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WO2009114011A1 - Protéines de déméthylation d’histone et leurs procédés d’utilisation - Google Patents

Protéines de déméthylation d’histone et leurs procédés d’utilisation Download PDF

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Publication number
WO2009114011A1
WO2009114011A1 PCT/US2008/056516 US2008056516W WO2009114011A1 WO 2009114011 A1 WO2009114011 A1 WO 2009114011A1 US 2008056516 W US2008056516 W US 2008056516W WO 2009114011 A1 WO2009114011 A1 WO 2009114011A1
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utx
histone
protein
cell
jmjd3
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PCT/US2008/056516
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English (en)
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Yang Shi
Fei Lan
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President And Fellows Of Harvard College
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
    • G01N2333/91011—Methyltransferases (general) (2.1.1.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention is related to the areas of post-translational modifications and gene regulation.
  • it relates to the area of modification of chromosome structure, specifically histone methylation, as a means of regulating gene transcription.
  • Histone amino-terminal tails are subject to multiple post-translational modifications including methylation that determine chromatin structure and regulate gene transcription.
  • Methylation of histone H3, particularly H3K27 methylation has been recognized as important in epigenetic repression of transcription.
  • H3K27 methylation is catalyzed by the histone methyltransferase EZH2, a mammalian homolog of Drosophila polycomb group protein Ez (enhancer of zeste).
  • H3K27 methylation regulates transcription of tumor suppressor genes and has been implicated in tumorigenesis and cancer development.
  • H3K27 methylation also impacts stem- cell pluripotency regulation. Thus, there is a continuing need in the art to regulate histone methylation status in order to control gene expression.
  • Figure 1 demonstrates histone demethylation mediated by the UTX.
  • H3K27me3 by UTX Tri-methylated synthetic histone peptides were incubated with UTX purified from Sf9 cells. Demethylated peptides were detected as mass peaks with the molecular weight that is 14 daltons (Da) smaller than the input. Demethylated peptides are marked with stars. Among the tri-methylated residues examined (H3K4, 9, 27, and 36, and H4K20), UTX only catalyzed demethylation of H3K27me3. (B) UTX demethylates H3K27me3/2 on bulk histones.
  • Calf thymus histones were incubated with purified, full-length UTX, and subjected to Western blot analysis using antibodies that specifically recognize methylated histones. Mock-purified material was used as a control. Reduced signals were found only for H3K27me3 and H3K27me2.
  • C JMJD3 catalytic domain demethylates H3K27me3/2 on nucleosomal substrates. Mono-nucleosomes purified from HeLa cells were incubated with purified catalytic domain of JMJD3 (aa 1164-1682). The demethylation of H3K27me3/2 was detected by Western blotting.
  • FIG. 2 demonstrates that UTX regulates H3K27me3 at the Hox gene locus
  • A Schematic diagram of the mammalian HoxD gene cluster. The primer sets used for ChIP are shown. The direction of transcription of the Hox genes shown in the diagram is from right to left.
  • B Specific shRNAs effectively knocked down the transcripts of UTX and JMJDi in HeLa cells.
  • C ChIP analysis of H3K27me3 at the HoxD gene cluster. ChIP results with standard deviation using the H3K27me3 antibody and the primer sets in (A) are shown. ChIP results are expressed as relative fold of enrichment over input, by comparing the Hox gene locus with a control ChIP of an unaffected region.
  • Figure 3 demonstrates that UTX binds near Hox gene start sites to facilitate H3K27 demethylation and gene activation.
  • A UTX occupancy over -100 kilobases of the HoxA locus in lung fibroblasts.
  • High confidence UTX occupancy sites (FDR ⁇ 0.01, ChIP/IgG > 1.5) are shown as tick marks over the locus.
  • Hox genes represent -20% of probes on the Hox tiling array but account for 92% of UTX binding events (p ⁇ 10-64).
  • C Average UTX occupancy profiles for Hox genes bound by UTX aligned by Hox transcriptional start sites (left axis). Average of H3K27me3 occupancy profiles of Hox genes bound by both UTX and H3K27me3 (right axis). The genes and ChIP profiles used are detailed in Table 1.
  • D UTX is excluded from Hox loci of ES cells. Occupancy of UTX, PRC2, and H3K27me3 in ES cells and differentiated fibroblasts is shown as a matrix; red indicates occupancy.
  • E siRNA-mediated depletion of UTX decreases HoxA9 transcription. Relative transcript levels (mean + s.e.) by Taqman qRT-PCR are shown. Top: corresponding UTX and actin protein levels.
  • Figure 4 demonstrates that knockdown of zebrafish UTXl results in improper development of the posterior trunk.
  • A Prior to 24 hours post- fertilization (hpf) development of UTX morpholino (MO) injected embryos is slightly delayed. After 48hpf, lack of posterior extension is obvious and the embryos have slightly larger heads than controls.
  • B Evidence of notochord degeneration (between arrows) can be observed at 72hpf between approximately somites 13 to 23. However, the neural floor plate in this area (arrowheads) remains. The somites in the affected region are very short and compact. In most knockdown embryos, the most distal portion of the notochord (between somites 24-30) remains fairly normal.
  • Figure 5 shows a phylogenic tree of the UTX and JMJD3 family proteins. All Amino acid sequences are retrieved from NCBI GenBank, except for ENSDART00000088452, which is from Ensembl database. The conserved domains are identified by the NCBI conserved domain search. The scale bar denotes 100 amino acids (aa).
