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WO2001016368A1 - Systemes de transcription in vitro et ses utilisations - Google Patents

Systemes de transcription in vitro et ses utilisations Download PDF

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Publication number
WO2001016368A1
WO2001016368A1 PCT/US2000/017800 US0017800W WO0116368A1 WO 2001016368 A1 WO2001016368 A1 WO 2001016368A1 US 0017800 W US0017800 W US 0017800W WO 0116368 A1 WO0116368 A1 WO 0116368A1
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Prior art keywords
rxr
transcription
dimer
chromatin
receptor
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PCT/US2000/017800
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English (en)
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WO2001016368A9 (fr
Inventor
F. Jeffrey Dilworth
Catherine Fromental-Ramain
Pierre Chambon
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Bristol-Myers Squibb Company
Centre National De La Recherche Scientifique
Universite Louis Pasteur
Institut National De La Sante Et De La Recherche Medicale
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Application filed by Bristol-Myers Squibb Company, Centre National De La Recherche Scientifique, Universite Louis Pasteur, Institut National De La Sante Et De La Recherche Medicale filed Critical Bristol-Myers Squibb Company
Priority to AU62010/00A priority Critical patent/AU6201000A/en
Priority to CA002383698A priority patent/CA2383698A1/fr
Publication of WO2001016368A1 publication Critical patent/WO2001016368A1/fr
Publication of WO2001016368A9 publication Critical patent/WO2001016368A9/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors

Definitions

  • the invention relates to methods of identifying agents that interact with retinoid X receptor dimers.
  • the invention also relates to in vitro chromatin based
  • RAs Retinoic acids
  • NRs nuclear receptors
  • RAR ⁇ , RAR ⁇ and RAR ⁇ Two families of receptors, the retinoic acid receptor isotypes (RAR ⁇ , RAR ⁇ and RAR ⁇ ) and the retinoid X receptors isotypes (RXR ⁇ , RXR ⁇ and RXR ⁇ ) are implicated in the transduction of the RA signal (Chambon, P., FASEB J. 70:940-954 (1996), and references therein).
  • RARs bind all-trans RA (tRA) and 9-cis RA (9cRA), whereas RXRs respond exclusively to 9cRA (Allenby, G., et al, Proc. Natl. Acad. Sci. USA 90:30-34 (1993), and references therein).
  • tRA 9-cis RA
  • RXRs respond exclusively to 9cRA (Allenby, G., et al, Proc. Natl. Acad. Sci. USA 90:30-34 (1993), and references therein).
  • RXRs contains both the ligand binding domain (LBD), which functions as a ligand-dependent transactivation domain (activation function 2 (AF-2)), and surfaces for both homo- and hetero-dimerization as well as for interaction with other factors (see below).
  • LBD ligand binding domain
  • AF-2 activation function 2
  • An additional ligand-independent activation function, AF-1 is present within the N-terminal region (see, Chambon, P., FASEB J.
  • RARs and RXRs can bind as dimers to RA response elements (RAREs) consisting of two hexameric motifs [PuG(G/A)(T/A)CA] usually arranged as direct repeats.
  • RXRs readily heterodimerize with RARs, and RXR/RAR heterodimers bind to and transactivate from RAREs made up of direct repeat motifs separated by 5 (DR5) and 2 (DR2) bp much more efficiently than RXR homodimers on their own. This indicates that RXR/RAR heterodimers might be the functional units transducing the retinoic acid signals in vivo
  • ACTR Chonda, H., et al, Cell 90:569-580 (1997)
  • histone acetyltransferase p/CAF Yang, X-J., et al, Nature 382:319-324 ( 1996)
  • CBP and p300 also interact with RNA helicase A, which in turn binds RNA polymerase II (Nakajima, T., et al, Cell 90:1107-1112 (1997)).
  • RXRs form heterodimers in solution with either RARs, TRs or VDR and that the receptor domains required for heterodimeric interactions overlap with the LBD of each receptor.
  • Ligand dependent transcription activation by the RXR VDR heterodimer has been shown (Rachez, C. et al, Nature 595:824-828 (1999)). The formation of heterodimers between
  • RXRs and PPARs was also demonstrated (Kliewer, S.A. et al. Nature 555:771- 774 (1992); Bardot, O. etal, Biochem. Biophys. Res. Comm. 792:37-45 (1993)).
  • RXR also heterodimerizes with liver X receptors (LXRs; Apfel et al, Mol. Cell Biol. 7 ⁇ :7025-7035 (1994), farnesoid X receptor (FXR; Forman et al, Cell 57:687-693 (1995)), benzoate X receptor (BXR; Blumberg et al, Genes Dev.
  • CARs constitutively active receptor or constitutive androstane receptors
  • SXR steroid and xenobiotic receptor
  • the first class consists of several ATP-driven chromatin remodeling complexes that modify chromatin structure by affecting the position and/or stability of nucleosomes.
  • the second class corresponds to a variety of core histone acetyltransferases (HAT) (recent reviews on chromatin modifying activities include Varga-Weisz, P.D. and Becker, P.B., Curr. Opin. Cell Biol. 70:346-353 (1998); Workman, J.L. and guitarist, R.E., Ann. Rev. Biochem. 67:545-579 (1998); guitarist, R.E. and Narlikar, G. J.,
  • the invention is directed to methods of identifying agents that interact with retinoid X receptor dimers.
  • the invention is also directed to in vitro chromatin based DNA template transcription systems.
  • FIG. 1 DNA templates and SI nuclease probe.
  • the structures of the (DR5)5 ⁇ 2G, (17m)5 ⁇ 2G and internal control pGl reporter templates are schematically represented with the positioning of the response elements relative to the transcription start site.
  • FIG.2. Analysis of RAR ⁇ /RXR ⁇ heterodimers and chromatin structure.
  • FIG.2(A) Purification of RAR ⁇ /RXR ⁇ heterodimers: FhRAR ⁇ and HmRXR ⁇ were co-expressed in Sf9 cells and affinity-purified using a Ni 2+ column followed by anti-Flag agarose column that bind the HmRXR moiety and the FhRAR moiety of the heterodimer, respectively.
  • nM and tRA were digested with varying concentrations of micrococcal nuclease in a final volume of 80 ⁇ l, separated on a 1.5% agarose gel, and Southern blotted using a [ 32 P] probe corresponding to the -40 to +5 region of the (DR5)5 ⁇ 2G promoter.
  • DNA supercoiling was estimated as described (Becker, P.B., et al, Methods Cell Biol. ⁇ :207-223 (1994)) on DNA (200 ng) treated (or not treated) by topoisomerase 1(10 units; final volume of 45 ⁇ l) .
  • DNA was separated on a 1% agarose gel in the presence or absence of 1.2 ⁇ M chloroquine. Migration of relaxed and supercoiled template DNA is indicated.
  • FIG. 3 RAR ⁇ /RXR ⁇ heterodimers activate transcription from chromatin templates in a ligand- and template-specific manner.
  • FIG. 3(A) tRA-induced derepression of transcription from chromatin templates by RAR ⁇ /RXR ⁇ . In vitro transcription was performed on chromatin or naked (DR5)5 ⁇ 2G templates (200 pM) using a HeLa cell nuclear extract (100 ⁇ g) for 45 min in the presence or absence of FhRAR ⁇ HmRXR ⁇ (1 nM) and tRA (1 ⁇ M) in a final reaction volume of 50 ⁇ l as indicated. SI nuclease analysis was carried out after deproteinization.
  • FIG. 3(B) Template specificity of activated transcription.
  • Activation of transcription on chromatin (DR5)5 ⁇ 2G or (17M)5 ⁇ 2G templates was determined in the presence of 1 nM of activator (either Gal4(l-147), Gal4- VP 16 or FhRAR ⁇ /HmRXR ⁇ ) with or without tRA ( 1 ⁇ M) as above.
  • S 1 nuclease digestion of RNA transcripts originating from ⁇ 2G and pGl templates generated
  • FIG. 4 RAR ⁇ /RXR ⁇ heterodimers bind all five RAREs in the promoter region of the (DR5)5 ⁇ 2G chromatin template, irrespective of the presence of tRA.
  • Chromatin or naked (DR5)5 ⁇ 2G templates 250 ng were incubated in the presence or absence of FhRAR ⁇ /HmRXR ⁇ and tRA (IO "6 M) (under the conditions described above for transcription reactions) for 30 min, subjected to
  • DNase I digestion (5 units; final volume 50 ⁇ l), then analyzed by primer extension foot printing (.see Materials and Methods, Example 1 ). Sites of increased (closed triangle) or decreased (open triangle) sensitivity to DNase I are shown.
  • FIG. 5 Dose-dependent synergistic effects of specific retinoids on activation of transcription by RAR ⁇ /RXR ⁇ heterodimers.
  • FIG. 5(A) Dose-dependent activation by tRA and 9cRA. Transcription reactions were performed as described in FIG. 3 on (DR5)5 ⁇ 2G template by using FhRAR ⁇ /HmRXR ⁇ in presence of varying concentrations (5x10 " 10 to IO "6 M) of tRA (open circles) or 9cRA (closed squares).
  • FIG.5(B) Receptor-selective and synergistic activation of transcription. Transcription reactions were performed as described above using synthetic retinoid agonists and antagonists at the concentrations indicated.
  • the receptor specificity of retinoids used are as follows: tRA (panRAR-specific ligand), 9cRA (panRAR- and panRXR-ligand), Compound I (RAR ⁇ -specific agonist), Compound IV (RAR ⁇ -specific agonist), SRI 1237 (panRXR-specific agonist), and Compound II (RAR ⁇ -specific antagonist).
