JP4665190B2 - Gene transcription regulation method - Google Patents

Description

  The present invention relates to a gene transcriptional regulation method.

  In recent years, genome analysis has progressed and the whole genome has been analyzed in many organisms. Along with this, genes that were previously unknown have been identified on the genome, but in many cases their functions are not clear.

One means of clarifying the function of an unknown gene is to forcibly express the gene in cells or individuals and examine its phenotype. Conventionally, in order to forcibly express a gene, the cDNA of the gene is cloned, connected downstream of the constitutive promoter or conditional promoter, returned to the cell or individual, and transiently expressed or transformed. (For example, refer nonpatent literature 1). According to this method, it was necessary to clone the gene to be forcibly expressed step by step.
Annual Review of Physiology vol.66, pp647-63, 2004

  Thus, until now, there has been no system that allows for forced expression of any gene on the genome using only its base sequence, and there is only a time-consuming method for forced expression of a gene. I did not.

  Therefore, the present invention has been made for the purpose of providing a method that can easily regulate transcription of an arbitrary gene.

  The method according to the present invention is a gene transcriptional regulation method, comprising: a promoter region of the gene; a first region that forms a triple-stranded nucleic acid; and a second region that specifically binds to a predetermined RNA binding domain. And an RNA having the RNA and a polypeptide having the RNA binding domain and the transcriptional activation domain or the transcriptional repression domain are allowed to act on the gene.

  The RNA according to the present invention has a promoter region of a gene whose transcription is regulated, a first region that forms a triple-stranded nucleic acid, and a second region that specifically binds to a predetermined RNA binding protein. The polypeptide according to the present invention has an RNA binding domain that specifically binds to a predetermined RNA region, and a transcription activation domain or a transcription repression domain. The DNA according to the present invention encodes these.

  The first region has a length of 16 bases or more, and preferably consists of either one of purine or pyrimidine. More preferably, the first region has 34 bases or more. The RNA binding domain may include the RNA binding domain of the coat protein of MS2 phage, and the second region may include the RNA binding sequence of the coat protein of MS2 phage. The transcription activation domain may comprise a herpes simplex virus VP16 transcription activation domain.

  According to the present invention, it has become possible to provide a method capable of easily regulating transcription of an arbitrary gene.

  Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Triple-stranded nucleic acid forming RNA ==
Triplex nucleic acid forming RNA, which is the first constituent molecule of the transcriptional regulatory machinery of the present invention, is specific to a promoter region of a target gene to be transcriptionally regulated, a first region forming a triplex nucleic acid, and a predetermined RNA binding domain. And a second region that is bonded together.

  The first region preferably has a sequence that forms a triple-stranded nucleic acid with the promoter region of the gene of interest, and preferably consists of either a purine or a pyrimidine. The triple-stranded nucleic acid is preferably formed stably. For example, in terms of length, it is preferably 16 bases or more, more preferably 34 bases or more. The sequence of the base sequence itself is not particularly limited, and a promoter region may be selected so as to satisfy the above conditions, and the sequence of the first region may be determined according to the sequence. In the promoter, the position of the region for forming the triple-stranded nucleic acid is not particularly limited and may be anywhere, but taking into account the translation from the start codon, it is preferable to be away from the start codon.

  The second region has a sequence that specifically binds to a predetermined RNA binding domain. That is, this region has a sequence to which the RNA binding domain of the transcription regulatory peptide, which is another constituent molecule, specifically binds. Here, as the RNA binding domain and the sequence that specifically binds thereto, for example, the RNA binding domain of the coat protein of MS2 phage and the sequence that specifically binds thereto can be used.

  A sequence serving as a linker may be inserted between these two regions as appropriate, and extra sequences may be present at both ends. This RNA may be chemically synthesized, but may also be synthesized by preparing DNA such as a plasmid encoding RNA by gene recombination and transcribed in vitro or in vivo such as in a cell. I do not care.

== Transcriptional regulatory peptide ==
The transcriptional regulatory peptide that is the second constituent molecule of the transcriptional regulatory machinery of the present invention comprises an RNA binding domain that specifically binds to the second region of the triple-stranded nucleic acid-forming RNA, and transcription of a gene that is a transcriptional regulatory target. It has an activation domain or a repression domain for activating or suppressing.

  The type of RNA binding domain is determined in pairs with the second region of triplex nucleic acid forming RNA.

  In addition, as the transcription activation domain or repression domain, for example, VP16 and GAL4 activation domain or Cyc8 and Tup1 repression domain can be used, but not limited thereto.

