Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA.

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N6,N6-Dimethyladenosine (m6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m6,6A) method for the site-specific detection and quantification of m6,6A in RNA. The N6,N6-dimethyl groups in m6,6A can cause reverse transcription to stall at the m6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m6,6A in RNA. Using the AD-m6,6A method, we successfully detected and quantified m6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m6,6A in RNA, which can aid in uncovering the functions of m6,6A in human diseases.

Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution.

DNA methylation (5-methylcytosine, 5mC) is the most important epigenetic modification in mammals. Deciphering the roles of 5mC relies on the quantitative detection of 5mC at the single-base resolution. Bisulfite sequencing (BS-seq) is the most often employed technique for mapping 5mC in DNA. However, bisulfite treatment may cause serious degradation of input DNA due to the harsh reaction conditions. Here, we engineered the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3C (A3C) protein to endow the engineered A3C (eA3C) protein with differential deamination activity toward cytosine and 5mC. By the virtue of the unique property of eA3C, we proposed an engineered A3C sequencing (EAC-seq) method for the bisulfite-free and quantitative mapping of 5mC in DNA at the single-base resolution. In EAC-seq, the eA3C protein can deaminate C but not 5mC, which is employed to differentiate C and 5mC in sequencing. Using the EAC-seq method, we quantitatively detected 5mC in genomic DNA of lung cancer tissue. In contrast to the harsh reaction conditions of BS-seq, which could lead to significant degradation of DNA, the whole procedure of EAC-seq is carried out under mild conditions, thereby preventing DNA damage. Taken together, the EAC-seq approach is bisulfite-free and straightforward, making it an invaluable tool for the quantitative detection of 5mC in limited DNA at the single-base resolution.

Enzymatic Cleavage-Mediated Extension Stalling Enables Accurate Recognition and Quantification of Locus-Specific Uracil Modification in DNA.

Chemical modifications in DNA have profound influences on the structures and functions of DNA. Uracil, a naturally occurring DNA modification, can originate from the deamination of cytosine or arise from misincorporation of dUTP into DNA during DNA replication. Uracil in DNA will imperil genomic stability due to their potential in producing detrimental mutations. An in-depth understanding of the functions of uracil modification requires the accurate determination of its site as well as content in genomes. Herein, we characterized that a new member of the uracil-DNA glycosylase (UDG) family enzyme (UdgX-H109S) could selectively cleave both uracil-containing single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Based on this unique property of UdgX-H109S, we developed an enzymatic cleavage-mediated extension stalling (ECES) method for the locus-specific detection and quantification of uracil in genomic DNA. In the ECES method, UdgX-H109S specifically recognizes and cleaves the N-glycosidic bond of uracil from dsDNA and generates an apurinic/apyrimidinic (AP) site, which could be broken by APE1 to form a one-nucleotide gap. The specific cleavage by UdgX-H109S is then evaluated and quantified by qPCR. With the developed ECES approach, we demonstrated that the level of uracil at position Chr4:50566961 in genomic DNA of breast cancer tissues was significantly decreased. Collectively, the ECES method has been proved to be accurate and reproducible in the locus-specific quantification of uracil in genomic DNA from biological and clinical samples.

Bisulfite-Free and Single-Base Resolution Detection of Epigenetic DNA Modification of 5-Methylcytosine by Methyltransferase-Directed Labeling with APOBEC3A Deamination Sequencing.

