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  • C12N2330/51—Specially adapted vectors

Definitions

• the present invention relates to a method of reducing or silencing expression of genes in mammalian cells.
• RNA interference was first identified as a process by which double strand (ds) RNA is introduced into cells/organisms which can result in the inhibition of genes containing homologous sequence. It is now appreciated that most eukaryotes display some facet of RNAi. RNAi appears to operate at two levels of gene expression inhibition. On one level the gene may be switched off by the induction of repressive chromatin structures, a process called transcriptional gene silencing (TGS). Alternatively the messenger RNA made by the gene may be blocked from being translated either through its direct degradation or through blocking its access to protein synthesis, a process called post transcriptional gene silencing (PTGS).
• TGS transcriptional gene silencing
• TGS appears to be exclusively used in some fungi such as fission yeast (S. pombe) as well as in plants. Previous studies in fission yeast have demonstrated that natural transcriptional gene silencing is a cis specific process. Some organisms use both TGS and PTGS mechanisms such as C. elegans and Drosophila. Mammals appear to use only PTGS mechanisms. Such post transcriptional gene silencing may be achieved using either exogenous siRNAs or endogenous micro RNAs. This type of gene silencing is transient in nature.
• RNAi small dsRNAs either synthetic or alternatively gene constructions that produce small hairpin RNAs.
• Small dsRNAs or hairpin RNA so introduced into cells by transfection are processed in the cytoplasm into microRNAs that then target specific mRNA inactivation.
• RNAi using longer dsRNAs is not believed to function in mammals because dsRNA will activate a nonspecific viral defence mechanism, the interferon response, which leads to an arrest of protein synthesis and nonspecific mRNA degradation in the affected cells (Tuschl, 2001).
• Interferons are a group of signalling molecules which are induced and secreted when cells are infected by R A viruses or exposed to dsR A.
• PKR A sensitive protein kinase called PKR that is known to inhibit cellular translation with resulting cell death.
• the interferon response is not sequence specific and PKR is potentially activated by any cytoplasmic dsRNA greater than 30 bp in length (Clemens et al, 1997; Cole, 2007).
• CGs convergent genes
• S. pombe display a regulated transcription termination process during the cell cycle.
• CGs convergent genes
• CGs no longer generate read-through transcription as heterochromatin recruited cohesin acts to block G2 read-through transcription so that in G2, CGs form shorter mRNA transcripts that do not overlap to form dsRNA.
• TGS is not considered a viable gene silencing approach for mammalian cells
• some studies described in the literature do employ convergent transcription to induce RNAi in other organisms.
• studies have employed artificial convergent transcription units comprising convergent bacteriophage T7 promoters flanking a gene sequence to be silenced that was then trans fected into the parasite.
• trypanosomes were also engineered to express the T7 phage RNA polymerase and show significant gene silencing effects assumed to be PTGS (Alibu et al, 2005; Shi et al, 2000; Wang et al, 2000).
• Giordano et al (2002) describe a study in Drosophila, where TGS is known to occur, in which convergent transcription was engineered by use of heterologous Gal4 regulated RNA polymerase II promoters (from budding yeast) placed convergently flanking a test gene sequence. Following transfection into flies, also expressing Gal4 transcription factor, significant gene silencing was observed and assumed to be of a PTGS nature.
• Tran et al (2003) used a gene construct containing two U6 R A polymerase III convergent promoters (DualU6) to generate very short dsRNA less than 30 bps. They indicate that it is essential to use such short dsRNA to avoid activation of the cytoplasmic dsRNA interferon response which was predicted to cause general inhibition of longer dsRNA expressing cells. Significant gene silencing of target genes was again observed.
• WO 01/77350 proposes the use of convergent transcription to inhibit gene expression particularly in cereal crops.
• the invention provides a method of reducing or preventing the expression of a gene of interest comprising providing a DNA molecule having a sequence which comprises in a 5' to 3' direction (i) an RNA polymerase II (Pol II) promoter element in sense orientation, (ii) the gene of interest or a portion thereof comprising 40 nucleotides or more in length, and (iii) a Pol II promoter element in antisense orientation, and expressing the gene of interest incorporated into the DNA molecule in a mammalian cell, for example in a mammal or in a mammalian expression system.
• this method does not induce an interferon response.
• Pol II promoter elements (i) and (iii) may be the same or different.
• the Pol II promoter element selected is either a highly active viral or composite promoter.
• Preferred promoters are the CMV promoter and the SV40 late promoter. It may also be useful in some applications to have a regulatable promoter or one that is only active in particular tissues. For example an inducible system may be used using one of the above-mentioned promoters in combination with one of tetracycline (tet) regulatable, Doxycycline or oestrogen.
• the gene of interest or the portion thereof comprises 50 nucleotides or more, more preferably 60, 70, 80, 90, 100, 150, 200 or 500 nucleotides or more, and most preferably a substantial part or the full sequence of the gene.
• a substantial part we mean at least about 50% of the gene sequence, more preferably at least about 60%, 70%), 80%o, 90%), 95%o or 99% of the gene sequence. Using a longer sequence will provide more specificity to the gene silencing effect, while still avoiding any interferon response.
• the DNA molecule does not contain termination sequences associated with the gene of interest in either the sense or antisense orientations.
• the expression system may be selected from a range of tissue culture cells, for example any human primary cell line, or cells isolated from patients, cancer cell lines, and stem cells, for example Hela cells, 293T cells, CHO cells, HEP G2 cells, HEK293, cos7 cells.
• the mammal may be a human, cow, pig or sheep.
• expression of the gene of interest is reduced or prevented for at least about 72 hours, more preferably at least about 96 hours or at least about 120 hours. Such a long-lasting effect has not previously been demonstrated using RNAi techniques such as siRNA or shRNA.
• expression of the gene of interest is decreased at least 10-fold.
• the gene of interest may be selected as one which it is desirable to silence for therapeutic purposes or may be one which it is desirable to silence in order to investigate its function.
• the gene of interest is one or more genes selected from viral genes, for example from HIV, herpes or CMV, such as env, gag, pol, tat, rev, nef or vif; genes required for the exosome to function efficiently, such as yeast Rrp6 or any homolog thereof, or yeast Rrp44 or any homolog thereof; genes required for the proteosome to function efficiently, such as ubiquitine activating enzyme, ubiquitine conjugating enzymes or ubiquitine ligase; a region of translocation associated with a cancer, for example all or part of the c-myc fusion protein on chromosome 8; 15-200 trinucleotide repeats specific for a particular trinucleotide repeat disorder, preferably 15-100 trinucleotide repeats, for example 15-100 repeats of the sequence -CAG-
• the invention provides a DNA molecule having a sequence which comprises in a 5' to 3 ' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation, for use in reducing or preventing the expression of the gene of interest in a mammalian cell, for example in a mammal or a mammalian expression system.
• This aspect of the invention has the advantages set out above. Furthermore, preferred features set out above and herein are also preferable in this aspect of the invention.
