International Journal of Molecular Sciences

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11 pages, 2270 KiB

Open AccessArticle

Identifying N6-Methyladenosine Sites in HepG2 Cell Lines Using Oxford Nanopore Technology

by Viktoriia A. Arzumanian, Ilya Y. Kurbatov, Konstantin G. Ptitsyn, Svetlana A. Khmeleva, Leonid K. Kurbatov, Sergey P. Radko and Ekaterina V. Poverennaya

Int. J. Mol. Sci. 2023, 24(22), 16477; https://doi.org/10.3390/ijms242216477 - 18 Nov 2023

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Abstract

RNA modifications, particularly N6-methyladenosine (m6A), are pivotal regulators of RNA functionality and cellular processes. We analyzed m6A modifications by employing Oxford Nanopore technology and the m6Anet algorithm, focusing on the HepG2 cell line. We identified 3968 potential m6A modification sites in 2851 transcripts, [...] Read more.

RNA modifications, particularly N6-methyladenosine (m6A), are pivotal regulators of RNA functionality and cellular processes. We analyzed m6A modifications by employing Oxford Nanopore technology and the m6Anet algorithm, focusing on the HepG2 cell line. We identified 3968 potential m6A modification sites in 2851 transcripts, corresponding to 1396 genes. A gene functional analysis revealed the active involvement of m6A-modified genes in ubiquitination, transcription regulation, and protein folding processes, aligning with the known role of m6A modifications in histone ubiquitination in cancer. To ensure data robustness, we assessed reproducibility across technical replicates. This study underscores the importance of evaluating algorithmic reproducibility, especially in supervised learning. Furthermore, we examined correlations between transcriptomic, translatomic, and proteomic levels. A strong transcriptomic–translatomic correlation was observed. In conclusion, our study deepens our understanding of m6A modifications’ multifaceted impacts on cellular processes and underscores the importance of addressing reproducibility concerns in analytical approaches. Full article

(This article belongs to the Special Issue Epigenetic Regulation of Gene Expression)

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17 pages, 13299 KiB

Open AccessArticle

Characteristics of Shisa Family Genes in Zebrafish

by Yansong Liu, Na Du, Beibei Qian, Congcong Zou, Zhouxin Yu, Fei Xu, Lijuan Wang, Sishi Qin, Feng You and Xungang Tan

Int. J. Mol. Sci. 2023, 24(18), 14062; https://doi.org/10.3390/ijms241814062 - 14 Sep 2023

Viewed by 1017

Abstract

Shisa represents a type of single-transmembrane adaptor protein containing an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Nine shisa subfamily genes have been proposed in most vertebrates; however, some might be species-specific. The number of shisa genes present in zebrafish remains unclear. [...] Read more.

Shisa represents a type of single-transmembrane adaptor protein containing an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Nine shisa subfamily genes have been proposed in most vertebrates; however, some might be species-specific. The number of shisa genes present in zebrafish remains unclear. This study aimed to investigate the evolutionary relationships among shisa family genes in zebrafish (TU strain) using phylogenetic and syntenic analyses. The function of shisa-2 was preliminarily examined via CRISPR/Cas13d-mediated knockdown. Following identification in zebrafish, 10 shisa family genes, namely shisa-1, 2, 3, 4, 5, 6, 7, 8, 9a, and 9b, were classified into three main clades and six subclades. Their encoding proteins contained a cysteine-rich N-terminal domain and a proline-rich C-terminal region containing different motifs. A specific syntenic block containing atp8a2 and shisa-2 was observed to be conserved across all species. Furthermore, all these genes were expressed during embryogenesis. Shisa-2 was expressed in the presomitic mesoderm, somites, and so on. Shisa-2 was identified as a regulator of the expression of the somite formation marker mesp-ab. Overall, our study provides new insights into the evolution of shisa family genes and the control of shisa-2 over the convergent extension cells of somitic precursors in zebrafish. Full article

(This article belongs to the Special Issue Epigenetic Regulation of Gene Expression)