DNA methylation (5-methylcytosine, 5mC) is the most prevalent epigenetic modification that is predominantly found in CG dinucleotides in mammalian genomes. In-depth investigation of the functions of 5mC heavily relies on the quantitative measurement of 5mC at single-base resolution in genomes. Here, we proposed a methyltransferase-directed labeling with APOBEC3A (A3A) deamination sequencing (MLAD-seq) method for the single-base resolution and quantitative detection of 5mC in DNA. In MLAD-seq, a mutant of DNA methyltransferase, M.MpeI-N374K, is utilized to selectively transfer a carboxymethyl group to the 5 position of cytosine in the CG dinucleotide to form 5-carboxymethylcytosine (5camC) using carboxy-S-adenosyl-l-methionine (caSAM) as the cofactor. After A3A treatment, 5camC is resistant to the deamination and base pairs with guanine. Thus, the cytosines in CG sites are read as C in sequencing. On the contrary, the methyl group in 5mC inhibits its carboxymethylcytosine by M.MpeI-N374K and therefore is readily deaminated by A3A to produce thymine that pairs with adenine and is read as T in sequencing. The differential readouts from C and 5mC in the MLAD-seq enable the single-base resolution mapping of 5mC in CG sites in DNA. With the developed MLAD-seq method, we observed the hypermethylation in the promoter region of retinoic acid receptor ß (RARB) gene from human nonsmall cell lung tumor tissue. Compared to harsh reaction conditions in bisulfite sequencing that could lead to significant degradation of DNA, the whole procedure of MLAD-seq is carried out under mild conditions, which will avoid DNA damage. Thus, MLAD-seq is more suitable in the scenario where only limited input DNA is available. Taken together, the MLAD-seq offers a valuable tool for bisulfite-free, single-base resolution and quantitative detection of 5mC in limited DNA.

WRKY13 Enhances Cadmium Tolerance by Promoting D-CYSTEINE DESULFHYDRASE and Hydrogen Sulfide Production.

Hydrogen sulfide (H2S), a plant gasotransmitter, functions in the plant response to cadmium (Cd) stress, implying a role for cysteine desulfhydrase in producing H2S in this process. Whether d -CYSTEINE DESULFHYDRASE (DCD) acts in the plant Cd response remains to be identified, and if it does, how DCD is regulated in this process is also unknown. Here, we report that DCD-mediated H2S production enhances plant Cd tolerance in Arabidopsis (Arabidopsis thaliana). When subjected to Cd stress, a dcd mutant accumulated more Cd and reactive oxygen species and showed increased Cd sensitivity, whereas transgenic lines overexpressing DCD had decreased Cd and reactive oxygen species levels and were more tolerant to Cd stress compared with wild-type plants. Furthermore, the expression of DCD was stimulated by Cd stress, and this up-regulation was mediated by a Cd-induced transcription factor, WRKY13, which bound to the DCD promoter. Consistently, the higher Cd sensitivity of the wrky13-3 mutant was rescued by the overexpression of DCD Together, our results demonstrate that Cd-induced WRKY13 activates DCD expression to increase the production of H2S, leading to higher Cd tolerance in plants.

Synthesis and BK channel-opening activity of 2-amino-1,3-thiazole derivatives.

A series of 2-amino-5-arylmethyl- or 5-heteroarylmethyl-1,3-thiazole derivatives were synthesized and evaluated for BK channel-opening activities in cell-based fluorescence assay and electrophysiological recording. The assay results indicated that the activities of the investigated compounds were influenced by the physicochemical properties of the substituent at benzene ring.

Simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine at specific genomic loci by engineered deaminase-assisted sequencing.

Cytosine modifications, particularly 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), play crucial roles in numerous biological processes. Current analytical methods are often constrained to the separate detection of either 5mC or 5hmC, or the combination of both modifications. The ability to simultaneously detect C, 5mC, and 5hmC at the same genomic locations with precise stoichiometry is highly desirable. Herein, we introduce a method termed engineered deaminase-assisted sequencing (EDA-seq) for the simultaneous quantification of C, 5mC, and 5hmC at the same genomic sites. EDA-seq utilizes a specially engineered protein, derived from human APOBEC3A (A3A), known as eA3A-M5. eA3A-M5 exhibits distinct deamination capabilities for C, 5mC, and 5hmC. In EDA-seq, C undergoes complete deamination and is sequenced as T. 5mC is partially deaminated resulting in a mixed readout of T and C, and 5hmC remains undeaminated and is read as C. Consequently, the proportion of T readouts (P T) reflects the collective occurrences of C and 5mC, regulated by the deamination rate of 5mC (R 5mC). By determining R 5mC and P T values, we can deduce the precise levels of C, 5mC, and 5hmC at particular genomic locations. We successfully used EDA-seq to simultaneously measure C, 5mC, and 5hmC at specific loci within human lung cancer tissue and their normal counterpart. The results from EDA-seq demonstrated a strong concordance with those obtained from the combined application of BS-seq and ACE-seq methods. EDA-seq eliminates the need for bisulfite treatment, DNA oxidation or glycosylation and uniquely enables simultaneous quantification of C, 5mC and 5hmC at the same genomic locations.