  • Fl RAR ⁇ /HmRXR ⁇ is expressed relative to that observed from the internal control template (pGl). Induction by tRA (IO "6 M) was arbitrarily set to 100%. All points are the average of at least two independent experiments run in duplicate.
  • Transcription reactions were performed in the presence or absence of acety ICo A ( 1 ⁇ M) on naked or chromatin (17M)5 ⁇ 2G or (DR5)5 ⁇ 2G templates plus or minus 1 nM activator (either Gal4(l-147), Gal4-VP16 or FhRAR ⁇ /HmRXR ⁇ ) and/or tRA (1 ⁇ M), as described in FIG. 3.
  • FIG.6(B) Addition of acetylCoA does not further enhance p300-activated transcription. Transcription was performed on (DR5)5 ⁇ 2G -7-
  • Histone HI increases nucleosome repeat length from 170 to 200 bp in a chromatin structure that is more resistent to micrococcal nuclease (MNase I) digestion.
  • Chromatin (DR5)5 ⁇ 2G template (160 ng) with (+H1) or without (-HI) histone HI were digested with 6.5 units MNasel (final volume 55 ⁇ l, 24 °C) for the time indicated in seconds (sec). Following deproteinization and separation on a 1.2% agarose gel in 0.5x TBE, DNA was stained with ethidium bromide.
  • FIG. 7(B) Incorporation of Histone HI into
  • FIG. 7(C) Histone HI remains associated with chromatin template after size-exclusion chromatography .
  • FIG. 7(D) SWI/SNF endogenous to the chromatin assembly extract is removed during size exclusion chromatography.
  • "Crude” or “purified” chromatin (DR5)5 ⁇ 2G templates (70 ng) assembled in presence or absence of histone HI were applied to a nitrocellulose membrane using a slot blot apparatus, and then probed for the presence of dSWI/SNF using antibodies directed against dBRM.
  • FIG. 7(E) Purification of human SWI/SNF complexes Human SWI/SNF SWI/SNF complexes were purified from HeLa cells expressing Flag-tagged Ini 1. Purified SWI/SNF was separated on a 10% SDS-PAGE gel before staining with
  • FIG. 7(F) Chromatin purification removes some HAT activity associated with the "crude” chromatin preparation.
  • "Crude” or “purified” chromatin (DR5)5 ⁇ 2G templates (30 ng) assembled in presence or absence of histone HI were incubated with free core histones (2 ⁇ g) and 0.1 ⁇ Ci of [Acetyl- 1- 14 C] Acetyl CoA at 30°C Ovalbumin and p300 (1 pmol of each) were used as a negative and positive controls, respectively.
  • FIG.7(G) Both TIF2 and p300 possess intrinsic HAT activity. HAT assays were as described in panel D using 2 ⁇ g of free core histones or purified nucleosomes in the presence of either TIF2 (1 or 3 pmol), p300 (1 or 3 pmol), or ovalbumin (1 pmol), as indicated. A 25-fold excess of cold Acetyl CoA was added to reactions as indicated to confirm that transfer of 14 C to the histones was specific.
  • FIG.8 Requirements for activation of transcription on purified chromatin templates.
  • FIG.8(A). Transcriptional flow chart.
  • FIG.8(B). Ligand-dependent activation of transcription by RAR ⁇ /RXR ⁇ heterodimers on purified chromatin templates required coactivator acetyltransferase and ATP-dependent chromatin remodeling activities. Transcription was performed using a HeLa nuclear extract on "crude” or "purified” chromatin (DR5)5 ⁇ 2G templates assembled in the presence or absence of histone HI. RAR ⁇ /RXR ⁇ heterodimers, tRA/SR 11237, p300, TIF2, hSWI/SNF, ATP, and/or Acetyl CoA were included as indicated. Transcription reactions was analyzed as described in Figure 7(B).
  • Acetyl CoA and ATP are required for transactivation on "purified” chromatin. Transcription was performed on “purified” (DR5)5 ⁇ 2G chromatin templates assembled in the presence of histone HI using a HeLa nuclear extract, RAR ⁇ /RXR ⁇ heterodimers, and tRA/SR 11237 ligands. Different combination of p300, TIF2, hSWI/SNF, ATP and Acetyl CoA were included as indicated.
  • FIG 8(D) TIF2 and p300 stimulate activation of transcription by RAR ⁇ /RXR ⁇ heterodimers. Transcription was performed on "purified" (DR5)5 ⁇ 2G chromatin templates assembled in the presence of histone HI using a HeLa nuclear extract, RAR ⁇ /RXR ⁇ heterodimers, ATP, and hS WI/SNF. Acetyl CoA, p300, TIF2, and tRA SR 11237 were included as indicated.
  • FIG. 9 Both ATP-driven chromatin remodeling factors and coactivator acetyltransferase activities synergistically contribute to stimulation of transcription -9-
  • Rounds of transcription was calculated by dividing the intensity of the signal in the absence of sarkosyl (multiple rounds) by that in the presence of sarkosyl (single round) and is rounded off to a whole number. Similar results were obtained in several experiments carried out with different chromatin preparations and sarkosyl concentrations.
  • FIG. 10 ATP and ISWI-containing complexes are required for efficient binding of RAR ⁇ /RXR ⁇ heterodimers to their response elements (RARE) on chromatin templates.
  • FIG.10(A) "Purified" (DR5)5 ⁇ 2G chromatin templates (1 nM) assembled in the presence of histone HI were incubated as indicated in the presence or absence of RAR ⁇ /RXR ⁇ (5 nM), tRA (1 ⁇ M), SR 11237 (1 ⁇ M), TIF2 (2.5 nM), p300 (2.5 nM), hSWI/SNF (10 ng/ ⁇ l), ATP (0.1 mM), and/or Acetyl CoA (2 ⁇ M) for 30 min at 27°C, and then processed for DNAse I footprinting (Experimental Procedures).
  • FIG. 10(B) Same as (A) except that hISWI (5 ng/ ⁇ l) was added to reactions were indicated.
  • FIG.11 ATP-dependent remodeling of(DR5)5 ⁇ 2G chromatin templates in the presence of RAR/RXR heterodimers is limited to the region containing the REs.
  • "Purified" (DR5)5 ⁇ 2G chromatin templates (1 nM) were incubated as indicated in the presence or absence of RAR ⁇ /RXR ⁇ a (5 nM), and/or ATP (0.1 mM) at 27 °C After 30 min chromatin templates were digested with 1 unit MNasel (final volume 60 ml, 24 °C) for the time indicated in seconds (sec).
  • FIG.12. RAR/RXR heterodimers target histone acetyltransferase activity to (DR5)5 ⁇ 2G promoter region in a ligand-dependent manner.
  • "Purified" chromatin (DR5)5 ⁇ 2G (200 pM) and pSG5 (200 pM) templates were incubated in the presence of RAR ⁇ /RXR ⁇ heterodimers (1 nM), hS WI/SNF (2 ng/ ⁇ l), ATP (0.1 mM), Acetyl CoA (2 ⁇ M), tRA and SR 11237 (1 ⁇ M each), and/or TIF2 and ⁇ 300 (500 pM each) as indicated.
  • ChIP assays were then performed (Example 2, Experimental Procedures). The data displayed is representative of three independent experiments.
  • FIG.13 ATP-driven chromatin remodeling activities and coactivators act sequentially to mediate the ligand-dependant stimulatory effect of RAR ⁇ /RXR ⁇ heterodimers on transcription initiation.
  • FIG.13(A) Transcriptional timeline and schematic diagram indicating the "normal" time of addition of the different components of the in vitro transcription system.
  • FIG. 13(B) Effect of varying the time of addition of RAR ⁇ /RXR ⁇ heterodimers and tRA/SR 11237. Transcription was performed on "purified" (DR5)5 ⁇ 2G HI -containing chromatin templates using a HeLa nuclear extract, ATP, hSWI/SNF, AcetylCoA, p300, and TIF2.
  • the activators, cofactors, coactivators or agonistic ligands were introduced as indicated at either the same time as the chromatin template (- 40 min relative to transcription initiation), 20 min later (-20 min), at the same time as (-10 min) or 5 min after (-5 min) the HeLa nuclear extract, 0.5 min before the rNTPs (-0.5 min), or not at all (none or -).
  • the experiments displayed in panels B-E were repeated at least three times with different chromatin preparations and yielded similar results.
  • RXR/RAR heterodimers mimics the effects of retinoids on gene transactivation as observed in vivo.
  • Activation of transcription by RXR/RAR heterodimers depends on packaging of the template into a nucleosomal structure and that it is specific, in that it requires the heterodimer, a cognate ligand, and a cognate response element.
  • the agonist-bound transcription activation function of RXR can act synergistically with that of RAR but that the binding of an agonist to RAR is a prerequisite for effective activation of transcription by agonist-bound RXR.
  • RAR/RXR heterodimers cannot efficiently initiate transcription from a chromatin template from which transcription co- regulators have been removed, unless the chromatin template is exposed to two types of chromatin modifying activities that synergize to activate transcription: ATP-driven chromatin remodeling activities and histone acetyltransferase activities.
  • the invention is directed to a method of identifying an agent which interacts with a retinoid X receptor (RXR) dimer, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of the RXR dimer; and (b) measuring activation of transcription, thereby determining whether the agent interacts with the RXR dimer.
  • activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known agent.
