Reinvent Aliphatic Arsenicals as Reversible Covalent Warheads toward Targeted Kinase Inhibition and Non-acute Promyelocytic Leukemia Cancer Treatment.

The success of arsenic in acute promyelocytic leukemia (APL) treatment is hardly transferred to non-APL cancers, mainly due to the low selectivity and weak binding affinity of traditional arsenicals to oncoproteins critical for cancer survival. We present herein the reinvention of aliphatic trivalent arsenicals (As) as reversible covalent warheads of As-based targeting inhibitors toward Bruton's tyrosine kinase (BTK). The effects of As warheads' valency, thiol protection, methylation, spacer length, and size on inhibitors' activity were studied. We found that, in contrast to the bulky and rigid aromatic As warhead, the flexible aliphatic As warheads were well compatible with the well-optimized guiding group to achieve nanomolar inhibition against BTK. The optimized As inhibitors effectively blocked the BTK-mediated oncogenic signaling pathway, leading to elevated antiproliferative activities toward lymphoma cells and xenograft tumor. Our study provides a promising strategy enabling rational design of new aliphatic arsenic-based reversible covalent inhibitors toward non-APL cancer treatment.

Cytokine status and significant increase of IL-6 and sIL-6R in the aqueous humor of diabetic cataract patients revealed by quantitative multiplexed assays.

OBJECTIVE: This study aimed to investigate inflammatory cytokine expression profiles in the aqueous humor (AH) of diabetic cataract (DC) patients. METHODS: A quantitative multiplexed antibody assay was performed to measure the expression levels of 40 inflammatory cytokines in AH samples from DC and age-related cataract (ARC) patients. Bioinformatics analysis was used to examine the functions of the cytokines. Enzyme-linked immunosorbent assays (ELISAs) and western blots were performed to verify the data. RESULTS: The multiplexed antibody assay revealed that the expression levels of IL-6, sIL-6R, IL-17A, IL-8, MCP-1, TNF-ß, RANTES, TIMP-1, and TIMP-2 were higher in the AH of DC patients compared with ARC patients. However, IL-1ra and IL-1a expression levels were lower in the DC patient AH samples. Pathway analysis indicated that IL-6 and sIL-6R belong to the class I helical cytokine family, which is associated with many biological functions. ELISA and western blot results confirmed that IL-6R and IL-6 expression levels were significantly higher in DC patients compared with ARC patients. CONCLUSIONS: Our results revealed the status of 40 inflammatory cytokines in the AH by quantitative multiplexed assays. Additionally, IL-6 and sIL-6R were expressed markedly higher in DC compared with ARC, which may play critical roles in DC pathophysiology.

Circadian Regulator-Mediated Molecular Subtypes Depict the Features of Tumor Microenvironment and Indicate Prognosis in Head and Neck Squamous Cell Carcinoma.

Introduction: Circadian rhythm is involved in multiple biological activities and implicated in cancer development. However, the role of circadian rhythm in head and neck squamous cell carcinoma (HNSCC) has not been fully interpreted yet. Herein, the present study set out to explore the significance of circadian regulator genes (CRGs) in HNSCC. Materials and Methods: The molecular landscape and clinical significance of 13 CRGs in HNSCC were explored based on The Cancer Genome Atlas (TCGA). The biological functions of PER3, a key CRG, were validated by cellular experiments. The correlation of CRGs with microenvironment, pathway activities, and prognosis was determined by bioinformatic algorithms. A novel circadian score was introduced to evaluate the circadian modification pattern of each patient and further validated in an independent cohort from the Gene Expression Omnibus (GEO) dataset. Results: CRGs presented high heterogeneity in HNSCC at both genomic and transcriptomic levels. Specifically, PER3 indicated a better prognosis and inhibited HNSCC cell proliferation. Moreover, HNSCC tissues displayed three circadian regulator patterns with distinct clinical outcomes, transcriptomic characteristics, and microenvironment features. Circadian score was an independent risk factor and exhibited excellent predictive efficiency in both the training cohort from the TCGA database and the validation cohort from the GEO database. Conclusions: CRGs played an indispensable role in HNSCC development. An in-depth exploration of circadian rhythm would improve the understanding of HNSCC carcinogenesis and confer novel insights for future clinical practices.

Intrahepatic transcriptomics reveals gene signatures in chronic hepatitis B patients responded to interferon therapy.

