Semimethylation is a feature of diffuse large B-cell lymphoma, and subgroups with poor prognosis are characterized by global hypomethylation and short telomere length.

BACKGROUND: Large B-cell lymphoma (LBCL) is the most common lymphoma and is known to be a biologically heterogeneous disease regarding genetic, phenotypic, and clinical features. Although the prognosis is good, one-third has a primary refractory or relapsing disease which underscores the importance of developing predictive biological markers capable of identifying high- and low-risk patients. DNA methylation (DNAm) and telomere maintenance alterations are hallmarks of cancer and aging. Both these alterations may contribute to the heterogeneity of the disease, and potentially influence the prognosis of LBCL. RESULTS: We studied the DNAm profiles (Infinium MethylationEPIC BeadChip) and relative telomere lengths (RTL) with qPCR of 93 LBCL cases: Diffuse large B-cell lymphoma not otherwise specified (DLBCL, n = 66), High-grade B-cell lymphoma (n = 7), Primary CNS lymphoma (n = 8), and transformation of indolent B-cell lymphoma (n = 12). There was a substantial methylation heterogeneity in DLBCL and other LBCL entities compared to normal cells and other B-cell neoplasms. LBCL cases had a particularly aberrant semimethylated pattern (0.15 ≤ ß ≤ 0.8) with large intertumor variation and overall low hypermethylation (ß > 0.8). DNAm patterns could not be used to distinguish between germinal center B-cell-like (GC) and non-GC DLBCL cases. In cases treated with R-CHOP-like regimens, a high percentage of global hypomethylation (ß < 0.15) was in multivariable analysis associated with worse disease-specific survival (DSS) (HR 6.920, 95% CI 1.499-31.943) and progression-free survival (PFS) (HR 4.923, 95% CI 1.286-18.849) in DLBCL and with worse DSS (HR 5.147, 95% CI 1.239-21.388) in LBCL. These cases with a high percentage of global hypomethylation also had a higher degree of CpG island methylation, including islands in promoter-associated regions, than the cases with less hypomethylation. Additionally, telomere length was heterogenous in LBCL, with a subset of the DLBCL-GC cases accounting for the longest RTL. Short RTL was independently associated with worse DSS (HR 6.011, 95% CI 1.319-27.397) and PFS (HR 4.689, 95% CI 1.102-19.963) in LBCL treated with R-CHOP-like regimens. CONCLUSION: We hypothesize that subclones with high global hypomethylation and hypermethylated CpG islands could have advantages in tumor progression, e.g. by inactivating tumor suppressor genes or promoting treatment resistance. Our findings suggest that cases with high global hypomethylation and thus poor prognosis could be candidates for alternative treatment regimens including hypomethylating drugs.

Novel genome-wide DNA methylation profiling reveals distinct epigenetic landscape, prognostic model and cellular composition of early-stage lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) has been a leading cause of cancer-related mortality worldwide. Early intervention can significantly improve prognosis. DNA methylation could occur in the early stage of tumor. Comprehensive understanding the epigenetic landscape of early-stage LUAD is crucial in understanding tumorigenesis. METHODS: Enzymatic methyl sequencing (EM-seq) was performed on 23 tumors and paired normal tissue to reveal distinct epigenetic landscape, for compared with The Cancer Genome Atlas (TCGA) 450K methylation microarray data. Then, an integrative analysis was performed combined with TCGA LUAD RNA-seq data to identify significant differential methylated and expressed genes. Subsequently, the prognostic risk model was constructed and cellular composition was analyzed. RESULTS: Methylome analysis of EM-seq comparing tumor and normal tissues identified 25 million cytosine-phosphate-guanine (CpG) sites and 30,187 differentially methylated regions (DMR) with a greater number of untraditional types. EM-seq identified a significantly higher number of CpG sites and DMRs compared to the 450K microarray. By integrating the differentially methylated genes (DMGs) with LUAD-related differentially expressed genes (DEGs) from the TCGA database, we constructed prognostic model based on six differentially methylated-expressed genes (MEGs) and verified our prognostic model in GSE13213 and GSE42127 dataset. Finally, cell deconvolution based on the in-house EM-seq methylation profile was used to estimate cellular composition of early-stage LUAD. CONCLUSIONS: This study firstly delves into novel pattern of epigenomic DNA methylation and provides a multidimensional analysis of the role of DNA methylation revealed by EM-seq in early-stage LUAD, providing distinctive insights into its potential epigenetic mechanisms.

