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Microorganisms Sep 2023When compared with bacteria, relatively little is known about the restriction-modification (RM) systems of archaea, particularly those in taxa outside of the...
When compared with bacteria, relatively little is known about the restriction-modification (RM) systems of archaea, particularly those in taxa outside of the haloarchaea. To improve our understanding of archaeal RM systems, we surveyed REBASE, the restriction enzyme database, to catalog what is known about the genes and activities present in the 519 completely sequenced archaeal genomes currently deposited there. For 49 (9.4%) of these genomes, we also have methylome data from Single-Molecule Real-Time (SMRT) sequencing that reveal the target recognition sites of the active mA and mC DNA methyltransferases (MTases). The gene-finding pipeline employed by REBASE is trained primarily on bacterial examples and so will look for similar genes in archaea. Nonetheless, the organizational structure and protein sequence of RM systems from archaea are highly similar to those of bacteria, with both groups acquiring systems from a shared genetic pool through horizontal gene transfer. As in bacteria, we observe numerous examples of "persistent" DNA MTases conserved within archaeal taxa at different levels. We experimentally validated two homologous members of one of the largest "persistent" MTase groups, revealing that methylation of C(mC)WGG sites may play a key epigenetic role in Crenarchaea. Throughout the archaea, genes encoding mA, mC, and mC DNA MTases, respectively, occur in approximately the ratio 4:2:1.
PubMed: 37894082
DOI: 10.3390/microorganisms11102424 -
International Journal of Molecular... Jul 2021Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA...
Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in locus and promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.
Topics: DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Fibroblasts; Gene Editing; Genomic Imprinting; HEK293 Cells; Humans; Insulin-Like Growth Factor II; Locus Control Region; Promoter Regions, Genetic; RNA, Long Noncoding; Transcription Factors; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; DNA Methyltransferase 3B
PubMed: 34299205
DOI: 10.3390/ijms22147587 -
Nature Communications May 2023Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here,...
Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular consensus sequencing (CCS) to achieve high-throughput and high-accuracy single-cell RNA isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy long-reads in a single PacBio Sequel II SMRT Cell 8M. We also report the development of scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell cDNA reads with >99.99% accuracy and specificity. We apply HIT-scISOseq to characterize the transcriptomes of 3375 corneal limbus cells and reveal cell-type-specific isoform expression in them. HIT-scISOseq is a high-throughput, high-accuracy, technically accessible method and it can accelerate the burgeoning field of long-read single-cell transcriptomics.
Topics: RNA; RNA Isoforms; High-Throughput Nucleotide Sequencing; Consensus; Protein Isoforms; Sequence Analysis, DNA; Sequence Analysis, RNA
PubMed: 37149708
DOI: 10.1038/s41467-023-38324-9 -
Nucleic Acids Research Dec 2020Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are...
Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are plasmid-borne ORFs, carried by pBCA072 for B. cenocepacia J2315 and pESBL for E. coli O104:H4. Pacific Biosciences SMRT sequencing was used to investigate DNA methyltransferases M.BceJIII and M.EcoGIX, using artificial constructs. Mating properties of engineered pESBL derivatives were also investigated. Both MTases yield promiscuous m6A modification of single strands, in the context SAY (where S = C or G and Y = C or T). Strikingly, this methylation is asymmetric in vivo, detected almost exclusively on one DNA strand, and is incomplete: typically, around 40% of susceptible motifs are modified. Genetic and biochemical studies suggest that enzyme action depends on replication mode: DNA Polymerase I (PolI)-dependent ColE1 and p15A origins support asymmetric modification, while the PolI-independent pSC101 origin does not. An MTase-PolI complex may enable discrimination of PolI-dependent and independent plasmid origins. M.EcoGIX helps to establish pESBL in new hosts by blocking the action of restriction enzymes, in an orientation-dependent fashion. Expression and action appear to occur on the entering single strand in the recipient, early in conjugal transfer, until lagging-strand replication creates the double-stranded form.
