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KMID : 1034820200160010051
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Molecular & Cellular Toxicology
2020 Volume.16 No. 1 p.51 ~ p.61
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Lipopolysaccharide-dependent transcriptional regulation of PU.1 in microglial cells
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Mandal Chanchal
Yoon Tae-Ho
Park Ji-Yoon
Jung Kyoung-Hwa
Jung Kyoung-Hwa
Chai Young-Gyu
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Abstract
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Backgrounds: PU.1 is a pioneer transcription factor and a master regulator of the myeloid lineage. However, the role of PU.1 in lipopolysaccharide-dependent microglial activation has yet to be investigated. So, this study was conducted to determine the effects of PU.1 in LPS-induced activation of microglial cells.
Methods: We knocked out PU.1 in murine BV-2 cells using the CRISPR-Cas9 system to investigate the role of PU.1 in the expression of immune-related genes. We performed RNA sequencing (RNA-seq) of PU.1 KO and BV-2 cells to analyze the gene expression patterns in PU.1 KO cells and compare them to those in wild-type BV-2 cells. The validation of differential expressions was achieved by qRT-PCR. To explore this regulatory role of PU.1, ChIP sequencing for PU.1 and H3K27Ac was performed. The sequencing result was further confirmed by ChIP-qPCR.
Results: RNA sequencing and subsequent bioinformatic analysis revealed that the expression of most of the immune-related genes was suppressed in the absence of PU.1. Proinflammatory chemokine genes were differentially expressed in LPS-treated PU.1 KO cells. The ChIP sequencing result followed by ChIP-qPCR revealed a LPS-mediated increase in the enrichment of PU.1 binding in pro-inflammatory chemokine gene promoters and enhancer regions in wild-type BV-2 cells. There was no enrichment of PU.1 in PU.1 KO cells.
Conclusion: The above-mentioned results suggest that PU.1 is directly involved in regulating the immune response and that this regulation of inflammatory chemokines is LPS-dependent. We hope that PU.1 would be an option for limiting neurodegeneration in a diverse range of neurological disorders.
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KEYWORD
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LPS, Microglia, Immune response, Chemokine genes
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