Targeted histone acetylation

一种组蛋白、靶向的技术，应用在含有定位/靶向基序的多肽、酰基转移酶、化学仪器和方法等方向，能够解决非特异性等问题

Active Publication Date: 2016-06-08
SIGMA ALDRICH CO LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although sodium butyrate acts to inhibit histone deacetylase activity and thus indirectly increases histone acetylation, this method of histone acetylation is global and nonspecific

Method used

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  • Targeted histone acetylation
  • Targeted histone acetylation
  • Targeted histone acetylation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Histones acetylated by the ZF(Oct4)-p300 fusion protein associated with the Oct4 gene

[0075] The fusion protein was designed to target specific sequences near the transcription start site of the human OCT4 gene. The fusion protein contains zinc finger (ZF) DNA binding domain and human p300HAT domain. The ZF binding domain was designed to bind a specific sequence 5'-GGAGGGcCAGGAATCGGGC-3' (SEQ ID NO: 1) starting at +181 nucleotides downstream of the transcription start site of OCT4. 被称为ZF(Oct4+181)的这种ZFDNA结合结构域具有以下氨基酸序列:AAMAERPFQCRICMRNFSDRSHLTRHIRTHTGEKPFACDICGRKFARNDDRKKHTKIHTGSQKPFQCRICMRNFSQSGNLARHIRTHTGEKPFACDICGRKFAAKWNLDAHTKIHTHPRAPIPKPFQCRICMRNFSRSAHLSRHIRTHTGEKPFACDICGRKFAQSGHLSRHTKIHLRQKDAAR(SEQIDNO:2)。 The HAT domain corresponds to amino acids 1284-1673 of human p300 (accession number: NM_001429).

[0076] The expression vector encoding the fusion protein ZF(Oct4+181)-p300 was transfected into HEK293 cells. Negative control cells were transfecte...

Embodiment 2

[0077] Example 2: ZF(Oct4+181)-p300 fusion protein increases Oct4 expression

[0078] Additional ZF-p300 fusion proteins were designed: ZF(Oct4-720)-p300 and ZF(Oct4-615)-p300, which target the upstream nucleotides -720 or - of the transcription initiation site of OCT4 The sequence at 615. A second set of control fusion proteins containing the NF-κB subunit p65 was also constructed for use as positive controls: ZF(Oct4+181)-p65 and ZF(Oct4-720)-p65.

[0079] Expression vectors encoding ZF(Oct4+181)-p300 or one of the control fusion proteins described above were transfected into HEK293 cells. Control cells were transfected with vectors encoding GFP, p65 (no ZF) or p300 (no ZF). After 48 hours, cells were harvested and total RNA was isolated. Levels of OCT4 mRNA and cyclophilin A (ie, PPIA) endogenous control mRNA were measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR) (Life Technologies, Carlsbad, CA). OCT4 / cyc ratio was used to normalize OCT4 ex...

Embodiment 3

[0080] Example 3: ZF(PEDF)-p300 fusion protein increases PEDF expression

[0081] To test whether ZF fusion proteins containing the p300HAT domain could activate other genes, ZF DNA binding domain fusions were designed to target the human PEDF (pigment epithelium-derived factor) gene. The ZF binding domain was designed to bind a specific sequence 5'-GGATGGtGGTGCAGCAGTG-3' (SEQ ID NO: 7) starting at nucleotide -92 upstream of the transcription start site of the sequence encoding PEDF. 所述ZF(PEDF)DNA结合结构域被称为ZF6961并且具有以下氨基酸序列:AAMAERPFQCRICMRNFSRSDALSRHIRTHTGEKPFACDICGRKFAQSGDLTRHTKIHTGGQRPFQCRICMRNFSQSGDLTRHIRTHTGEKPFACDICGRKFATSGHLSRHTKIHTGGGGSQKPFQCRICMRNFSRSDHLSNHIRTHTGEKPFACDICGKKFAQSATRITHTKIHLRQKDAAR(SEQIDNO:8)。 The ZF (PEDF) DNA binding domain is linked to the p300HAT domain or the NF-κB p65 subunit.

[0082] The expression vector encoding ZF(PEDF)-p300 or ZF(PEDF)-p65 was transfected into HEK293 cells. Control cells were transfected with vectors encoding GFP, p65 (no ZF) ...

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PUM

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Abstract

The present disclosure provides compositions and methods for acetylating histones at targeted chromosomal locations in a cell. In particular, the disclosure provides a fusion protein comprising a DNA binding domain and at least one histone acetyltransferase (HAT) domain, such that the DNA binding domain targets the fusion protein to a targeted chromosomal location and the HAT domain acetylates histones at the targeted location.

Description

field of invention [0001] The present disclosure generally relates to means for acetylation targeting histone proteins at chromosomal locations. Background of the invention [0002] It is well established that histone acetylation correlates with transcriptional activity in eukaryotic cells. Acetylated core histones are known to be preferentially associated with transcriptionally active chromatin. Acetylation occurs at lysine residues on the amino-terminal tail of histone proteins, thereby neutralizing the positive charge of the histone tail and reducing its affinity for DNA. Thus, histone acetylation alters nucleosome conformation, thereby increasing the accessibility of transcriptional regulatory proteins to the chromatin template. [0003] Despite progress in identifying a large number of proteins with histone acetyltransferase or histone deacetylase activity and elucidating their role in regulating gene expression, there is currently no way to modify histone acetylation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C07K14/47
CPCC12Y203/01048C07K2319/80C07K2319/915C12N9/1029C07K2319/81C07K14/47C07K2319/09
Inventor 季清洲C.克里德
Owner SIGMA ALDRICH CO LLC
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