Identification of DNA methylation characteristics associated with metastasis and prognosis in colorectal cancer.

Colorectal cancer (CRC) is prone to metastasis and recurrence after surgery, which is one of the main causes for its poor treatment and prognosis. Therefore, it is essential to identify biomarkers associated with metastasis and recurrence in CRC. DNA methylation has a regulatory role in cancer metastasis, tumor immune microenvironment (TME), and prognosis and may be one of the most valuable biomarkers for predicting CRC metastasis and prognosis. We constructed a diagnostic model and nomogram that can effectively predict CRC metastasis based on the differential methylation CpG sites (DMCs) between metastatic and non-metastatic CRC patients. Then, we identified 17 DMCs associated with progression free survival (PFS) of CRC and constructed a prognostic model. The prognosis model based on 17 DMCs can predict the PFS of CRC with medium to high accuracy. The results of immunohistochemical analysis indicated that the protein expression levels of the genes involved in prognostic DMCs were different between normal and colorectal cancer tissues. According to the results of immune-related analysis, we found that the low-risk patients had better immunotherapy response. In addition, high risk scores were negatively correlated with high tumor mutation burden (TMB) levels, and patients with low TMB levels in the high-risk group had the worst PFS. Our work shows the clinical value of DNA methylation in predicting CRC metastasis and PFS, as well as their correlation with TME, immunotherapy, and TMB, which helps understand the changes of DNA methylation in CRC metastasis and improving the treatment and prognosis of CRC.

Promoter hypermethylation as a novel regulator of ANO1 expression and function in prostate cancer bone metastasis.

Despite growing evidence implicating the calcium-activated chloride channel anoctamin1 (ANO1) in cancer metastasis, its direct impact on the metastatic potential of prostate cancer and the possible significance of epigenetic alteration in this process are not fully understood. Here, we show that ANO1 is minimally expressed in LNCap and DU145 prostate cancer cell lines with low metastatic potential but overexpressed in high metastatic PC3 prostate cancer cell line. The treatment of LNCap and DU145 cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) potentiates ANO1 expression, suggesting that DNA methylation is one of the mechanisms controlling ANO1 expression. Consistent with this notion, hypermethylation was detected at the CpG island of ANO1 promoter region in LNCap and DU145 cells, and 5-Aza-CdR treatment resulted in a drastic demethylation at promoter CpG methylation sites. Upon 5-Aza-CdR treatment, metastatic indexes, such as cell motility, invasion, and metastasis-related gene expression, were significantly altered in LNCap and DU145 cells. These 5-Aza-CdR-induced metastatic hallmarks were, however, almost completely ablated by stable knockdown of ANO1. These in vitro discoveries were further supported by our in vivo observation that ANO1 expression in xenograft mouse models enhances the metastatic dissemination of prostate cancer cells into tibial bone and the development of osteolytic lesions. Collectively, our results help elucidate the critical role of ANO1 expression in prostate cancer bone metastases, which is epigenetically modulated by promoter CpG methylation.

Machine learning unveils an immune-related DNA methylation profile in germline DNA from breast cancer patients.

BACKGROUND: There is an unmet need for precise biomarkers for early non-invasive breast cancer detection. Here, we aimed to identify blood-based DNA methylation biomarkers that are associated with breast cancer. METHODS: DNA methylation profiling was performed for 524 Asian Chinese individuals, comprising 256 breast cancer patients and 268 age-matched healthy controls, using the Infinium MethylationEPIC array. Feature selection was applied to 649,688 CpG sites in the training set. Predictive models were built by training three machine learning models, with performance evaluated on an independent test set. Enrichment analysis to identify transcription factors binding to regions associated with the selected CpG sites and pathway analysis for genes located nearby were conducted. RESULTS: A methylation profile comprising 51 CpGs was identified that effectively distinguishes breast cancer patients from healthy controls achieving an AUC of 0.823 on an independent test set. Notably, it outperformed all four previously reported breast cancer-associated methylation profiles. Enrichment analysis revealed enrichment of genomic loci associated with the binding of immune modulating AP-1 transcription factors, while pathway analysis of nearby genes showed an overrepresentation of immune-related pathways. CONCLUSION: This study has identified a breast cancer-associated methylation profile that is immune-related to potential for early cancer detection.

