Epigenetic regulatory complex for control of gene expression

A technology for polypeptide complexes and expression vectors, which is applied in the field of compositions with histone methyltransferase activity and can solve problems such as failure

Inactive Publication Date: 2013-05-15
CAMBRIDGE ENTERPRISE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, recipient oocytes may contain factors that exert epigenetic effects that also contribute to the failure of the method

Method used

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  • Epigenetic regulatory complex for control of gene expression
  • Epigenetic regulatory complex for control of gene expression
  • Epigenetic regulatory complex for control of gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Identification of Blimp1 activity

[0076] Significant epigenetic modifications occur immediately after mouse PGC specification, including methylation and acetylation of histone termini by methyltransferases (HMTases) and acetyltransferases (HATs), respectively. Among the candidate genes that may regulate these epigenetic changes in PGCs are HMTases belonging to the conserved SET / PR domain protein family. Expression analysis of 25 candidate genes containing SET / PR domains was performed in E7.5 PGCs and peripheral somatic cells, and Blimp1, G9a, Set1, Ezh2, and Pfml were found to be expressed in embryonic regions containing PGCs ( figure 1 b and Figure 7 ). However, only Blimp1 expression was restricted to PGCs at E7.5 and persisted in germ cells thereafter.

[0077] To investigate the activity of the Blimpl SET / PR domain, it was first determined that the transiently expressed Myc-tagged mouse Blimp1 in 293T cells could be efficiently immunoprecipitated us...

Embodiment 2

[0079] Example 2: Identification of the Blimp1 / Prmt5 complex

[0080] Since the SET / PR domains are associated with histone methyltransferase activity only on lysine residues, it can be assumed that the arginine methyltransferase activity detected above may not be attributable to Blimp1 and must imply Other HMTases exist. It has been previously reported that two protein arginine methyltransferases, Prmt1 and Prmt5, mediate the methylation of histone H4 R3. Prmt1 is a type I arginine methyltransferase that produces NG-monomethylarginine (Rme1) and asymmetric NG,N'G-dimethylarginine (Rme2a) on different types of substrates. As mentioned above, Prmt5 belongs to the type II arginine methyltransferases responsible for monomethylation of arginine (Rme1) and symmetric NG, N′G-dimethylation (Rme2s) ( Figure 8 ) 20 . Expression of these proteins in nascent PGCs and peripheral somatic cells was analyzed using the single-cell cDNA library described above to test whether one of these ...

Embodiment 3

[0082] Example 3: Activity of the Blimp1 / Prmt5 complex in vivo

[0083] The specificity of the antibody raised against H4R3me2s was tested, and the results showed that the antibody could efficiently recognize histones H2A and H4 from calf thymus ( Figure 8 b Left; H2A and H4 also appear as contaminants in H3 and H2B preparations). In a competition assay, this antibody was efficiently titrated by the R3me2s-containing H4(1-9C) polypeptide (Fig. S2c). In addition, this antibody also recognized the Blimp1 / Prmt5 HMTase assay product of H4 and H2A (Fig. S2b right), thus further demonstrating that the complex is responsible for the symmetric dimethylation activity of Prmt5 rather than the asymmetric dimethylation activity of Prmt1. methylation activity.

[0084] PGCs isolated from early embryos were analyzed by immunocytochemistry using an antibody specific for H2 / H4R3me2s modification. Significant H2A / H4R3me2s were seen in both PGCs and somatic cells at E8.5 ( image 3 f), but...

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Abstract

An epigenetic regulatory polypeptide complex comprises at least a first domain having site-specific DNA binding activity and at least a second domain having an arginine methyltransferase activity, wherein the second domain is capable of methylating an arginine residue located in the tail region of a histone H2A. The complex is able to regulate gene expression in cells, particularly in mammalian stem cells by controlling the methylation of R3 in the tail regions of histones H2A and H4. The complex is exemplified by a polypeptide complex comprising the DNA binding activity of Blimpi and the arginine methyltransferase activity of Prmt5.

Description

technical field [0001] The present invention relates to the field of epigenetic regulation of gene expression. In particular, the present invention relates to compositions and methods having histone methyltransferase activity for the purpose of controlling gene expression in vivo and in vitro. Background technique [0002] Epigenetics involves the transmission of information by a cell or multicellular tissue to its progeny that is not encoded by the nucleotide sequence of a gene. Epigenetic control is usually achieved through chemical modifications to DNA or chromatin structure. For example, gene expression can be regulated by methylating and acetylating histones that bind to genomic DNA. Methylation and acetylation of histones often occur at the ends of histones, which are the exposed surfaces and have a net positive charge. The chemical modification of histone termini is regulated by enzymes with histone methyltransferase activity (MTase) and histone acetyltransferase ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C07K14/47
Inventor 阿齐姆·苏拉尼乌尔丽克·朗格彼得拉·哈伊科娃卡蒂娅·安瑟兰
Owner CAMBRIDGE ENTERPRISE LTD
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