Whole-Genome Sequencing of 5-Hydroxymethylcytosine at Base Resolution by Bisulfite-Free Single-Step Deamination with Engineered Cytosine Deaminase.

The epigenetic modification 5-hydroxymethylcytosine (5hmC) plays a crucial role in the regulation of gene expression. Although some methods have been developed to detect 5hmC, direct genome-wide mapping of 5hmC at base resolution is still highly desirable. Herein, we proposed a single-step deamination sequencing (SSD-seq) method, designed to precisely map 5hmC across the genome at single-base resolution. SSD-seq takes advantage of a screened engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein, known as eA3A-v10, to selectively deaminate cytosine (C) and 5-methylcytosine (5mC) but not 5hmC. During sequencing, the deaminated C and 5mC are converted to uracil (U) and thymine (T), read as T in the sequencing data. However, 5hmC remains unaffected by eA3A-v10 and is read as C during sequencing. Consequently, the presence of C in the sequence reads indicates the original 5hmC. We applied SSD-seq to generate a base-resolution map of 5hmC in human lung tissue. Our findings revealed that 5hmC was predominantly localized to CpG dinucleotides. Furthermore, the base-resolution map of 5hmC generated by SSD-seq demonstrated a strong correlation with prior ACE-seq results. The advantages of SSD-seq are its single-step process, absence of bisulfite treatment or DNA glycosylation, cost effectiveness, and ability to detect and quantify 5hmC directly at single-base resolution.

Bisulfite-free and single-nucleotide resolution sequencing of DNA epigenetic modification of 5-hydroxymethylcytosine using engineered deaminase.

The discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes is a landmark in epigenomics study. Similar to 5-methylcytosine (5mC), 5hmC is viewed as a critical epigenetic modification. Deciphering the functions of 5hmC necessitates the location analysis of 5hmC in genomes. Here, we proposed an engineered deaminase-mediated sequencing (EDM-seq) method for the quantitative detection of 5hmC in DNA at single-nucleotide resolution. This method capitalizes on the engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein to produce differential deamination activity toward cytosine, 5mC, and 5hmC. In EDM-seq, the engineered A3A (eA3A) protein can deaminate C and 5mC but not 5hmC. The original C and 5mC in DNA are deaminated by eA3A to form U and T, both of which are read as T during sequencing, while 5hmC is resistant to deamination by eA3A and is still read as C during sequencing. Therefore, the remaining C in the sequence manifests the original 5hmC. By EDM-seq, we achieved the quantitative detection of 5hmC in genomic DNA of lung cancer tissue. The EDM-seq method is bisulfite-free and does not require DNA glycosylation or chemical treatment, which offers a valuable tool for the straightforward and quantitative detection of 5hmC in DNA at single-nucleotide resolution.

Early renal injury indicators can help evaluate renal injury in patients with chronic hepatitis B with long-term nucleos(t)ide therapy.