  • transcription co-regulators are not removed from the chromatin based DNA template prior to adding an agent.
  • the method further comprises removing one or more or all transcription co-regulators.
  • a co-activator can be added before or after adding the agent.
  • the agent can be a co-activator or an agent that mediates an interaction between a RXR dimer and a co-activator.
  • this method is useful in identifying agents that regulate interactions between RXR dimers and co-activators.
  • Another embodiment of the invention is directed to a method of identify ing a retinoic acid receptor (RAR) agonist, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of an RXR/RAR dimer; and (b) measuring activation of transcription, thereby determining whether the agent is an RAR agonist.
  • RAR retinoic acid receptor
  • activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known RAR agonist.
  • transcription co-regulators are not removed from the chromatin based DNA template prior to adding an agent.
  • the method further comprises removing one or more or all transcription co-regulators.
  • a co-activator can be added before or after adding the agent.
  • the agent can be a co-activator or an agent that mediates an interaction between a RXR dimer and a co-activator. Thus, this method is useful in identifying agents that regulate interactions between RXR dimers and co-activators.
  • the invention is also directed to a method of identifying an RXR agonist, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of an RXR/RAR dimer and an RAR agonist; and (b) measuring activation of transcription, thereby determining whether the agent is an RXR agonist.
  • activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known RXR agonist.
  • transcription co-regulators are not removed from the chromatin based DNA template prior to adding an agent.
  • the method further comprises removing one or more or all transcription co- regulators.
  • a co-activator can be added before or after adding the agent.
  • the agent can be a co-activator or an agent that mediates an interaction between a RXR dimer and a co-activator. Thus, this method is useful in identifying agents that regulate interactions between RXR dimers and coactivators.
  • the invention is further directed to a method of identifying an RAR antagonist, the method comprising: (a) adding an agent to a chromatin based
  • DNA template in the presence of an RXR/RAR dimer and an RAR agonist and (b) measuring activation of transcription, thereby determining whether the agent is an RAR antagonist.
  • activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known RAR antagonist.
  • transcription co-regulators are not removed from the chromatin based DNA template prior to adding an agent.
  • the method further comprises removing one or more or all transcription co-regulators.
  • a co-activator can be added before or after adding the agent.
  • the agent can be a co-activator or an agent that mediates an interaction between RXR dimers and a co-activator.
  • this method is useful in identifying agents that regulate interactions between RXR dimers and co-activators.
  • the method further comprises adding a co-repressor.
  • a co-repressor can be added before or after adding the agent.
  • the agent can be a co-repressor or an agent that mediates an interaction between a RXR dimer and a co-repressor.
  • this method is useful in identifying agents that regulate interactions between RXR dimers and co- repressors.
  • the invention is directed to a method of identifying an RXR antagonist, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of a RXR/RAR dimer, an RAR agonist, and an RXR agonist; and (b) measuring activation of transcription, thereby determining whether the agent is an RXR antagonist.
  • activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known RXR antagonist.
  • transcription co-regulators are not removed from the chromatin based DNA template prior to adding an agent.
  • the method further comprises removing one or more or all transcription co-regulators.
  • a co-activator can be added before or after adding said agent.
  • the agent can be a co-activator or an agent that mediates an interaction between a RXR dimer and a co-activator.
  • this method is useful in identifying agents that regulate interactions between RXR dimers and co-activators.
  • the method further comprises adding a co-repressor.
  • a co-repressor can be added before or after adding said agent.
  • the agent can be a co-repressor or an agent that mediates an interaction between a RXR dimer and a co-repressor.
  • this method is useful in identifying agents that regulate interactions between RXR dimers and co- repressors.
  • the invention is directed to a method of identifying a co-activator of an RXR dimer, the method comprising: (a) adding a first agent to a chromatin based DNA template in the presence of the RXR dimer and an agonist of the RXR dimer; and (b) measuring activation of transcription, thereby determining whether the first agent is a co-activator of the RXR dimer.
  • activation of transcription can be compared to the method performed in the absence of the first agent or in the presence of a known co-activator.
  • this method can be used for identifying a co-repressor of the RXR dimer.
  • activation of transcription can be compared to the method performed in the absence of the first agent or in the presence of a known co-repressor.
  • transcription co-regulators are not removed from the chromatin based DNA template prior to adding the first agent and a RXR dimer agonist.
  • the chromatin based DNA template is purified such that one or more or all transcription co-regulators are removed prior to adding the first agent and the agonist of the RXR dimer.
  • the invention is further directed to a method of identifying a modulator which modulates interactions between a RXR dimer and a co-activator of the RXR dimer, the method comprising: (a) adding an agent to a chromatin based
  • DNA template in the presence of the RXR dimer, an agonist of the RXR dimer, and a co-activator of the RXR dimer ; and (b) measuring activation of transcription, thereby determining whether the agent modulates interactions between the RXR dimer and the co-activator of the RXR dimer.
  • activation of transcription can be compared to the method performed in the absence of the agent.
  • this method can be used for identifying a co-repressor of the RXR dimer.
  • activation of transcription can be compared to the method performed in the absence of the agent.
  • transcription co-regulators are not removed from the chromatin based DNA template prior to adding an agent, RXR dimer agonist, and a co-activator.
  • the chromatin based DNA template is purified such that one or more or all transcription co-regulators are removed prior to adding the first agent, the agonist of the RXR dimer, and a co-activator.
  • Agents identified by the above methods can be useful in treating a variety of conditions and diseases including, but not limited to, lung cancer, mesothelioma, photodamaged skin, fine and course wrinkling, improper skin pigmentation, skin roughness, premalignant skin growths such as actinic keratoses, diseases of the nervous system, improper regulation of cellular growth and differentiation, visual impairment, acute promyelocytic leukemia, and basal cell carcinoma.
  • the invention is directed to an in vitro chromatin based DNA template transcription system comprising: (a) a chromatin based DNA template; and (b) an RXR dimer.
  • transcription co-regulators are not removed from the chromatin based DNA template.
  • the chromatin based DNA template is purified such that one or more or all transcription co- regulators are removed.
  • the system can further comprise a co-activator and/or a co-repressor.
  • the invention is also directed to a kit comprising the in vitro chromatin based DNA template transcription system.
  • nuclear receptor or nuclear receptor superfamily receptor
  • steroid/thyroid hormone receptor superfamily is intended a ligand-dependent transcription factor that regulates the expression of target genes involved in metabolism, development, and reproduction.
  • Nuclear receptors include receptors for which specific ligands have not yet been identified (termed “orphan receptors”). These hormone binding proteins can bind to specific DNA sequences to modulate transcriptional activity of a target gene, upon binding of a ligand to the receptor.
  • Exemplary nuclear receptors include, but are not limited to, retinoic acid receptors (RARs; ⁇ , ⁇ and ⁇ ), retinoid X receptors (RXRs; ⁇ , ⁇ and ⁇ ), vitamin D 3 receptor (VDR), thyroid receptors (TRs; ⁇ and ⁇ ), peroxisome proliferator activated receptors (PPARs; ⁇ , ⁇ , ⁇ and ⁇ ), liver X receptors (LXRs; ⁇ and ⁇ )
  • RARs retinoic acid receptors
  • RXRs retinoid X receptors
  • VDR vitamin D 3 receptor
  • TRs thyroid receptors
  • PPARs peroxisome proliferator activated receptors
  • LXRs liver X receptors
  • CARs constitutively active receptor or constitutive androstane receptors
  • SXR steroid and xenobiotic receptor
  • RXR dimer is intended a dimer formed by an RXR ( ⁇ , ⁇ or ⁇ ) and a second nuclear receptor, and includes an RXR homodimer and RXR heterodimer.
  • RXR homodimer is intended a dimer of an RXR ( ⁇ , ⁇ or ⁇ ) and another RXR ( ⁇ , ⁇ or ⁇ ).
  • RXR heterodimer a dimer of an RXR ( ⁇ , ⁇ or ⁇ ) and a non-RXR nuclear receptor capable of dimerizing with an RXR, including, but not limited to, an RAR ( ⁇ , ⁇ or ⁇ ), VDR, TR ( ⁇ or ⁇ ), PPAR ( ⁇ , ⁇ , ⁇ or ⁇ ), LXR ( ⁇ or ⁇ ), BXR, CAR ( ⁇ or ⁇ ), SXR and FXR.
  • Preferred non-RXR nuclear receptors capable of dimerizing with an RXR include RARs, TRs, PPARs, LXRs, BXR, CARs, SXR and FXR. More preferred non-RXR nuclear receptors capable of dimerizing with an RXR include RARs, TRs and PPARs.
  • the nuclear receptor structure contains an amino-terminal activation function (AF-1; A/B domain), the DNA-binding domain (DBD; C domain), a hinge region (D domain), and a carboxy-terminal ligand-binding domain, LBD (E domain), which includes the activation function AF-2, required for ligand-dependent activation by nuclear receptors.
  • an "agent which interacts with an RXR dimer” is intended a compound which binds to an RXR dimer to mediate transcription of a target gene, i.e., "RXR dimer mediated transcription.”
  • the agent can mediate an interaction between -17-
  • the agent can activate or repress transcription of the target gene.
  • agents can be, but are not limited to, peptides, carbohydrates, steroids and vitamin derivatives, which may each be natural or synthetic (prepared, for example, using methods of synthetic organic and inorganic chemistry that are well-known in the art).
  • such an agent includes a "retinoid” which is a compound which binds to one or more of the retinoid receptors (RAR ⁇ , RAR ⁇ , RAR ⁇ , RXR ⁇ , RXR ⁇ and RXR ⁇ ).