Specific CpG sites methylation is associated with hematotoxicity in low-dose benzene-exposed workers.

Benzene is a broadly used industrial chemicals which causes various hematologic abnormalities in human. Altered DNA methylation has been proposed as epigenetic biomarkers in health risk evaluation of benzene exposure, yet the role of methylation at specific CpG sites in predicting hematological effects remains unclear. In this study, we recruited 120 low-level benzene-exposed and 101 control male workers from a petrochemical factory in Maoming City, Guangdong Province, China. Urinary S-phenylmercapturic acid (SPMA) in benzene-exposed workers was 3.40-fold higher than that in control workers (P < 0.001). Benzene-induced hematotoxicity was characterized by reduced white blood cells counts and nuclear division index (NDI), along with an increased DNA damage and urinary 8-hydroxy-2'-deoxyguanosine (all P < 0.05). Methylation levels of TRIM36, MGMT and RASSF1a genes in peripheral blood lymphocytes (PBLCs) were quantified by pyrosequencing. CpG site 6 of TRIM36, CpG site 2, 4, 6 of RASSF1a and CpG site 1, 3 of MGMT methylation were recognized as hot CpG sites due to a strong correlation with both internal exposure and hematological effects. Notably, integrating hot CpG sites methylation of multiple genes reveal a higher efficiency in prediction of integrative damage compared to individual genes at hot CpG sites. The negative dose-response relationship between the combined methylation of hot CpG sites in three genes and integrative damage enabled the classification of benzene-exposed individuals into high-risk or low-risk groups using the median cut-off value of the integrative index. Subsequently, a prediction model for integrative damage in benzene-exposed populations was built based on the methylation status of the identified hot CpG sites in the three genes. Taken together, these findings provide a novel insight into application prospect of specific CpG site methylation as epi-biomarkers for health risk assessment of environmental pollutants.

Comprehensive DNA methylation profiling by MeDIP-NGS identifies potential genes and pathways for epithelial ovarian cancer.

Ovarian cancer, among all gynecologic malignancies, exhibits the highest incidence and mortality rate, primarily because it is often presents with non-specific or no symptoms during its early stages. For the advancement of Ovarian Cancer Diagnosis, it is crucial to identify the potential molecular signatures that could significantly differentiate between healthy and ovarian cancerous tissues and can be used further as a diagnostic biomarker for detecting ovarian cancer. In this study, we investigated the genome-wide methylation patterns in ovarian cancer patients using Methylated DNA Immunoprecipitation (MeDIP-Seq) followed by NGS. Identified differentially methylated regions (DMRs) were further validated by targeted bisulfite sequencing for CpG site-specific methylation profiles. Furthermore, expression validation of six genes by Quantitative Reverse Transcriptase-PCR was also performed. Out of total 120 differentially methylated genes (DMGs), 68 genes were hypermethylated, and 52 were hypomethylated in their promoter region. After analysis, we identified the top 6 hub genes, namely POLR3B, PLXND1, GIGYF2, STK4, BMP2 and CRKL. Interestingly we observed Non-CpG site methylation in the case of POLR3B and CRKL which was statistically significant in discriminating ovarian cancer samples from normal controls. The most significant pathways identified were focal adhesion, the MAPK signaling pathway, and the Ras signaling pathway. Expression analysis of hypermethylated genes was correlated with the downregulation of the genes. POLR3B and GIGYF2 turned out to be the novel genes associated with the carcinogenesis of EOC. Our study demonstrated that methylation profiling through MeDIP-sequencing has effectively identified six potential hub genes and pathways that might exacerbate our understanding of underlying molecular mechanisms of ovarian carcinogenesis.

Tuning CpG motif position in nanostructured DNA for efficient immune stimulation.