  • FIG. 6 demonstrates that over-expression of JMJD3 results in reduction of H3K27me3 and H3K27 me2 signal in 293T cells.
  • Expression construct carrying HA-JMJD3 was transiently transfected into 293T cells, stained by anti-HA (green) and appropriate antibodies against methylated histone (H3K27me3, H3K27me2, H3K27mel, and H3K36me3; red). The nuclei were counter-stained by Hoechst (blue). The percentages of change in cell numbers with moderate to high HA-JMJD3 expression were listed in the Table.
  • Figure 7 demonstrates that UTX binding to the Hox loci and relationship with H3K4 methylation.
  • A UTX occupancy over -100 kilobases of the HoxA locus in foot fibroblasts.
  • High confidence UTX occupancy sites (FDR ⁇ 0.01, ChIP/IgG > 1.5) are shown as tick marks over the locus.
  • Hox transcriptional start sites and locus-wide profiles of raw UTX chlP-chip hybridization signal shown as Iog2 ratios of UTX ChIP/IgG ChIP are shown for comparison.
  • ChIP signals are presented as the percentage of input DNA (mean + standard deviation). Three predicted UTX occupancy events at the start of HoxA9 and HoxD13 in two different cell types show strong enrichment over IgG chIP while a predicted negative control in the intergenic region between HoxA9 and HoxAlO show no such enrichment.
  • C Comparison of average UTX occupancy profiles versus H3K4me2 profiles in the Hox loci. Hox genes are aligned by their transcriptional start sites. ChlP-chip profiles used for this analysis are detailed in Table 1.
  • Figure 8 shows the identification of UTX occupancy sites in mES cells.
  • UTX occupancy genome-wide We focused our analysis on 21,600 promoters corresponding to well-annotated genes. The most significant class of UTX-occupied genes was the odorant receptor genes (OR). Of the 454 high confidence occupancy sites (> 1.5 fold enrichment over IgG ChIP and FDR ⁇ 0.05), 81 UTX occupancy sites were in genes encoding OR. (B) Gene Ontology enrichment of UTX occupied genes in mES cells. Consistent with the over-representation of odorant receptor genes, the most significant p-values were for biological processes associated with the sensory perception of smell.
  • Figure 9 demonstrates that the UTX H1226A catalytic domain is inactive towards
  • H3K27me3/2/l histone peptides The critical His 1226 residue involved in Fe2+ chelating was changed to Ala by site direct mutagenesis, and this mutant protein was purified and assayed under the same condition as the wt catalytic domain in Figure ID. No demethylated peptides were detected as mass peaks with the molecular weight that is 14 Da (removal of one methyl group) smaller than the input. Comparable amount of the wildtype UTX (analyzed alongside the mutant) showed clear demethylation activity (not shown). Note, the small peaks are background, and they are not 14Da less than the input.
  • Figure 10 provides a whole mount in situ experiment showing mis-regulation of zHox genes in UTXl morphants at 36 hpf.
  • zHoxClla staining is significantly stronger in the control morphants in the tail regions (arrows) than in the zUTXl morphants (box arrowheads), (e) Reduced expression of zHoxDl 2a in the pectoral fin buds in zUTXl morphants, which was observed in 5 out of 10 embryos. Arrows mark the pectoral fin bud staining in the control morphants, which was significantly reduced in the zUTXl morphants (box arrowheads), while the overall tail staining was comparable between the control and the zUTXl morphants.
  • Figure 11 shows the quantitative ChIP analyses of UTX, ALR, RBQ3 and H3K4me3 at selected Hox loci in lung fibroblasts. Co-localization of UTX, ALR and RBQ3, as well as H3K4me3, was identified at HoxA9, AlO and Dl 3 transcription start sites, but not at other neighboring regions. ChIP signals are presented as fold of enrichment (mean + standard deviation).
  • Figure 12 illustrates that no global change of H3K27me and H3K4me3 levels occurs in
  • UTX knocking down cells and no in vitro activity change of immunoprecipitated ALR/MLL2 from UTX knocking down cells.
  • A UTX in mES cells and 293T cells were knocked down by siRNAs and shRNAs, respectively. Whole cell lysates were subjected to Western blotting analyses by indicated antibodies recognizing specific histone modifications.
  • B ALR/MLL2 was immunoprecipitated from 293T cells treated with control and UTX shRNAs, and bulk histone and histone H3 peptides were used as substrates in the in vitro HMT assay.
  • JmjC domain-containing proteins as histone demethylases that mediate the reversal of methylation at histone H3K4, H3K9 and H3K36.
  • UTX, UTY and JMJD3 comprise a subfamily of JmjC domain-containing proteins, which are evolutionarily conserved from C. elegans to human.
  • UTX and UTY, but not JMJD3 also contain tetratricopeptide repeats (TPR) at their N-terminal regions, which are predicted protein interaction motifs ( Figure 5). All three proteins contain a Treble-clef zinc finger at their C-terminus.
  • UTX resides on the X chromosome, escapes X-inactivation and is ubiquitously expressed. UTY is a male-specific protein and may contribute to sex-specific tissue transplantation rejection response. It is a discovery of the present inventors that UTX and JMJD3 function as transcriptional activators of gene expression. Using recombinant UTX and a collection of methylated histone peptides as substrates, it has been determined that UTX specifically mediates the methylation status of H3K27, particularly H3K27me3. See Lan et al. Nature (2007) 449: 689-94, the contents of which are hereby incorporated by reference in their entirety.