• the invention provides the use of a DNA molecule having a sequence which comprises in a 5' to 3' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation, for reducing or preventing the expression of the gene of interest in a mammalian cell, for example in a mammal or a mammalian expression system.
• the invention provides a DNA molecule having a sequence which comprises in a 5' to 3 ' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation, for use in therapy.
• the invention provides the use of a DNA molecule having a sequence which comprises in a 5' to 3' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation, for the manufacture of a medicament for the treatment of cancer or a trinucleotide repeat disorder or a viral infection.
• a DNA molecule having a sequence which comprises in a 5' to 3' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation, for the manufacture of a medicament for the treatment of cancer or a trinucleotide repeat disorder or a viral infection.
• Preferred features set out above and herein are also preferable in these aspects of the invention.
• the invention provides a method of reducing or preventing the expression of a gene of interest in a mammalian cell comprising integrating a Pol II promoter element in antisense orientation downstream (3') of the gene of interest and inducing expression from the antisense promoter.
• this method is an in vitro method.
• This aspect of the invention has the advantages set out above. Additionally, this approach may be simpler from the cloning point of view and one can integrate such antisense promoters behind any gene or genes in a cell. Furthermore, preferred features set out above and herein are also preferable in this aspect of the invention.
• the invention provides an in vitro method of preparing a pool of siRNA molecules specific for a gene of interest comprising:
• a DNA molecule having a sequence which comprises in a 5 ' to 3 ' direction (i) a Pol II promoter element in sense orientation, (ii) the gene of interest or a portion thereof comprising 100 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation,
• Dicer preferably recombinant Dicer
• the transcription is achieved using nuclear extract or in presence of active Pol II.
• this provides a simple and cheap in vitro method to prepare a pool of siRNA specific to a gene of interest and which may be used to reduce or prevent expression of the gene of interest.
• the invention provides a method of reducing or preventing the expression of a gene of interest in a mammalian cell comprising transfecting the cell with siR A molecules produced according to the above method.
• the invention provides siRNA molecules produced according to the above method, for use in therapy, as well as the use of siRNA molecules produced according to the above method, for the manufacture of a medicament for the treatment of cancer or a trinucleotide repeat disorder or a viral infection. Preferred features set out above and herein are also preferable in these aspects of the invention.
• Figure 1 shows data indicating that convergent genes in plasmids induce transcriptional cis silencing.
• Figure 2 shows data indicating that plasmid derived convergent genes induce transcriptional trans silencing.
• Figure 3 shows data indicating that convergent transcription induces trans silencing.
• Figure 4 shows data indicating that trans silencing of essential genes is induced by convergent transcription.
• Figure 5 shows data indicating that convergent transcription induces trans silencing of mammalian genes.
• Figure 6 shows data indicating that transcriptional silencing induced by the CT cassette leads to more efficient inhibition of test gene expression than siRNA or shRNA treatment.
• Figure 7 shows a model of induced transcriptional silencing in trans.
• Figure 8 shows data indicating that convergent transcription induces trans silencing of mammalian genes.
• Figure 9 shows examples of other cell lines and other target genes.
• Figure 10 shows in vitro experiments proving that Dicer interacts with pol II, dsRNA formation derived from CT plasmid and siRNA formation derived from CT plasmid.
• Figure 11 shows gene silencing effect of CT, sense, antisense and sense+antisense transcription. Only CT induces most efficient TGS. Full gene inserted in CT plasmid is more efficient to induce TGS than exon or intron only. CT without terminators (polyA signals) is more efficient in inducing TGS.
• Figure 12 shows CT induced TGS on TDP43 gene.
• Figure 13 shows limited spreading of CT induced heterochromatin and long lasting effect.
• Figure 14 shows different CT gene constructs used. Detailed description of the invention
• the invention is based on the approach of inducing nuclear TGS either by transfecting long convergent transcription units into mammalian cell nuclei or by integrating an antisense promoter downstream of an endogenous target gene in a mammalian cell.
• This approach since the dsRNAs produced as a result of convergent transcription remain in the nuclei, activation of the interferon dsR A response is avoided.
• the benefits of this procedure are that convergent transcription constructs are cheap and easy to make and use.
• TGS so induced produces a longer term gene silencing effect unlike more transient PTGS.
• the invention provides a method of reducing or preventing the expression of a gene of interest comprising providing a DNA molecule having a sequence which comprises in a 5' to 3 ' direction (i) a Pol II promoter element in sense orientation, (ii) the gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation, and expressing the gene of interest incorporated into the DNA molecule in a mammalian cell.
• the invention provides a DNA molecule having a sequence which comprises in a 5' to 3' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation, for use in reducing or preventing the expression of the gene of interest in a mammalian cell.
• the invention provides the use of a DNA molecule having a sequence which comprises in a 5' to 3' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation, for reducing or preventing the expression of the gene of interest in a mammalian cell.
• the invention provides a method of reducing or preventing the expression of a gene of interest in a mammalian cell comprising integrating a Pol II promoter element in antisense orientation downstream (3') of the gene of interest and inducing expression from the antisense promoter.
• RNA polymerase II (Pol II) promoter may be used in accordance with the invention.
• the Pol II promoter element selected is either a highly active viral or composite promoter.
• Preferred promoters are the CMV promoter and the SV40 late promoter. It may also be useful in some applications to have a regulatable promoter or one that is only active in particular tissues. For example an inducible system may be used using one of the above-mentioned promoters in combination with one of tetracycline (tet) regulatable, Doxycycline or oestrogen.
• tet tetracycline
• Doxycycline or oestrogen tetracycline
• the same Pol II promoter may be used in (i) and (iii) or alternatively different promoter combinations may also be used effectively.
• promoters (i) and (iii) are inducible as this will allow the control of target gene (ii) at anytime, for example by adding the appropriate drug.
• a Pol II promoter is integrated directly downstream of an endogenous gene of interest then the Pol II promoter is preferably a regulatable promoter or one that is only active in particular tissues.
• an inducible system may be used using one of the above-mentioned promoters (e.g. a highly active viral or composite promoter such as the CMV promoter or SV40 late promoter) in combination with one of tetracycline (tet) regulatable, Doxycycline or oestrogen.
• the gene of interest may be any gene which it is desired to reduce or prevent expression from.
• the gene of interest may be a single gene or may be 2 or more genes, for example 3, 4 or 5 genes, or portions thereof.
• one or more different genes from the same pathway for example 2, 3 or 4 genes, may be cloned in sequence between (i) and (iii) which may provide a maximal effect on the pathway.
• one or more HIV genes such as gag, pol, env, tat, rev, nef and vif, may be cloned in sequence between (i) and (iii) in order to target HIV.
• the DNA molecules described herein have a sequence which consists essentially of in a 5' to 3' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation. Termination sequences provide polyadenylation of the mRNA and/or transcriptional termination signal.
