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WO2007046803A1 - Dosage de promoteurs méthylés actifs en transcription - Google Patents

Dosage de promoteurs méthylés actifs en transcription Download PDF

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Publication number
WO2007046803A1
WO2007046803A1 PCT/US2005/037673 US2005037673W WO2007046803A1 WO 2007046803 A1 WO2007046803 A1 WO 2007046803A1 US 2005037673 W US2005037673 W US 2005037673W WO 2007046803 A1 WO2007046803 A1 WO 2007046803A1
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WIPO (PCT)
Prior art keywords
gene
interest
methylated
dna
cell
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PCT/US2005/037673
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English (en)
Inventor
George Blanck
Melissa I. Niesen
Aaron R. Osborne
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University Of South Florida
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Priority to PCT/US2005/037673 priority Critical patent/WO2007046803A1/fr
Priority to US11/735,706 priority patent/US20070184481A1/en
Publication of WO2007046803A1 publication Critical patent/WO2007046803A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the present invention relates to the field of gene regulation.
  • the invention provides novel screening systems for identifying test agents that modulate expression of methylation-dependent repressed genes.
  • Methylation of DNA cytosine residues is strongly associated with higher order chromatin formation, heterochromatin, and repression of RNA transcription.
  • Several molecular mechanisms leading to transcriptional repression, as a result of DNA methylation, have been elucidated.
  • MeCP2 one of a family of proteins with a methyl-DNA binding domain (MBDs)
  • MBDs methyl-DNA binding domain
  • DNA methylation has been shown to enhance transcription.
  • DNA methylation of the far upstream region of the IL-8 gene and methylation of the egr-2 intron are associated with increased promoter activity.
  • the mechanism of these effects is unknown, and no DNA binding proteins involved in these processes have been identified.
  • Other studies have indicated that methylated DNA can be transcribed, apparently in the absence of proteins that directly mediate transcriptional repression.. However, none of these studies involve a transition from repressed to activated methylated DNA.
  • Lembo et al. have identified a protein that facilitates a transition from MBD-mediated repression to MBD-mediated activation.
  • the molecular mechanism that specifies a transition from repressed methylated DNA to methylated DNA that becomes or remains transcriptionally active, for a given promoter remains unknown.
  • assaying for transcription of the methylated HLA-DRA gene may provide a way to obtain novel anti-inflammatory drugs because the gene is sometimes methylated and transcribed.
  • the invention includes an assay for drugs that interfere with HLA-DRA gene expression, thus reducing inflammation.
  • the invention includes an assay for transcriptional activation of methylated DNA for any gene. Accordingly, the present invention provides a method of identifying genes that can be activated when methylated; as well as proteins that mediate such activation.
  • the assay is used as a positive control for future experiments, ie, to ensure validation and quality control for future tests, experiments, assays, etc.
  • the inventors show that the sequence specific DNA binding protein, RFX, previously shown to mediate the transition from an inactive to an active chromatin structure, preferentially activates a methylated promoter.
  • RFX is capable of mediating enhanceosome formation on a methylated promoter, thereby mediating a transition from a methylation-dependent repression of the promoter to a methylation-dependent activation of the promoter.
  • Figure 1 is a graph of experiment results showing DNA methylation represses transfected HLA-DRA promoter.
  • Figures 2A through 2D are graphs of experiment results showing RFX activation of methylated pDRA.
  • Figures 3 A and 3B are graphs of experiment results showing RFX facilitates CIITA activation of pDRA.
  • Figures 4A through 4C are graphs of experiment results showing RFX does not activate unmethylated pDRA and does not decrease the level of pDRA methylation.
  • Figure 4D is a pDRA map indicating the relative positions of the HLA-DRA promoter, the RFX binding site, the luciferase gene, the Hgal and Aval restriction enzyme sites used in the methylation sensitive Real-Time PCR experiments of panels F-I, and of primers a-f used in the Real-Time PCR experiments.
  • Figure 4E shows the HLA-DRA promoter sequence of pDRA indicating Sssl CG methylation sites (in larger font) and the binding sites of RFX, Oct-1 and YYl. The X2- and Y-elements are also required for HLA-DRA promoter activity.
  • Figures 4F through 41 are graphs of experiment results showing methylation sensitive Real-Time PCR.