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15 pages, 4596 KiB

Open AccessArticle

HCN2 Channels in the Ventral Hippocampal CA1 Regulate Nociceptive Hypersensitivity in Mice

by Yawen Zheng, Shan Shao, Yu Zhang, Shulu Yuan, Yuanwei Xing, Jiaxin Wang, Xuetao Qi, Kun Cui, Jifu Tong, Fengyu Liu, Shuang Cui, You Wan and Ming Yi

Int. J. Mol. Sci. 2023, 24(18), 13823; https://doi.org/10.3390/ijms241813823 - 7 Sep 2023

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Abstract

Chronic pain is a significant health problem worldwide. Recent evidence has suggested that the ventral hippocampus is dysfunctional in humans and rodents, with decreased neuronal excitability and connectivity with other brain regions, parallel pain chronicity, and persistent nociceptive hypersensitivity. But the molecular mechanisms [...] Read more.

Chronic pain is a significant health problem worldwide. Recent evidence has suggested that the ventral hippocampus is dysfunctional in humans and rodents, with decreased neuronal excitability and connectivity with other brain regions, parallel pain chronicity, and persistent nociceptive hypersensitivity. But the molecular mechanisms underlying hippocampal modulation of pain remain poorly elucidated. In this study, we used ex vivo whole-cell patch-clamp recording, immunofluorescence staining, and behavioral tests to examine whether hyperpolarization-activated cyclic nucleotide-gated channels 2 (HCN2) in the ventral hippocampal CA1 (vCA1) were involved in regulating nociceptive perception and CFA-induced inflammatory pain in mice. Reduced sag potential and firing rate of action potentials were observed in vCA1 pyramidal neurons from CFA-injected mice. Moreover, the expression of HCN2, but not HCN1, in vCA1 decreased in mice injected with CFA. HCN2 knockdown in vCA1 pyramidal neurons induced thermal hypersensitivity, whereas overexpression of HCN2 alleviated thermal hyperalgesia induced by intraplantar injection of CFA in mice. Our findings suggest that HCN2 in the vCA1 plays an active role in pain modulation and could be a promising target for the treatment of chronic pain. Full article

(This article belongs to the Special Issue Epigenetic Regulation of Gene Expression)

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25 pages, 18713 KiB

Open AccessArticle

Dysregulation of Histone Deacetylases Inhibits Trophoblast Growth during Early Placental Development Partially through TFEB-Dependent Autophagy-Lysosomal Pathway

by Peixin Wang, Chenqiong Zhao, Hanjing Zhou, Xiaona Huang, Hanqi Ying, Songying Zhang, Yibin Pan and Haiyan Zhu

Int. J. Mol. Sci. 2023, 24(15), 11899; https://doi.org/10.3390/ijms241511899 - 25 Jul 2023

Cited by 1 | Viewed by 1301

Abstract

Dysregulated biological behaviors of trophoblast cells can result in recurrent spontaneous abortion (RSA)—whose underlying etiology still remains insufficient. Autophagy, a conserved intracellular physiological process, is precisely monitored throughout whole pregnancy. Although the exact mechanism or role remains elusive, epigenetic modification has emerged as [...] Read more.

Dysregulated biological behaviors of trophoblast cells can result in recurrent spontaneous abortion (RSA)—whose underlying etiology still remains insufficient. Autophagy, a conserved intracellular physiological process, is precisely monitored throughout whole pregnancy. Although the exact mechanism or role remains elusive, epigenetic modification has emerged as an important process. Herein, we found that a proportion of RSA patients exhibited higher levels of autophagy in villus tissues compared to controls, accompanied with impaired histone deacetylase (HDAC) expression. The purpose of this study is to explore the connection between HDACs and autophagy in the pathological course of RSA. Mechanistically, using human trophoblast cell models, treatment with HDAC inhibitor (HDACI)-trichostatin A (TSA) can induce autophagy by promoting nuclear translocation and transcriptional activity of the central autophagic regulator transcription factor EB (TFEB). Specifically, overactivated autophagy is involved in the TSA-driven growth inhibition of trophoblast, which can be partially reversed by the autophagy inhibitor chloroquine (CQ) or RNA interference of TFEB. In summary, our results reveal that abnormal acetylation and autophagy levels during early gestation may be associated with RSA and suggest the potential novel molecular target TFEB for RSA treatment. Full article