BACKGROUND: Patients with chronic hepatitis B (CHB) with long-term nucleos(t)ide therapy may experience renal insufficiency. Traditional renal function indicators, such as urine protein, serum urea nitrogen (BUN), and serum creatinine, are normal when early mild lesions occur. Therefore, more sensitive renal function indicators are needed. AIM: To investigate the significance of early renal injury indicators in evaluating renal injury in patients with CHB with long-term nucleos(t)ide therapy. METHODS: We collected the clinical data of 69 outpatients with CHB at Peking University First Hospital from March 2018 to January 2020 who had been treated with long-term nucleos(t)ide therapy and analyzed the results of early renal injury indicators. Continuous normal distribution data were analyzed by the t-test to determine the difference between two groups. Continuous non-normally distributed data were analyzed by the Mann-Whitney U-test between two groups. The Kruskal-Wallis H test was used to determine the differences among multiple groups. Enumeration data were analyzed by the chi-square test. The related factors of early renal injury indicators were analyzed by logistic regression analysis. RESULTS: The average treatment duration with nucleos(t)ide analogs of the 69 patients with CHB was 99.7 ± 28.7 mo. The cases of patients with elevated BUN and hypophosphatemia were 6 (8.7%) and 13 (18.8%), respectively; 31 (44.9%) patients had abnormal early renal injury indicators, including 9 patients with abnormal urine microalbumin, 7 patients with abnormal urine immunoglobulin, 6 patients with abnormal urine transferrin, and 19 patients with abnormal α1 microglobulin. There were no significant differences in the mean values of age, sex, BUN, estimated glomerular filtration rate (eGFR), serum uric acid, serum calcium, or serum phosphorus between the two groups of patients with and without early renal injury indicators. However, the mean levels of serum creatinine and urine creatinine, N-acetyl-ß-D-glucosidase enzyme, α1 microglobulin, and urine immunoglobulin in the former group of patients were significantly higher than those in the latter group of patients (P < 0.05). The incidence of early renal injury in patients with eGFR ≥ 90, 60-89, and 30-59 mL/(min·1.73 m2) was 36.4% (8/22), 47.6% (20/42), and 60% (3/5), respectively. Logistic regression analysis results showed that gamma-glutamyl transpeptidase [odds ratio (OR) = 1.05 (1.008-1.093), P = 0.020], direct bilirubin [OR = 1.548 (1.111-2.159), P = 0.010], serum creatinine [OR = 1.079 (1.022-1.139), P = 0.006], and age [OR = 0.981 (0.942-1.022), P = 0.357] were independent predictors of early renal injury. CONCLUSION: Patients with CHB treated with long-term nucleos(t)ide analog therapy had a high probability of early renal injury, and early renal injury indicators were highly sensitive and could be used to monitor early renal impairment.

Jasmonic acid promotes leaf senescence through MYC2-mediated repression of CATALASE2 expression in Arabidopsis.

Plants relocate nutrients and energy from aging leaves to developing tissues during leaf senescence, which is important for plant growth, development, and responses to various environmental stimuli. Both jasmonic acid (JA) and H2O2 are two crucial signalling molecules positively regulating leaf senescence, whereas whether and how they are coordinated in leaf senescence remains elusive. Here, we report that H2O2 accumulates in JA-treated leaves, while scavenging the increased H2O2 can significantly suppresses JA-induced leaf senescence and the expression of senescence-associated genes (SAGs). The mutant myc2 with a mutation of MYC2, a master transcription factor in JA signalling pathway, exhibits delayed leaf senescence with increased catalase activity and decreased H2O2 accumulation compared with the wild type upon JA treatment. Further study showed that MYC2 downregulates CATALASE 2 (CAT2) expression by binding to its promoter, thus promoting JA-induced H2O2 accumulation and leaf senescence. Moreover, the delayed leaf senescence with reduced H2O2 accumulation and SAGs expression of the myc2 mutant is significantly reverted by the cat2-1 mutation in myc2 cat2-1 double mutant. Thus, our study reveals that JA represses CAT2 expression to increase H2O2 accumulation, thus promoting leaf senescence in a MYC2 dependent manner in Arabidopsis.