• one or more termination sequences may be associated with the gene of interest in the sense orientation and/or one or more termination sequences may be associated with the gene of interest in the antisense orientation, but preferably no termination sequences are associated with the gene of interest in either orientation. It may also be desired to reduce or prevent the expression of a gene for a therapeutic purpose, for example by gene therapy of a patient. For example a particular gene may be known to be overexpressed or expressed anomalously in a particular disease condition. In such a case the gene may be silenced in accordance with the invention in order to provide a therapeutic effect. An example of this might be the expression of a particular gene in cancer cells.
• the gene of interest is one or more genes selected from viral genes, for example from HIV, herpes or CMV, such as env, gag, pol, tat, rev, nef or vif; genes required for the exosome to function efficiently, such as yeast Rrp6 or any homolog thereof, or yeast Rrp44 or any homolog thereof; genes required for the proteosome to function efficiently, such as ubiquitine activating enzyme, ubiquitine conjugating enzymes or ubiquitine ligase; a region of translocation associated with a cancer, for example all or part of the c-myc fusion protein on chromosome 8; 15-200 trinucleotide repeats specific for a particular trinucleotide repeat disorder, preferably 15-100 trinucleotide repeats,
• chromosome translocations i.e. abnormal rearrangement between non-homologous chromosomes
• Such a translocation can lead to the fusion of normally separated genes, if the joining of chromosomes happens in coding regions.
• translocation of c-myc on chromosome 8 causes a fusion protein, which gives lymphocyte proliferation ability.
• Such occurrence of fusion proteins can be fatal for cell and lead to various phenotypes, typical of cancer cells. For example it has been described that several leukemias are caused by acquired translocations.
• the present invention could be used to silence the regions of translocations and so kill the cancer cells, thereby allowing normal cells (which do not have regions of translocation) to proliferate.
• the gene of interest may comprise a region of translocation associated with cancer, such regions being known to the skilled person.
• the gene of interest may comprise the whole or part of the c-myc fusion protein on chromosome 8.
• Other translocations which may be targeted may be found for example in Igbokwe and Lopez-Terrada (Molecular Testing of Solid Tumors. Archives of Pathology & Laboratory Medicine: January 2011, Vol. 135, No. 1, pp. 67-82) and Sandberg and Avery (Cancer Genetics and Cytogenetics, 2003 (2010) 102-126).
• the gene of interest encodes a protein comprising a growth hormone, a clotting factor, a viral antigen, an antibody, an ion channel or an enzyme.
• convergent transcription in accordance with the invention can be used with advantage in any case where the value of an organism or cell line to commerce, including agriculture, is determined by the level of expression of one of its natural genes or of an artificially introduced gene.
• the invention could be used to treat, prevent or reduce viral infection by targeting one or more viral genes, for example from HIV-1, Herpes or CMV, for example env, gag, pol, tat, rev, nef or vif
• the invention could also be used to increase the yield of transgene expression by inactivating the exosome (RNA level) or the proteasome (protein level) by targeting genes required for these complexes to function efficiently.
• Specific genes which may be targeted include any homolog of yeast Rrp6 or rrp44, in order to inactivate the exosome.
• many genes may be targeted, for example ubiquitine activating enzyme, a ubiquitine conjugating enzyme or ubiquitine ligase.
• the gene of interest is cloned into a DNA molecule sandwiched between convergent Pol II promoters.
• the whole coding sequence of the gene is included in the DNA construct.
• the sequence of gene preferably at least about 40 nucleotides or more, more preferably about 50 60, 70, 80, 90, 100, 125, 150, 200, 250, 500 or 1000 nucleotides.
• the maximum length of gene insert is about 10 kb as beyond this size gene cloning and cell transfection are less efficient. In other embodiments the maximum length of gene insert may be about 5kb or about 2 kb.
• the DNA molecule may be made by any method known to the skilled person (such as restriction digestion/PCR/ligation). For example standard cloning techniques may be used as described in Molecular Cloning: A Laboratory Manual (Maniatis et al., published by Cold Spring Harbor Laboratory Press).
• the gene is preferably located adjacent to the promoter but there may be a portion of non-coding sequence between the promoter and the gene provided that the promoter is able to direct expression of the gene.
• the portion of non-coding sequence between the promoter and the gene may be up to 2kb.
• the DNA molecule is isolated, in the sense that the invention is not intended to encompass naturally-occurring DNA molecules that include convergent Pol II promoters.
• the DNA molecule is usually in the form of an expression vector.
• An expression vector is any vector capable of expressing those DNA sequences contained therein which are operably linked to other sequences capable of effecting their expression.
• An example of an expression vector is a plasmid.
• the DNA In the case of stable expression the DNA must be replicable in the host either as an episome or as an integral part of the chromosomal DNA.
• a eukaryotic cell There are various methods of introducing foreign DNA into a eukaryotic cell: some rely on physical treatment (electroporation, nanoparticles, magnetofection), other on chemical materials or biological particles (viruses) that are used as carriers. Any of these methods, or others, may be used as known to the skilled person.
• the construct may be transfected into the cell using calcium phosphate, by electroporation, or using liposomes.
• Transfection into the mammalian cell may be transient or stable.
• Transient transfection/ expression of the convergent gene transcription construct may be suitable in order to achieve short or mid term gene silencing applications (for example in the region of 3, 4, 5, 6 or 7 days).
• long term gene silencing for example longer than 7 days, more preferably longer than 14 or 28 days
• stable transfection is preferred and this may be conveniently achieved by stable integration of the DNA construct into the host cell chromosomes.
• the addition of a selectable marker gene on the DNA construct (such as neomycin) will allow for the selection of stable transfected cells that will be permanently silenced at the target genes.
• use of inducible/repressible promoters would allow to switch integrated CT construct on or off, just by adding an appropriate drug, with no need of transfection.
• Expression from the gene of interest can be induced in the usual way and this will depend on the type of promoter element(s) used. Expression from the gene of interest will result in the production of dsRNA molecules. Since transcription occurs from both the sense and antisense promoters, sense and antisense RNA molecules are produced which then hybridise together to form dsRNA molecules. These dsRNA molecules then act to suppress further transcription from the gene, both from the construct and any other copy of the gene found in the cell, by heterochromatin modification and by other RNAi pathways. Any mammalian cell or expression system may be used in accordance with the invention. For example the mammal may be a human, cow, pig, sheep or chicken.
• Preferred expression systems are human and mammalian tissues and cell lines, for example any human primary cell line, or cells isolated from patients, cancer cell lines, and stem cells, for example Hela cells, 293T cells, CHO cells, HEP G2 cells, HEK293, cos7 cells.
• the mammal may be a human, cow, pig, sheep or chicken.
• RNA and protein can be quantified by numerous techniques known to the skilled person, including real-time PCR, northern blot, RNAse protection and SI nuclease analysis for mRNA yields, and Western blot for protein yields.
• a Pol II promoter element is integrated in antisense orientation downstream (3') of a gene of interest.