It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine-phosphate-guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2-5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5'-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.

Genome-wide screening and functional validation of methylation barriers near promoters.

CpG islands near promoters are normally unmethylated despite being surrounded by densely methylated regions. Aberrant hypermethylation of these CpG islands has been associated with the development of various human diseases. Although local genetic elements have been speculated to play a role in protecting promoters from methylation, only a limited number of methylation barriers have been identified. In this study, we conducted an integrated computational and experimental investigation of colorectal cancer methylomes. Our study revealed 610 genes with disrupted methylation barriers. Genomic sequences of these barriers shared a common 41-bp sequence motif (MB-41) that displayed homology to the chicken HS4 methylation barrier. Using the CDKN2A (P16) tumor suppressor gene promoter, we validated the protective function of MB-41 and showed that loss of such protection led to aberrant hypermethylation. Our findings highlight a novel sequence signature of cis-acting methylation barriers in the human genome that safeguard promoters from silencing.

Characterization of the genetic determinants of context-specific DNA methylation in primary monocytes.

To better understand inter-individual variation in sensitivity of DNA methylation (DNAm) to immune activity, we characterized effects of inflammatory stimuli on primary monocyte DNAm (n = 190). We find that monocyte DNAm is site-dependently sensitive to lipopolysaccharide (LPS), with LPS-induced demethylation occurring following hydroxymethylation. We identify 7,359 high-confidence immune-modulated CpGs (imCpGs) that differ in genomic localization and transcription factor usage according to whether they represent a gain or loss in DNAm. Demethylated imCpGs are profoundly enriched for enhancers and colocalize to genes enriched for disease associations, especially cancer. DNAm is age associated, and we find that 24-h LPS exposure triggers approximately 6 months of gain in epigenetic age, directly linking epigenetic aging with innate immune activity. By integrating LPS-induced changes in DNAm with genetic variation, we identify 234 imCpGs under local genetic control. Exploring shared causal loci between LPS-induced DNAm responses and human disease traits highlights examples of disease-associated loci that modulate imCpG formation.

Epigenetic Activation of Cytochrome P450 1A2 Sensitizes Hepatocellular Carcinoma Cells to Sorafenib.

Cytochrome P450 1A2 (CYP1A2) is a known tumor suppressor in hepatocellular carcinoma (HCC), but its expression is repressed in HCC and the underlying mechanism is unclear. In this study, we investigated the epigenetic mechanisms of CYP1A2 repression and potential therapeutic implications. In HCC tumor tissues, the methylation rates of CYP1A2 CpG island (CGI) and DNA methyltransferase (DNMT) 3A protein levels were significantly higher, and there was a clear negative correlation between DNMT3A and CYP1A2 protein expression. Knockdown of DNMT3A by siRNA significantly increased CYP1A2 expression in HCC cells. Additionally, treating HCC cells with decitabine (DAC) resulted in a dose-dependent upregulation of CYP1A2 expression by reducing the methylation level of CYP1A2 CGI. Furthermore, we observed a decreased enrichment of H3K27Ac in the promoter region of CYP1A2 in HCC tissues. Treatment with the trichostatin A (TSA) restored CYP1A2 expression in HCC cells by increasing H3K27Ac levels in the CYP1A2 promoter region. Importantly, combination treatment of sorafenib with DAC or TSA resulted in a leftward shift of the dose-response curve, lower IC50 values, and reduced colony numbers in HCC cells. Our findings suggest that hypermethylation of the CGI at the promoter, mediated by the high expression of DNMT3A, and hypoacetylation of H3K27 in the CYP1A2 promoter region, leads to CYP1A2 repression in HCC. Epigenetic drugs DAC and TSA increase HCC cell sensitivity to sorafenib by restoring CYP1A2 expression. Our study provides new insights into the epigenetic regulation of CYP1A2 in HCC and highlights the potential of epigenetic drugs as a therapeutic approach for HCC. SIGNIFICANCE STATEMENT: This study marks the first exploration of the epigenetic mechanisms underlying cytochrome P450 (CYP) 1A2 suppression in hepatocellular carcinoma (HCC). Our findings reveal that heightened DNA methyltransferase expression induces hypermethylation of the CpG island at the promoter, coupled with diminished H3K27Ac levels, resulting in the repression of CYP1A2 in HCC. The use of epigenetic drugs such as decitabine and trichostatin A emerges as a novel therapeutic avenue, demonstrating their potential to restore CYP1A2 expression and enhance sorafenib sensitivity in HCC cells.

iChIP-SILAC analysis identifies epigenetic regulators of CpG methylation of the p16INK4A gene.

