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Differential impacts of DNA methylation on pH dependent i-motifs formation in rice [Bisulfite-seq]

ABSTRACT: I-motifs (iMs) are four-stranded non-B DNA structures containing C-rich DNA sequences, which can be formed under various pH conditions. DNA methylation exhibits complex impacts on iM formation in vitro. However, how DNA methylation differentially affects pH dependent iM formation on a genome wide scale is completely uncharacterized in both humans and plants. To this end, we conducted iM-IP-seq under pH 5.5 and 7.0 conditions using CK and hyper/hypomethylated DNA in combination with BS-seq in rice. We found that pH 7.0 and 5.5 biased iMs had distinct genomic features and differential DNA methylation levels, the former having higher DNA methylation levels than the latter. Moreover, DNA demethylation and hyper methylation had more impacts on a subset of iM formation under pH 7.0 and 5.5, respectively, indicating that DNA de/hyper-methylation exhibits pH dependent impacts on iM formation. Importantly, we found that CG hypo-DMRs and CHH hyper-DMRs alone or coordinated with CG/CHG hyper-DMRs may play determinant roles in the regulation of iM formation under pH 5.5 and 7.0. Thus, our study shows that intrinsic DNA sequences alone or in combination with DNA methylation play vital roles in determining pH-dependent formation of iMs. It will contribute to in-depth understanding of DNA methylation in the modulation of pH dependent dynamics of iM conformation, therefore broadening its biological implications and practical application ranges.

ORGANISM(S): Oryza sativa Japonica Group

PROVIDER: GSE234750 | GEO | 2024/02/15

REPOSITORIES: GEO

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Differential impacts of DNA methylation on pH dependent i-motifs formation in rice [ChIP-seq]

Project description:I-motifs (iMs) are four-stranded non-B DNA structures containing C-rich DNA sequences, which can be formed under various pH conditions. DNA methylation exhibits complex impacts on iM formation in vitro. However, how DNA methylation differentially affects pH dependent iM formation on a genome wide scale is completely uncharacterized in both humans and plants. To this end, we conducted iM-IP-seq under pH 5.5 and 7.0 conditions using CK and hyper/hypomethylated DNA in combination with BS-seq in rice. We found that pH 7.0 and 5.5 biased iMs had distinct genomic features and differential DNA methylation levels, the former having higher DNA methylation levels than the latter. Moreover, DNA demethylation and hyper methylation had more impacts on a subset of iM formation under pH 7.0 and 5.5, respectively, indicating that DNA de/hyper-methylation exhibits pH dependent impacts on iM formation. Importantly, we found that CG hypo-DMRs and CHH hyper-DMRs alone or coordinated with CG/CHG hyper-DMRs may play determinant roles in the regulation of iM formation under pH 5.5 and 7.0. Thus, our study shows that intrinsic DNA sequences alone or in combination with DNA methylation play vital roles in determining pH-dependent formation of iMs. It will contribute to in-depth understanding of DNA methylation in the modulation of pH dependent dynamics of iM conformation, therefore broadening its biological implications and practical application ranges.

2024-02-15 | GSE234751 | GEO

iMbody - a nanobody for i-Motif research

Project description:i-Motifs (iMs) are four-stranded structures formed by certain cytosine-rich nucleic acids. They may be involved in regulation of cellular processes such as transcription. In addition, the pH dependency of iMs makes them suitable for certain nanobiotechnological applications. Probes that recognize these structures with high specificity and affinity can significantly advance the iM research field. Here we report the generation of an iM specific nanobody (iMbody) and describe its applications for the in vitro characterization of a potential iM-forming oligonucleotide, strand-specific pull-down of iMs present in the human genome at near physiological pH and temperature followed by sequencing, and visualization of iMs in the nuclei of human cells. Utilization of iMbody could be beyond above-mentioned applications and it can be used to investigate iMs in other organisms. The plasmid encoding iMbody for the bacterial expression is available via Addgene (Plasmid #184496).