• the Pol II promoter element is an inducible element, such as TetO.
• the promoter element may be integrated downstream of a gene of interest using standard site specific genome integration using homology regions for recombination, as is well known to the skilled person. This approach may be used to reduce or prevent the expression of one or more genes of interest.
• the method may be used to create a stable cell line with an inducible knock down system.
• reducing or preventing gene expression refers to a decrease in the mRNA and/or protein levels which are observed when a particular gene is expressed in a particular expression system from a DNA molecule in accordance with the invention, as compared to expression of the same gene in the same expression system which does not contain a DNA molecule in accordance with the invention.
• the DNA molecule induces a trans effect on any copy of the gene found in the cells of the expression system.
• the term 'silence' is also used herein and is intended to have the same meaning as 'reduce or prevent'.
• the term "reducing or preventing gene expression” is used to refer to a decrease in the mRNA and/or protein levels which are observed when transcription of the gene of interest is induced from the antisense promoter in accordance with the invention, as compared to when there is no transcription of the gene of interest from the antisense promoter.
• the level of silencing is likely to vary between genes, preferably, according to the invention expression of the gene of interest is reduced by from about 2-fold to about 10-fold or more. Most preferably the gene is silenced entirely in that no protein encoded by the gene is produced or is capable of detection.
• the amount of nuclear mRNA and cytoplasmic mRNA produced according to the invention is at least 2- to 3 -fold lower, preferably at least 10-fold lower, than the amount produced in the absence of providing such a DNA construct.
• the amount of nuclear mRNA and cytoplasmic mRNA produced according to the invention may be from 2-fold to 20-fold lower, or from 4-fold to 12- fold lower, than previous methods.
• mRNA in the cytoplasm is also down-regulated.
• the amount of protein produced according to the invention is at least 2- to 3-fold lower, preferably at least 10-fold lower, than the amount produced in the absence of providing such a DNA construct.
• the amount of protein produced according to the invention may be from 2-fold to 20-fold lower, or from 4- fold to 12-fold lower, than previous methods.
• the gene is silenced entirely in that no protein encoded by the gene is produced. This may be determined by quantitating mRNA levels, from situations plus the DNA construct according to the invention compared with situations minus the DNA construct according to the invention, using RT-PCR (or other such techniques known to the skilled person). Following this, a western blot may be performed to detect the target protein produced from the two situations (plus and minus the DNA molecule).
• the gene silencing effect brought about in accordance with the invention may vary and it likely to last at least about 72 hours, more preferably at least about 96 hours or at least about 120 hours. Furthermore the gene silencing effect may be permanent.
• the present invention may be used to reduce or prevent the expression of genes integrated into chromosomal locations in cells by providing the same gene, or a portion thereof, contained in an expression vector, such as a plasmid, in vitro. Because reduced protein production can be achieved by simply inserting the gene of interest between convergent promoter sequences, this technique is an incredibly cheap and easy technology to implement and requires nothing more than cloning techniques. Further, no alterations in the coding portion of the gene are required.
• the present invention may be used to reduce or prevent the expression of genes integrated into chromosomal locations in cells by integrating a Pol II promoter element in antisense orientation downstream of the selected genes. Because reduced protein production can be achieved by simply integrating a promoter element downstream the gene of interest, this technique is an incredibly cheap and easy technology to implement and requires nothing more than cloning techniques. Further, no alterations in the coding portion of the gene are required.
• a further great advantage of the invention is for the production of stable cell lines, where promoters (i) and (iii) are inducible and are integrated into a chromosome thereby allowing the silencing of target gene (ii) at anytime, just by adding an appropriate drug. Alternatively the same effect may be achieved if the sense promoter is constitutive and the antisense promoter is inducible. Furthermore the invention provides therapeutic uses.
• the invention also provides a DNA molecule having a sequence which comprises in a 5' to 3 ' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 (preferably 100) nucleotides or more, and (iii) the Pol II promoter element in antisense orientation, for use in therapy.
• the invention provides the use of a DNA molecule having a sequence which comprises in a 5' to 3' direction (i) a Pol II promoter element in sense orientation, (ii) a gene of interest or a portion thereof comprising 40 (preferably 100) nucleotides or more, and (iii) a Pol II promoter element in antisense orientation, for the manufacture of a medicament for the treatment of cancer or a trinucleotide repeat disorder or a viral infection.
• Any type of cancer may be treated as discussed above, and in particular those cancers which have a translocation event as an early indicator.
• any viral infection may be treated, for example HIV, herpes, CMV.
• the preferred features of the DNA molecules mentioned above also apply to this method and use.
• the type of therapy or disease to be treated or prevented will depend on the selected gene of interest.
• the gene of interest may encode a protein, for example interferon protein, insulin, a growth hormone, a clotting factor, a viral antigen, an antibody or an enzyme, which has a known effect on a particular disease and the expression of said gene may be decreased or prevented in accordance with the invention in order to prevent or treat said disease.
• the invention may have an application in gene therapy of a patient where it is desired to decrease or prevent the expression of a particular protein which is produced excessively by the patient.
• This technology could also be useful in gene therapy of a patient where it is desired to decrease or prevent the expression of a particular protein where excessive production of certain protein (gene expression) is unwanted in the cell. Examples of this approach are discussed above.
• inventive approach could also be used for treatment of other trinucleotide repeat disorders in a corresponding manner, for example myotonic dystrophy, myoclonic epilepsy, spinocerebellar ataxias or Friedreich's ataxia.
• this technology could be used is in the treatment of Huntington's chorea.
• This neurodegenerative genetic disorder affects muscle coordination and leads to cognitive decline and dementia.
• the Huntingtin gene normally encodes a protein called Huntingtin.
• the mutation of the Huntingtin gene leads to the production of different forms of the protein, which cause gradual damage to specific areas of the brain.
• the Huntingtin gene contains repeated sections called a trinucleotide repeat (...(CAG) n ). These repeats vary in length. When the length of this repeated section reaches a certain threshold, a mutant form of the protein is produced.
• the normal number of trinucleotide repeats is less than 28.
• An intermediate phenotype is caused by 28-35 repeats and full disease is developed in cells with more than 40 repeats.
• the present invention could be used to target excessive repeats by incorporating 15-100 trinucleotide repeats as the gene of interest and so decrease effect of the disease.
• trinucleotide repeat disorders which may be treated in accordance with the invention are described by Orr and Zoghbi (Annual Review of Neuroscience Vol. 30: 575-621), and are set out in the tables below (split into polyQ and non-polyQ disorders).
• the treatment strategy would be similar to that discussed above for Huntington's chorea and the skilled person would readily be able to determine such a suitable strategy.
• the strategy may be to include 15-200 trinucleotide repeats specific for a particular trinucleotide repeat disorder, preferably 15-100 trinucleotide repeats, as the gene of interest.