Allele-specific epigenetic events regulate the expression of specific genes such as tumor suppressor genes. Methods to biochemically identify epigenetic regulators remain limited. Here, we used insertional chromatin immunoprecipitation (iChIP) to address this issue. iChIP combined with quantitative mass spectrometry identified DNA methyltransferase 1 (DNMT1) and epigenetic regulators as proteins that potentially interact with a region of the p16INK4A gene that is CpG-methylated in one allele in HCT116 cells. Some of the identified proteins are involved in the CpG methylation of this region, and of these, DEAD-box helicase 24 (DDX24) contributes to CpG methylation by regulating the protein levels of DNMT1. Thus, iChIP is a useful method to identify proteins which bind to a target locus of interest.

Molecular pathological approach to cancer epigenomics and its clinical application.

Careful microscopic observation of histopathological specimens, accumulation of large numbers of high-quality tissue specimens, and analysis of molecular pathology in relation to morphological features are considered to yield realistic data on the nature of multistage carcinogenesis. Since the morphological hallmark of cancer is disruption of the normal histological structure maintained through cell-cell adhesiveness and cellular polarity, attempts have been made to investigate abnormalities of the cadherin-catenin cell adhesion system in human cancer cells. It has been shown that the CDH1 tumor suppressor gene encoding E-cadherin is silenced by DNA methylation, suggesting that a "double hit" involving DNA methylation and loss of heterozygosity leads to carcinogenesis. Therefore, in the 1990s, we focused on epigenomic mechanisms, which until then had not received much attention. In chronic hepatitis and liver cirrhosis associated with hepatitis virus infection, DNA methylation abnormalities were found to occur frequently, being one of the earliest indications that such abnormalities are present even in precancerous tissue. Aberrant expression and splicing of DNA methyltransferases, such as DNMT1 and DNMT3B, was found to underlie the mechanism of DNA methylation alterations in various organs. The CpG island methylator phenotype in renal cell carcinoma was identified for the first time, and its therapeutic targets were identified by multilayer omics analysis. Furthermore, the DNA methylation profile of nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma was clarified in groundbreaking studies. Since then, we have developed diagnostic markers for carcinogenesis risk in NASH patients and noninvasive diagnostic markers for upper urinary tract cancer, as well as developing a new high-performance liquid chromatography-based diagnostic system for DNA methylation diagnosis. Research on the cancer epigenome has revealed that DNA methylation alterations occur from the precancerous stage as a result of exposure to carcinogenic factors such as inflammation, smoking, and viral infections, and continuously contribute to multistage carcinogenesis through aberrant expression of cancer-related genes and genomic instability. DNA methylation alterations at the precancerous stages are inherited by or strengthened in cancers themselves and determine the clinicopathological aggressiveness of cancers as well as patient outcome. DNA methylation alterations have applications as biomarkers, and are expected to contribute to diagnosis, as well as preventive and preemptive medicine.

CheekAge: a next-generation buccal epigenetic aging clock associated with lifestyle and health.

Epigenetic aging clocks are computational models that predict age using DNA methylation information. Initially, first-generation clocks were developed to make predictions using CpGs that change with age. Over time, next-generation clocks were created using CpGs that relate to both age and health. Since existing next-generation clocks were constructed in blood, we sought to develop a next-generation clock optimized for prediction in cheek swabs, which are non-invasive and easy to collect. To do this, we collected MethylationEPIC data as well as lifestyle and health information from 8045 diverse adults. Using a novel simulated annealing approach that allowed us to incorporate lifestyle and health factors into training as well as a combination of CpG filtering, CpG clustering, and clock ensembling, we constructed CheekAge, an epigenetic aging clock that has a strong correlation with age, displays high test-retest reproducibility across replicates, and significantly associates with a plethora of lifestyle and health factors, such as BMI, smoking status, and alcohol intake. We validated CheekAge in an internal dataset and multiple publicly available datasets, including samples from patients with progeria or meningioma. In addition to exploring the underlying biology of the data and clock, we provide a free online tool that allows users to mine our methylomic data and predict epigenetic age.