2022-12-06 | GSE212765 | GEO

Genome-wide mapping of i-Motifs reveals their association with transcription regulation in live human cells [RNA-seq]

Project description:i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences and in acidic conditions in vitro. However, their formation and distribution in cells is still under debate. Here we performed CUT&Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped native iMs in two different human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters and that their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs throughout the genome. By simultaneously mapping G-quadruplexes (G4s), four-stranded structures that form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent genomic regions; however, when both iMs and G4s are present in the same genomic tract, their formation is favored. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome.

2023-08-02 | GSE220881 | GEO

Genome-wide mapping of i-Motifs reveals their association with transcription regulation in live human cells [CUT&Tag]

2023-08-02 | GSE220878 | GEO

Single-cell gene expression profiling of interstitial macrophages exposed to hypoxia

Project description:Employing single-cell transcriptomic analyses, we investigated the repertoire and functional profiles of pulmonary interstitial macrophages in response to exposure at 0, 1, 3, 7, and 21 days of hypobaric hypoxia (at an altitude of 5,486 meters). The repertoire and functional profiles of interstitial macrophages (IMs) undergo dynamic changes based on the duration of hypoxia exposure. Acute hypoxia leads to type 1 acute inflammatory responses in most IM populations, which largely subside by Day 7. The resolution of the inflammatory phase is succeeded by the accumulation of dysregulated IMs, contributing to abnormal pulmonary vascular repair and remodeling. The classical and alternative complement pathways may play distinct roles in regulating early inflammation and later vascular remodeling phases in pulmonary hypertension (PH) pathogenesis. These findings highlight the dynamic nature of IMs, emphasizing that understanding the timing and mechanisms through which they influence PH pathogenesis will contribute to the development of IM-specific therapeutic targets.

2024-04-15 | GSE254606 | GEO

Single-cell RNAseq analysis (10X Genomics Chromium) of refilled lung interstium macrophages on day 4 after depletion

Project description:Lung interstitium macrophages (IMs) are non-alveolar resident tissue macrophages which contribute to the lung homeostasis. These cells were reported to be heterogeneous by our group and other teams, which contains two main distinct subpopulations: CD206+ IMs and CD206- IMs. However, the exact origin of IMs and the transcriptional programs that regulate IM differentiation remains unclear. In recent report, we analyzed the refilled IMs in the course of time after induced IM depletion with single-cell RNA sequencing (10X Genomics Chromium) and bulk RNA sequencing. The IMs in Day 4 post-depletion were compared to the those without depletion. Results showed that refilled IMs had a lower ratio of CD206+ IM vs CD206- subpopulation comparing to IMs without depletion, but they shared high similarity to each other, indicating that the de novo IM population had been established before Day 4 post-depletion.

2023-02-07 | GSE193894 | GEO

Maternal obesity induced mouse embryonic defects derive from Stella insufficiency in oocytes

Project description:In order to obtain genome-wide profiles, base-resolution methylomes of zygotes were generated using the bisulfite sequencing (BS-seq) method for small samples. We find that global loss of DNA methylation during zygotic development in HFD mice. A total of 412 DMRs were identified, of which 294 were hypomethylated (hypo-DMRs; 71.4%) and 118 were hypermethylated (hyper-DMRs; 28.6%) (Fig. 6A and 6B), showing a predominance of hypo-DMRs. Furthermore, we show that hyper-DMRs are significantly depleted from CGI, but enriched in short interspersed elements (SINEs) and non-CGI regions. Strikingly, hypo-DMRs are specifically depleted from SINEs, with a concurrent enrichment in DNA transposons.

2017-03-28 | GSE97109 | GEO

Differential roles of Stella in the modulation of DNA methylation during oocyte and zygotic development

Project description:In order to obtain genome-wide profiles, base-resolution methylomes of oocytes and zygotes were generated using the bisulfite sequencing (BS-seq) method for small samples. We find that global loss of DNA methylation during zygotic development in HFD mice. A total of 412 DMRs were identified, of which 294 were hypomethylated (hypo-DMRs; 71.4%) and 118 were hypermethylated (hyper-DMRs; 28.6%) (Fig. 6A and 6B), showing a predominance of hypo-DMRs. Furthermore, we show that hyper-DMRs are significantly depleted from CGI, but enriched in short interspersed elements (SINEs) and non-CGI regions. Strikingly, hypo-DMRs are specifically depleted from SINEs, with a concurrent enrichment in DNA transposons.