• FRAXA Frazier X FMR1, on the X-
• FRAXE Frazier XE AFF2 or FMR2, on the
• the invention also provides such treatment by integrating a Pol II promoter element in antisense orientation downstream (3') of the endogenous gene and inducing expression from the antisense promoter.
• the Pol II promoter is preferably a regulatable promoter or one that is only active in particular tissues.
• an inducible system may be used using e.g. a highly active viral or composite promoter, such as the CMV promoter or SV40 late promoter, in combination with one of tetracycline (tet) regulatable, Doxycycline or oestrogen.
• the invention provides an in vitro method of preparing a pool of siR A molecules specific for a gene of interest comprising:
• a DNA molecule having a sequence which comprises in a 5 ' to 3 ' direction (i) a Pol II promoter element in sense orientation, (ii) the gene of interest or a portion thereof comprising 40 nucleotides or more, and (iii) a Pol II promoter element in antisense orientation,
• the description and preferred features of the DNA molecule set out hereinabove apply also to this aspect of the invention.
• the method may be carried out in the presence of a nuclear extract or purified RNA polymerase II to provide the required transcription. If purified Pol II is used then the skilled person will know to include all essential components for efficient transcriptions, like transcription factors, dNTPs etc. Dicer present in nuclear extract may be sufficient but it is preferable to use additional recombinant Dicer to provide for a more efficient reaction. Reaction conditions can be readily determined by those of skill in the art.
• RNA molecules Transcription of RNA from the gene of interest will result in the production dsRNA molecules. Since transcription occurs from both the sense and antisense promoters, sense and antisense RNA molecules are produced which then hybridise together to form double-stranded RNA (dsRNA) molecules. These dsRNA molecules are then cleaved by Dicer, an endoribonuclease in the RNase III family, to form a pool of short double-stranded RNA fragments called small interfering RNA (siRNA) molecules, each about 20-25 nucleotides in length.
• siRNA small interfering RNA
• siRNA molecules may be used to reduce or prevent the expression of a gene of interest in a mammalian cell.
• the siRNA molecules may first be isolated, for example by standard RNA isolation methods (e.g. phenol/chloroform or Trizol).
• the cell may then be transfected with the siRNA molecules using any technique known to the skilled person, for example Lipofectamine 2000.
• the invention provides siRNA molecules produced according to the above method, for use in therapy, which may be prophylactic or therapeutic.
• the invention also provides the use of siRNA molecules produced according to the above method, for the manufacture of a medicament for the treatment of cancer or a trinucleotide repeat disorder or a viral infection.
• the details discussed above in relation to therapies, including the diseases that may be treated and the genes of interest, are relevant to these aspects also.
• Figure 1 shows:
• Plasmid design pYL16 (Werner Bio Agents), containing resistance gene for nourseothricin, was used as vector. Further ura4 or ade6 genes with endogenous promoters and terminators sequences were inserted into pYL16 single (S). Additionally ura4 and ade6 were cloned into the vector in tandem (T) or convergent orientation (C) as depicted.
• H3K9me3 ChIP analysis Levels of H3K9me3 analysed in samples (as in B) and probed in ura4 (first graph), ade6 (second graph) and centromeric dg (third graph). Bars represent the levels of H3K9me3 as % of input. Error bars as in B.
• H3K9me3 ChIP analysis Levels of H3K9me3 analysed in samples as in A and probed in ura4 (left graph), ade6 (centre graph) and centromeric dg (right graph). Bars represent the levels of H3K9me3 as % of input. Error bars as in Figure ID.
• E. C plasmid was transformed into Adcrl and Aagol strains. Wt C and wt V plasmids were used as controls.
• CT convergent transcription cassette
• RNA isolated from 972- cells transformed with pYL16 vector, ura4 or ade6 single genes in pYL16 vector, and ura4 or ade6 in CT cassette were growing in EMM (CT cassette on) or YES (CT cassette off) media until log phase. PCR performed done with primers specific for ura4 or ade6.
• RNA isolated from 972- cells transformed with pYL16 vector or rad21 in CT cassette were growing in EMM (CT cassette on) or YES (CT cassette off) media until log phase.
• RT primers were specific for sense (left graph) and antisense (right graph) rad21 mRNA. PCR was carried out with primers specific to rad21.
• RT primers were specific for sense (left graph) and antisense (right graph) esol mRNA. PCR performed with primers specific for esol.
• Pol II ChIP chromatin was isolated from HeLa cells transfected with either pCI plasmid alone (vector) or with pCI containing yACTl gene fragment (exon 4) between CMV promoters (CTyACTl). Untransfected HeLa cells were used as a control. PCR was performed using yACTl exon 4 primers and primers for GAPDH.
• Figure 6 shows: A. qRT-PCR analysis using yACTl reverse primer for reverse transcription, probing for yACTl sense mRNA levels (left graph).
• Total RNA was isolated from HeLa cells transfected with siRNA (specific for yACTl gene, ThermoFishers), shRNA (specific for yACTl, Sigma) or a yACTl fragment convergently expressed from pCI plasmid (CTyACTl), 48, 72 and 96 hours after transfection. Untransfected cells were used as a control. Levels of sense mRNA signals were normalized to untransfected cells, which were set as 1. Levels of GAPDH mRNA were used as a control (right graph).
• B qRT-PCR analysis as in A, except using yACTl forward primer for reverse transcription to detect yACTl antisense transcripts (left graph) and GAPDH forward primer detecting antisense potential transcripts from GAPDH gene (right graph).
• CT Convergent transcription
• C Measurement of mRNA levels using oligo dT primed qRT/PCR by from HeLa cells as in B.
• H3K9me2 ChIP was performed as in Fig. 8 but using additional y-ACTl (Ex2-6) expression constructs: sense (S) or antisense (AS) alone y-ACTl transcription plasmids or S and AS plasmids cotransfected. Note only CT plasmid is effective in inducing full levels of heterochromatm and maximum reduction in mRNA levels.
• CT constructs containing y-ACTl intron 3 or exon 4 only were compared for TGS effects versus the full CT yACT!Ex2-6 sequence measuring polyA+ mRNA and nascent (intronic) transcript.
• CT yACT!Ex2-6 plasmid was modified by positioning PAS derived from SV40 at either end of the y-ACTl sequence so that both S and AS transcripts are polyadenylated. The coTGS effects were then determined for both nascent and steady state y-ACTl transcripts by qRT/PCR analysis.
• Figure 12 shows:
• TDP-43 gene map is depicted with exons as boxes and introns as lines. Positions of PCR amlpicons used are indicated. Diagram depicting TDP-43 insert into CT plasmid is also presented.
• Figure 13 shows:
• TDP-43 gene map is shown below with exons as boxes and introns as lines. Positions of PCR amlpicons used are indicated.
• CG heterochromatin is dependent on chromosomal structure or is a more general feature of convergent transcription
• the pYL16 expression plasmid was employed, carrying a resistance gene to nourseothricin antibiotic.
• ura4 and ade6 genes with their own promoter and terminator regions were inserted into pYL16 as single or double gene constructs.
