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. 2020 Jul 27;15(7):e0236403.
doi: 10.1371/journal.pone.0236403. eCollection 2020.

Histone lysine demethylase 3B (KDM3B) regulates the propagation of autophagy via transcriptional activation of autophagy-related genes

Hyeonsoo Jung  1 , Sang-Beom Seo  1
Affiliations

Histone lysine demethylase 3B (KDM3B) regulates the propagation of autophagy via transcriptional activation of autophagy-related genes

Hyeonsoo Jung et al. PLoS One. .

Abstract

Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The changes of the expression of epigenetic enzymes in nutrient-deprived HCT116 cells.
HCT116 cells were starved using the starvation medium. After 4, 6, and 8 hours, cells were harvested. Extracts were eluted, resolved by SDS-PAGE, and immunoblotted with each specified antibody.
Fig 2
Fig 2. KDM3B regulates the transcription of autophagy-related genes.
(A) The differential expression of autophagy-related genes in KDM3B wildtype and KO HepG2 cells was visualized and analyzed. (B-C) The changes in target gene expression were measured by qPCR. (B) HCT116 cells were incubated in starvation medium for 8 hours. (C) Stable shKDM3B CDS and shKDM3B 3′UTR HCT116 cells were harvested and RNA of the cells was isolated. Knockdown of KDM3B was confirmed by immunoblotting. (D) Stable shKDM3B HCT116 cells were co-transfected with pGL3.0-ATG5 or pGL3.0-ATG7 promoters. Luciferase activities were measured 48 hours after transfection and normalized to that of β-galactosidase. (B-D) All results represent the data from at least three independent experiments (± SEMs). * P < 0.05, ** P < 0.01, *** P < 0.001. (E) The occupancies of KDM3B and H3K9me2 at the promoters of autophagy-related genes in nutrient starved-HCT116 cells were analyzed. The data were normalized by input. The results are shown as mean ± SD (n = 3).
Fig 3
Fig 3. Rapamycin increased KDM3B recruitment to the promoters of autophagy-related target genes.
(A) HCT116 cells were treated with 1, 2, or 5 μM of rapamycin or DMSO for 8 hours. Cell extracts were eluted and immunoblotted using KDM3B antibody and LC3B. Antibodies against mTOR and mTOR pS2448 were used to confirm the activity of rapamycin. (B) mRNA levels of autophagy-related genes in HCT116 cells treated with 2 μM of rapamycin for 8 hours were confirmed by qPCR. All results represent the data obtained from at least three independent experiments (± SEMs). * P < 0.05, ** P < 0.01. (C) ChIP analyses of the promoters of the target genes in HCT116 cells treated with 2 μM of rapamycin for 8 hours were examined using antibodies against KDM3B and H3K9me2 and rabbit IgG by qPCR. The results are shown as mean ± SD (n = 3).
Fig 4
Fig 4. KDM3B is a prerequisite for the autophagic flux.
(A-B) The proteins from the stable shKDM3B HCT116 cells were extracted, resolved by SDS-PAGE, and immunoblotted with an anti-LC3B antibody to measure the ratio of the conversion of LC3B-I to LC3B-II. The ratio of LC3B conversion was measured by Image J. (A) Cells were incubated in starvation medium for 8 hours. (B) KDM3B depleted HCT116 cells were treated with 2 μM rapamycin for 8 hours. CQ was also added to the cells for 4 hours. (C) The formations of the GFP-LC3B puncta were measured by fluorescent microscopy in GFP-LC3B overexpressed HCT116 cells treated with 2 μM rapamycin for 8 hours. (D) The GFP-LC3B puncta per cells were measured by Image J. The numbers of GFP-LC3B puncta were counted at least 5 cells, and this data represent the data obtained from at least three independent experiments (± SEMs). * P < 0.05, ** P < 0.01.
Fig 5
Fig 5. Autophagy inhibits the proteasomal degradation of KDM3B induced by VCP/p97 in HCT116 cells.
(A) mRNA levels of KDM3B in HCT116 cells incubated in starvation medium for 4, 6, and 8 hours were confirmed by qPCR. (B) Stable VCP-depleted HCT116 cells were harvested (C) Mock and plenti-Flag-VCP HCT116 cells were treated with 20 μM of MG132 or DMSO for 4 hours. (B-C) Cell extracts were eluted and immunoblotted using antibodies against KDM3B and VCP or Flag. (D) The KDM3B transcript level was measured by qPCR in Flag-VCP-overexpressing HCT116 cells. (E) Mock and plenti-Flag-VCP overexpressing HCT116 cells were treated with rapamycin for 8 hours and/or CQ for 4 hours. The cell extracts were resolved by SDS-PAGE and immunoblotted with anti-KDM3B, anti-LC3B, and anti-Flag antibodies. The ratio of LC3B conversion was measured by Image J. (F) The protein level of VCP in HCT116 cells incubated with starvation media or treated with 2 μM of rapamycin for 8 hours, was measured by immunoblotting. (G) plenti-Flag-VCP-overexpressing HCT116 cells were treated with 2 μM of rapamycin for 8 hours and the cell extracts were immunoprecipitated with anti-KDM3B antibodies. The changes in the differential interaction were detected by immunoblotting with anti-KDM3B and anti-Flag antibodies. * indicates non-specific bands.
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This work was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF-2019R1A4A2001609 and NRF-2017R1A2B4004407) to Sang Beom Seo. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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