Search for: All records

Abstract The mammalian DNA methylome is formed by two antagonizing processes, methylation by DNA methyltransferases (DNMT) and demethylation by ten-eleven translocation (TET) dioxygenases. Although the dynamics of either methylation or demethylation have been intensively studied in the past decade, the direct effects of their interaction on gene expression remain elusive. Here, we quantify the concurrence of DNA methylation and demethylation by the percentage of unmethylated CpGs within a partially methylated read from bisulfite sequencing. After verifying ‘methylation concurrence’ by its strong association with the co-localization of DNMT and TET enzymes, we observe that methylation concurrence is strongly correlated with gene expression. Notably, elevated methylation concurrence in tumors is associated with the repression of 40~60% of tumor suppressor genes, which cannot be explained by promoter hypermethylation alone. Furthermore, methylation concurrence can be used to stratify large undermethylated regions with negligible differences in average methylation into two subgroups with distinct chromatin accessibility and gene regulation patterns. Together, methylation concurrence represents a unique methylation metric important for transcription regulation and is distinct from conventional metrics, such as average methylation and methylation variation. 

Abstract Promoter DNA methylation is a well-established mechanism of transcription repression, though its global correlation with gene expression is weak. This weak correlation can be attributed to the failure of current methylation quantification methods to consider the heterogeneity among sequenced bulk cells. Here, we introduce Cell Heterogeneity–Adjusted cLonal Methylation (CHALM) as a methylation quantification method. CHALM improves understanding of the functional consequences of DNA methylation, including its correlations with gene expression and H3K4me3. When applied to different methylation datasets, the CHALM method enables detection of differentially methylated genes that exhibit distinct biological functions supporting underlying mechanisms. 

Cell‐free metabolic engineering is an emerging and promising alternative platform for the production of fuels and chemicals. In recent years, macromolecular crowding effect, which is an important function in living cells but ignored in cell‐free systems, has been transferred to cell‐free protein synthesis (CFPS). However, inhibitory effects of crowding agents on CFPS were frequently observed, and the mechanism is unclear. In this study, free Mg²⁺was found to be a key factor that can regulate the macromolecular crowding effect onin vitrotranscription,in vitrotranslation, and coupled transcript/translation. Addition of crowding agents (20% of Ficoll‐70 or Ficoll‐400) enhancedin vitrotranscription at an index of free Mg²⁺concentration (IFMC) below 2 mM but inhibited the transcription when the IFMC was higher than 2 mM. Similarly, Ficoll‐400 enhancedin vitrotranslation and coupled transcription/translation at a lower IFMC (0.1–2 mM) and inhibited the reactions at higher IFMC (>2 mM). Based on the results, CFPS systems could be further optimized by adjusting the content of crowding agents and the IFMC. Besides, the results also indicate that macromolecular crowding effect is important for maintaining the efficiency ofin vivotranscription and translation which occur at a low intracellular IFMC (<1 mM).

 

Reconstructing the chemical and structural characteristics of the plant cell wall represents a promising solution to overcoming lignocellulosic biomass recalcitrance to biochemical deconstruction. This study aims to leverage hydroxyproline (Hyp)‐O‐glycosylation, a process unique to plant cell wall glycoproteins, as an innovative technology for de novo design and engineering in planta of Hyp‐O‐glycosylated biopolymers (HypGP) that facilitate plant cell wall reconstruction. HypGP consisting of 18 tandem repeats of “Ser–Hyp–Hyp–Hyp–Hyp” motif or (SP4)₁₈was designed and engineered into tobacco plants as a fusion peptide with either a reporter protein enhanced green fluorescence protein or the catalytic domain of a thermophilic E1 endoglucanase (E1cd) fromAcidothermus cellulolyticus. The engineered (SP4)₁₈module was extensively Hyp‐O‐glycosylated with arabino‐oligosaccharides, which facilitated the deposition of the fused protein/enzyme in the cell wall matrix and improved the accumulation of the protein/enzyme in planta by 1.5–11‐fold. The enzyme activity of the recombinant E1cd was not affected by the fused (SP4)₁₈module, showing an optimal temperature of 80°C and optimal pH between 5 and 8. The plant biomass engineered with the (SP4)₁₈‐tagged protein/enzyme increased the biomass saccharification efficiency by up to 3.5‐fold without having adverse impact on the plant growth.

 

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)₁₈and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)₃₂. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGPmodule secreted up to 56‐fold greater levels ofEGFP, compared with anEGFPcontrol lacking any HypGPmodule, supporting the function of HypGPmodules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈and (SP)₃₂modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fusedEGFPprotein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFPand (SP)₃₂‐EGFPintermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGPmodules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈module andAGP‐styled (SP)₃₂module is proposed.