2018-11-22 | GSE122829 | GEO

Leukocyte Recruitment and iBALT Formation Require Chemokine-Secreting Pf4+ Interstitial Macrophages [scRNA-seq: mTumor]

Project description:Pulmonary resident macrophages consist of alveolar macrophages (AMs) and interstitial macrophages (IMs). While much is known about the role of AMs, the function of IMs, which are conserved across multiple organs and species, remains unclear. Using single-cell RNA sequencing (scRNA-seq) after inducing acute lung injury, we identified at least six distinct subtypes of pulmonary IMs, based on the chemokines they express. These subtypes are: IMck1 (Ccl2, Ccl7, Ccl12, a few Cxcl14); IMck2-4 (Ccl3, Ccl4, Ccl5, Cxcl2, Cxcl3); IMck5-7 (Ccl6, Ccl8, Ccl9); IMck8 (Cxcl9, Cxcl10); IMck9 (Cxcl13); and IMck10 (Ccl24). Based on the selective chemokine expression profiles observed, we hypothesized that pulmonary IMs play a critical role in recruiting leukocytes and forming inducible bronchus-associated lymphoid tissue (iBALT) in the lung. First, we examined multiple Cre-expressing mice (Lyve1, Nes, Pdpn, and Pf4) for IM specificity by crossing them with Rosa26EYFP. Only the Pf4Cre mice selectively induced EYFP expression in CD206hiCD64hi IMs, where no other immune cell in the lung expressed EYFP. We then created a CD206hiCD64hi IM-depleting mouse by crossing Pf4Cre with Rosa26DTR and CX3CR1_001DTR. In a bacterial infection and allergic airway disease, depletion of CD206hiCD64hi IMs resulted in diminished leukocyte recruitment and iBALT formation. Overall, our study highlights the increased granularity in IM populations and demonstrates a division of labor for cellular recruitment and iBALT formation in the lung. Furthermore, the study suggests that the specific combination of chemokines expressed by IMs are tightly regulated, like Th cytokine-producing cells.

2023-03-03 | GSE225667 | GEO

Leukocyte Recruitment and iBALT Formation Require Chemokine-Secreting Pf4+ Interstitial Macrophages [scRNA-seq: mSk]

Project description:Pulmonary resident macrophages consist of alveolar macrophages (AMs) and interstitial macrophages (IMs). While much is known about the role of AMs, the function of IMs, which are conserved across multiple organs and species, remains unclear. Using single-cell RNA sequencing (scRNA-seq) after inducing acute lung injury, we identified at least six distinct subtypes of pulmonary IMs, based on the chemokines they express. These subtypes are: IMck1 (Ccl2, Ccl7, Ccl12, a few Cxcl14); IMck2-4 (Ccl3, Ccl4, Ccl5, Cxcl2, Cxcl3); IMck5-7 (Ccl6, Ccl8, Ccl9); IMck8 (Cxcl9, Cxcl10); IMck9 (Cxcl13); and IMck10 (Ccl24). Based on the selective chemokine expression profiles observed, we hypothesized that pulmonary IMs play a critical role in recruiting leukocytes and forming inducible bronchus-associated lymphoid tissue (iBALT) in the lung. First, we examined multiple Cre-expressing mice (Lyve1, Nes, Pdpn, and Pf4) for IM specificity by crossing them with Rosa26EYFP. Only the Pf4Cre mice selectively induced EYFP expression in CD206hiCD64hi IMs, where no other immune cell in the lung expressed EYFP. We then created a CD206hiCD64hi IM-depleting mouse by crossing Pf4Cre with Rosa26DTR and CX3CR1DTR. In a bacterial infection and allergic airway disease, depletion of CD206hiCD64hi IMs resulted in diminished leukocyte recruitment and iBALT formation. Overall, our study highlights the increased granularity in IM populations and demonstrates a division of labor for cellular recruitment and iBALT formation in the lung. Furthermore, the study suggests that the specific combination of chemokines expressed by IMs are tightly regulated, like Th cytokine-producing cells.

2023-03-03 | GSE225666 | GEO

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