• ura4 and ade6 genes were positioned in either tandem or convergent orientation ( Figure 1A). These plasmids were then transformed into S. pombe A2 strain, which lacks both ura4 and ade6. Positive transformants were selected on plates with nourseothricin.
• Pol II levels over ura4 or ade6 in A2 cells carrying either vector only (V), plasmids with single ura4 or ade6 (S), tandem ura4-ade6 (T) or convergent ura4-ade6 (C) gene pairs were analysed ( Figure 1A).
• V cells were used as a negative control, since they lack endogenous ura4 and ade6.
• Pol II signals at ura4 or ade6 in V cells were at background levels.
• Cells containing plasmids with single ura4 or ade6 (S) gave significant Pol II signal over either ura4 or ade6 genes, confirming the plasmid based expression of these genes.
• tandem arranged ura4 and ade6 on plasmid result in similar Pol II levels over ura4 and ade6. This suggests that the expression of tandem arranged genes is efficient. Significantly, Pol II levels over convergent ura4 and ade6 were significantly decreased (C) ( Figure IB). This result suggests that transcription of CGs shows similar behaviour regardless of their endogenous or episomal context. It should be noted that CGs on plasmids appear not to be regulated during the cell cycle, as the Pol II ChIP experiments described here were performed on cycling cells, which are predominantly in G2 phase of the cell cycle.
• H3K9me3 ChIP analysis on V, S, T and C transformed cells was performed and high H3K9me3 levels were detected only in C cells, indicative of heterochromatin formation.
• a centromeric dg repeat probe was used as a positive control ( Figure ID).
• the effect of decreased ura4 and ade6 mRNAs on cell growth in selective media lacking uracil or adenine was finally tested. Since A2 cells are deficient in endogenous ura4 or ade6, in selective media, they rely on plasmid genes expression. Serial dilutions of V, S, T and C cells were prepared and spotted on non-selective, ura- selective or ade- selective plates.
• Example 2 Plasmid derived convergent genes induce transcriptional trans silencing.
• Pol II levels in S or T cells were somewhat higher, corresponding to the sum of Pol II at the endogenous alleles plus additional copies of plasmid ura4 or ade6. Surprisingly, a substantial reduction in Pol II occupancy was observed with C cells, when compared to V cells. Pol II levels over actl were also analyzed, and no significant level changes were observed between V, S, T and C cells. This suggests that the effect of plasmid expression is specific to the tested genes and has no general effect on transcription. These data predict that siRNAs produced from plasmids have a trans effect on expression of their endogenous allele.
• Phased oligo d(T) with a linker sequence was used to prime transcription, followed be PCR using a reverse primer specific for the linker sequence and a forward primer in each ORF.
• all polyadenylated mRNAs produced could be detected.
• 3 'RACE analysis using total RNA isolated from V, S and T cells resulted in only one major band, corresponding to mRNA with proximal polyA site usage for both tested ura4 and ade6.
• larger bands were detected when analyzing RNA from C cells. These correspond to long polyadenylated mRNAs, using more distal cryptic polyA sites, presumably derived from the plasmid CGs.
• actl was also tested as a control and only one band was observed in all analyzed samples corresponding to proximal polyA site usage. These results confirm that transcriptional read-through occurs on both DNA strands when the genes are in convergent arrangement. Further, it was tested whether CG induced trans silencing occurs at the transcriptional or mRNA level.
• H3K9me3 ChIP was performed on chromatin isolated from V, S, T and C strains, probing in either ura4 or ade6 ORFs ( Figure 2D). As expected no positive H3K9me3 signals were detected on ura4 and ade6 (either endogenous or exogenous) with V, S or T chromatin.
• Heterochromatin and gene silencing should induce a growth defect phenotype, when cells are grown on selective plates.
• Serial dilutions of V, S, T and C strain cells were therefore performed on either non-selective media (to test viability) or on media lacking uracil or adenine ( Figure 2E). All strains grew similarly on non-selective plates. In contrast, C cells show a clear growth defect on plates without uracil or adenine. It is concluded that expression of ura4 and ade6 as plasmid CGs inhibits endogenous gene expression in trans through heterochromatin induction. This results in a defective growth phenotype, when the cells are grown in selective media.
• plasmid expression of ura4 or ade6 has no effect on endogenous transcription, when these genes are present singly or in tandem arrangement.
• plasmid expression of ura4 and ade6 in convergent orientation blocks the expression of the endogenous alleles of these two genes.
• transcriptional read-through on both DNA strands leads to production of long dsRNA molecules, which is processed by Dicer into siRNAs. This induces RNAi and leads to silencing of endogenous copies of tested genes through heterochromatin formation in trans.
• Such gene silencing leads to less ura4 or ade6 mRNA, causing a growth defect, when C cells are grown on selective media.
• qRT-PCR analysis was performed on steady state ura4 or ade6 mRNA levels under conditions where convergent transcription (CT) is switched on or off ( Figure 3B).
• CT convergent transcription
• V S or C strains were grown in either EMM (CT on) or YES medium (CT off).
• Total RNA was isolated and reverse transcribed followed by PCR amplification to detect sense ura4 or ade6 mRNA levels. All tested strains yielded positive sense mRNA signals when grown in YES medium.
• V and S cells showed similar sense mRNA levels for both ura4 and ade6, when cells were grown in EMM medium with the CT cassette switched on.
• ura4 or ade6 sense mRNA levels were clearly substantially reduced in CT cells when CT cassette was switched on, suggesting that convergent transcription from the CT plasmid leads to endogenous gene silencing.
• nmtl promoter is stronger than ura4 or ade6 promoters and will therefore induce a higher transcriptional rate in sense and antisense direction. This may cause the production of more siRNA from CT than C plasmids and so be more efficient and faster in the induction of transcriptional gene silencing in trans.
• Example 4 Trans silencing of essential genes induced by convergent transcription
• Rad21 is a subunit of the cohesin protein complex, which is involved in many regulatory mechanisms, such as gene expression, morphogenesis, cell proliferation, DNA repair and chromosome segregation.
• CTrad21 cells were grown in EMM and YES media.
• Total RNA was isolated and analysed by qRT-PCR (Figure 4A). Similar levels of sense rad21 mRNA were detected in V and CTrad21 cells, when the cassette was switched off. However when switched on a strong reduction (10% of V signal) of rad21 sense mRNA levels in CTrad21 cells was observed. The same approach was used to detect the antisense rad21 mRNA levels. As expected, no increase above background levels was observed in V and CTrad21 cells, when grown in YES medium.
• CT cassette it was possible to manipulate mRNA levels of these two essential genes, by simply growing cells in either YES or EMM media, to switch the CT cassette either on or off.
• the growth phenotype of CTrad21 and CTesol cells was looked at.