  A linker may be appropriately provided between these two domains, and extra amino acids may be added to both ends of the domain. This peptide may be chemically synthesized, but may also be synthesized by producing a DNA such as a plasmid encoding the peptide by gene recombination and expressing it in vitro or in vivo such as in a cell. I do not care.

== Transcriptional control method ==
The triplex nucleic acid-forming RNA and the transcription regulatory peptide are allowed to act on the promoter of the gene to be regulated (see FIG. 1). The place of action may be in vivo or in vitro.

  When the site of action is in vivo, for example, when the gene to be regulated by transcription is present in the genome, expression vectors such as plasmids encoding triple-stranded nucleic acid-forming RNA and transcription regulatory peptide are prepared, and It may be introduced into cells by the method. A gene to be regulated by transcription may be introduced into a cell together with DNA encoding a transcriptional regulatory machinery as a foreign gene. At that time, the gene to be regulated by transcription is incorporated on a vector such as a plasmid. Moreover, the promoter on the expression vector for expressing the triple-stranded nucleic acid-forming RNA and the transcription regulatory peptide may be constitutive or conditional depending on the purpose. That is, if a constitutive promoter is used for both promoters, the target gene can be forcibly expressed regardless of the cell environment, and if either one of the promoters is conditional, for example, time-specific or tissue Specifically, the target gene can be expressed.

  When the place of action is in vitro, for example, the transcription of the gene of interest can be regulated by adding a vector having a gene to be regulated for transcription and a transcriptional regulation machinery to the nuclear extract. Regarding the transcriptional regulatory machinery, it is preferable to synthesize triple-stranded nucleic acid-forming RNA and regulatory peptide in vitro in advance.

== Construction of VP16-MS2 fusion protein expression plasmid ==
MS2 coat protein is known to bind with high affinity to RNA (5'-ACAUGAGGAUUACCCAUGU-3 '), which is 19 bases long in a sequence-specific manner (RNA. 7: 1616- 27, 2001). Therefore, in this example, a VP16-MS2 fusion protein in which the transcription activation domain of herpes simplex virus VP16 and the RNA binding domain of the coat protein of MS2 phage were fused was used as the transcription regulatory peptide.

  In order to express this VP16-MS2 fusion protein in cells, an expression vector encoding the VP16-MS2 fusion protein was constructed as follows.

  A BamHI-MunI fragment containing a DNA sequence encoding the RNA binding domain of the coat protein of MS2 phage was excised from pHybLex / Zeo-MS2 (InVitrogen), introduced into the BamHI-EcoRI site of the pYESTrp3 vector (InVitrogen), and VP16 -An expression vector pVP16-MS2 encoding the MS2 fusion protein was constructed.

  As a control vector, pYESTrp3 vector (VP16-) from which VP16 transcription activation domain was removed was prepared by digesting pYESTrp3 vector with SacI and self-ligating.

== Construction of plasmid for expressing TFO-MS2 RNA fusion RNA ==
In this example, the triple-stranded nucleic acid-forming RNA was fused with the TFO sequence, which was thought to be capable of forming a triple-stranded nucleic acid with respect to the yeast ADH promoter sequence, and the RNA binding sequence of the coat protein of MS2 phage. The fusion RNA “TFO-MS2RNA” was used. As the TFO sequence, the following sequences from TFO 1 to 5 were used. FIG. 2 shows the promoter region that is the basis of each TFO sequence on the yeast ADH promoter.

TFO1: 5′-GAAGAAAAGAAAAAAAAAGAAAAGAGAGAGGGGG-3 ′ (SEQ ID NO: 1, 34b)
TFO2: 5′-AAAAAAGGAAA-3 ′ (SEQ ID NO: 2, 11b)
TFO3: 5′-GAAAAAGGAAGGAAG-3 ′ (SEQ ID NO: 3, 15b)
TFO4: 5′-GGAAGGAAGG-3 ′ (SEQ ID NO: 10b)
TFO5: 5′-AAGGGAAAGAAGGAATAAAGAAAAAGA-3 ′ (SEQ ID NO: 5, 27b)
In order to express these TFO-MS2 RNAs in cells, an expression vector encoding TFO-MS2 RNA was constructed as follows.

  An oligonucleotide with CTCGAG added to the 3 ′ end of the complementary sequence of each TFO sequence and an oligonucleotide with ACGT added to the 3 ′ end of each TFO sequence and CTCGAG added to the 5 ′ end were synthesized and annealed. To make TFO inserts. Each TFO insert is inserted into the PmeI-AatII site of the expression vectors pRH3 ′ and pRH5 ′ (both from InVitrogen), and p3′TFO1-MS2RNA to p3′TFO5-MS2RNA and p5′MS2RNA-TFO1 to p5′MS2RNA-TFO5 10 types of expression vectors were prepared. Each region is designed to be in the order of TFO-MS2RNA for p3 ′ series vectors and MS2RNA-TFO for p5 ′ series vectors. As a control vector, pURA3 ′ was digested with EcoRI and self-ligated to prepare a vector pURA from which the RNA binding sequence was removed.