(This article belongs to the Special Issue Epigenetic Regulation of Gene Expression)

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13 pages, 2276 KiB

Open AccessArticle

Rice OsMRG702 and Its Partner OsMRGBP Control Flowering Time through H4 Acetylation

by Feng Gong, Kaixin Zhang, Jing Wen, Shenbo Yu, Wenjin Li, Gaofeng Du, Cui Wu, Kangjing Zhu and Yifeng Xu

Int. J. Mol. Sci. 2023, 24(11), 9219; https://doi.org/10.3390/ijms24119219 - 25 May 2023

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Abstract

MORF-RELATED GENE702 (OsMRG702) regulates flowering time genes in rice, but how it controls transcription is not well known. Here, we found that OsMRGBP can directly interact with OsMRG702. Both Osmrg702 and Osmrgbp mutants show the delayed flowering phenotype with the reduction in the [...] Read more.

MORF-RELATED GENE702 (OsMRG702) regulates flowering time genes in rice, but how it controls transcription is not well known. Here, we found that OsMRGBP can directly interact with OsMRG702. Both Osmrg702 and Osmrgbp mutants show the delayed flowering phenotype with the reduction in the transcription of multiple key flowering time genes, including Ehd1 and RFT1. Chromatin immunoprecipitation study showed that both OsMRG702 and OsMRGBP bind to the Ehd1 and RFT1 loci and the absence of either OsMRG702 or OsMRGBP leads to a decrease of H4K5 acetylation at these loci, indicating OsMRG702 and OsMRGBP cooperatively together to promote the H4K5 acetylation. In addition, whilst Ghd7 are upregulated in both Osmrg702 and Osmrgbp mutants, only OsMRG702 binds to the loci, together with the global increased and Ghd7 locus-specific increased H4K5ac levels in Osmrg702 mutants, suggesting an additional negative effect of OsMRG702 on H4K5 acetylation. In summary, OsMRG702 controls flowering gene regulation by altering H4 acetylation in rice; it works either together with OsMRGBP to enhance transcription by promoting H4 acetylation or with other unknown mechanisms to dampen transcription by preventing H4 acetylation. Full article

(This article belongs to the Special Issue Epigenetic Regulation of Gene Expression)

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16 pages, 3364 KiB

Open AccessArticle

Chromatin Remodeling Enzyme Cluster Predicts Prognosis and Clinical Benefit of Therapeutic Strategy in Breast Cancer

by Chia-Yu Kuo, Sin-Hua Moi, Ming-Feng Hou, Chi-Wen Luo and Mei-Ren Pan

Int. J. Mol. Sci. 2023, 24(6), 5583; https://doi.org/10.3390/ijms24065583 - 15 Mar 2023

Cited by 1 | Viewed by 1533

Abstract

The treatment provided for breast cancer depends on the expression of hormone receptors, human epidermal growth factor receptor-2 (HER2), and cancer staging. Surgical intervention, along with chemotherapy or radiation therapy, is the mainstay of treatment. Currently, precision medicine has led to personalized treatment [...] Read more.

