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| Determining Histone Modifications on Encoded Region of Serum Amyloid A Activating Transcription Factor Gene in Inflammatory Stimulus Conditions by Chromatin Immunoprecipitation |
| CAO Xi-mei1, LUO Xu-guang2, LIANG Jun-hong3, ZHANG Chao3, BAI Li-juan3, GUO Da-wei3 |
1. Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, China;
2. Department of Microbiology and Immunology, Shanxi Medical University, Taiyuan 030001, China;
3. Department of Forensic Science, Shanxi Medical University, Taiyuan 030001, China |
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Abstract Objective:The aim is to probe the histone epigenetic information in encoded region of SAF gene after inflammatory cells model from human THP-1 monocytic leukemia cell line are constructed, which is expected to provide basis for the further research about the mechanism of transcriptional regulation of SAF gene. Methods:The LPS-stimulated or unstimulated THP-1 cells were colletcted and fixed by 1% formaldehyde for 10 minutes at room temperature. After that, nuclei were isolated. Chromatin was sheared by sonication, and samples were precleared for 1 hour at 4℃ with 50% protein A/G. Chromatin containing target proteins were precipitated overnight at 4℃ with 5μg antibodies or isotype-matched control IgG. Input and immunoprecipitated chromatin were incubated at 65℃ overnight to reverse crosslinks. After proteinase K digestion, DNA was extracted and purified by phenol-chloroform. The DNA was used as templates for PCR and qPCR to get the histone epigenetic signals. Antibodies included rabbit polyclonal to Histone H3, rabbit polyclonal to Histone H3 (tri methyl K36), rabbit polyclonal to Histone H3 (mono methyl K9) and rabbit polyclonal to RNA polymerase II CTD repeat YSPTSPS (phospho S5). Results:During inflammation, the total histone H3 signals in the 4A and 4B exon of SAF gene were significantly reduced. However, histone H3K36me3 didn't obviously diminish. The level of ser5 phosphorylation of the polymerase C-terminal domain (CTD) heptamer repeat was reduced in the 4A exon of SAF gene in inflammatory stimulus conditions. Expression of H3K9me1 decreased in the 4A exon of SAF gene. During inflammation, the level of H3K9me1 decreased and the enrichment of H3K36me3 increased in the single nucleosome in 4A exon of SAF gene. At the same time, the level of ser5 phosphorylation of the polymerase C-terminal domain (CTD) heptamer repeat decreased. Conclusion:The low level of histone H3 enrichment in SAF coding regions support the idea that histone H3 in SAF coding region could be evicted by LPS induction so as to pave the way for RNA polymerase II elongation. All of these effects might increase the elongation rate of the RNA polymerase II.
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Received: 17 September 2014
Published: 25 March 2015
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