• the CT cassette efficiency was also compared to commonly used ts mutants of rad21 and esol. Serial dilutions of wt, CTrad21, rad21ts, CTesol and esolts cells were plated on either YES or EMM plates and incubated at permissive temperature (25°C) for four days (Figure 4C).
• CT cassette offers a new and efficient system to decrease mR A levels of essential genes. This can be achieved without the necessity to grow cells at higher temperatures that could otherwise induce confusing side effects.
• Example 5 Convergent transcription induces trans silencing of mammalian genes
• Pol II ChIP was performed on chromatin isolated from untransfected HeLa cells (UN), HeLa cells transfected with empty vector (V) or cells transfected with CTyACTl (CT).
• PCR primers were designed to amplify either yACTl exon 4 or GAPDH as a control, similar Pol II occupancy was observed over in yACTl in UN and V cells, but only low Pol II levels in CT cells ( Figure 5B).
• Pol II levels over GAPDH were similar in all three samples, excluding a general effect of CT on transcription.
• qRT-PCR was used to detect yACTl and GAPDH mRNA levels in UN, V and CT cells ( Figure 5C).
• ⁇ actin protein level reduction following CT transfection was finally investigated. Total proteins were isolated from UN, V and CT cells after 24, 48 and 72 hours of transfection and analysed by Western blot using a specific ⁇ actin antibody (Sigma). The blot was visualized by radioautography and quantified using ImageQuant software. Signals were normalized to the UN signal, set at 100%. Levels of ⁇ actin in UN and V cells were similar at all time points. However with CT transfected cells, a decrease in ⁇ actin protein levels was observed after 24 hours of transfection, with further reductions at 48 and 72 hours post transfection (Figure 5E).
• ⁇ actin is an essential gene. Transfection of HeLa cells with CT construct leads to silencing of the endogenous yACTl allele and consequent cell death. It is therefore difficult to collect cells at later time points. Even so it was desired to define how long the CT induced heterochromatin mark persists and so transfected HeLa cells with CT cassette but maintained their survival by splitting them every three days. Aliquots of cells were selected at 96, 168 and 264 hours post transfection and subjected to ChIP analysis with antibody to H3K9me2. High levels of H3K9me3 were detected 96 hours post transfection.
• Knock down of mammalian gene expression is a commonly used procedure. The usual approach is to design specific siRNA or shRNA and transfect these reagents into mammalian cells. The efficiency of such knock down experiments varies, depending on the expression of the tested gene. Usually a "two hit" transfection is experiment is necessary to achieve a significant knock down effect.
• transcriptional gene silencing induced by a convergent gene cassette works in higher eukaryotes; human HeLa cells.
• Example 7 Convergent transcription induces trans silencing of mammalian genes
• the experimental system is to insert test gene fragments between convergent CMV promoters so generating convergent transcription (CT) plasmids (Fig. 8A).
• CT convergent transcription
• Fig. 8A convergent transcription
• TGS transcriptional gene silencing
• CT plasmids employed in these experiments do not contain polyA signals (PAS) so mRNA levels detected can only derive from endogenous genes.
• PAS polyA signals
• These results proved general as CT constructs containing 3 other gene sequences (from CYPA, PGK1 and GAPDH) each gave similar endogenous gene silencing effects at both Pol II and mRNA levels (Fig. 9C-F).
• Importantly robust heterochromatic marks (H3K9me3) were also detected over endogenous y-ACTl following CT transfection (Fig. 8D). Note that primers used in this ChIP analysis will not detect ⁇ -ACTl sequence on the CT plasmid. A further 2 fold reduction in ⁇ -actin protein levels was detected after 3 days transfection (Fig.
• CTyACTl was therefore transfected into ES cells lacking dicer expression. Although transfection efficiencies for these cells is lower than for HeLa a 2 fold ⁇ -ACTl gene silencing effect of both nascent and steady state RNA was still detected. Significantly dicer knock out ES cells (ADCRl) lost this silencing effect (Fig. 8F and 9B). It is concluded from the above data that CT plasmid expression induces TGS of endogenous target genes through an RNAi mechanism.
• Example 8 use of CT plasmid transfection can induce effective coTGS
• CT ACT 1 can generate dsRNA and consequent dicer dependent siRNA formation by in vitro transcription in nuclear extracts with added 32 P UTP.
• a control template that yields a 350 nt single strand RNA following in vitro transcription was also employed (+) as was empty CT vector (V).
• RNA isolated from untreated extracts gave heterogeneous RNA species for CT and V templates and a single RNA product for +.
• Single strand specific SI nuclease treated extracts degraded most transcripts implying that they are predominantly single stranded.
• Fig. IOC Single strand specific SI nuclease treated extracts degraded most transcripts implying that they are predominantly single stranded.
• Fig. IOC This effect was controlled by use of VI nuclease which is dsRNA specific.
• CTyACTl The stable silencing by CTyACTl was again shown to correlate with TGS as H3K9me3 chromatin marks were confirmed for this treatment but were not seen with siRNA or shRNA which induce PTGS. It is concluded that CT induced coTGS may have considerable utility as an alternative gene silencing method to siRNA treatment.
• ⁇ -ACTl mRNA was also only effectively reduced by coTGS with CTyACZY transfected cells.
• the weaker TGS effect of S+AS versus CT reflects the fact that separately synthesized complementary RNAs do not anneal as effectively as co -transcribed transcripts.
• the even weaker TGS effects seen with AS alone transfected cells may reflect low level recognition of endogenous ⁇ -ACTl mRNA by this antisense transcript.
• CT plasmids containing only intron sequence Notably intron sequence was as effective as exon sequence in reducing levels of either nascent ⁇ -ACTl transcript or mRNA (Fig. 11D).
• CT plasmid transfection induces coTGS since introns are nuclear restricted.
• PAS convergent transcript
• a CT construct designed to target the pre-mRNA splicing associated TDP- 43 gene was employed.
• a CT construct containing TDP-43 cDNA exons 2-6 Using a CT construct containing TDP-43 cDNA exons 2-6, a clear coTGS effect was again shown; reduced Pol II occupancy over the endogenous gene with commensurate reduction in gene expression at the mRNA and protein levels (Fig. 12).
• the profile of induced heterochromatin marks across TDP-43 was also investigated (Fig. 13 A). While exonic regions cloned into the CT vector showed substantial H3K9me3 marks, above the vector only transfection control, adjacent intronic sequence showed reduced heterochromatin marks. These results indicate that heterochromatin spreading is locally restricted.
• S. pombe A2 and 972 K was used in this study as wt. Growth conditions and all genetic manipulations were carried out as described previously (Moreno et al. 1991). Plasmid pYL16 containing nourseothricin resistance gene, was used as backbone for experiments in fission yeast. Single gene ura4 or ade6 were cloned into rnulti cloning site. Furthermore, both genes ura4 and ade,6 were cloned into pYI.,16 in tandem or convergent orientation. CT plasmid was constructed by cloning nmtl promoter sequence in sense and antisense orientation, with, multi cloning site in the middle. Ura4, ade6, radii or eso genes were inserted in muiti cloning site on CT plasmid.