The treatment provided for breast cancer depends on the expression of hormone receptors, human epidermal growth factor receptor-2 (HER2), and cancer staging. Surgical intervention, along with chemotherapy or radiation therapy, is the mainstay of treatment. Currently, precision medicine has led to personalized treatment using reliable biomarkers for the heterogeneity of breast cancer. Recent studies have shown that epigenetic modifications contribute to tumorigenesis through alterations in the expression of tumor suppressor genes. Our aim was to investigate the role of epigenetic modifications in genes involved in breast cancer. A total of 486 patients from The Cancer Genome Atlas Pan-cancer BRCA project were enrolled in our study. Hierarchical agglomerative clustering analysis further divided the 31 candidate genes into 2 clusters according to the optimal number. Kaplan–Meier plots showed worse progression-free survival (PFS) in the high-risk group of gene cluster 1 (GC1). In addition, the high-risk group showed worse PFS in GC1 with lymph node invasion, which also presented a trend of better PFS when chemotherapy was combined with radiotherapy than when chemotherapy was administered alone. In conclusion, we developed a novel panel using hierarchical clustering that high-risk groups of GC1 may be promising predictive biomarkers in the clinical treatment of patients with breast cancer. Full article

(This article belongs to the Special Issue Epigenetic Regulation of Gene Expression)

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14 pages, 26540 KiB

Open AccessArticle

CRISPR-Cas9-Mediated Mutation of Methyltransferase METTL4 Results in Embryonic Defects in Silkworm Bombyx mori

by Hao Guo, Feng Chen, Mingyi Zhou, Weiqun Lan, Wenchang Zhang, Guanwang Shen, Ping Lin, Qingyou Xia, Ping Zhao and Zhiqing Li

Int. J. Mol. Sci. 2023, 24(4), 3468; https://doi.org/10.3390/ijms24043468 - 9 Feb 2023

Cited by 2 | Viewed by 1840

Abstract

DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It [...] Read more.

DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm. Full article

(This article belongs to the Special Issue Epigenetic Regulation of Gene Expression)

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14 pages, 4042 KiB

Open AccessArticle

METTL3 Regulates the Inflammatory Response in CPB2 Toxin-Exposed IPEC-J2 Cells through the TLR2/NF-κB Signaling Pathway

by Juanli Zhang, Jiaojiao Yang, Xiaoli Gao, Xiaoyu Huang, Ruirui Luo, Qiaoli Yang, Zunqiang Yan, Pengfei Wang, Wei Wang, Kaihui Xie, Jie Li, Bo Zhang and Shuangbao Gun

Int. J. Mol. Sci. 2022, 23(24), 15833; https://doi.org/10.3390/ijms232415833 - 13 Dec 2022

Cited by 1 | Viewed by 1388

Abstract

Clostridium perfringens beta2 (CPB2) toxin is one of the main pathogenic toxins produced by Clostridium perfringens, which causes intestinal diseases in animals and humans. The N6-methyladenosine (m6A) modification is the most common reversible modification in eukaryotic disease processes. Methyltransferase-like 3 (METTL3) regulates [...] Read more.

Clostridium perfringens beta2 (CPB2) toxin is one of the main pathogenic toxins produced by Clostridium perfringens, which causes intestinal diseases in animals and humans. The N6-methyladenosine (m6A) modification is the most common reversible modification in eukaryotic disease processes. Methyltransferase-like 3 (METTL3) regulates immunity and inflammatory responses induced by the bacterial infections in animals. However, METTL3′s involvement in CPB2-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) remains unclear. In the current study, we used methylated RNA immunoprecipitation-quantitative polymerase chain reaction, Western blotting and immunofluorescence assay to determine the role of METTL3 in CPB2-exposed IPEC-J2 cells. The findings revealed that m6A and METTL3 levels were increased in CPB2 treated IPEC-J2 cells. Functionally, METTL3 overexpression promoted the release of inflammatory factors, increased cytotoxicity, decreased cell viability and disrupted tight junctions between cells, while the knockdown of METTL3 reversed these results. Furthermore, METTL3 was involved in the inflammatory response of IPEC-J2 cells by activating the TLR2/NF-κB signaling pathway through regulating TLR2 m6A levels. In conclusion, METTL3 overexpression triggered the TLR2/NF-κB signaling pathway and promoted CPB2-induced inflammatory responses in IPEC-J2 cells. These findings may provide a new strategy for the prevention and treatment of diarrhea caused by Clostridium perfringens. Full article

(This article belongs to the Special Issue Epigenetic Regulation of Gene Expression)

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