  • retinoid a compound which binds to one or more of the retinoid receptors (RAR ⁇ , RAR ⁇ , RAR ⁇ , RXR ⁇ , RXR ⁇ and RXR ⁇ ).
  • Compounds can be either "RAR retinoids” or "RXR retinoids” depending on their binding characteristics (RAR retinoids bind to one or more RARs; RXR retinoids bind to one or more RXRs (also referred to as
  • RAR and RXR agonists and antagonists are known in the art, such as, for example, 4-[[(2,3-Dihydro-l,l,3,3-tetramethyl-2-oxo-lH-inden-5-yl) carbonyl]amino]ber ⁇ zoic acid (Compound I; WO 98/47861), 4-[[[5,6-Dihydro-5,5- dimethyl-8-(3-quinolinyl)-2-naphthalenyl]carbonyl]amino]benzoic acid (Compound II; U.S. PatentNo.5,559,248; U.S.
  • PatentNo.5,849,923 3-Fluoro- 4[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)hydroxyacetyl] amino]benzoic acid (Compound IV; U.S. Patent No. 5,624,957), 4-[2-(5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)- 1 ,3-dioxolan-2-yl]benzoic acid
  • Agents that interact with RXR dimers can be screened using the methods of the present invention.
  • agents that interact with only one partner or both partners of the RXR dimer can be identified.
  • the invention mimics the effects of retinoids on gene transactivation in vivo, the invention provides more accurate methods of identifying RXR dimer agonists and antagonists.
  • the action of the identified agent can be further confirmed by binding assays known in the art to determine which partner of the RXR dimer is bound by the identified agent (Rochel, N. et al, Biochem. Biophys, Res. Comm. 230:293-296 (1997)).
  • agents which cause transactivation via their receptors are examples of "agonists,” while agents which do not cause transactivation, but instead block the transactivation caused by other agonists, are examples of “antagonists.”
  • agonists agents which cause transactivation via their receptors
  • antagonists agents which block the transactivation caused by other agonists
  • CARs are constitutively expressed
  • a reverse agonist is needed to deactivate transcription
  • a reverse antagonist is needed to activate transcription.
  • Agents can have the ability to bind to multiple receptors.
  • agents that are "specific" for a nuclear receptor are intended compounds that only bind to one, two or three particular nuclear receptor(s) and not to others.
  • agents that are "selective" for a nuclear receptor are intended compounds that preferably bind to one, two or three particular nuclear receptor(s) over others by a magnitude of approximately five-fold or greater than to other retinoid receptors, preferably eight-fold or greater, more preferably, ten-fold or greater.
  • a ligand for a member of the nuclear receptor an agent, compound or hormone that binds to a nuclear receptor, which in turn can activate an appropriate hormone response element.
  • a ligand acts to modulate transcription of a gene maintained under the control of a hormone response element.
  • Ligands include hormones, steroid or steroid-like compounds, retinoids, thyroid hormones, pharmaceutically active compounds, and the like.
  • exemplary ligands include ligands for retinoid receptors (e.g., all-trans retinoic acid, 9-cis retinoic acid, etc.), ligands for thyroid hormone receptors (e.g., thyroid hormone), and ligands for vitamin D 3 receptor (e.g., 1,25-dihydroxyvitamin D 3 ).
  • Other ligands which bind to nuclear receptors can be identified by the present invention.
  • a hormone is intended a substance produced in a gland of an animal, human and nonhuman, which exerts specific effects on other parts of the body.
  • a "co-regulator” is intended a “co-activator” or a “co-repressor.”
  • a "co-activator” is intended a molecule or factor, generally a protein or RNA, that interacts with nuclear receptors (e.g., RARs, RXRs) and enhance their transactivation.
  • the co-regulator can complex with other molecules or factors to interact with nuclear receptors.
  • Exemplary co-activators include, but are not limited to, ERAP-160 (GRIP-170; pl60), ERAP-140, RIP-140, RIP-160,
  • TBP/TAF ⁇ s SRC-1 (hSRC-l;NCoA-l/mSRC-l),hSRC-3,Trip-l (Sug-1), Trips, TIF1 ⁇ , TIFl ⁇ , ⁇ , ARA-70, TRAPs (DRIPs), CBP, p300, PCAF (hGCN5), TIF2/hSRC-2 (GRIP-l/mSRC-2; NCoA-2, pi 60), mSRC-3/hSRC-3, TRIP230, L7/SPA,p/CIP/mSRC-3 (ACTR/hSRC-3; RAC3/hSRC-3; AIB-L/hSRC; TRAM- l/hSRC-3; pl60; SRC-3), E6-AP, RPF-1 (hRSP5), BRG-1 (SWI2/SNF2), Brahma, NSD-1, PGC-1, HMG-1, HMG-2, NCoA-62, BX42, TSC-2 (Tuberin),
  • co-repressor a molecule or factor, generally a protein or RNA, that interacts with nuclear receptors (e.g. RARs, RXRs) and lowers the transcription rate at their target genes.
  • exemplary co-repressors include, but are not limited to, NCoR (RIP- 13), SMRT (silencing mediator for retinoic acid and thyroid receptors; TRAC2), repressor domains of SMRT (e.g., SRD-1, SRD-2, amino acids 1-981 thereof, etc), TRUP (SURF-3; PLA-X; L7a), SUNCoR, NURD, mSin3A, protein-protein interaction domains of mSin3A (e.g., PAH-1,
  • PAH-2 PAH-3, PAH-4, combinations thereof, etc.
  • N-CoR Mad/Mxi-1
  • mSin3B mSin3B
  • Sin3 Sin3, etc.
  • methods are provided for identifying a "modulator" which promote dissociation of the co-activator or co-repressor complex from the nuclear receptors (e.g., retinoid and/or thyroid hormone receptors) or promote association of co-activator or co-repressor complexes with the nuclear receptors.
  • nuclear receptors e.g., retinoid and/or thyroid hormone receptors
  • an "agent” is alternatively intended a molecule, factor, substance or compound which is screened for an intended function, such as co-activator, co-repressor, or modulator function, as it will be clear from the context in which the term is used.
  • the RXR dimers of the invention can be obtained by expressing the receptors proteins in eukaryotic or bacteria cells and purifying the receptors.
  • the receptor is purified from tissues or cells which naturally produce the receptor.
  • the receptor can be expressed recombinantly, for example, by inserting the gene encoding the receptor into the baculovirus or vaccinia virus genome and infecting the baculovirus or vaccinia virus, respectively, into insect or human cells, respectively.
  • the receptors can also be expressed in yeast.
  • Exemplary constructs for production of the receptor can be obtained from, for example, human, mouse or chicken, and include, but are not limited to, human Flag-tagged RAR ⁇ and mouse His-tagged RXR ⁇ (Dilworth, et al, Proc. Natl. Acad. Sci. USA 96:2000-2004 (1999)), human Flag-tagged VDR and human Flag- tagged RXR ⁇ (Rachez et al, Nature 595:824-828 (1999)), and human Flag- tagged TR ⁇ (Fondell et al, Proc. Natl. Acad. Sci. USA 96:1959-1964 (1996)).
  • constructs can be generated by subcloning the cDNA from existing DNA vectors of, for example, LXRs (Willy et al, Genes Dev. 9:1033-1045 (1995)), PPAR ⁇ (Isseman and Green, Nature 347:645-650, FXR (Forman et al, Cell
  • LXR ⁇ (Apfel etal, Mol. Cell Biol. 74:7025-7035 (1994)), BXR (Blumberg et al, Genes Dev. 72:1269-1277 (1998)), CAR ⁇ and ⁇ (Choi et al, J. Biol Chem. 272:23565-23571 (1997); Forman et al, Nature 595:612- 615 (1998)), and SXR (Blumberg et al, Genes Dev. 72:3195-3205 (1998)).
  • a variety of methodologies are known in the art that can be used to obtain, isolate or purify the nuclear receptors, including, but not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and affinity chromatography.
  • Nuclear receptors bind to specific DNA sequences known as response elements (REs) or hormone response elements (HREs).
  • REs response elements
  • HREs hormone response elements
  • Those of skill in the art can readily determine suitable hormone response elements (HREs) for use in the practice of the present invention, such as, for example, the response elements described in U.S. Patent No. 5,091,518 and WO 92/16546.
  • the recognition of REs by a given RXR dimer is dependent on the actual sequence, orientation and spacing of the repeated motifs.
  • Naturally occurring HREs are composed of direct repeats (DRs; Umesono et al, Cell 65:1255-1266 (1991)), and inverted repeats (IRs; Umesono et al, Nature 336:262-265 (1988)).
  • DRs direct repeats
  • IRs inverted repeats
  • Direct repeats and inverted repeats can have a gap which separates the two core-binding sites.
  • spacers of 1, 3, 4 and 5 nucleotides serve as preferred DR response elements for heterodimers of RXR with PPAR, VDR, T 3 RandRAR, respectively (Naax etal, Cell 65:1267-1279(1991); Klieweret ⁇ /., Nature 355:771-774 (1992); andlssemannet ⁇ /., Biochimie 75:251-256 (1993)).
  • the optimal gap length for each heterodimer is determined by protein-protein contacts which appropriately position the DNA binding domains (DBDs) of RXR and its partner (Kurokawa et al, Genes Dev. 7:1423-1435 (1993); Perlmann etal, Genes Dev. 7:1411-1422 (1993); Towers etal, Proc. Natl. Acad. Sci. USA
  • Exemplary DR1 is provided in Vivat et al, EMBO J. 16:5697-5709 (1997).