• CMV promoter in antisense orientation replaced SV40 poiyA signal in pCJ plasmid.
• Gamma actin gene was cloned in. multi cloning site between sense and antisense CMV promoters on pCI. Transformation and transfection
• Transformation of fission yeast was done using Li-acetate method.
• Exponentially growing cells were serially diluted and dropped onto selective -ura and - ade plates. Growth of cells was checked on EMM complete plates.
• Chromatin immunoprecipitation (ChIP)
• Cells were grown to OD 6 oo 0.5, cross-linked with 1% formaldehyde and incubated for 10 min at 25°C with gentle shaking. These were chilled on ice for 30 min with occasional shaking and harvested by centrifugation at 1000 g for 5 min at 4°C. Pellets were washed 4 times with ice cold buffer I (50 mM Hepes/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 7.5, 1% Triton X-100, 0.1% sodium deoxycholate) and resuspended in 500 ⁇ of buffer I containing protease inhibitors (Roche).
• buffer I 50 mM Hepes/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 7.5, 1% Triton X-100, 0.1% sodium deoxycholate
• Antibodies (Pol II, H3K9me3 and H3K9me2, all from Abeam) were added to the whole cell extracts and incubated over night at 4°C on a rotating wheel. Agarose beads were added to cell extracts and incubated by rotation at 4°C for 2 hr.
• Beads were washed twice in ice cold buffer I, once in ice cold buffer II (50 mM Hepes/KOH pH 7.5, 500 mM NaCl, 1 mM EDTA pH 7.5, 1% Triton X-100, 0.1% sodium deoxycholate) and once in ice cold buffer III (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA pH 7.5, 0.5%) Nonidet P-40, 0.5%> sodium deoxycholate).
• Chromatin immunoprecipitation Hela cells.
• Transfected HeLa cells were collected from 10 cm plates. Formaldehyde was added directly, at 20°C to tissue culture medium at 1%>: 250ul of 40%>w/v, followed by incubation lOmin 20°C on gently shaking platform. Formaldehyde was inactivated by adding glycine to a final concentration of 0.125M. Medium was aspirated and cells washed twice with 5ml ice cold PBS, containing protease and phosphatase inhibitors and scraped into 2ml tubes. Samples were centrifuged for 4min, 700xg (2800rpm) at 4°C.
• Cells were gently resuspended in 300ul of cell lysis buffer [5mM PIPES, pH8.0; 85mM KC1; 0.5% nonidet P-40; lmM PMSF; lug/ml pepstatin A; lug/ml leupeptin; 5mM sodium butyrate] and incubated on ice for lOmin.
• cell lysis buffer [5mM PIPES, pH8.0; 85mM KC1; 0.5% nonidet P-40; lmM PMSF; lug/ml pepstatin A; lug/ml leupeptin; 5mM sodium butyrate]
• Nuclei were collected by centrifugation at 550g/2400rpm at 4°C and resuspended in ice-cold 400ul nuclear lysis buffer (1% SDS, lOmM EDTA, 50mM Tris-HCl, pH8.0, 0.5mM PMSF, 0.8ug/ml pepstatin A, lug/ml leupeptin, 5mM sodium pyruvate), followed by incubation on ice for lOmin.
• nuclear lysis buffer 1% SDS, lOmM EDTA, 50mM Tris-HCl, pH8.0, 0.5mM PMSF, 0.8ug/ml pepstatin A, lug/ml leupeptin, 5mM sodium pyruvate
• IP dilution buffer 0.01% SDS, 1,1% Triton X100, 1.2mM EDTA, 16.7mM Tris-HCl pH 8.1, 167mN NaCl, 0.5mM PMSF, 0.8ug/ml pepstatin A, lug/ml leupeptin, 5mM sodium butyrate
• Antibodies were added to samples and incubated o/n at 4°C on rotating wheel.
• A 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH8.0, 150mM NaCl.
• B 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH8.0, 500mM NaCl
• C 0.25M LiCl, 1% NP40, 1% sodium dexoycholate, lmM EDTA, lOmM Tris-HCl pH8.0.
• Immune complexes were eluted with 250ul IP elution buffer (1% SDS, 0.1M NaHC03) and spun down 3min, 13000rpm. Reversal of cross links was performed by initially adding to 3ug/ml RNaseA, 0.3M NaCl, at 65°C 4-5hours, followed by addition of lOul of 0.5M EDTA, 20ul of 1M Tris-HCl, pH 6.5, 2ul lOmg/ml proteinase K and incubation at 45. C for 2 hours.
• DNA was purified by Qiagen PCR clean up columns and eluted by lOOul of elution buffer. RNA isolation and RT-PCR
• RNA from S. pombe, HeLa, HEK293 and mouse ES cells was isolated using phenol/chloroform or Trizol (Gibco). RNA was dissolved in sterile water and treated with RNase-free DNase (Promega) for 30 min at 37°C. 100 ng of total RNA was reverse transcribed using Superscript III system (Invitrogen) with oligo dTi 5 or specific primers. Phased oligodT was used for 3 'RACE experiment. cDNA was diluted to 100 ⁇ in TE buffer and 10 ⁇ was used for PCR. Genomic DNA was employed as a positive control for primer pair efficiency. Results were quantified by real-time PCR with SYBR Green dye and Rotor-Gene 6 software.
• Western blots employed the following antibodies: anti-yactin (Sigma) anti-tubulin (Sigma), anti-OASl (Abeam), anti-TDP43 (gift from Ashish Dhir), anti- Dicer (Abeam), anti-Pol II 8WG16 (Abeam) and anti-Hsp70 (Abeam). Immunodepletion and immunoprecipitation
• Nuclear extract was incubated with anti-Dicer and anti-Pol II antibodies (Abeam) in presence of Protein G beads at 4°C for 90 minutes. Beads with bound protein complexes were washed with 1 ml of washing buffer (50mM Tris-HCl, pH 7.5, 200mM NaCl, 0.1% TritonX, 0.025% NP40).
• DNA templates + (linearized fragment under CMV promoter, 363 nt long), V (CT cassette with no insert) and CT (CTyACTlEx4, 390 nt long) were incubated with nuclear extract and Dicer depleted nuclear extract in presence of 32 P-UTP for 1 hour at 30°C.
• Total RNA was isolated by phenol/chloroform. Low molecular weight RNA were separated from high molecular weigh RNA by 20%PEG8000/2M NaCl on ice for 30 minutes. Long RNAs were treated with SI or VI nucleases for 10 minutes at r.t. and separated on 6% PAGE gel. Small RNAs were visualized on 20% PAGE gel.
• MicroRNAs target recognition and regulatory functions. Cell 136, 215-233.
• Cohesin complex promotes transcriptional termination between convergent genes in S. pombe. Cell 132, 983-995.
• RNAi complexes RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119, 789-802.
• RNA 6, 1069-1076 Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.

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