  • Figures 5 A and 5B are graphs of experiment results showing the Oct-1 and YYl repression mechanisms do not function with methylated pDRA. 5B. Repeat of Figure 5A, except with methylated pDRA (Me-pDRA), methylated pDRA ⁇ Oct (Me- pDRA ⁇ Oct), or methylated pDRA ⁇ YYl (Me-pDRA ⁇ YYl).
  • Figures 5C and 5D are graphs of experiment results showing two examples of a repeat of Figure 5B except the Me-pDRA and Me-pDRA mutated constructs were transfected with the RFX expression vectors.
  • Figures 6A and 6B are two models for how RFX could mediate enhanceosome formation on methylated HLA-DRA promoter DNA.
  • MBD methyl DNA binding domain protein
  • HDAC histone deacetylase
  • Nuc nucleosome.
  • Figure 7 represents repressosome and enhanceosome states and transitions for methylated and nonmethylated HLA-DRA promoter DNA.
  • a "test agent” refers to an agent that is to be screened in one or more of the assays described herein.
  • the agent can be virtually any compound and can exist as a single isolated compound or can be a member of a chemical library.
  • a "reporter gene” refers to any gene or DNA that expresses a product that is detectable by spectroscopic, photochemical, biochemical, enzymatic, immunochemical, electrical, optical or chemical means.
  • the preferred reporter gene to which a promoter element is ligated is luciferase.
  • reporter genes for use for this purpose include, for example, .beta.-galactosidase gene (.beta.gal) and chloramphenicol acetyltransferase gene (CAT)
  • Assays for expression produced in conjunction with each of these reporter gene elements are well-known to those skilled in the art. Assay in connection with the luciferase gene are described below.
  • Expression constructs are prepared by ligating transcriptional promoter elements to the reporter genes by methods well-known in the art, e.g., by utilizing restriction enzymes to cut the reporter gene in appropriate portion to provide binding sites for the transcriptional promoter elements, incubating the restriction enzyme treated reporter gene with the transcriptional promoter elements and screening for the recombinants.
  • gene of interest refers to a nucleic acid which can be of any origin and isolated from a genomic DNA, a cDNA, or any DNA encoding a RNA, such as a genomic RNA, a mRNA, an anti-sense RNA, a ribosomal RNA, a ribozyme or a transfer RNA.
  • the gene of interest can also be an oligonucleotide (i.e., a nucleic acid having a short size of less than 100 bp). It can be engineered from genomic DNA to remove all or part of one or more intronic sequences (i.e., minigene).
  • the gene of interest in use in the present invention encodes a target gene product, such as a protein, of therapeutic interest.
  • a gene product of therapeutic interest, or target gene protein is one which has a therapeutic or protective activity when administered appropriately to a patient, especially a patient suffering from a disease or illness condition or who should be protected against this disease or condition.
  • a therapeutic or protective activity can be correlated to a beneficial effect on the course of a symptom of said disease or said condition. It is within the reach of the man skilled in the art to select a gene encoding an appropriate gene product of therapeutic interest, depending on the disease or condition to be treated. In a general manner, his choice may be based on the results previously obtained, so that he can reasonably expect, without undue experimentation, i.e., other than practicing the invention as claimed, to obtain such therapeutic properties.
  • the inventors show that the sequence specific binding protein, RFX, mediates the transcriptional activation of a methylated major histocompatibility (MHC) gene promoter that was repressed by the methylation. This indicates that DNA demethylation is not a necessary step in promoter activation and shows that methylated DNA can be transcribed. Furthermore, the RFX-related results show that a sequence specific DNA binding protein can mediate a transition between a repressed and active promoter.
  • MHC major histocompatibility
  • RPX The methyl-DNA binding protein, RPX, was first identified as a protein defective in Bare Lymphocyte Syndrome, a rare immunodeficiency disease that is due to the lack of transcription of the MHC class II genes, which encode the class II antigen presenting molecules.
  • RFX has two apparent functions in facilitating MHC class II promoter activation. RFX participates in the formation of an MHC class II enhanceosome, by binding specifically to the MHC class II promoter and interacting with other MHC class II promoter binding proteins as well as with the MHC class II specific coactivator (CIITA).
  • CIITA MHC class II specific coactivator
  • RFX also mediates the establishment of an MHC class II DNase I hypersensitive site, thus facilitating a transition from an inactive, condensed form of chromatin to a form of promoter chromatin that permits enhanceosome assembly and transcriptional activation.