Topics: Bacterial Proteins; Burkholderia cenocepacia; DNA Methylation; DNA Polymerase I; DNA Replication; DNA, Single-Stranded; Escherichia coli O104; Escherichia coli Proteins; Genome, Bacterial; Methyltransferases; Plasmids; Ribosomal Proteins
PubMed: 33270887
DOI: 10.1093/nar/gkaa1163 -
American Journal of Clinical and... 2014Nuclear receptor corepressor (NCoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) function as corepressors for diverse transcription factors... (Review)
Review
Nuclear receptor corepressor (NCoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) function as corepressors for diverse transcription factors including nuclear receptors such as estrogen receptors and androgen receptors. Deregulated functions of NCoR and SMRT have been observed in many types of cancers and leukemias. NCoR and SMRT directly bind to transcription factors and nucleate the formation of stable complexes that include histone deacetylase 3, transducin b-like protein 1/TBL1-related protein 1, and G-protein pathway suppressor 2. These NCoR/SMRT-interacting proteins also show deregulated functions in cancers. In this review, we summarize the literature on the mechanism, regulation, and function of the core components of NCoR/SMRT complexes in the context of their involvement in cancers and leukemias. While the current studies support the view that the corepressors are promising targets for cancer treatment, elucidation of the mechanisms of corepressors involved in individual types of cancers is likely required for effective therapy.
PubMed: 25374920
DOI: No ID Found -
Communications Biology Oct 2023Talaromyces marneffei (T. marneffei) immune escape is essential in the pathogenesis of talaromycosis. It is currently known that T. marneffei achieves immune escape...
Talaromyces marneffei (T. marneffei) immune escape is essential in the pathogenesis of talaromycosis. It is currently known that T. marneffei achieves immune escape through various strategies. However, the role of cellular alternative splicing (AS) in immune escape remains unclear. Here, we depict the AS landscape in macrophages upon T. marneffei infection via high-throughput RNA sequencing and detect a truncated protein of NCOR2 / SMRT, named NCOR2-013, which is significantly upregulated after T. marneffei infection. Mechanistic analysis indicates that NCOR2-013 forms a co-repression complex with TBL1XR1 / TBLR1 and HDAC3, thereby inhibiting JunB-mediated transcriptional activation of pro-inflammatory cytokines via the inhibition of histone acetylation. Furthermore, we identify TUT1 as the AS regulator that regulates NCOR2-013 production and promotes T. marneffei immune evasion. Collectively, these findings indicate that T. marneffei escapes macrophage killing through TUT1-mediated alternative splicing of NCOR2 / SMRT, providing insight into the molecular mechanisms of T. marneffei immune evasion and potential targets for talaromycosis therapy.
Topics: Humans; Alternative Splicing; Macrophages; Inflammation
PubMed: 37845378
DOI: 10.1038/s42003-023-05409-6 -
PloS One 2022Tokay Gecko (Gekko gecko) is a rare and endangered medicinal animal in China. Its dry body has been used as an anti-asthmatic agent for two thousand years. To date, the...
Tokay Gecko (Gekko gecko) is a rare and endangered medicinal animal in China. Its dry body has been used as an anti-asthmatic agent for two thousand years. To date, the genome and transcriptome of this species remain poorly understood. Here, we adopted single molecule real-time (SMRT) sequencing to obtain full-length transcriptome data and characterized the transcriptome structure. We identified 882,273 circular consensus (CCS) reads, including 746,317 full-length nonchimeric (FLNC) reads. The transcript cluster analysis revealed 212,964 consensus sequences, including 203,994 high-quality isoforms. In total, 111,372 of 117,888 transcripts were successfully annotated against eight databases (Nr, eggNOG, Swiss-Prot, GO, COG, KOG, Pfam and KEGG). Furthermore, 23,877 alternative splicing events, 169,128 simple sequence repeats (SSRs), 10,437 lncRNAs and 7,932 transcription factors were predicted across all transcripts. To our knowledge, this report is the first to document the G. gecko transcriptome using SMRT sequencing. The full-length transcript data might accelerate transcriptome research and lay the foundation for further research on G. gecko.
Topics: Alternative Splicing; Animals; Genome; High-Throughput Nucleotide Sequencing; Lizards; Microsatellite Repeats; Protein Isoforms; RNA; RNA, Long Noncoding; Sequence Analysis, RNA; Transcription Factors; Transcriptome
PubMed: 35213661
DOI: 10.1371/journal.pone.0264499 -
Frontiers in Microbiology 2022The mite is distributed worldwide and parasitism the ear canals of cats and dogs, causing otitis externa. Molecular biology of is poorly understood, with only a few...