  • histone demethylase protein and " JmjC domain-containing protein” (and similar terms) as used herein encompass any JmjC domain-containing demethylase (including histone demethylases), which includes without limitation proteins in the UTX, UTX and JMJD3 families, and further includes variants, functional fragments and "catalytic analogs" of any of the foregoing that retain substantial demethylase activity (e.g., at least about 5%, 10%, 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 100% or more demethylase activity as compared with the native protein).
  • UTX and JMJD3 are preferred histone demethylase proteins.
  • NM_001080193, ENSDART00000088462, and XP_684619 UTY sequences include BC071744, NM_001093305, NM_001009002, EF491796, EF491815, NM_009484, NM_007125, NM_182659, and NM_182660.
  • the amino acid sequence of human UTX is provided in Accession No. NP 066963 is shown below as SEQ ID NO: 1.
  • NM 021140 is shown below as SEQ ID NO: 2.
  • the amino acid sequence of human JMJD3 is provided in Accession No. NP OO 1073893 is shown below as SEQ ID NO: 3.
  • the nucleic acid sequence of human JMJD3 is provided in Accession No. NM OO 1080424 is shown below as SEQ ID NO: 4.
  • a homolog may also be a protein that is encoded by a nucleic acid that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity with a nucleotide sequence described herein.
  • a homolog may also be a protein that is encoded by a nucleic acid that hybridizes, e.g., under stringent hybridization conditions, to a nucleic acid consisting of a nucleotide sequence described herein or the coding sequence thereof.
  • homologs may be encoded by nucleic acids that hybridize under high stringency conditions of 0.2 to 1 x SSC at 65oC followed by a wash at 0.2 x SSC at 65oC to a nucleic acid containing a sequence described herein.
  • Nucleic acids that hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature to nucleic acid consisting of a sequence described herein or a portion thereof can be used.
  • Other hybridization conditions include 3 x SSC at 40 or 50oC, followed by a wash in 1 or 2 x SSC at 20, 30, 40, 50, 60, or 65oC.
  • Hybridizations can be conducted in the presence of formaldehyde, e.g., 10%, 20%, 30% 40% or 50%, which further increases the stringency of hybridization. Theory and practice of nucleic acid hybridization is described, e.g., in S.
  • Homologs of a protein of interest also include portions thereof, such as portions comprising one or more conserved domains, such as those described herein.
  • the demethylase proteins of the invention can be derived from any species of interest, including without limitation, mammalian ⁇ e.g., human, non- human primate, mouse, rat, lagomorph, bovine, ovine, caprine, porcine, equine, feline, and canine), insect (e.g., Drosophila), avian, fungal, plant, yeast ⁇ e.g., S. pombe or S. cerevisiae), C. elegans, D. rerio (zebrafish), etc. as well as allelic variations, isoforms, splice variants and the like.
  • the demethylase sequences can further be wholly or partially synthetic.
  • a catalytic analog of UTX or JMJD3 may be a portion of the wild type UTX or JMJD3 protein including one or more of the conserved domains.
  • a catalytic analog of UTX or JMJD3 may comprise at least a portion of the JmjC domain, the Treble-clef zinc finger domain, and/or the TPR repeat.
  • a catalytic analog of a histone demethylase protein includes a JmjC domain and, optionally, further includes a JmjN domain, a zinc finger domain (e.g., a Treble- clef zinc finger motif), zinc finger-like domain, a PHD domain, a tetratricopeptide repeat (TPR), an FBOX domain, a Vietnamese domain, an AT-Rich Interactive Domain (Arid/Bright), a coiled coil motif and/or a Leucine Rich Repeat (LRR) domain.
  • a zinc finger domain e.g., a Treble- clef zinc finger motif
  • TPR tetratricopeptide repeat
  • FBOX domain e.g., an FBOX domain
  • a Jewish domain e.g., an AT-Rich Interactive Domain (Arid/Bright) domain
  • LRR Leucine Rich Repeat
  • analogs can differ from naturally occurring proteins by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. Any number of procedures may be used for the generation of mutant, derivative or variant forms of a protein of interest using recombinant DNA methodology well known in the art such as, for example, that described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, Green & Wiley, New York).
  • conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function, e.g., its demethylase activity.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine (in positions other than proteolytic enzyme recognition sites); phenylalanine, tyrosine.
  • Whether an analog is a catalytic analog can be determined according to methods known in the art. For example, a demethylase activity can be determined as described in the Examples.
  • An illustrative example for determining whether a demethylase analog has demethylase activity includes contacting the demethylase analog with a target peptide that is methylated, and determining whether the demethylase analog is capable of demethylating the target peptide.
  • the assay may further comprise one or more other components, such as other proteins.
  • a target peptide may be a histone peptide. Any histone peptide can be used. Preferably it is used with a histone demethylase enzyme that recognizes the histone peptide as a substrate.
  • the full histone protein can be used or a peptide comprising only a portion of the histone protein can be used, so long as that portion contains the methylated residue upon which the demethylase enzyme acts and the portion contains sufficient contextual residues to permit its recognition by the enzyme. Typically at least 3, at least 4, at least 5, at least 6, or at least 7 residues on either side of the methylated residue are believed to be sufficient for recognition.
