Substances for regulating chromatin histone methylation level of rDNA gene and application thereof

A technology of chromatin and trimethylation, applied in the field of molecular biology

Active Publication Date: 2019-01-11
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above results suggest that the regulation of rDNA transcription by PHF6 may be involved in the occurrence and development of leukemia

Method used

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  • Substances for regulating chromatin histone methylation level of rDNA gene and application thereof
  • Substances for regulating chromatin histone methylation level of rDNA gene and application thereof
  • Substances for regulating chromatin histone methylation level of rDNA gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. PHF6 binds to the pre-rRNA transcription region of rDNA

[0033] 1. Design detection primers according to the sequence of the pre-rRNA transcription region on rDNA, a total of 8 pairs of primers, namely H0, H1, H4, H8, H13, H18, H23, H42.9, spread throughout the pre-rRNA transcription region ( figure 1 A).

[0034] H0 primer sequences are SEQ ID No.4 and SEQ ID No.5

[0035] F: GGTATATCTTTCGCTCCGAG SEQ ID No.4

[0036] R:GACGACAGGTCGCCAGAGGA SEQ ID No.5

[0037] H1 primer sequences are SEQ ID No.6 and SEQ ID No7 respectively

[0038] F: GGCGGTTTGAGTGAGACGAGA SEQ ID No.6

[0039] R: ACGTGCGCTCACCGAGAGCAG SEQ ID No7

[0040] H4 primer sequences are SEQ ID No.8 and SEQ ID No.9 respectively

[0041] F: CGACGACCCATTCGAACGTCT SEQ ID No.8

[0042] R:CTCTCCGGAATCGAACCCTGA SEQ ID No.9

[0043] H8 primer sequences are SEQ ID No.10 and SEQ ID No.11 respectively

[0044] F:AGTCGGGTTGCTTGGGAATGC SEQ ID No.10

[0045] R:CCCTTACGGTACTTGTTGACT SEQ ID No.11

[0046...

Embodiment 2

[0062] Example 2, PHF6 can bind to histone H3 methylation mimic peptide

[0063]Detection was performed by co-immunoprecipitation and immunoblotting. Artificially synthesized some histone H3 methylation modification site mimic peptides, including H3K9, H3K9me1, H3K9me2, H3K9me3, H3K27, H3K27me1, H3K27me2 and H3K27me3, these mimic peptides have biotin (Biotin) tags, can use antibiotics Primary antibody for detection. Transfer plasmid Flag-PHF6 into HEK 293 cells to express Flag-PHF6 protein, extract cell lysate after 48h, add anti-Flag primary antibody and synthetic H3K9, H3K9me1, H3K9me2, H3K9me3, H3K27, H3K27me1, H3K27me2 and H3K27me3 mimic peptides After co-incubating overnight, the Flag-antibody conjugates were precipitated with proteinA / G beads, and then analyzed by Western blotting. The experimental results show that Flag-PHF6 binds to H3K9me1 / H3K9me2 / H3K9me3, among which the binding to H3K9me1 and H3K9me2 is weak, and the binding of PHF6 to H3K9me3 is more ( figure 2...

Embodiment 3

[0064] Example 3, PHF6 can bind to the H3K9 (me1 / 2 / 3) site and H3K27 (me1 / 2 / 3) site in the rDNA transcription initiation region

[0065] HeLa cells were selected and the experiment was carried out using the Re-ChIP method, which required two chromatin immunoprecipitation steps. First, collect the cell lysate after fixation with formaldehyde, collect the supernatant after sonication and centrifugation, incubate the supernatant with the primary antibody against PHF6, and add magnetic beads to obtain the small DNA fragment bound by the primary antibody against PHF6; Add 10mM dithiothreitol (DL-Dithiothreitol, DTT) to the magnetic beads bound to the small fragments of DNA bound by the primary antibody against PHF6 for elution operation, divide the eluate into the required aliquots, add cell lysis After diluting to an appropriate volume, the corresponding negative control IgG and anti-PHF6 / H3K9me1 / H3K9me2 / H3K9me3 / H3K27me1 / H3K27me2 / H3K27me3 primary antibodies were added to carry out...

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Abstract

The invention discloses substances for regulating expression of PHF6, and provides an application of substances for preparing and regulating the level of rDNA transcription initiation region H3K9me3 in animal cells. The experiments of the present application demonstrate that the level of rDNA transcription initiation region h3k9me3 can be influenced by regulating the expression level of PHF6, andthe overexpression of PHF6 could significantly increase the level of H3K9me3 and reduction of PHF6 can significantly reduce the level of H3K9me3, so as to realize the apparent modification effect on rDNA histone.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a new application of substances for regulating PHF6 to regulate the methylation level of rDNA chromatin histones. Background technique [0002] The plant homeodomain finger protein 6 (Plant Homeodomain Finger Protein 6, PHF6) gene is located on the X chromosome and consists of 11 exons. Among them, exon 2-10 is coded to contain 365 amino acids with a molecular weight of 41kDa. PHF6 protein exists widely in human tissues, among which the expression level is highest in thymus, ovary and thyroid gland, and it was initially identified as a family genetic disease "Borjeson-Forssman-Forssman syndrome" linked to an X chromosome. -Lehmann Syndrome, BFLS) are closely related. With the in-depth study of PHF6, it was found that some BFLS patients suffered from T-cell leukemia at the same time, and PHF6 gene mutations were also found in non-BFLS leukemia patients. These studies indicated t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7105A61P35/00A61P35/02
CPCA61K31/7105A61P35/00A61P35/02
Inventor 陈红波黄来强吴彦萍蔡湘仪王丽君
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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