  • Exemplary DR3 is provided in Rachez et al, Nature 595:824-828 (1999).
  • Exemplary DR4 is provided in Fondellet /., Proc. Natl. Acad. Sci. USA 96:1959- 1964 (1996).
  • Exemplary DR5 is provided herein and in Dilworth et al, Proc.
  • RXR/RAR heterodimer binding repertoire in vitro to DNA (DR1, DR2, and DR5, in order of increasing efficiency) is similar to the "natural" RARE repertoire, which suggest that RXR/RAR heterodimers are the functional units that transduce the retinoid signal in vivo.
  • RXR/TR The preferential RXR/RAR heterodimer binding repertoire in vitro to DNA (DR1, DR2, and DR5, in order of increasing efficiency) is similar to the "natural" RARE repertoire, which suggest that RXR/RAR heterodimers are the functional units that transduce the retinoid signal in vivo.
  • RXR/TR
  • RXR/VDR, and RXR/PPAR bind preferentially to DR4, DR3 , and DR1 elements, respectively (Giguere, V. Endocr. Rev. 75:61-79 (1994); Glass, C.K. Endocr. Rev. 75:391-407 (1994); and Mader, S. et al. J. Biol. Chem. 265:591-600 (1993)).
  • RXRs also bind as homodimers to a DR1 element (Nakshatri, H., and Chambon, P. J Biol Chem. 269:890-902 (1994)).
  • RXR/LXR binds to DR4
  • RXR/BXR binds to modified DR4
  • RXR/CAR binds to DR5
  • RXR/SXR binds to DR4
  • RXR/FXR binds to IRI (inverted repeat with a 1 bp spacer).
  • Direct repeat hormone response elements contemplated for use in the practice of the invention are composed of at least one direct repeat of two or more half sites, optionally separated by one or more spacer nucleotides (with spacers of 1-5 preferred).
  • the spacer nucleotides can be selected from any one of A, C, G or T.
  • Each half site of direct repeat HREs contemplated for use in the practice of the invention comprises the sequence -RGBNNM- wherein R is selected from A or G; B is selected from G, C, or T; each N is independently selected from A, T, C, or G; and M is selected from A or C; with the proviso that at least 4 nucleotides of said -RGBNNM- sequence are identical with the nucleotides at corresponding positions of the sequence -AGGTCA-.
  • Response elements employed in the practice of the invention can optionally be preceded by N x , wherein x falls in the range of 0 up to 5.
  • Exemplary hormone response elements include, but are not limited to, direct repeats of -PuG(G/A)(T/A)CA- (Mader, S. et al, J. Biol. Chem. 265:591-600 (1993)).
  • reporter elements are operatively linked to a reporter or target gene, whereby expression of the reporter or target gene indicates the action of a ligand, RXR dimer and/or the response element.
  • reporter genes include, but are not limited to, chloramphenicol acetyl transferase (CAT), ⁇ -galactbsidase ( ⁇ -gal), luciferase (LUC), and ⁇ -globin.
  • nucleosomes In a steady state, eukaryotic chromosomes ("chromatin") are organized into a repeating protein DNA unit, the nucleosome.
  • the basic protein unit of the nucleosome is the histone, a small, highly basic, globular moiety.
  • a nucleosome core particle contains a histone octamer, made up of two copies of each of histones H2A, H2B, H3 and H4, around which is wrapped 1.7 turns of a left- handed DNA superhelix (-200 bp of DNA).
  • chromatin based DNA template or "chromatin template” or “chromatin assembled template” is intended an in vitro nucleosomal array generated by complexing an oligonucleotide (linear DNA or plasmid) with histone octamers (H2A, H2B, H3 and H4) and/or histone HI.
  • the oligonucleotide sequence or DNA template comprises a hormone response element, at least a minimal promoter element (including a TATA box and a transcription start site, i.e., -35 to +80 of any natural eukaryotic or viral gene promoter), and a reporter gene, as described above.
  • a "naked" oligonucleotide sequence or DNA template is not complexed with histone octamers.
  • a chromatin based DNA template is prepared by adding a chromatin assembly extract to the oligonucleotide (linear DNA or plasmid) in the presence of histones.
  • a chromatin assembly extract contains the proteins and factors necessary for assembly of the DNA template around the histones into nucleosomes and for movement of the nucleosome along the DNA template to allow transcriptionally repressive and permissive states.
  • Methods for preparing chromatin assembly extracts useful in the present invention are known in the art (Becker, P.B. et al, Meth. Cell Biol. 44:207-223 (1994); Pazin, M.J. etal, Science 266:2007-2011 (1994); Kamakaka, R.T. etal, Genes Dev.
  • Chromatin assembly extracts can be prepared, for example, from tissue culture cells (Banerjee, S. and Cantor, C.R., Mol. Cell. Biol. 70:2863-2873 (1990)), Xenopus eggs and oocytes (Almouzni, G. and Mechali, M., EMBO J. 9:573-582 (1988)); Shimamura, A. etal, Mol. Cell Biol. 5:4257-4269 (1988)), Drosophila ISWI (Ito et al, Genes Dev. 75: 1529-1539 (1999); Carona et al Mol. Cell. 5:239-245 (1999)), human SNF2h (Leroy et al, Science 252:1900-1904
  • the chromatin based DNA template can be purified such that one or more or all transcription co-regulators are removed.
  • Methods of removing a transcription co-regulator include, but are not limited to, chromatography and immunoprecipitation.
  • Chromatographic methods for removing a transcription co-regulator include, but are not limited to, size-exclusion chromatography, affinity chromatography, heparin chromatography, DEAE chromatography, ion exchange chromatography, phenyl sepharose chromatography, phosphocellulose chromatography, and hydroxy-apatite.
  • Removal of a transcription co-regulator from a chromatin based DNA template by size-exclusion chromatography is a function of the resin employed, the size of the template, and the size of the co- regulators) to be removed.
  • the resin is selected such that factors greater in size than the exclusion limit of the resin are isolated.
  • the template in order to remove a transcription co-regulator from a chromatin based DNA template, the template must be larger in size than the exclusion limit of the resin used, and the transcription co-regulator must be smaller in size than the exclusion limit of the resin used.
  • the size of the template is a function of the size of the DNA, the chromatin extract complexed thereto, and the histones present.
  • the size of the DNA is itself a function of the length (in base pairs) of the hormone response element, the minimal promoter element, and the reporter gene.
  • SWI/SNF 2000 kilodaltons
  • p300 300 kDa
  • TIF2 160 kDa
  • ATP ATP
  • Other co-regulators that can be removed are provided above.
  • the pre-equilibration and elution buffer can contain KC1 or a sodium monovalent salt such as NaCl in a concentration of 1-100 mM, preferably 1-50 mM, more preferably 10-40 mM, still more preferably 10-30 mM, and even more preferably 10-20 mM.
  • concentration of this salt in the final in vitro transcription reaction can be 40-100 mM, preferably 50- 100 mM, more preferably 50-75 mM, and still more preferably 50-60 mM.
  • RAR ⁇ /RXR ⁇ heterodimers cannot efficiently initiate transcription from chromatin templates from which transcription co-regulators have been removed unless initiation of transcription is preceded by a preincubation period during which the chromatin template is exposed to ATP -driven remodeling activities of complexes containing SNF2 family members including, but not limited to, SWI/SNF, CHRAC, ACF, NURF, WCRF, and RSF.
  • ATP-driven remodeling activity or "ATP-driven chromatin remodeling activity” is intended an ATP-requiring activity that modifies chromatin and/or nucleosomal structure such that ligand-dependent transcriptional activation is enhanced. Examples of compounds that confer such activity include, but are not limited to, SWI/SNF, CHRAC, ACF, NURF, WCRF, and RSF.
  • HAT activity or "histone acetyltransferase activity” is meant a histone acetyltransferase-requiring activity that modifies chromatin and/or nucleosomal structure by interacting with DNA-binding proteins that regulate transcription.
  • Examples of compounds that confer such activity include, but are not limited to, p300, TIF2, pl60, CBP, pCAF, GCN5, ACTR, and SRC-1.
  • Chromatin can be assembled on relaxed or supercoiled circular DNA by preincubating the extract with histones to assemble histone octamers and ' adding the template of interest.
  • Core histones can be purified according to the method of Simon, R.H., & Felsenfeld, G., Nucl. Acids. Res. 6:689-696 (1979), or calf thymus histones are commercially available (Boehringer Mannheim).
  • the appropriate amount of histones can be determined empirically, using as a guide a stoichiometry of histones to DNA of -0.8:1 (w/w) (Albright, S.C et al, J. Biol. Chem. 254:1065-1073 (1979)). Details of a method for chromatin assembly on a DNA template are provided in Example 1, infra.
  • DNA supercoiling assay is based on topological changes that accompany the wrapping of DNA around a nucleosome core (Becker, P.B. et al, Meth. Cell Biol. 44:207-223 (1994)). Winding ofDNA around a nucleosome core introduces one positive superhelical turn in the plasmid DNA, which is relaxed by topoisomerase I activity present in the embryo extracts. When nucleosomes are removed by proteinase K digestion and DNA purification, one negative superhelical turn corresponding to each assembled nucleosome appears in the closed circular DNA.
  • the superhelical density of a plasmid i.e., the absolute number of superhelical turns, can be directly counted by visualization of the plasmid topoisomers on two-dimensional agarose gels or by resolving duplicate samples on multiple agarose gels containing different chloroquine concentrations.