  • the methylated residue is preferably a lysine.
  • the histone peptide and the histone demethylase are derived from the same species of organism.
  • Measurement of the reaction between a histone and a eukaryotic histone demethylase protein can be accomplished by any means known in the art.
  • protein or histone "substrate” refers to a starting reagent in an enzymatic reaction that is acted upon to produce the reaction product(s).
  • the protein or histone substrate can be directly acted upon by the demethylase (typically by binding to the active site and undergoing a chemical reaction catalyzed by the enzyme) or can first be modified prior to being acted upon by the enzyme.
  • a typical a histone substrate is a H3K27 histone peptide substrate (e.g., H3K27me3).
  • H3K27me indicates methylation of a histone H3 peptide at lysine residue 27, when numbered in accordance with the amino acid sequence of the H3 protein.
  • Methylated H3K27 includes mono-, di- and trimethylated H3K27 (i.e., H3K37mel, H3K37me2, and H3K37me3).
  • the terms “modulate,” “modulates” or “modulation” or grammatical variations thereof refer to enhancement (e.g., an increase) or inhibition (e.g., a reduction) in the specified activity.
  • the terms “increases,” “enhancement,” “enhance,” “enhances,” or “enhancing” or grammatical variations thereof refers to an increase in the specified activity (e.g., at least about a 1.1 -fold, 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, or even 15- fold or more increase).
  • the terms “decreases,” “"inhibition,” “inhibit”, “reduction,” “reduce,” “reduces” or grammatical variations thereof as used herein refer to a decrease or diminishment in the specified activity of at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.
  • the decrease, inhibition or reduction results in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectible activity.
  • treat By the terms “treat,” “treating” or “treatment of it is meant that the severity of the subject's condition is reduced or at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness.
  • the terms “treat,” “treating” or “treatment of refer to both prophylactic and therapeutic regimens.
  • the phrase "reversing a tumorigenic state" of a cell or plurality of cells includes the act of a providing some modification to a cell such that it ceases to become tumorigenic or reduces the tumorigenic potential of the cell. Such a cell may become quiescent, senescent, or apoptotic.
  • a "tumor suppressor gene” includes a gene that protects a cell from one or more modifications that result in an increased tumorigenic potential of the cell. Tumor suppressor genes may promote apoptosis or repress cell cycle progression, or both.
  • An "embryonic stem cell” includes a pluripotent stem cell derived from the inner cell mass of an embryo, such as a blastocyst.
  • a "retinoblastoma susceptibility gene” includes the human retinoblastoma susceptibility gene (RB) and its gene product (pRB). See Lee et al. (1987), Science 235: 1394 - 1399.
  • compositions and complexes containing one or more proteins described herein may be a pharmaceutical composition.
  • the invention provides an isolated complex including a UTX protein or a catalytic analog thereof (such as a catalytic analog containing a JmjC domain and a treble-clef zinc finger domain), and a JMJD3 protein or a catalytic analog thereof (such as a catalytic analog containing a JmjC domain and a treble-clef zinc finger domain).
  • the isolated complex may also include a histone H3 peptide.
  • the invention also provides an isolated complex including a histone H3 peptide and either a UTX protein (or a catalytic analog thereof) or a JMJD3 protein (or a catalytic analog thereof).
  • Nucleic acids e.g., those encoding a protein of interest or functional homolog thereof, or a nucleic acid intended to inhibit the production of a protein of interest (e.g., siRNA or antisense RNA) can be delivered to cells, e.g., eukaryotic cells, in culture, to cells ex vivo, and to cells in vivo.
  • the cells can be of any type including without limitation cancer cells, stem cells, neuronal cells, and non-neuronal cells.
  • the delivery of nucleic acids can be by any technique known in the art including viral mediated gene transfer, liposome mediated gene transfer, direct injection into a target tissue, organ, or tumor, injection into vasculature which supplies a target tissue or organ.
  • Polynucleotides can be administered in any suitable formulations known in the art. These can be as virus particles, as naked DNA, in liposomes, in complexes with polymeric carriers, etc. Polynucleotides can be administered to the arteries which feed a tissue or tumor. They can also be administered to adjacent tissue, whether tumor or normal, which could express the demethylase protein.
  • Nucleic acids can be delivered in any desired vector. These include viral or non- viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.
  • the nucleic acids encoding a protein or nucleic acid of interest may be in a plasmid or viral vector, or other vector as is known in the art. Such vectors are well known and any can be selected for a particular application.
  • the gene delivery vehicle comprises a promoter and a demethylase coding sequence.
  • Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters.
  • promoters which are activatable by infection with a virus such as the ⁇ - and ⁇ -interferon promoters, and promoters which are activatable by a hormone, such as estrogen.
  • promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter.
  • a promoter may be constitutive or inducible.
  • naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Patent 5,580,859.
  • gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et al., Hum. Gene. Ther. 3:147-154, 1992.
  • Other vehicles which can optionally be used include DNA- ligand (Wu et al., J. Biol. Chem. 264:16985-16987, 1989), lipid-DNA combinations (Feigner et al., Proc. Natl. Acad. Sci.
  • a gene delivery vehicle can optionally comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus.
  • the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci.
  • a polynucleotide of interest can also be combined with a condensing agent to form a gene delivery vehicle.