== Construction of LacZ expression plasmid ==
As a reporter, a plasmid having a LacZ marker downstream of the yeast ADH promoter was constructed as follows.

  Using the genomic DNA extracted from Escherichia coli W3110 as a template, the entire length of the LacZ gene was amplified by PCR using the following primers 1 and 2. In the reaction, a cycle of 96 ° C. 1 minute-55 ° C. 2 minutes-72 ° C. 4 minutes was repeated 25 times, and Pfu Turbo DNA polymerase (Stratagene) was used as the enzyme. The PCR product was cleaved with KpnI-PstI and inserted into the KpnI-PstI site of pAD-GAL4-2.1 (Stratagene) to prepare pADH-LacZ, which is a LacZ reporter.

Primer 1: 5'-CGGGGTACCGCCATGACCATGATTACGGATTCACTGGCCG-3 '(SEQ ID NO: 6)
Primer 2: 5'-AAAACTGCAGTTATTATTATTTTTGACACCAGACCAACTGGTAATGG-3 '(SEQ ID NO: 7)

== Construction of transcriptional regulation machinery ==
The above plasmids were introduced into the yeast FY23 strain, which is Ura-Trp-Leu-requiring, in the combinations shown in Table 1. Colonies were selected on a selective medium plate, and single grown colonies were picked up and cultured in a selective liquid medium. When the turbidity OD600 of the medium reached about 1, the cells were collected and suspended in a phosphate buffer for pH adjustment. This was treated with chloroform and SDS, ONPG which is a substrate of β-Gal was added, and 1M NaHCO ₃ was added after 10 minutes to stop the reaction. This was centrifuged, the supernatant was collected, OD420 was measured, and β-Gal activity (unit / ml) was determined from the following Miller formula.

β-Gal activity = OD420 x 1000 / OD600 x reaction time (min) x reaction volume (ml)
The results are shown in Table 1.

  Among TFO1-5, TFO1 in the position (442-475 bp) farthest from the start codon showed the most remarkable activity. In both p3 'series and p5' series, this activity is controlled by reporter only control (indicated as “(reporter only)” in the table), reporter + pVP16-MS2 (indicated as “VP16MS2” in the table), reporter + P3'TFO1-MS2RNA (indicated in the table as "3'TFO1 (without VP16MS2)") or reporter + p5'MS2RNA-TFO1 (indicated in the table as "5'TFO1 (without VP16MS2)") On the other hand, when Mann-Whitney U test was performed, a significant difference was recognized with a risk rate of 1% in any combination.

  Since this TFO1 has the longest sequence used (34b), it is considered that the longer the sequence, the more stable triple-stranded nucleic acid is constructed and transcription is remarkably activated. TFO5 was also suggested that the sequence of consecutive purine bases was divided into 15 and 11 bases by T in the middle, and continuity was required. These results suggested that TFO2-5 may not bind to the ADH promoter stably enough to activate at least downstream transcription. Thus, in this study, it was revealed that stable formation of triple-stranded nucleic acid is important for successful transcriptional regulation.

  In this example, the transcriptional activation domain of VP16 was used as the transcriptional activation domain of the transcriptional regulatory peptide, but by using a transcriptional repression domain instead, transcription can be efficiently repressed against a promoter that originally has transcriptional activity. it is conceivable that.

It is a figure showing the conceptual diagram of the transcriptional regulation machinery of this invention. It is a figure which shows the position in the ADH promoter considered that each TFO used in one Example concerning this invention can couple | bond.

Claims (5)

A gene transcription control method (except for methods that target humans) ,
A first region that forms a triple-stranded nucleic acid with the promoter region of the gene;
A second region that specifically binds to a predetermined RNA binding domain;
RNA having
The RNA binding domain;
A polypeptide having a transcriptional activation domain or a transcriptional repression domain;
To act on the gene.   The method according to claim 1, wherein the first region has a length of 16 bases or more and consists of one of purine and pyrimidine.   3. The method of claim 2, wherein the first region is 34 bases or more.   The RNA binding domain includes an RNA binding domain of a coat protein of MS2 phage, and the second region includes an RNA binding sequence of a coat protein of MS2 phage. Method.   The method according to claim 1, wherein the transcription activation domain comprises a herpes simplex virus VP16 transcription activation domain.