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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  • Abstract
  • Description
  • Claims
  • Application Information

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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
CPCC12Q1/6806A61P43/00C12Q2537/164C12Q2525/207A61K31/7088C12N15/1135
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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