  • the condensing agent may be a polycation, such as polylysine, polyarginine, polyornithine, protamine, spermine, spermidine, and putrescine. Many suitable methods for making such linkages are known in the art.
  • a polynucleotide of interest is associated with a liposome to form a gene delivery vehicle.
  • Liposomes are small, lipid vesicles comprised of an aqueous compartment enclosed by a lipid bilayer, typically spherical or slightly elongated structures several hundred Angstroms in diameter. Under appropriate conditions, a liposome can fuse with the plasma membrane of a cell or with the membrane of an endocytic vesicle within a cell which has internalized the liposome, thereby releasing its contents into the cytoplasm.
  • the liposome membrane acts as a relatively impermeable barrier that sequesters and protects its contents, for example, from degradative enzymes.
  • a liposome is a synthetic structure, specially designed liposomes can be produced which incorporate desirable features. See Stryer, Biochemistry, pp. 236-240, 1975 (W.H. Freeman, San Francisco, CA); Szoka et al., Biochim. Biophys. Acta 600:1, 1980; Bayer et al., Biochim. Biophys. Acta. 550:464, 1979; Rivnay et al., Meth. Enzymol. 149:119, 1987; Wang et al., PROC. NATL.
  • Liposomes can encapsulate a variety of nucleic acid molecules including DNA, RNA, plasmids, and expression constructs comprising growth factor polynucleotides such those disclosed in the present invention.
  • Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.
  • Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7416, 1987), mRNA (Malone et al., Proc. Natl. Acad. Sci. USA 86:6077-6081, 1989), and purified transcription factors (Debs et al., J. Biol. Chem. 265:10189-10192, 1990), in functional form. Cationic liposomes are readily available.
  • N[l-2,3-dioleyloxy)propyl]-N,N,N- triethylammonium (DOTMA) liposomes are available under the trademark LIPOFECTIN®, from GIBCO BRL, Grand Island, NY. See also Feigner et al, Proc. Natl. Acad. Sci. USA 91 : 5148- 5152.87, 1994.
  • Other commercially available liposomes include TransfectACE (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).
  • Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA 75:4194-4198, 1978; and WO 90/11092 for descriptions of the synthesis of DOTAP (1,2- bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
  • anionic and neutral liposomes are readily available, such as from Avanti Polar
  • Lipids can be easily prepared using readily available materials.
  • Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphosphatidyl ethanolamine (DOPE), among others.
  • DOPC dioleoylphosphatidyl choline
  • DOPG dioleoylphosphatidyl glycerol
  • DOPE dioleoylphosphatidyl ethanolamine
  • These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
  • One or more protein (e.g., a demethylase) or nucleic acid (e.g., siRNA) of interest may be encoded by a single nucleic acid delivered.
  • nucleic acids may encode different protein or nucleic acids of interest.
  • Different species of nucleic acids may be in different forms; they may use different promoters or different vectors or different delivery vehicles.
  • the same protein or nucleic acid of interest may be used in a combination of different forms.
  • Antisense molecules, siRNA or shRNA molecules, ribozymes or triplex molecules may be contacted with a cell or administered to an organism. Alternatively, constructs encoding these may be contacted with or introduced into a cell or organism. Antisense constructs, antisense oligonucleotides, RNA interference constructs or siRNA duplex RNA molecules can be used to interfere with expression of a protein of interest, e.g., a histone demethylase. Typically at least 15, 17, 19, or 21 nucleotides of the complement of the mRNA sequence are sufficient for an antisense molecule. Typically at least 19, 21, 22, or 23 nucleotides of a target sequence are sufficient for an RNA interference molecule.
  • RNA interference molecule will have a 2 nucleotide 3 ' overhang. If the RNA interference molecule is expressed in a cell from a construct, for example from a hairpin molecule or from an inverted repeat of the desired histone demethylase sequence, then the endogenous cellular machinery will create the overhangs.
  • siRNA molecules can be prepared by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, or other methods known in the art. See Hannon, GJ, 2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002, The rest is silence.
  • RNA 7 1509-1521; Hutvagner G et al., RNAi: Nature abhors a double-strand. Curr. Opin. Genetics & Development 12: 225-232; Brummelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553; Lee NS, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J. (2002). Expression of small interfering RNAs targeted against HIV-I rev transcripts in human cells. Nature Biotechnol. 20:500- 505; Miyagishi M, and Taira K. (2002).
  • U6-promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnol. 20:497-500; Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, and Conklin DS. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul CP, Good PD, Winer I, and Engelke DR. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnol.
  • Antisense or RNA interference molecules can be delivered in vitro to cells or in vivo, e.g., to tumors of a mammal. Typical delivery means known in the art can be used. For example, delivery to a tumor can be accomplished by intratumoral injections. Other modes of delivery can be used without limitation, including: intravenous, intramuscular, intraperitoneal, intra-arterial, local delivery during surgery, endoscopic, subcutaneous, and per os. In a mouse model, the antisense or RNA interference can be administered to a tumor cell in vitro, and the tumor cell can be subsequently administered to a mouse. Vectors can be selected for desirable properties for any particular application. Vectors can be viral or plasmid.
  • Adenoviral vectors are useful in this regard.
  • Tissue-specific, cell-type specific, or otherwise regulatable promoters can be used to control the transcription of the inhibitory polynucleotide molecules.
  • Non-viral carriers such as liposomes or nanospheres can also be used.
  • a method may comprise administering to a subject, e.g., a subject in need thereof, a therapeutically effective amount of an agent described herein.