  • the introduction of supercoils into a plasmid can simply be visualized by agarose gel electrophoresis as a rapid indicator of nucleosome reconstitution.
  • supercoiling of the chromatin can be assayed by incubating the assembled chromatin with the heterodimer (e.g., Flag-tagged human RAR ⁇ /His- tagged mouse RXR ⁇ ) in the presence of ligand.
  • the heterodimer e.g., Flag-tagged human RAR ⁇ /His- tagged mouse RXR ⁇
  • Supercoiling can be determined by adding topoisomerase I and/or chloroquine and resolving the DNA on an agarose gel. Details of a supercoiling assay are provided in the "Materials and Methods" section in Example 1, infra.
  • DNA supercoiling measures the wrapping of DNA around a particle but does not necessarily indicate the reconstitution of a full octamer of core histones
  • nuclease digestion assay is used to provide information on the nature of the nucleosome core particle ' as well as on the average distance between particles.
  • nucleases include, for example,
  • DNase I DNase I
  • DNase II micrococcal nuclease
  • SI nuclease SI nuclease
  • copper/phenanthroline and restriction enzymes.
  • Micrococcal nuclease (MNase) assay relies on the ability of MNase to preferentially cleave the linker DNA between nucleosome core particles. After the initial endonucleolytic attack of linker DNA, the trimming activity associated with the enzyme progressively removes the linker DNA. Extensive digestion of chromatin with MNase can bring the size of the mononucleosome from 160-220 bp to the 147 bp DNA fragment protected by the nucleosome core particle whereas a partial digest results in a ladder of fragments representing oligonucleosomal DNAs. Details of a MNase assay is provided in the "Materials and Methods" section in Example 1 , infra.
  • kits containing the elements necessary to carry out the methods disclosed herein can comprise a carrier being compartmentalized to receive in close confinement therein one or more contain means, such as tubes or vials.
  • One of the container means can contain the DNA template.
  • One of the container means can contain the chromatin extract.
  • One or more of the container means can contain the histones.
  • One or more of the containers can contain known agonists, antagonists, co-activators, co-repressors or modulators which can be used as controls.
  • the kit can also include a "catalog” defined broadly as a booklet, book pamphlet, computer disk or the like, which can assist in carrying out the invention.
  • the kit can contain all of the additional elements necessary to carry out the method of the invention, such as buffers, enzymes, pipettes, tubes, nucleic acids, nucleoside triphosphates, and the like.
  • a “compound” is intended a protein, nucleic acid, carbohydrate, lipid or a small molecule. It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of the invention or any embodiment thereof.
  • the supercoiled plasmid (DR5)5 ⁇ 2G that contains the RAR ⁇ 2 core promoter (- 35 to +85) and five copies of the RA response element (RARE) of the RAR ⁇ 2 gene was used to study activation of transcription by RAR/RXR heterodimers (see Materials and Methods, Example 1 ; see FIGs. 1 and 2B).
  • RARE RA response element
  • Periodic nucleosomal arrays (FIG. 2B) were generated using supercoiled (DR5)5 ⁇ 2G plasmid and a chromatin-assembly extract (see,
  • the response element specificity of transcriptional activation by RAR ⁇ /RXR heterodimers was examined by comparing transcription from the cognate (DR5)5 ⁇ 2G template and the (17M)5 ⁇ 2G template, in which the five DR5 RAREs have been replaced by five copies of the 17-mer binding site for the
  • 9cRA that binds both RARs and RXRs was more efficient than tRA at limiting concentrations, with ED 50 of approximately 9xl0 -10 M and 4x10 "9 M for 9cRA and tRA, respectively (FIG. 5 A). Because these differential effects of 9cRA and tRA suggested that synergistic activation of transcription might occur when both RAR ⁇ and RXR ⁇ are liganded, transcriptional activation by RAR ⁇ /RXR ⁇ heterodimers upon addition of receptor-specific synthetic retinoids (FIG.5B) was investigated.
  • p300 enhanced the activation of transcription by the heterodimers -4-fold in the presence of tRA, while transcription of the chromatin template remained repressed in the absence of the agonistic ligand, irrespective of the presence of the heterodimers (FIG.6B).
  • No p300 effect was seen on naked DNA templates.
  • the further addition of acetylCoA had no effect on the extent of transcriptional enhancement, even though the purified p300 coactivator exhibited histone acetyltransferase activity.
  • the in vitro system contains some endogenous histone acetyltransferase activity that was not further enhanced by the addition of p300 to the transcription reaction.
  • the plasmids (DR5)5 ⁇ 2G and (17M)5 ⁇ 2G (-5.2 kb) were constructed by inserting five copies of the DR5 RA response element from the mouse RAR ⁇ 2 promoter or the 17-mer GAL4 binding site, respectively, upstream of the mouse RAR ⁇ 2 core promoter [-35 to +85] which had been previously linked to the -9 to +1516 chicken ⁇ -globin gene sequence (FIG. 1).
  • the most 3 ' DR5 element is positioned at approximately the same distance from the TATA box as the DR5 RARE found in the natural RAR ⁇ 2 promoter (Zelent, A., et al, EMBO J. 9:71-81 (1991)).
  • Chromatin assembly extracts were prepared from Drosophila embryos (0-6 hr) as described in Kamakaka, R.T. et al. , Genes Dev. 7: 1779- 1795 ( 1993)). Canton-S wild-type flies were grown at 25 °C at 70-80% humidity in population cages. The embryos were collected on apple juice-agar plates covered with yeast.
  • HEPES K +
  • 10 mM KC1 1.5 mM MgCl 2
  • 0.5 mM EGTA 10% (vol/vol) glycerol
  • 10 mM ⁇ -glycerophosphate 1 mM DTT
  • 0.2 mM phenylmethylsulfonyl fluoride (PMSF) In the final wash, the embryos take longer to settle (-10 min), and the final volume of the embryo suspension before homogenization is roughly twice of that of the loosely packed volume of dechorionated embryos.
  • the embryos were then transferred to a Wheaton Dounce homogenizer (40 ml) and disrupted by 15 strokes with the B pestle followed by 40 strokes with the A pestle.
  • the homogenate was subjected to centrifugation in a Falcon 2059 tube in a Sorvall SS-34 rotor at 8000 rpm for 5 min. at 4 ° C
  • the cloudy, yellow cytoplasmic fraction was collected with a syringe
  • MgCl 2 from a 1 M stock solution was added to increase the Mg(II) concentration from 1.5 mM to a final concentration of 7 mM.
  • the extract was then subjected to centrifugation in a Beckman SW55 rotor at 45,000 rpm (192,000g) for 2 hr at 4°C After centrifugation, the white upper layer was removed with a spatula and the yellow-brown liquid was collected.
  • This supernatant fraction was frozen in liquid nitrogen, thawed in water (at room temperature), and then subjected to a second centrifugation in the Beckman S W55 rotor at 45,000 rpm for 2 hr at 4°C
  • the resulting chromatin reconstitution extract (also referred to as the Drosophila S-190 extract) was frozen in liquid nitrogen and stored at - 80°C
  • the extracts remain active for >1 year at - 80°C Chromatin was assembled on supercoiled circular DNA (see FIG. 2B) as follows.
  • the chromatin assembly extract was preincubated with 3 ⁇ g of calf thymus core histones (Boehringer Mannheim) at room temperature to assemble histone octamers.
  • Micrococcal digestion analysis of reconstituted chromatin was performed essentially as previously described (Bellard, M., et al, in Methods Enzymol.
  • Micrococcal nuclease digestion (Bellard, M., et al, in Methods Enzymol. 170:317-346 (1989)) showed that they had a periodicity of approximately 160 bp (FIG. 2B). DNase I footprinting was performed essentially as previously described
  • Chromatin was assembled on 250 ng of (DR5)5 ⁇ 2G plasmid then incubated alone or with 1 nM FhRAR ⁇ /HmRXR ⁇ heterodimer in the presence or absence of IO "6 M RA for 30 min at 27 °C CaCl 2 was added to a final concentration of 3 mM along with 5 U of DNase I (Boehringer Mannheim) for 90 sec. DNA fragments were amplified with VentR (exo-) [New England Biolabs, Beverley, MA] using a 30 bp primer complementary to a sequence located between -280 and -250 upstream of the RAR ⁇ 2 promoter start site.
  • the spodoptera frugipenda cell line Sf9 was co-infected with baculoviruses expressing His-tagged mouse RXR ⁇
  • HmRXR ⁇ Flag-tagged human RAR ⁇
  • FhRAR ⁇ Flag-tagged human RAR ⁇
  • Sf9 cells expressing the heterodimeric proteins were lysed by homogenization in a low salt buffer (20 mM Hepes pH 7.6, 100 mM KC1, 10 mM imidazole, lx PIC [2.5 ⁇ g/mL leupeptin, 2.5 ⁇ g/mL pepstatin, 2.5 ⁇ g/mL aprotinin, 2.5 ⁇ g/mL antipain, 2.5 ⁇ g/mL chymostatin], and 1 mM PMSF).
  • the FhRAR ⁇ /HmRXR ⁇ heterodimer was partially purified by chromatography using a Ni 2+ column (Amersham Pharmacia) and eluted with a low salt buffer containing 300 mM imidazole. The heterodimer was then further purified from the Ni 2+ column eluate by affinity purification using agarose-coupled M2 anti-Flag antibodies (Sigma), as specified in the manufacturer's instructions.