Investigating the potential of single-cell DNA methylation data to detect allele-specific methylation and imprinting.

Allele-specific methylation (ASM) is an epigenetic modification whereby one parental allele becomes methylated and the other unmethylated at a specific locus. ASM is most often driven by the presence of nearby heterozygous variants that influence methylation, but also occurs somatically in the context of genomic imprinting. In this study, we investigate ASM using publicly available single-cell reduced representation bisulfite sequencing (scRRBS) data on 608 B cells sampled from six healthy B cell samples and 1,230 cells from 11 chronic lymphocytic leukemia (CLL) samples. We developed a likelihood-based criterion to test whether a CpG exhibited ASM, based on the distributions of methylated and unmethylated reads both within and across cells. Applying our likelihood ratio test, 65,998 CpG sites exhibited ASM in healthy B cell samples according to a Bonferroni criterion (p < 8.4 × 10-9), and 32,862 CpG sites exhibited ASM in CLL samples (p < 8.5 × 10-9). We also called ASM at the sample level. To evaluate the accuracy of our method, we called heterozygous variants from the scRRBS data, which enabled variant-based calls of ASM within each cell. Comparing sample-level ASM calls to the variant-based measures of ASM, we observed a positive predictive value of 76%-100% across samples. We observed high concordance of ASM across samples and an overrepresentation of ASM in previously reported imprinted genes and genes with imprinting binding motifs. Our study demonstrates that single-cell bisulfite sequencing is a potentially powerful tool to investigate ASM, especially as studies expand to increase the number of samples and cells sequenced.

Multi-omics analysis reveals epigenetically regulated processes and patient classification in lung adenocarcinoma.

Aberrant DNA methylation is a hallmark of many cancer types. Despite our knowledge of epigenetic and transcriptomic alterations in lung adenocarcinoma (LUAD), we lack robust multi-modal molecular classifications for patient stratification. This is partly because the impact of epigenetic alterations on lung cancer development and progression is still not fully understood. To that end, we identified disease-associated processes under epigenetic regulation in LUAD. We performed a genome-wide expression-methylation Quantitative Trait Loci (emQTL) analysis by integrating DNA methylation and gene expression data from 453 patients in the TCGA cohort. Using a community detection algorithm, we identified distinct communities of CpG-gene associations with diverse biological processes. Interestingly, we identified a community linked to hormone response and lipid metabolism; the identified CpGs in this community were enriched in enhancer regions and binding regions of transcription factors such as FOXA1/2, GRHL2, HNF1B, AR, and ESR1. Furthermore, the CpGs were connected to their associated genes through chromatin interaction loops. These findings suggest that the expression of genes involved in hormone response and lipid metabolism in LUAD is epigenetically regulated through DNA methylation and enhancer-promoter interactions. By applying consensus clustering on the integrated expression-methylation pattern of the emQTL-genes and CpGs linked to hormone response and lipid metabolism, we further identified subclasses of patients with distinct prognoses. This novel patient stratification was validated in an independent patient cohort of 135 patients and showed increased prognostic significance compared to previously defined molecular subtypes.

Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.

The presence of CpGs in AAV gene therapy vectors induces a plasmacytoid dendritic cell-like population very early after administration.

AAV-mediated gene transfer is a promising platform still plagued by potential host-derived, antagonistic immune responses to therapeutic components. CpG-mediated TLR9 stimulation activates innate immune cells and leads to cognate T cell activation and suppression of transgene expression. Here, we demonstrate that CpG depletion increased expression of an antibody transgene product by 2-3-fold as early as 24 h post-vector administration in mice. No significant differences were noted in anti-transgene product/ anti-AAV capsid antibody production or cytotoxic gene induction. Instead, CpG depletion significantly reduced the presence of a pDC-like myeloid cell population, which was able to directly bind the antibody transgene product via Fc-FcγR interactions. Thus, we extend the mechanisms of TLR9-mediated antagonism of transgene expression in AAV gene therapy to include the actions of a previously unreported pDC-like cell population.

