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Lipopolysaccharide-dependent transcriptional regulation of PU.1 in microglial cells

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Abstract

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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Acknowledgements

This work was supported by National Research Foundation of Korea (NRF) Grants 2017M3A9G7073033, 2017R1A2B4012905, and 2011-0030049 (to YGC) and 2016R1D1A1B04934970 (to KHJ) from the Korean government. The authors declare that they have no conflicts of interest with the contents of this article.

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Correspondence to Kyoung Hwa Jung or Young Gyu Chai.

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Mandal, C., Yoon, T., Park, J.Y. et al. Lipopolysaccharide-dependent transcriptional regulation of PU.1 in microglial cells. Mol. Cell. Toxicol. 16, 51–61 (2020). https://doi.org/10.1007/s13273-019-00057-6

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Keywords

  • LPS
  • Microglia
  • Immune response
  • Chemokine genes