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Methods of detecting long range chromosomal interactions

A long-distance, chromosomal technology, used in biochemical equipment and methods, pharmaceutical formulations, and microbial determination/inspection, which can solve problems such as histone acetylation

Active Publication Date: 2011-05-04
牛津生物动力公开有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, levels of antisense transcripts correlated strongly with levels of the associated Hda1 at this locus, but not with histone acetylation itself

Method used

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  • Methods of detecting long range chromosomal interactions
  • Methods of detecting long range chromosomal interactions
  • Methods of detecting long range chromosomal interactions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Epigenetic control of the GAL locus

[0093] Conditional antisense transcript at GAL10.

[0094] Addition of glucose to cultures of cells grown in galactose resulted in a rapid repression of transcription. As expected, within 15 minutes of glucose addition, the 2.25 kb GAL10 transcript and the longer 4.1 kb GAL10-7 fusion transcript (Greger and Proudfoot, 1998, Embo J 17, 4771-4779; St. John and Davis, 1981 , J Mol Biol 152, 285-315) the levels of both decreased significantly ( figure 1 A). Antisense transcripts have been reported at the repressed GAL10 gene (Perocchi et al, 2007, Nucleic Acids Res 35, e128; Samanta et al, 2006, Proc Natl Acad Sci USA 103, 4192-4197; David et al, 2006 , Proc Natl Acad Sci USA 103, 5320-5325; Miura et al, 2006, PNAS 103, 17846-17851). The inventors investigated whether antisense transcripts are a general feature of the GAL10 gene and, if so, when these transcripts arise.

[0095] In induced cultures, three antisense trans...

Embodiment 2

[0241] Example 2: Control of long-range interactions by CTCF

[0242] Several transcription factors have been shown to play a role in long-range DNA interactions and transcription and thus may be good candidates to provide a link between these processes. Examples include the essential transcription factor TFIIB, which has been shown to organize the looping of several genes in yeast (Mol Cell, 27, 806-16); and the transcription factors EKLF, GATA-1 and FOG-1, which are responsible for β-globin Long-range DNA interactions in genes (Drissen et al, 2004, Genes Dev, 18, 2485-90; Vakoc, et al, 2005, Mol Cell, 17, 453-62).

[0243] The inventors identified the CCCTC-binding protein (CTCF) as another candidate for carrying out these functions throughout the genome. CTCF is involved in the regulation of transcription and the formation of higher-order conformational intrachromosomal and interchromosomal structures (Klenova, 2002, Semin Cancer Biol, 12, 399-414; Kurukuti et al, 2006, Pr...

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Abstract

The present invention relates to a method of monitoring epigenetic changes comprising monitoring changes in conditional long range chromosomal interactions at at least one chromosomal locus where the spectrum of long range interaction is associated with a specific physiological condition, the method comprising the steps of:- (i) in vitro crosslinking of said long range chromosomal interactions present at the at least one chromosomal locus; (ii) isolating the cross linked DNA from said chromosomal locus; (iii) subjecting said cross linked DNA to restriction digestion with an enzyme that cuts at least once within the at least one chromosomal locus; (iv) ligating said cross linked cleaved DNA ends to form DNA loops; (v) identifying the presence of said DNA loops; wherein the presence of DNA loops indicates the presence of a specific long range chromosomal interaction.

Description

field of invention [0001] The present invention relates to methods of monitoring epigenetic changes, diagnosing specific physiological conditions and the use of antisense RNA for the treatment of physiological conditions. Background of the invention [0002] Eukaryotic genomes are organized into complex higher-order structures; indeed, earlier electron micrographs of chromatin revealed non-histone scaffolds forming radial loops (Earnshaw and Laemmli, 1983, J Cell Bio 96, 84-93). Unconstrained negative supercoilability in mammalian genomes suggests that chromosomal "domain" sizes are on the order of tens of kilobases (Kramer and Sinden, 1997, Biochem 36, 3151-3158). More recently, the use of 3C technology (capturing chromosome conformation) has demonstrated that high-level long-range interactions exist in a wide variety of eukaryotic cells, including S. cerevisiae (Dekker et al, 2002, Science 295, 1306-1311) , flies (Blanton et al, 2003 Genes Dev 17, 664-675), mice (Tolhuis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6806A61P43/00C12Q2537/164C12Q2525/207A61K31/7088C12N15/1135C12Q1/6844C12Q1/6809C12Q2523/101C12Q2521/301C12Q2521/501C12Q2531/113
Inventor 阿鲁尔·S·拉马达斯亚历山大·阿孔利切夫伊丽莎白·J·米勒
Owner 牛津生物动力公开有限公司
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