  • Diseases such as cancers can be treated by administration of modulators of histone methylation, e.g., modulators of histone demethylase enzyme activity.
  • H3K27 methylation has been reported to be involved in overexpression of certain genes in cancers.
  • Modulators that are identified by the disclosed methods or modulators that are described herein can be used to treat these diseases, i.e., to restore normal methylation to affected cells.
  • a method for treating cancer in a subject may comprise administering to the subject a therapeutically effective amount of one or more agents that decrease methylation or restores methylation to its level in corresponding normal cells.
  • modulators of methylation can be used for modulating cell proliferation generally. Excessive proliferation may be reduced with agents that decrease methylation, whereas insufficient proliferation may be stimulated with agents that increase methylation. Accordingly, diseases that may be treated include hyperproliferative diseases, such as benign cell growth and malignant cell growths. Exemplary cancers that may be treated include leukemias, e.g., acute lymphoid leukemia and myeloid leukemia, and carcinomas, such as colorectal carcinoma and hepatocarcinoma.
  • leukemias e.g., acute lymphoid leukemia and myeloid leukemia
  • carcinomas such as colorectal carcinoma and hepatocarcinoma.
  • cancers include Acute Lymphoblastic Leukemia; Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Acute Myeloid Leukemia; Adrenocortical Carcinoma Adrenocortical Carcinoma; AIDS- Related Cancers; AIDS-Related Lymphoma; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Basal Cell Carcinoma, see Skin Cancer (non-Melanoma); Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer; Bone Cancer, osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma; Brain Tumor; Brain Tumor, Brain Stem Glioma; Brain Tumor, Cerebellar Astrocytoma; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma; Brain Tumor, Ependymoma; Brain Tumor
  • screening methods for identifying agents that modulate methylation of a target protein such as a histone, e.g., lysine 27 (K27) of histone 3.
  • One method involves screening for an enhancer or inhibitor of histone demethylase activity, including the steps of contacting a histone H3 peptide with a histone demethylase protein (such as UTX, JMJD3, or a catalytic analog thereof), in the presence and in the absence of a test substance; determining the methylation status of the histone H3 peptide at a lysine 27 position; and identifying a test substance as an enhancer of histone demethylase activity if less mono-, di- or trimethylated H3K27 is found in the presence than in the absence of the test substance, and identifying a test substance as an inhibitor of histone demethylase protein activity if more mono-, di- or trimethylated H3K27 is found in the presence than in the absence of the
  • Test agents (or substances) for screening as inhibitors or enhancers of the demethylase enzymes can be from any source known in the art. They can be natural products, purified or mixtures, synthetic compounds, members of compound libraries, etc. The compounds to be tested may be chosen at random or may be chosen using a filter based on structure and/or mechanism of the enzymes. The test substances can be selected from those that have previously identified to have biological or drug activity or from those that have not. In some embodiments a natural substrate is the starting point for designing an inhibitor. Modifications to make the substrate non-modifiable by the enzyme can be used to make an inhibitor.
  • EXAMPLE 1 A histone H3 27 demethylase regulates animal posterior development.
  • the UTX JmjC catalytic domain alone also mediated demethylation of H3K27me3, me2 and mel, when the methylated histone peptides were used as substrates ( Figure ID).
  • Figure ID When bulk histones were analyzed, only H3K27me3 and me2, but not H3K27mel, levels were reduced ( Figure IE).
  • JMJD3 catalytic domain displayed similar specificity, demethylating H3K27me3, me2 and mel of the histone peptides ( Figure ID) but only H3K27me3 and me2 on native histones and nucleosomal substrates ( Figure ID and 1C).
  • JMJD3 significantly reduced the levels of H3K27me3 and me2 in approximately 78% and 56% of the transfected cells, respectively ( Figure 6, marked by arrowheads), but not H3K36me3, H3K9me3 or H3K4me3 ( Figure 6 and data not shown).
  • Over-expression of JMJD3 did not reduce the H3K27mel level, and in fact, an increase in H3K27mel was observed in 24% of the transfected cells ( Figure 6). This accumulation of H3K27mel is probably a result of the conversion of H3K27me3 and me2 to H3K27mel due to over-expression of JMJD3.
  • H3K27 trimethylation has been shown to be critical for the regulation of the Hox gene cluster
  • H3K27 methylation at the Hox gene locus was investigated and compared their levels in HeLa cell in the presence and absence of shRNA plasmids that inhibited the expression of UTX or JMJD3 (Figure 2B).
  • RNAi inhibition of the endogenous UTX resulted in H3K27me3 increases in some but not all Hox D genes.
  • H3K27me3 level was clearly elevated in the UTX knockdown cells ( Figure 2C).
  • the endogenous UTX in fibroblasts was isolated by chromatin immunoprecipitation (ChIP) followed by hybridization to ultra-dense tiling microarrays (ChIP-chip) that interrogated all four human Hox loci at 5 base pair resolution and 2 megabases of control regions including portions of X chromosome, chromosome 22, the beta-globin locus, and many transcribed genes in fibroblasts 27. Strikingly, UTX was selectively localized to the Hox loci of fibroblasts; over 90% of all UTX binding events were in the Hox loci ( Figure 3B, p ⁇ 10-64, hypergeometric distribution).
  • UTX was selectively targeted to narrow windows within 500 base pairs downstream of the transcriptional start site of HOX genes ( Figure 3 A and C).