  • RFX5 one of the three subunits of RFX, is a member of a family of methyl-DNA binding proteins, some of which also have a connection with the regulation of transcription of DNA that is in a condensed form. For example, drosophila RFX functions in sperm, and RFX2 and RFX4 function in the testis.
  • Example I DNA methylation represses transcriptional activity of the HLA-DRA promoter Repression of Endogenous HLA-DRA Promoter
  • RFX-defective B-cells specifically lacking RFX, but possessing the other required transactivators, do not have a transcriptionally active HLA-DRA gene (the human MHC class II gene that represents the prototype for MHC class II gene regulation).
  • RFX-defective B-cells have a condensed, inaccessible HLA-DRA promoter chromatin conformation.
  • the inventors treated two RFX-negative cell lines with Azacytidine (AzaC), which inhibits DNA methyltransferase activity, and assayed the cells for HLA- DRA mRNA by RT-PCR (Fig. IA) to determine whether DNA methylation plays a role in preventing transcription of HLA-DRA in RFX-negative cells.
  • Azacytidine Azacytidine
  • Fig. IA RT-PCR
  • the inventors methylated a pDRA (luciferase) construct with Sssl methylase, which methylates all plasmid CpG dinucleotides, to determine whether an HLA-DRA promoter-reporter construct could be repressed by methylation.
  • Methylated pDRA was transfected into 5637 cells, which require IFN- ⁇ treatment for the activation of the pDRA promoter.
  • the HLA-DRA expression construct (promoter-luciferase), pDRA derived from pGL3, was methylated using Sssl methylase (20ug pDRA, 5mM S-adenosyl methionine (SAM), 8 units of Sssl, 1OX New England BioLabs buffer #2, H 2 O to a total volume of 50 ul). Mock methylated pDRA was prepared in the same way but without Sssl methylase in the reaction.
  • Methylated pDRA or mock methylated DNA, was extracted with phenol: chloroform, ethanol precipitated, resuspended in water, and quantified by absorbance at A 260 and by agarose gel electorphoresis. Fifty nanograms of methylated pDRA were added to each of six wells containing 5 x 10 4 5637 bladder carcinoma cells. DNA transfection was performed using the Transit Reagent according to the vendor's instructions. IFN- ⁇ was added to 400 units/ml following the transfection and luciferase assays were performed 24 hours following the transfection. Each bar graph, Figure 1, represents the average and standard deviations for six transfections.
  • Methylated pDRA is insensitive to Aval digestion.
  • pDRA has two Aval sites: one site in the HLA-DRA promoter region cloned upstream of the luciferase coding region and one site in the luciferase coding region. (See pDRA map, Fig. 2D.)
  • Example II - RFX facilitates activation of a methylated HLA-DRA promoter reporter construct
  • Methylated pDRA was co-transfected into 5637 cells with equal amounts of either (a) empty vector or (b) the RPXAP, RFXB, and RFX5 expression vectors, representing the three subunits of RFX. Cells were treated with IFN- ⁇ for 24 hours following transfection. The methylated pDRA co-transfected with RFX showed a ten- fold increase (Fig. 2A) in luciferase activity compared with the controls samples lacking the exogenous RFX.
  • Me-pDRA methylated pDRA
  • the methylated or mock methylated promoter luciferase contructs were transfected into cells as indicated above.
  • the pGL3-Basic (with no promoter activity) was used to construct pDRA.
  • the pGL3 -Control contains the SV40 promoter and enhancer. P-values are indicated in boxes ( Figure 2B).
  • RFX was co-transfected with methylated pDRA (Me-pDRA) as a positive control for the experiment ( Figure 2C).
  • Relative luciferase activity is indicated to the right of the bar graphs, with the results from pDRA co-transfected with RFX arbitrarily set at 1.0, for comparison with panel C.
  • luciferase quantification is to the left of the bar graphs and the p-value is indicated in the box.
  • the methylated pGL3-Control luciferase construct was then co-transfected into cells with the RFXAP, RFXB, and RFX5 expression vectors or with empty vector.
  • Methylated pDRA co-transfected with the RFX expression vectors represented a positive control for the RFX effect (Fig. 2D).
  • Methylated pGL3-Control co-transfected with the RFX expression vectors, or with an equivalent amount of empty vector. Relative luciferase activity is indicated to the right ( Figure 2C). Thus, results indicated essentially no increase in activity due to RFX expression. No increase in transcriptional activity was observed for the pGL3-Control luciferase construct ( Figure. 2C); consistent with the conclusion that RFX activates methylated pDRA by binding to its cognate site in the DRA promoter (Fig. 4E).