Chronic hepatitis B virus (HBV) infection remains a substantial public health burden worldwide. Alpha-interferon (IFNα) is one of the two currently approved therapies for chronic hepatitis B (CHB), to explore the mechanisms underlying IFNα treatment response, we investigated baseline and 24-week on-treatment intrahepatic gene expression profiles in 21 CHB patients by mRNA-seq. The data analyses demonstrated that PegIFNα treatment significantly induced antiviral responses. Responders who achieved HBV DNA loss and HBeAg or HBsAg seroconversion displayed higher fold change and larger number of up-regulated interferon-stimulated genes (ISGs). Interestingly, lower expression levels of certain ISGs were observed in responders in their baseline biopsy samples. In HBeAg+ patients, non-responders had relative higher baseline HBeAg levels than responders. More importantly, HBeAg- patients showed higher HBsAg loss rate than HBeAg+ patients. Although a greater fold change of ISGs was observed in HBeAg- patients than HBeAg+ patients, upregulation of ISGs in HBeAg+ responders exceeded HBeAg- responders. Notably, PegIFNα treatment increased monocyte and mast cell infiltration, but decreased CD8 T cell and M1 macrophage infiltration in both responders and non-responders, while B cell infiltration was increased only in responders. Moreover, co-expression analysis identified ribosomal proteins as critical players in antiviral response. The data also indicate that IFNα may influence the production of viral antigens associated with endoplasmic reticulum. Collectively, the intrahepatic transcriptome analyses in this study enriched our understanding of IFN-mediated antiviral effects in CHB patients and provided novel insights into the development of potential strategies to improve IFNα therapy.

WDR62 variants contribute to congenital heart disease by inhibiting cardiomyocyte proliferation.

BACKGROUND: Congenital heart disease (CHD) is the most common birth defect and has high heritability. Although some susceptibility genes have been identified, the genetic basis underlying the majority of CHD cases is still undefined. METHODS: A total of 1320 unrelated CHD patients were enrolled in our study. Exome-wide association analysis between 37 tetralogy of Fallot (TOF) patients and 208 Han Chinese controls from the 1000 Genomes Project was performed to identify the novel candidate gene WD repeat-containing protein 62 (WDR62). WDR62 variants were searched in another expanded set of 200 TOF patients by Sanger sequencing. Rescue experiments in zebrafish were conducted to observe the effects of WDR62 variants. The roles of WDR62 in heart development were examined in mouse models with Wdr62 deficiency. WDR62 variants were investigated in an additional 1083 CHD patients with similar heart phenotypes to knockout mice by multiplex PCR-targeting sequencing. The cellular phenotypes of WDR62 deficiency and variants were tested in cardiomyocytes, and the molecular mechanisms were preliminarily explored by RNA-seq and co-immunoprecipitation. RESULTS: Seven WDR62 coding variants were identified in the 237 TOF patients and all were indicated to be loss of function variants. A total of 25 coding and 22 non-coding WDR62 variants were identified in 80 (6%) of the 1320 CHD cases sequenced, with a higher proportion of WDR62 variation (8%) found in the ventricular septal defect (VSD) cohort. WDR62 deficiency resulted in a series of heart defects affecting the outflow tract and right ventricle in mouse models, including VSD as the major abnormality. Cell cycle arrest and an increased number of cells with multipolar spindles that inhibited proliferation were observed in cardiomyocytes with variants or knockdown of WDR62. WDR62 deficiency weakened the association between WDR62 and the cell cycle-regulated kinase AURKA on spindle poles, reduced the phosphorylation of AURKA, and decreased expression of target genes related to cell cycle and spindle assembly shared by WDR62 and AURKA. CONCLUSIONS: WDR62 was identified as a novel susceptibility gene for CHD with high variant frequency. WDR62 was shown to participate in the cardiac development by affecting spindle assembly and cell cycle pathway in cardiomyocytes.

Tumour suppressor TET2 safeguards enhancers from aberrant DNA methylation and epigenetic reprogramming in ERα-positive breast cancer cells.