  • the purified heterodimer was eluted from the resin in a buffer consisting of 20 mM Hepes pH 7.6, 100 mM KC1, 1.5 mM MgCl 2 , 0.5 mM EGTA, 50 ⁇ M ZnCl 2 , 15% glycerol, 500 ⁇ g/mL competitor peptide (DYKDDDDK) (SEQ ID NO:l), 1 mM DTT, 1 mM PMSF and lx PIC.
  • a buffer consisting of 20 mM Hepes pH 7.6, 100 mM KC1, 1.5 mM MgCl 2 , 0.5 mM EGTA, 50 ⁇ M ZnCl 2 , 15% glycerol, 500 ⁇ g/mL competitor peptide (DYKDDDDK) (SEQ ID NO:l), 1 mM DTT, 1 mM PMSF and lx PIC.
  • the DNA binding properties of the heterodimer were examined by electrophoretic mobility shift analysis (Kumar, V. & Chambon, P., Cell 55:145- 156 (1988)). Briefly, the purified heterodimer was incubated with lO ⁇ MtRAon ice. After 15 min., the [ 32 P]-labeled DR5 oligonucleotide
  • 5'-TCGGGAGGGTTCACCGAAAGTTCACTCGCC-3' (SEQ ID NO:2) hybridized to its unlabeled compliment were added to the reaction in the presence of 20 mM Hepes pH 7.6, 10 mM KC1, 10% Glycerol, 1.5 mM MgCl 2 , 0.5 mM EGTA, 1 mg/mL BSA, and 2 ⁇ g poly dldC The reaction was then allowed to proceed for another 15 min at 22 °C. Reaction mixtures were resolved by polyacrylamide gel electrophoresis (5% in 0.5x TBE) for 4 hr at 150 volts, dried and visualized by autoradiography.
  • RAR ⁇ The integrity of the ligand binding domain of RAR ⁇ was examined using a ligand binding assay (Rochel, N., et al, Biochem. Biophys. Res. Comm. 250:293-296 (1997)).
  • the RAR ⁇ /RXR ⁇ heterodimer (10 fmol) was diluted to
  • Full-length p300 was prepared from Sf9 cells infected with a p300- expressing baculovirus (Kraus, W.L. & Kadonaga, J.T., Genes Dev. 72:331-342 (1998)), its purification was monitored using a rabbit polyclonal anti-p300 (C-20) antibody (Santa Cruz Biotechnology), and the histone acetyltransferase activity of the purified protein was confirmed as described (Ogryzko, V.V., Cell 87:953- 959 (1996)).
  • His-tagged Gal(l-147) and GAL-VP16 (Tora, L., et al, Cell 59:477-487 (1989)) were expressed from pET3 expression vectors in the BL-21 pLysS bacterial strain and purified by Ni 2+ column chromatography.
  • Retinoids 4-[[(2,3-Dihydro-l,l,3,3-tetramethyl-2-oxo-lH-inden-5-yl) carbonyl]amino]benzoic acid (Compound I; WO 98/47861 ), 4-[[[5,6-Dihydro-5,5- dimethyl-8-(3-quinolinyl)-2-naphthalenyl]carbonyl]amino]benzoic acid
  • ER ⁇ estrogen receptor ⁇
  • p300 may further enhance ligand-induced activation of transcription on chromatin templates by bridging RXR ⁇ /RAR ⁇ heterodimers to RNA polymerase II through its interaction with RNA helicase A (Nakajima, T., et al, Cell 90:1107-1112 (1997)), rather than by locally remodelling the chromatin structure through histone acetylation.
  • RNA helicase A Nakajima, T., et al, Cell 90:1107-1112 (1997)
  • CoA and ATP are all required for efficient retinoid-dependent activation of transcription by RARaRXRa heterodimers on a "purified" cognate chromatin template, irrespective of the presence of histone HI
  • the (DR5)5 ⁇ 2G template was purified by size-exclusion chromatography. This separated the chromatin template and its associated proteins away from "low” molecular weight components, including ATP that is required for chromatin assembly (Pazin, M. J. and Kadonaga, J.T., " Transcriptional and Structural Analysis of Chromatic Assembled In Vitro," in Chromatin: A Practical Approach, Gould, ed., Oxford University Press, Oxford, UK (1998), pp. 173-194).
  • RAR ⁇ /RXR ⁇ may have been rendered dependent on ATP-driven chromatin remodeling activities and coactivator HAT activities and their cofactors.
  • the addition of tRA/SR 11237 did not activate transcription by RAR ⁇ /RXR ⁇ heterodimers on "purified” chromatin templates (FIG.8(B), compare lanes 1 -3).
  • the re-addition of Drosophila embryo chromatin assembly extract during the preincubation period partially restored transactivation, while the addition of Acetyl CoA and/or ATP had little effect on their own (FIG. 8(B), lane 4; Fig.
  • the intrinsic HAT activity of purified p300 has been established using either free or nucleosomal core histones, as substrates (Ogryzko et al. 1996; see also FIG. 7(G)). Furthermore, the present data (FIG. 7(G)) show that purified TIF2 also exhibits an intrinsic HAT activity similar to that reported for the other NR coactivators (SRC- 1 , ACTR) that belong to the same p 160 family (Spencer,
  • the almost absolute requirement for a HAT activity is demonstrated by the dependence of transcriptional activation on addition of Acetyl CoA (FIG. 8(B), compare lanes 12 and 14; FIG. 8(C), compare lanes 12 and 15; FIG. 8(D), compare lanes 14 and 16), as well as by a drastic decrease of activation in the absence of both p300 and TIF2 (FIG. 8(C), compare lanes 11 and 15; FIG. 8(D), compare lanes 4 and 16).
  • both ATP-utilizing chromatin remodeling activities and coactivator HAT activities appear to synergistically contribute to stimulation of transcription by enhancing the initial formation of productive PICs, rather than by increasing the relative frequency of reinitiation events. This raises the question of which steps preceding initiation of transcription are actually affected to increase the efficiency of PIC formation by RXR ⁇ /RAR ⁇ heterodimers on chromatin templates.
  • Probe A compare lanes 1-6 with 7 and 8) while the nucleosomal structure appears unchanged around the transcription start site (FIG. 5 Probe B compare lanes 1-6 with 7 and 8), or at the 3' end of the globin reporter gene.
  • Probe B compare lanes 1-6 with 7 and 8
  • the addition of p300/TIF2 and Acetyl CoA did not result in "tight" binding of the heterodimers in the absence of
  • chromatin immunoprecipitation (ChlP) assays were performed using a monoclonal antibody directed against acetylated histone H4.
  • the "purified” cognate (DR5)5 ⁇ 2G chromatin template and a "purified control" chromatin template similarly assembled in vitro with the pSG5 plasmid that contains the SV40 early promoter (Green, S., et al, Nucleic Acids Res.
  • the acetylation of the (DR5)5 ⁇ 2G template at a distance may be due to the fact that a nucleosomal array on a template is not as rigid as a chromosome in vivo, and may permit distal regions to be in close proximity to the heterodimer-bound co-activators.
  • the above results demonstrate that RAR ⁇ /RXR ⁇ heterodimers bound to cognate REs recruit in a ligand-dependent manner the p300 and TIE2 coactivators whose HAT activity preferentially acetylate nucleosomes located in cis.
  • next steps most probably correspond to acetylation reactions catalyzed by p300/TIF2 recruited by heterodimers tightly bound to their REs. Indeed, there was no decrease in transcription when either p300/TIF2 (FIG. 13(C)), tRA/SR 11237 ligands (FIG. 13(B)) or Acetyl CoA (FIG. 13(D)), were added at - 20 min, whereas their addition at - 10 min (at the same time as HeLa nuclear extract) or at later times, resulted in severe decreases in activation of transcription.
  • hSWI/SNF remodeling complexes must act at a step subsequent to transacetylation, as their full stimulatory effect was still achieved upon addition at the same time as HeLa nuclear extract (- 10 min.), whereas no stimulation was observed upon addition, just before initiation of transcription (- 0.5 min) (FIG. 13(C), compare lanes 5 to 8).
  • two distinct chromatin remodeling activities appear to be required at two steps during the course of enhanced PIC formation by RAR ⁇ /RXR ⁇ heterodimers.
  • Nucleosomal arrays were assembled on (DR5)5 ⁇ 2G (Dilworth, F. J., et al. , Proc. Natl. Acad. Sci. USA 96:1995-2000 (1999)) or pSG5 (Green, S., et al, Nucleic Acids Res. 16:369 (1988)) DNA using core histones and assembly extracts prepared from Drosophila embryos (Pazin, M.J. and Kadonaga, J.T., "Transcriptional and Structural Analysis of Chromatic Assembled In Vitro," in Chromatin: A Practical Approach, Gould, ed., Oxford University Press, Oxford, UK (1998), pp. 173-194). Bovine thymus histone HI (Roche) was added to assembly reactions at 1:4 (w/w) histone H1:DNA ratio. "Crude" assembled
  • Micrococcal nuclease digestion was used as described (Dilworth, F. J., et al. , Proc. Natl. Acad. Sci. USA 96:1995-2000 (1999)) to examine the periodicity of the nucleosomal arrays.
  • DNase I footprinting analysis was according to Pazin, M.J. and Kadonaga,
  • TIF2 (2.5 nM), p300 (2.5 nM), hSWI/SNF (10 ng/ ⁇ l), hSNF2h complexes (5 ng/ ⁇ l), ATP (0. 1 mM) and Acetyl CoA (2 ⁇ M) at 270C.