  • Example III - RFX is required for the CIITA activation of methylated pDRA
  • sequence specific DNA binding proteins can occupy the HLA- DRA promoter without the advent of HLA-DRA transcription.
  • Transcriptional activation commences when the formation of the enhanceosome, including RFX, is completed by availability and the enhanceosome binding of CIITA, which in turn leads to the recruitment of histone deacetylase activity.
  • CIITA synthesis is induced by IFN- ⁇ , as a result of the IFN- ⁇ induction of activated STATl and IRF-I and the binding of these two transactivators to the CIITA promoter.
  • the inventors co-transfected the methylated pDRA with a CIITA expression vector and with either the RFX expression vectors or with control empty vector.
  • Panel A (i) Methylated pDRA (Me-pDRA), (ii) the RFX expression vectors or empty vector as indicated, and (iii) a CIITA expression vector were co-transfected as described in Figs. 1 and 2, except 50 ng of CIITA was used for each of six wells and the cells were not treated with IFN- ⁇ . P-value is indicated in the box.
  • Panel B. shows a separate, duplicate experiment, same as panel A. Greater absolute luciferase activity is observed with CIITA activation of pDRA vs. IFN- ⁇ activation in Figs. 1 and 2.
  • RFX expression strongly enhanced the CIITA activation of the methylated pDRA.
  • Example IV- RFX does not facilitate the activation of nonmethylated pDRA and does not facilitate demethylation of pDRA
  • RFX is capable of low affinity binding to nonmethylated HLA-DRA promoter DNA and of participating in enhanceosome formation in vitro in the absence of DNA methylation, it is not known whether RFX is important in the activation of nonmethylated DNA.
  • the inventors co-transfected either the RFX expression vectors or empty vector with nonmethylated pDRA ( Figures 4A ,B, C).
  • FIG. 4A cells were co-transfected with mock methylated pDRA and either empty vector or the three RFX expression vectors. P-value indicated in the box.
  • Figure 4B is a repeat of the experiment in Figure 4A.
  • Figure 4C is a repeat of the experiment in Figure 4A, except cells were co-tranfected with CIITA instead of treated with IFN- ⁇ and with individual RFX expression vectors as indicated.
  • the inventos recovered total DNA from cells co-transfected with either the RFX expression vectors or the empty vector in combination with either methylated pDRA or nonmethylated pDRA.
  • the recovered DNA was digested with either Hgal, the site for which overlaps the CG dinucleotide of the RFX binding site (Xl element) in the HLA- DRA promoter (Fig. 4E), or with Aval. Neither Hgal nor Aval cleave DNA when the C at the cleavage site is methylated.
  • the digested DNA was then assayed by Real- Time PCR using pDRA specific primers on either side of the pDRA promoter Hgal site or luciferase coding sequence primers on either side of an Aval site located in the luciferase coding region (Fig. 4D).
  • Primers e and f were used to determine the relative amounts of transfected plasmid present for each of the indicated transfections, as these primers amplify a segment of pDRA which does not include a site for either Hgal or Aval.
  • the results for Figure 4F and 4H were obtained by normalizing the results for each transfection using the results obtained with primers e and f.
  • the results for Figures 4G and 41 were not normalized and, in the case of Figure 41, include a "no-transfection control".
  • the y-axis numbers for the Figure 4H bar graph represent relative cycle numbers where the methylated pDRA co-transfected with empty vector was set to "1" for convenience in comparing the results for each set of transfected DNAs.
  • the actual cycle numbers for all Real-Time PCR results in Figures 4F-4I ranged from 21-26 with the exception of the "no-transfection" control in Figure 4H which averaged about 33 cycles. P-values are shown in boxes.
  • Example V The Oct-l/YY-1 repression mechanism does not function to repress either inactive or transcriptionally competent methylated pDRA Both Oct-1 and YY-I repress the IFN- ⁇ activation of pDRA.
  • this mechanism(s) functions to represses methylated pDRA
  • the inventors transfected methylated pDRA or the previously described, methylated pDRA ⁇ Oct or methylated pDRA ⁇ YYl into IFN- ⁇ treated 5637 cells.
  • the latter two pDRA luciferase constructs lack the Oct-1 and YYl binding sites respectively.