Cellular senescence gene TACC3 associated with colorectal cancer risk via genetic and DNA methylated alteration.

Cell senescence genes play a vital role in the pathogenesis of colorectal cancer, a process that may involve the triggering of genetic variations and reversible phenotypes caused by epigenetic modifications. However, the specific regulatory mechanisms remain unclear. Using CellAge and The Cancer Genome Atlas databases and in-house RNA-seq data, DNA methylation-modified cellular senescence genes (DMCSGs) were validated by Support Vector Machine and correlation analyses. In 1150 cases and 1342 controls, we identified colorectal cancer risk variants in DMCSGs. The regulatory effects of gene, variant, and DNA methylation were explored through dual-luciferase and 5-azacytidine treatment experiments, complemented by multiple database analyses. Biological functions of key gene were evaluated via cell proliferation assays, SA-ß-gal staining, senescence marker detection, and immune infiltration analyses. The genetic variant rs4558926 in the downstream of TACC3 was significantly associated with colorectal cancer risk (OR = 1.35, P = 3.22 × 10-4). TACC3 mRNA expression increased due to rs4558926 C > G and decreased DNA methylation levels. The CpG sites in the TACC3 promoter region were regulated by rs4558926. TACC3 knockdown decreased proliferation and senescence in colorectal cancer cells. In addition, subjects with high-TACC3 expression presented an immunosuppressive microenvironment. These findings provide insights into the involvement of genetic variants of cellular senescence genes in the development and progression of colorectal cancer.

Computationally inferred cell-type specific epigenome-wide DNA methylation analysis unveils distinct methylation patterns among immune cells for HIV infection in three cohorts.

BACKGROUND: Epigenome-wide association studies (EWAS) have identified CpG sites associated with HIV infection in blood cells in bulk, which offer limited knowledge of cell-type specific methylation patterns associated with HIV infection. In this study, we aim to identify differentially methylated CpG sites for HIV infection in immune cell types: CD4+ T-cells, CD8+ T-cells, B cells, Natural Killer (NK) cells, and monocytes. METHODS: Applying a computational deconvolution method, we performed a cell-type based EWAS for HIV infection in three independent cohorts (Ntotal = 1,382). DNA methylation in blood or in peripheral blood mononuclear cells (PBMCs) was profiled by an array-based method and then deconvoluted by Tensor Composition Analysis (TCA). The TCA-computed CpG methylation in each cell type was first benchmarked by bisulfite DNA methylation capture sequencing in a subset of the samples. Cell-type EWAS of HIV infection was performed in each cohort separately and a meta-EWAS was conducted followed by gene set enrichment analysis. RESULTS: The meta-analysis unveiled a total of 2,021 cell-type unique significant CpG sites for five inferred cell types. Among these inferred cell-type unique CpG sites, the concordance rate in the three cohorts ranged from 96% to 100% in each cell type. Cell-type level meta-EWAS unveiled distinct patterns of HIV-associated differential CpG methylation, where 74% of CpG sites were unique to individual cell types (false discovery rate, FDR <0.05). CD4+ T-cells had the largest number of unique HIV-associated CpG sites (N = 1,624) compared to any other cell type. Genes harboring significant CpG sites are involved in immunity and HIV pathogenesis (e.g. CD4+ T-cells: NLRC5, CX3CR1, B cells: IFI44L, NK cells: IL12R, monocytes: IRF7), and in oncogenesis (e.g. CD4+ T-cells: BCL family, PRDM16, monocytes: PRDM16, PDCD1LG2). HIV-associated CpG sites were enriched among genes involved in HIV pathogenesis and oncogenesis that were enriched among interferon-α and -γ, TNF-α, inflammatory response, and apoptotic pathways. CONCLUSION: Our findings uncovered computationally inferred cell-type specific modifications in the host epigenome for people with HIV that contribute to the growing body of evidence regarding HIV pathogenesis.