  • CaCl 2 was added to 3 mM and chromatin was digested using 0.75 units (5 ⁇ l) of DNase I (Roche) for 2 min at room temperature.
  • RNA and proteins were analyzed by PCR-based primer extension footprinting using a [ 32 P]-labelled oligonucleotide situated between -280 and -250 bp upstream of the (DR5)5 ⁇ 2G promoter transcription startsite.
  • RAR ⁇ /RXR ⁇ heterodimers were as described Dilworth, F.J., et al, Proc. Natl. Acad. Sci. USA 96:1995-2000 (1999)).
  • a cDNA baculovirus vector was used to express full length human TIF2 containing an amino-terminal His-tag (Voegel et al, EMBO J. 77:507-519 (1998)) in Sf9 cells.
  • TIF2 was purified from whole cell extracts using DEAE-sephacryl (Pharmacia) and Ni 2+
  • HAT assays were performed as described (Brand, M., et al, J. Biol. Chem. 274:18285-18289 (1999)). Briefly, recombinant p300 or TIF2 (1 or 3 pmol) were incubated with 2 ⁇ g of core histones or nucleosomes isolated from HeLa cells and 0.1 ⁇ Ci of [acetyl- 1- 14 C] Acetyl CoA (65 mCi/mmol - ICN) at 30 °C. Where indicated, cold Acetyl CoA (2 mM) was added to reactions to confirm the specificity of acetylation. After 1 hr, reactions were run on 12% SDS- PAGE gels, which were then treated with diphenyloxazol, dried and exposed to autoradiographic film for 5 days.
  • Chromatin immunoprecipitation ChlPs
  • Nucleosomal arrays were incubated O/N at 4°C with 30 ⁇ g of purified 3C10 antibody.
  • Antibody-bound nucleosomes were recovered by incubation with 30 ⁇ l of protein G-sepharose and washed with EX buffer (see above) containing 500 mM KCl.
  • Acetylated nucleosomes were eluted by incubating with 1% SDS for 30 min, and deproteinized using proteinase K.
  • Acetylated chromatin templates were quantitated by Southern blotting using [ 32 P]- labelled, 45-base-long, oligonucleotides recognizing the promoter regions of the (DR5)5 ⁇ 2G (-37 to +8) and pSG5 (-121 to -77) templates, respectively.
  • the limiting step may correspond to the possible recruitment targeting of ISWI-containing complexes through interaction with RAR ⁇ /RXR ⁇ heterodimers, as recently suggested in the case of progesterone receptor (Di Croce, L., et al, Mol. Cell 4:45-54 (1999)), and demonstrated for the SWI/SNF complexes that are recruited/targeted by a number of transactivators (see below).
  • SWI/SNF may be targeted to the nucleosomes acetylated by p300/TIF2 within the proximal promoter region through bromodomain of SNF2a/b, as bromodomains domains have previously been shown to have high affinity for acetylated lysine residues
  • the second step in the process leading to transcriptional initiation triggered by RAR/RXR heterodimers corresponds to the ligand-dependent recruitment/targeting of coactivators (p300 and TIF2) that acetylates histones through their intrinsic HAT activities.
  • coactivators p300 and TIF2
  • acetylates histones through their intrinsic HAT activities To occur optimally this step requires the presence of ATP in addition to liganded heterodimers, coactivators and Acetyl CoA (FIG. 12), indicating that it has to be preceded by the ATP-dependent ligand- independent receptor "tight" binding step.
  • this histone acetylation step is a prerequisite for subsequent efficient PIC formation upon HeLa nuclear extract addition, as activation of transcription is strongly decreased when either Acetyl CoA, ligands, and/or p300/TIF2, are added at the same time as HeLa nuclear extract (FIGs. 13(D), (B), (E)).
  • Acetyl CoA, ligands, and/or p300/TIF2 are added at the same time as HeLa nuclear extract.
  • yeast HAT activities through recruitement/targeting by transcriptional activators, facilitate transcription in vivo from nucleosomal templates in an Acetyl CoA-dependent fashion (Ikeda, K., et al. , Mol. Cell Biol. 79:855-863 (1999) and refs therein).
  • the first step corresponds to the recognition of cognate REs by unliganded RAR/RXR heterodimers that results in a "weak" interaction.
  • the third step is the ligand-dependent recruitment of coactivators (p300 and TIF2) by the receptors.
  • the fourth step that occurs upon addition of Acetyl CoA corresponds to histone acetylation by the intrinsic HAT activity of recruited coactivators. These first four steps take place in the absence of any of the transcription machinery components present in HeLa nuclear extract. Thus, the "raison d'etre" of these chromatin remodeling steps appears to be to create the proper environment for the next steps that lead to the formation of PICs.
  • Histone acetylation has been shown to increase the accessibility of regulatory molecules to nucleosomal DNA (Nightingale, K.P., et al. , EMBO J. 77:2865-2876 (1998); Steger et al, 1998; for additional refs see guitarist, R.E. and Narlikar, G.J., Genes Dev. 73:2339-2352 (1999)).
  • the liganded receptors bound to their cognate REs and associated withp300/CBP and pi 60 coactivators e.g. TIF2
  • GTFs general transcription factors
  • TFIID complexes RNA polymerase II
  • SMCC/TRAP Ito., M., et al, Mol Cell 3:361-370 (1999)
  • DRIP Rachez, C, et al, Nature 395:824-828 (1999)
  • ARC Naar, A.M., et al, N ⁇ twre 595:828-832 (1999)
  • SMCC/TRAP/DRIP/ARC complex may associate with most, if not all, nuclear receptors through a direct interaction between one of its subunits (TRAP220/DRIP205) and liganded LBDs (Yuan, C.X., et al, Proc. Natl. Acad. Sci. USA 95:7939-7944
  • SMCC/DRIP subunits belong to human and mouse mediator complexes and are homologs of components of the yeast SRB/mediator complex that is found associated with R ⁇ A polymerase II holoenzyme (Rachez, C., etal, Nature 595:824-828 (1999); Ito, M., et al, Mol. Cell 5:361-370 (1999)); guitarist, R.E., Nature 599: 199-200 (1999) and refs therein), one of the function of the SMCC/DRIP complex might be to recruit Pol II holoenzyme to promoters during PIC formation.
  • the fifth step in the process leading to ligand-dependent enhancement of transcriptional PIC formation mediated by retinoid receptors on chromatin templates may correspond to the binding of SMCC/DRIP complexes to liganded receptor LBDs through interaction with their TRAP220 subunit.
  • the next step would then be the recruitment of Pol II holoenzyme.
  • the liganded receptor might directly recruit preformed SMCC/DRIP-Pol II holoenzyme complexes. Further in vivo studies using isolated SMCC/DRIP complexes and purified components from HeLa nudear extract are required to discriminate between these possibilities and to reveal whether nucleosomal acetylation is a prerequisite for these recruitments.

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Abstract

La présente invention concerne des procédés d'identification d'agents qui interagissent avec les dimères de récepteurs X de rétinoïde. Cette invention concerne également des systèmes de transcription in vitro de modèles d'ADN reposant sur la chromatine.
PCT/US2000/017800 1999-09-01 2000-06-29 Systemes de transcription in vitro et ses utilisations WO2001016368A1 (fr)

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WO2003033529A3 (fr) * 2001-10-12 2003-11-20 Max Delbrueck Centrum Utilisation de tr4, d'activateurs de tr4, d'inhibiteurs de tr4 ou de molecules associees au tr4 pour traiter des leucemies
EP1402072A1 (fr) * 2001-05-30 2004-03-31 John A. Stamatoyannopoulos Quantification precise et efficace de la sensibilite d'adn par pcr en temps reel
EP1489098A1 (fr) * 2002-01-31 2004-12-22 Japan Science and Technology Agency Production d'un hybridome produisant un anticorps monoclonal dirige contre des peptides de la proteine cenp-a humaine, et methode d'utilisation correspondante
CN106480024A (zh) * 2015-08-30 2017-03-08 李小彦 受维甲酸调控的具有浓度依赖效应的重组启动子及其使用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1402072A1 (fr) * 2001-05-30 2004-03-31 John A. Stamatoyannopoulos Quantification precise et efficace de la sensibilite d'adn par pcr en temps reel
EP1402072A4 (fr) * 2001-05-30 2005-11-16 John A Stamatoyannopoulos Quantification precise et efficace de la sensibilite d'adn par pcr en temps reel
WO2003033529A3 (fr) * 2001-10-12 2003-11-20 Max Delbrueck Centrum Utilisation de tr4, d'activateurs de tr4, d'inhibiteurs de tr4 ou de molecules associees au tr4 pour traiter des leucemies
EP1489098A1 (fr) * 2002-01-31 2004-12-22 Japan Science and Technology Agency Production d'un hybridome produisant un anticorps monoclonal dirige contre des peptides de la proteine cenp-a humaine, et methode d'utilisation correspondante
EP1489098A4 (fr) * 2002-01-31 2006-01-25 Japan Science & Tech Agency Production d'un hybridome produisant un anticorps monoclonal dirige contre des peptides de la proteine cenp-a humaine, et methode d'utilisation correspondante
CN106480024A (zh) * 2015-08-30 2017-03-08 李小彦 受维甲酸调控的具有浓度依赖效应的重组启动子及其使用
CN106480024B (zh) * 2015-08-30 2020-10-20 南通大学附属医院 受维甲酸调控的具有浓度依赖效应的重组启动子及其使用

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