The mite is distributed worldwide and parasitism the ear canals of cats and dogs, causing otitis externa. Molecular biology of is poorly understood, with only a few genes being deposited in public databases. In the present study, we aimed to perform transcriptome analysis of using SMRT and Illumina sequencing of RNA from different development stages. SMRT-Seq of demonstrated 5,431 final transcripts, including 406 long non-coding RNAs and 2,698 differentially expressed genes (DEGs), including 1,357 up-regulated genes and 1,341 down-regulated genes between adult mites and nymph/larva. A total of 397 putative allergen genes were detected, 231 of which were DEGs. Among them, 77 were homologous of known mite allergens. The expression level of allergen genes hints at the pathogenicity of mites in different life stages, and the protein interaction network analysis could identify possible key genes in the pathogenic mechanism. Intriguingly, Gene Ontology analysis showed that most of the (DEGs) were associated with the terms hydrolase activity and proteolysis. Kyoto Encyclopedia of genes and genomes (KEGG) analysis identified drug metabolism-cytochrome P450 signal pathway as one of the top pathways. SMRT-Seq of the full-length transcriptome of was performed first, and a valuable resource was acquired through the combination analysis with the Illumina sequencing data. The results of our analyses provide new information for further research into .
PubMed: 35444625
DOI: 10.3389/fmicb.2022.687387 -
Nature Communications May 2023Placental development relies on coordinated cell fate decisions governed by signalling inputs. However, little is known about how signalling cues are transformed into...
Placental development relies on coordinated cell fate decisions governed by signalling inputs. However, little is known about how signalling cues are transformed into repressive mechanisms triggering lineage-specific transcriptional signatures. Here, we demonstrate that upon inhibition of the Fgf/Erk pathway in mouse trophoblast stem cells (TSCs), the Ets2 repressor factor (Erf) interacts with the Nuclear Receptor Co-Repressor Complex 1 and 2 (NCoR1/2) and recruits it to key trophoblast genes. Genetic ablation of Erf or Tbl1x (a component of the NCoR1/2 complex) abrogates the Erf/NCoR1/2 interaction. This leads to mis-expression of Erf/NCoR1/2 target genes, resulting in a TSC differentiation defect. Mechanistically, Erf regulates expression of these genes by recruiting the NCoR1/2 complex and decommissioning their H3K27ac-dependent enhancers. Our findings uncover how the Fgf/Erf/NCoR1/2 repressive axis governs cell fate and placental development, providing a paradigm for Fgf-mediated transcriptional control.
Topics: Mice; Animals; Female; Pregnancy; Trophoblasts; Fibroblast Growth Factor 2; Placenta; Cell Differentiation; Gene Expression Regulation; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Co-Repressor 2
PubMed: 37137875
DOI: 10.1038/s41467-023-38101-8 -
PloS One 2022The epigenetics of bacteria, and bacteria with a reduced genome in particular, is of great interest, but is still poorly understood. Mycoplasma gallisepticum, a...
The epigenetics of bacteria, and bacteria with a reduced genome in particular, is of great interest, but is still poorly understood. Mycoplasma gallisepticum, a representative of the class Mollicutes, is an excellent model of a minimal cell because of its reduced genome size, lack of a cell wall, and primitive cell organization. In this study we investigated DNA modifications of the model object Mycoplasma gallisepticum and their roles. We identified DNA modifications and methylation motifs in M. gallisepticum S6 at the genome level using single molecule real time (SMRT) sequencing. Only the ANCNNNNCCT methylation motif was found in the M. gallisepticum S6 genome. The studied bacteria have one functional system for DNA modifications, the Type I restriction-modification (RM) system, MgaS6I. We characterized its activity, affinity, protection and epigenetic functions. We demonstrated the protective effects of this RM system. A common epigenetic signal for bacteria is the m6A modification we found, which can cause changes in DNA-protein interactions and affect the cell phenotype. Native methylation sites are underrepresented in promoter regions and located only near the -35 box of the promoter, which does not have a significant effect on gene expression in mycoplasmas. To study the epigenetics effect of m6A for genome-reduced bacteria, we constructed a series of M. gallisepticum strains expressing EGFP under promoters with the methylation motifs in their different elements. We demonstrated that m6A modifications of the promoter located only in the -10-box affected gene expression and downregulated the expression of the corresponding gene.
Topics: Mycoplasma gallisepticum; DNA, Bacterial; DNA Restriction-Modification Enzymes; Tenericutes; DNA Methylation
PubMed: 36413541
DOI: 10.1371/journal.pone.0277819