DNA Methylation , Dioxygenases/metabolism , Animals , Dioxygenases/genetics , Humans , Iron/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Ketoglutaric Acids/metabolism , Models, Genetic , Oxidation-Reduction
18.
Biochem Biophys Res Commun ; 422(3): 369-74, 2012 Jun 08.
Article in English | MEDLINE | ID: mdl-22580280

ABSTRACT

Hypoxia-inducible-factor (HIF)-mediated expression of pro-angiogenic genes under hypoxic conditions is the fundamental cause of pathological neovascularization in retinal ischemic diseases and cancers. Recent studies have shown that histone lysine demethylases (KDMs) play a key role in the amplification of HIF signaling and expression of pro-angiogenic genes. Thus, the inhibitors of the HIF pathway or KDMs can have profound therapeutic value for diseases caused by pathological neovascularization. Here, we show that hypoxia-mediated expression of KDMs is a conserved process across multiple cell lines. Moreover, we report that honokiol, a biphenolic phytochemical extracted from Magnolia genus which has been used for thousands of years in the traditional Japanese and Chinese medicine, is a potent inhibitor of the HIF pathway as well as hypoxia-induced expression of KDMs in a number of cancer and retinal pigment epithelial cell lines. Further, treating the cells with honokiol leads to inhibition of KDM-mediated induction of pro-angiogenic genes (adrenomedullin and growth differentiation factor 15) under hypoxic conditions. Our results provide an evidence-based scientific explanation for therapeutic benefits observed with honokiol and warrant its further clinical evaluation for the treatment of pathological neovascularization in retinal ischemic diseases and cancers.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Biphenyl Compounds/pharmacology , Histone Demethylases/antagonists & inhibitors , Hypoxia-Inducible Factor 1/antagonists & inhibitors , Lignans/pharmacology , Neovascularization, Pathologic/metabolism , Cell Hypoxia , Cell Line , Cell Line, Tumor , Gene Expression/drug effects , Humans , Hypoxia/metabolism , Neovascularization, Pathologic/genetics , Oxygen/metabolism
19.
Biochem Biophys Res Commun ; 415(2): 373-7, 2011 Nov 18.
Article in English | MEDLINE | ID: mdl-22037463

ABSTRACT

Hypoxia inducible factor (HIF) plays a critical role in cellular adaptation to hypoxia by regulating the expression of essential genes. Pathological activation of this pathway leads to the expression of pro-angiogenic factors during the neovascularization in cancer and retinal diseases. Little is known about the epigenetic regulations during HIF-mediated transcription and activation of pro-angiogenic genes in oxygen-dependent retinal diseases. Here, we show that hypoxia induces the expression of a number of histone lysine demethylases (KDMs) in retinal pigment epithelial cells. Moreover, we show that the expression of pro-angiogenic genes (ADM, GDF15, HMOX1, SERPE1 and SERPB8) is dependent on KDMs under hypoxic conditions. Further, treating the cells with a general KDM inhibitor blocks the expression of these pro-angiogenic genes. Results from these studies identify a new layer of epigenetic transcription regulation under hypoxic conditions and suggest that specific inhibitors of KDMs such as JMJD1A can be a new therapeutic approach to treat diseases caused by the hypoxia induced neovascularization in cancer and retinal diseases.


Subject(s)
Epigenesis, Genetic , Histone Demethylases/biosynthesis , Oxygen/metabolism , Retinal Neovascularization/enzymology , Retinal Neovascularization/genetics , Retinal Pigment Epithelium/enzymology , Adrenomedullin/genetics , Amino Acids, Dicarboxylic/pharmacology , Cell Hypoxia/genetics , Cell Line, Tumor , Growth Differentiation Factor 15/genetics , Heme Oxygenase-1/genetics , Histone Demethylases/genetics , Humans , Retinal Pigment Epithelium/drug effects
20.
Antimicrob Agents Chemother ; 55(6): 2783-7, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21402836

ABSTRACT

Penicillin-binding protein 2a (PBP2a), the molecular determinant for high-level ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), is intrinsically resistant to most ß-lactam antibiotics. The development and characterization of new inhibitors targeting PBP2a would benefit from an effective and convenient assay for inhibitor binding. This study was directed toward the development of a fluorescently detected ß-lactam binding assay for PBP2a from MRSA. Biotinylated ampicillin and biotinylated cephalexin were tested as tagging reagents for fluorescence detection by using a streptavidin-horseradish peroxidase conjugate. Both bound surprisingly well to PBP2a, with binding constants of 1.6 ± 0.4 µM and 13.6 ± 0.8 µM, respectively. Two forms of the assay were developed, a one-step direct competition form of the assay and a two-step indirect competition form of the assay, and both forms of the assay gave comparable results. This assay was then used to characterize PBP2a binding to ceftobiprole, which gave results consistent with previous studies of ceftobiprole-PBP2a binding. This assay was also demonstrated for screening for PBP2a inhibitors by screening a set of 13 randomly selected ß-lactams for PBP2a inhibition at 750 µM. Meropenem was observed to give substantial inhibition in this screen, and a follow-up titration experiment determined its apparent K(i) to be 480 ± 70 µM. The availability of convenient and sensitive microtiter-plate based assays for the screening and characterization of PBP2a inhibitors is expected to facilitate the discovery and development of new PBP2a inhibitors for use in combating the serious public health problem posed by MRSA.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Discovery , Methicillin-Resistant Staphylococcus aureus/drug effects , Penicillin-Binding Proteins/antagonists & inhibitors , Cephalosporins/metabolism , Penicillin-Binding Proteins/metabolism
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