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Method for obtaining O-GlcNAc modified transcription factor binding chromatin DNA sequence based on glycometabolism labeling

A DNA sequence and transcription factor technology, applied in the field of glycobiochemical chromatin immunoprecipitation, can solve the problems of inability to study glycotranscription factors and target gene DNA sequences, and achieve strong biological stability, high chemical selectivity, and good The effect of compatibility

Pending Publication Date: 2020-08-11
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many transcription factors have been found to be O-GlcNAc-modified, but there is no way to enrich the DNA bound by all O-GlcNAc-modified transcription factors in the genome at one time, which means that it is impossible to analyze the O-GlcNAc-modified transcription factors at the omics level. Study on O-GlcNAc Sugar Transcription Factor and Binding Target Gene DNA Sequence

Method used

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  • Method for obtaining O-GlcNAc modified transcription factor binding chromatin DNA sequence based on glycometabolism labeling
  • Method for obtaining O-GlcNAc modified transcription factor binding chromatin DNA sequence based on glycometabolism labeling
  • Method for obtaining O-GlcNAc modified transcription factor binding chromatin DNA sequence based on glycometabolism labeling

Examples

Experimental program
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Embodiment 1

[0024] Example 1: This example provides a method for obtaining the O-GlcNAc glucose metabolism marker transcription factor group binding chromatin DNA sequence in MCF-7 cells.

[0025] The experiment includes the following steps:

[0026] S1. Cell labeling and cross-linking

[0027] MCF-7 cells were treated with 1mM GalNAz for 48h. Take out a plate of 10 cm dish cells and add formaldehyde for cross-linking, so that the final concentration of formaldehyde is 1%, incubate at room temperature for 10 min, add glycine with a final concentration of 0.125M to terminate cross-linking.

[0028] S2. Nuclei Preparation and Chromatin Digestion

[0029] Add 1 ml of ice-cold lysate containing 0.5 μM DTT and protease inhibitors to the cross-linked cells in S1. Incubate on ice for 10 min. Invert the tube every 3 minutes to mix. Nuclei were then pelleted by centrifugation. Resuspend the pellet in 1 ml ice-cold lysis buffer containing DTT. Resuspend in 100 μL of ice-cold lysate containin...

Embodiment 2

[0045] Example 2: This example provides a method for obtaining the O-GlcNAc glucose metabolism marker transcription factor group binding chromatin DNA sequence in Hela cells.

[0046] The experiment includes the following steps:

[0047] S1. Cell labeling and cross-linking

[0048] Hela cells were treated with 200μM Ac4GalNAz for 48h. Take out a plate of 10 cm dish cells and add formaldehyde for cross-linking, so that the final concentration of formaldehyde is 1%, incubate at room temperature for 15 minutes, add glycine with a final concentration of 0.125M to terminate cross-linking.

[0049] S2. Nuclei Preparation and Chromatin Digestion

[0050] Add 1 ml of ice-cold lysate containing 0.5 μM DTT and protease inhibitors to the cross-linked cells in S1. Incubate on ice for 10 min. Invert the tube every 3 minutes to mix. Nuclei were then pelleted by centrifugation. Resuspend the pellet in 1 ml ice-cold lysis buffer containing DTT. Resuspend in 100 μL of ice-cold lysate co...

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Abstract

The invention discloses a method for obtaining an O-GlcNAc modified transcription factor binding chromatin DNA sequence based on glycometabolism labeling. The method comprises the step of labeling allO-GlcNAc modified transcription factors in a cell with azide molecules by utilizing an azide labeled sugar probe; obtaining the azide-labeled O-GlcNAc modified transcription factor-chromatin fragmentthrough formaldehyde crosslinking and enzymolysis ultrasonic treatment; connecting biotin to the azide-labeled transcription factor and chromatin through a Click reaction, and precipitating a transcription factor chromatin compound by utilizing streptavidin magnetic beads and biotin affinity; and finally, carrying out decrosslinking to obtain the O-GlcNAc modified transcription factor binding chromatin DNA. The method for obtaining the O-GlcNAc modified transcription factor binding chromatin DNA in the whole genome range is developed for the first time, the method is high in specificity and low in cost, and the prepared O-GlcNAc sugar transcription factor binding DNA sample can be directly used for PCR detection or amplification to construct a DNA library.

Description

technical field [0001] The present invention relates to the technical field of glycobiochemical chromatin immunoprecipitation, in particular to a method for obtaining genome-wide O-GlcNAc modified transcription factor binding chromatin DNA sequences. Background technique [0002] O-GlcNAc modification is a post-translational modification of proteins ubiquitous in the cytoplasm and nucleus, and is linked to serine and threonine of many intracellular proteins through glycosidic bonds. So far, more than 4000 kinds of proteins modified by O-GlcNAc have been discovered, mainly including transcription factors, chromosome binding proteins, nucleoporins, cytoskeleton proteins, etc. Among them, the O-GlcNAc modification of nuclear proteins, especially transcription factors, plays an important role in gene transcription and stress response. Studies have shown that many transcription factors can undergo O-GlcNAc glycosylation modification, and the glycosylated transcription factors ma...

Claims

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Application Information

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IPC IPC(8): G01N33/539G01N33/543
CPCG01N33/539G01N33/54326
Inventor 刘宇博张娜娜张嘉宁
Owner DALIAN UNIV OF TECH
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