  • the raw UTX ChIP-chip profiles at both locus-wide and gene-specific resolutions are shown in Figure 7. Although focal, these UTX binding events are supported by hybridization of multiple contiguous probes and are thus of high statistical confidence and validated by additional quantitative PCR experiments (Figure 7).
  • UTX binds the start of both transcriptionally active and silent Hox genes in a manner largely independent of anatomic origins of cells ( Figure 3 A and D).
  • HOX genes in embryonic stem (ES) cells are largely occupied by H3K27me3 and transcriptionally silent 9.
  • UTX was entirely excluded from the Hox loci ( Figure 3D), although UTX is expressed and appeared to bind selected sets of genes at other genomic locations ( Figure 8).
  • Figure 3D The lack of UTX occupancy suggests a potential deficiency in the mechanisms important for targeting of UTX to the Hox gene locus in mES cells.
  • transcript levels of several 3' posterior ⁇ ox genes are modestly but consistently reduced at 36 hours dpf (Figure 4E).
  • the expression of more anterior zHox genes such as zHoxC ⁇ a, zHoxC ⁇ b and zHoxA3
  • the most posterior Hox genes appears unaffected ( Figure 4E and data not shown), which correspond well to the normal appearance of the anterior and most posterior tail regions of the mutant embryos ( Figure 4E and data not shown).
  • RNA in situ analysis showed modestly reduced transcript level of zHoxC8 and a posterior shift of its expression domain in most of the UTX morpholino-treated embryos (from somite 1-7 to somite 2-8) (Figure 4F).
  • Figure 4F we also observed reduced expression of zHoxCl Ia and zHoxC12b, loss of expression of zHoxDl 2a and zHoxA9b at the pectoral fin bud, and a shift of the zHoxD9 expression domain ( Figure 10).
  • This complex contains both UTX and WDR5, which is important for MLL complex regulating H3K4 methylation 29.
  • H3K4 and H3K27 methylation can be toggled independently for certain genes.
  • RNAi inhibition of UTX in 293T or mouse ES cells had no effect on global H3K4 methylation or the in vitro H3K4 methylation activity of ALR/MLL2 ( Figure 12), and UTX occupancy did not correspond to bulk H3K4 methylation level in the Hox loci ( Figure 7C).
  • UTX occupancy of the HOX locus may occur independently of ALR function.
  • UTX may act independently of ALR function in the HOX locus.
  • H0XA9 is also regulated by the MLLl complex, which is related to ALR/MLL2 but lacks UTX 30,31.
  • H3K27me3 9 activation of some of these genes is correlated with a loss of H3K27me3 9, suggesting possible dynamic regulation through demethylation during differentiation.
  • H3K27me demethylases combined with the observation that the JMJD3 expression is up-regulated during ES cell differentiation 9, supports the hypothesis that H3K27me demethylases play a role in the resolution of the "bivalent domain" and in the regulating transcription of these genes during ES differentiation.
  • UTX is excluded from the Hox gene locus identifies a possible mechanism that may help protect the bivalent domains at the Hox gene locus in ES cells. At the same time, UTX is involved in HOX gene locus regulation during development and differentiation.
  • UTX is in the same protein complex with enzymes that mediate ⁇ 3K4 trimethylation
  • important mechanisms must be in place to facilitate differential regulation of the K4 and K27 methylation states in ES versus differentiated cells.
  • These mechanisms may involve, but not limited to, possible differential MLL/UTX complex composition and/or differential regulation of MLL and/or UTX enzymatic activities at the target loci at different stages of cell differentiation.
  • Rb PcG proteins and H3K27 methylation in the transcriptional silencing of the pl6INK4Dtumor suppressor 33.
  • HOX genes are also candidate oncogenes and tumor suppressor genes in several types of human cancer 34,35.
  • UTX and or JMJD3 may play an antagonistic role to that of the PcG proteins in pi 6 and/or HOX regulation and therefore may function as putative tumor suppressors.
  • Antibodies (Ab) that recognize different histone modifications were purchased from Upstate Group INC.
  • MALDI-TOF mass spectrometry One microliter of the demethylation reaction mixture was desalted through a Cl 8 ZipTip (Millipore). The ZipTip was activated, equilibrated, and loaded as previously described by Shi et al. (2004). The bound material was then eluted with 10 mg/ml D- cyano-4-hydroxycinnamic acid MALDI matrix in 70% acetonitrile/0.1% TFA before being spotted and co-crystallized. The samples were analyzed by a MALDI-TOF/TOF mass spectrometer.
  • ChIP-chip Primary human lung, foot, and foreskin fibroblasts, custom human Hox tiling microarray, ChIP-chip analysis, and Hox loci-wide occupancy of Suzl2, H3K27me3, H3K4me2, and RNA pol II were as described (PMID: 17604720).
  • ChIP of mouse ES cells gifts of A. Wright and M. Scott
  • mouse promoter tiling array set (Nimblegen Systems, WI) which tiles 3.25 kilobases upstream and 0.75 kilobases downstream of promoters genome-wide.
  • UTX occupancy was determined by SignalMap peak calling algorithm comparing binding event to simulated data on shuffled probe sets; we chose peaks that had estimated false discovery rate of less than 0.01 and had signal intensity at least 1.5-fold over control chIP-chip experiment with IgG.