  • the nventors also transfected nonmethylated pDRA or pDRA ⁇ Oct or pDRA ⁇ YYl into IFN- ⁇ treated 5637 cells, a transfection series that serves as a positive control for the detection of Oct- 1 or YYl mediated repression.
  • Mock methylated pDRA or mock methylated pDRA mutants lacking the Oct-1 binding site or the YYl binding site were transfected into cells and assayed for luciferase activity.Results indicate that the nonmethylated DNA is repressed by Oct-1 and YYl ( Figure 5A).
  • RFX transactivator may either displace or prevent the binding of MBD proteins that form a repressosome complex (Fig. 6), leading to promoter activation.
  • the HLA-DRA promoter binding specificity for RFX requires all three subunits. Multiple RFX5 family members (RFXl-5) have been well-studied and have related "RFX-type" DNA binding domains. Because all three subunits of RFX are required for HLA-DRA promoter specificity, it is not likely that significant sequence specificity resides in the RFX-type DNA binding domain common to these RFX5 family members.
  • the other two RFX subunits, RFXAP and RFB/RFXANK/Tvl-1 have no known functional or structural homologs.
  • demethylation of the HLA-DRA promoter represents a transition from the methyl DNA binding protein-related repressosome that regulates methylated HLA-DRA DNA (Fig. 1) to the DRAN (Oct-1 related) or YYl-HDAC repressosomes that regulate demethylated HLA-DRA DNA ( Figure 7).

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Abstract

La présente invention a pour objet une méthode d'identification de gènes pouvant être activés lorsqu'ils sont méthylés ; ainsi que de protéines jouant le rôle d'intermédiaires dans une telle activation. La méthode permet l'identification d'agents inhibant le gène HLA-DRA de valeur thérapeutique supposée dans le traitement d'une inflammation associée à l'expression de HLA-DRA.
PCT/US2005/037673 2005-10-18 2005-10-18 Dosage de promoteurs méthylés actifs en transcription WO2007046803A1 (fr)

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US11/735,706 US20070184481A1 (en) 2005-10-18 2007-04-16 Assay For Transcriptionally Active Methylated Promoters

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008138572A1 (fr) * 2007-05-09 2008-11-20 Jacobs University Bremen Gmbh Procédé et réactifs pour l'étude des réactions de méthylation d'acide nucléique
EP2128259A1 (fr) * 2008-05-27 2009-12-02 Koninklijke Philips Electronics N.V. Administration de thérapie et surveillance utilisant un gène de protéine de fusion de rapporteur-intérêt et imagerie optique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DETICH ET AL.: "Promoter-specific activation and demethylation by MBD2/demethylase", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 39, September 2002 (2002-09-01), pages 35791 - 35794, XP003012093 *
REITH ET AL.: "The bare lymphocyte syndrome and the regulation of MHC expression", ANNUAL REVIEWS OF IMMUNOLOGY, vol. 19, 2001, pages 331 - 373, XP001121032 *
SENGUPTA ET AL.: "The RFX Family interacts at the collagen (COL1A2) start site and represses transcription", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 28, July 2002 (2002-07-01), pages 24926 - 24937, XP003012094 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008138572A1 (fr) * 2007-05-09 2008-11-20 Jacobs University Bremen Gmbh Procédé et réactifs pour l'étude des réactions de méthylation d'acide nucléique
EP2128259A1 (fr) * 2008-05-27 2009-12-02 Koninklijke Philips Electronics N.V. Administration de thérapie et surveillance utilisant un gène de protéine de fusion de rapporteur-intérêt et imagerie optique
WO2009147549A1 (fr) * 2008-05-27 2009-12-10 Koninklijke Philips Electronics N. V. Administration et surveillance de traitement utilisant une protéine de fusion pour gène rapporteur d'intérêt et imagerie optique
CN102046795A (zh) * 2008-05-27 2011-05-04 皇家飞利浦电子股份有限公司 使用感兴趣的基因-报道物融合蛋白和光学成像的治疗送递和监测
JP2011521637A (ja) * 2008-05-27 2011-07-28 コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ 対象のレポーター融合タンパク質の遺伝子及び光学撮像を使用した監視及び治療送出
US9528113B2 (en) 2008-05-27 2016-12-27 Koninklijke Philips N.V. Therapy delivery and monitoring using a gene of interest-reporter fusion protein and optical imaging

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