  • UTX was performed by injection of 2nL of a stock concentration of 250 ⁇ M antisense morpholinos (Gene-Tools, LLC) and 75ng/ ⁇ l of mRNA from human UTX constructs or a control EGFP into one- cell stage zebrafish embryos using a gas driven microinjector (Medical Systems Corp.).
  • the zUTXl morpholino sequence used in Figure 4 was 5'- AGCTCCGAGCGTCCAAAAGCCACAA -3' covering bases -55 to -31 in the 5' UTR, and a similar phenotype was observed at a lower frequency with a second morpholino (5'- CCACCGAC ACTCGGCACGGCTTCAT -3') covering ORF bases +1 to +25.
  • the sequence of the control morpholino was 5'-
  • CCTCTTACCTCAGTTACAATTTATA -3' Whole-mount in situ hybridization was done using digoxigenin and/or fluorescein-labeled antisense RNA probes (Roche).
  • Table 1 Summary of UTX occupancy in the Hox loci. Columns F-I give the genes that were used to create the average occupancy profiles shown in Figure 3C and Figure 7C. For example, 23 UTX occupancy profiles of 16 unique Hox genes from two sources of fibroblasts were averaged for the left axis of Fig. 3C. 12 H3K27me3 profiles of 10 unique Hox genes from the same two types of fibroblasts were averaged for the right axis of Fig. 3C.
  • EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc Natl Acad Sci USA lQQ, 11606-11 (2003).
  • Yamane, K. et al. PLU-I is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation. MoI Cell 25, 801-12 (2007).
  • Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth. Genes Dev 21, 537-51 (2007).
  • RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3. Cell 128, 1063-76 (2007).
  • Liang, G., Klose, R. J., Gardner, K. E. & Zhang, Y. Yeast Jhd2p is a histone H3 Lys4 trimethyl demethylase. Nat Struct MoI Biol 14, 243-5 (2007).

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Abstract

Selon l’invention, UTX et JMJD3 agissent en tant que histone H3 déméthylases, agissant spécifiquement sur H3K27. Une méthylation de l’histone H3K27 est critique pour la répression de l’expression génique de HOX et d’autres gènes. Une dérégulation de la méthylation de H3K27 est associée à des maladies comprenant le cancer.
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WO2011051269A1 (fr) 2009-10-27 2011-05-05 Glaxo Group Limited Procédé de traitement
WO2012052390A1 (fr) 2010-10-19 2012-04-26 Glaxo Group Limited Dérivés de n-2-(2-pyridinyl)-4-pyrimidinyl-bêta-alanine en tant qu'inhibiteurs d'histone déméthylase jmjd3
WO2012052391A1 (fr) 2010-10-19 2012-04-26 Glaxo Group Limited Polypeptide ayant l'activité catalytique de jmjd3
KR101304992B1 (ko) 2013-04-24 2013-09-17 한양대학교 에리카산학협력단 Ｊｍｊｄ３ 유전자의 발현을 억제시켜서 세포접착분자의 발현을 증가시키는 인공 마이크로 ｒｎａ
WO2013143597A1 (fr) 2012-03-29 2013-10-03 Glaxo Group Limited Inhibiteurs d'enzymes de déméthylase
US20140121201A1 (en) * 2012-09-24 2014-05-01 Dan Littman REGULATORY NETWORK FOR Th17 SPECIFICATION AND USES THEREOF
CN107299113A (zh) * 2017-06-12 2017-10-27 内蒙古大学 H3K27me3及其去甲基化酶KDM6A/B在小鼠核移植重构胚中的应用方法
CN107664698A (zh) * 2017-12-25 2018-02-06 阜阳师范学院 一种卵母细胞h3k27三甲基化分析指标体系

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011051269A1 (fr) 2009-10-27 2011-05-05 Glaxo Group Limited Procédé de traitement
WO2011051264A2 (fr) 2009-10-27 2011-05-05 Glaxo Group Limited Méthode de traitement
WO2011051270A1 (fr) 2009-10-27 2011-05-05 Glaxo Group Limited Procédé de traitement
WO2012052390A1 (fr) 2010-10-19 2012-04-26 Glaxo Group Limited Dérivés de n-2-(2-pyridinyl)-4-pyrimidinyl-bêta-alanine en tant qu'inhibiteurs d'histone déméthylase jmjd3
WO2012052391A1 (fr) 2010-10-19 2012-04-26 Glaxo Group Limited Polypeptide ayant l'activité catalytique de jmjd3
WO2013143597A1 (fr) 2012-03-29 2013-10-03 Glaxo Group Limited Inhibiteurs d'enzymes de déméthylase
US20140121201A1 (en) * 2012-09-24 2014-05-01 Dan Littman REGULATORY NETWORK FOR Th17 SPECIFICATION AND USES THEREOF
KR101304992B1 (ko) 2013-04-24 2013-09-17 한양대학교 에리카산학협력단 Ｊｍｊｄ３ 유전자의 발현을 억제시켜서 세포접착분자의 발현을 증가시키는 인공 마이크로 ｒｎａ
CN107299113A (zh) * 2017-06-12 2017-10-27 内蒙古大学 H3K27me3及其去甲基化酶KDM6A/B在小鼠核移植重构胚中的应用方法
CN107664698A (zh) * 2017-12-25 2018-02-06 阜阳师范学院 一种卵母细胞h3k27三甲基化分析指标体系

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