Aberrant DNA methylation is an epigenetic hallmark of malignant tumours. The DNA methylation level is regulated by not only DNA methyltransferases (DNMTs) but also Ten-Eleven Translocation (TET) family proteins. However, the exact role of TET genes in breast cancer remains controversial. Here, we uncover that the ERα-positive breast cancer patients with high TET2 mRNA expression had better overall survival rates. Consistently, knockout of TET2 promotes the tumorigenesis of ERα-positive MCF7 breast cancer cells. Mechanistically, TET2 loss leads to aberrant DNA methylation (gain of 5mC) at a large proportion of enhancers, accompanied by significant reduction in H3K4me1 and H3K27ac enrichment. By analysing the epigenetically reprogrammed enhancers, we identify oestrogen responsive element (ERE) as one of the enriched motifs of transcriptional factors. Importantly, TET2 loss impairs 17beta-oestradiol (E2)-induced transcription of the epigenetically reprogrammed EREs-associated genes through attenuating the binding of ERα. Taken together, these findings shed light on our understanding of the epigenetic mechanisms underlying the enhancer reprogramming during breast cancer pathogenesis.

Corrigendum: Mechanism of Growth Regulation of Yeast Involving Hydrogen Sulfide From S-Propargyl-Cysteine Catalyzed by Cystathionine-γ-Lyase.

[This corrects the article DOI: 10.3389/fmicb.2021.679563.].

Mechanism of Growth Regulation of Yeast Involving Hydrogen Sulfide From S-Propargyl-Cysteine Catalyzed by Cystathionine-γ-Lyase.

Pathogenic fungi are recognized as a progressive threat to humans, particularly those with the immunocompromised condition. The growth of fungi is controlled by several factors, one of which is signaling molecules, such as hydrogen sulfide (H2S), which was traditionally regarded as a toxic gas without physiological function. However, recent studies have revealed that H2S is produced enzymatically and endogenously in several species, where it serves as a gaseous signaling molecule performing a variety of critical biological functions. However, the influence of this endogenous H2S on the biological activities occurring within the pathogenic fungi, such as transcriptomic and phenotypic alternations, has not been elucidated so far. Therefore, the present study was aimed to decipher this concern by utilizing S-propargyl-cysteine (SPRC) as a novel and stable donor of H2S and Saccharomyces cerevisiae as a fungal model. The results revealed that the yeast could produce H2S by catabolizing SPRC, which facilitated the growth of the yeast cells. This implies that the additional intracellularly generated H2S is generated primarily from the enhanced sulfur-amino-acid-biosynthesis pathways and serves to increase the growth rate of the yeast, and presumably the growth of the other fungi as well. In addition, by deciphering the implicated pathways and analyzing the in vitro enzymatic activities, cystathionine-γ-lyase (CYS3) was identified as the enzyme responsible for catabolizing SPRC into H2S in the yeast, which suggested that cystathionine-γ-lyase might play a significant role in the regulation of H2S-related transcriptomic and phenotypic alterations occurring in yeast. These findings provide important information regarding the mechanism underlying the influence of the gaseous signaling molecules such as H2S on fungal growth. In addition, the findings provide a better insight to the in vivo metabolism of H2S-related drugs, which would be useful for the future development of anti-fungal drugs.

Enhancement of E-cadherin expression and processing and driving of cancer cell metastasis by ARID1A deficiency.

The ARID1A gene, which encodes a subunit of the SWI/SNF chromatin remodeling complex, has been found to be frequently mutated in many human cancer types. However, the function and mechanism of ARID1A in cancer metastasis are still unclear. Here, we show that knockdown of ARID1A increases the ability of breast cancer cells to proliferate, migrate, invade, and metastasize in vivo. The ARID1A-related SWI/SNF complex binds to the second exon of CDH1 and negatively modulates the expression of E-cadherin/CDH1 by recruiting the transcriptional repressor ZEB2 to the CDH1 promoter and excluding the presence of RNA polymerase II. The silencing of CDH1 attenuated the migration, invasion, and metastasis of breast cancer cells in which ARID1A was silenced. ARID1A depletion increased the intracellular enzymatic processing of E-cadherin and the production of C-terminal fragment 2 (CTF2) of E-cadherin, which stabilized ß-catenin by competing for binding to the phosphorylation and degradation complex of ß-catenin. The matrix metalloproteinase inhibitor GM6001 inhibited the production of CTF2. In zebrafish and nude mice, ARID1A silencing or CTF2 overexpression activated ß-catenin signaling and promoted migration/invasion and metastasis of cancer cells in vivo. The inhibitors GM6001, BB94, and ICG-001 suppressed the migration and invasion of cancer cells with ARID1A-deficiency. Our findings provide novel insights into the mechanism of ARID1A metastasis and offer a scientific basis for targeted therapy of ARID1A-deficient cancer cells.

MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus.

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

Quantitative acetylome analysis reveals histone modifications that may predict prognosis in hepatitis B-related hepatocellular carcinoma.

Lysine acetylation (Kac) as an important posttranslational modification of histones is essential for the regulation of gene expression in hepatocellular carcinoma (HCC). However, the atlas of whole acetylated proteins in HCC tissues and the difference in protein acetylation between normal human tissues and HCC tissues are unknown. In this report, we characterized the proteome and acetyl proteome (acetylome) profile of normal, paracancerous, and HCC liver tissues in human clinical samples by quantitative proteomics techniques. We identified 6781 acetylation sites of 2582 proteins and quantified 2492 acetylation sites of 1190 proteins in normal, paracancerous, and HCC liver tissues. Among them, 15 proteins were multiacetylated with more than 10 lysine residues. The histone acetyltransferases p300 and CBP were found to be hyperacetylated in hepatitis B virus pathway. Moreover, we found that 250 Kac sites of 214 proteins were upregulated and 662 Kac sites of 451 proteins were downregulated in HCC compared with normal liver tissues. Additionally, the acetylation levels of lysine 120 in histone H2B (H2BK120ac), lysine 18 in histone H3.3 (H3.3K18ac), and lysine 77 in histone H4 (H4K77ac) were increased in HCC. Interestingly, the higher levels of H2BK120ac, H3.3K18ac, and H4K77ac were significantly associated with worse prognosis, such as poorer survival and higher recurrence in an independent clinical cohort of HCC patients. Overall, this study lays a foundation for understanding the functions of acetylation in HCC and provides potential prognostic factors for the diagnosis and therapy of HCC.

TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T-cell numbers.

The host immune response is a fundamental mechanism for attenuating cancer progression. Here we report a role for the DNA demethylase and tumor suppressor TET2 in host anti-tumor immunity. Deletion of Tet2 in mice elevates IL-6 levels upon tumor challenge. Elevated IL-6 stimulates immunosuppressive granulocytic myeloid-derived suppressor cells (G-MDSCs), which in turn reduce CD8+ T cells upon tumor challenge. Consequently, systematic knockout of Tet2 in mice leads to accelerated syngeneic tumor growth, which is constrained by anti-PD-1 blockade. Removal of G-MDSCs by the anti-mouse Ly6g antibodies restores CD8+ T-cell numbers in Tet2-/- mice and reboots their anti-tumor activity. Importantly, anti-IL-6 antibody treatment blocks the expansion of G-MDSCs and inhibits syngeneic tumor growth. Collectively, these findings reveal a TET2-mediated IL-6/G-MDSCs/CD8+ T-cell immune response cascade that safeguards host adaptive anti-tumor immunity, offering a cell non-autonomous mechanism of TET2 for tumor suppression.

Nono deficiency compromises TET1 chromatin association and impedes neuronal differentiation of mouse embryonic stem cells.

NONO is a DNA/RNA-binding protein, which plays a critical regulatory role during cell stage transitions of mouse embryonic stem cells (mESCs). However, its function in neuronal lineage commitment and the molecular mechanisms of its action in such processes are largely unknown. Here we report that NONO plays a key role during neuronal differentiation of mESCs. Nono deletion impedes neuronal lineage commitment largely due to a failure of up-regulation of specific genes critical for neuronal differentiation. Many of the NONO regulated genes are also DNA demethylase TET1 targeted genes. Importantly, re-introducing wild type NONO to the Nono KO cells, not only restores the normal expression of the majority of NONO/TET1 coregulated genes but also rescues the defective neuronal differentiation of Nono-deficient mESCs. Mechanistically, our data shows that NONO directly interacts with TET1 via its DNA binding domain and recruits TET1 to genomic loci to regulate 5-hydroxymethylcytosine levels. Nono deletion leads to a significant dissociation of TET1 from chromatin and dysregulation of DNA hydroxymethylation of neuronal genes. Taken together, our findings reveal a key role and an epigenetic mechanism of action of NONO in regulation of TET1-targeted neuronal genes, offering new functional and mechanistic understanding of NONO in stem cell functions, lineage commitment and specification.

Correction to: DNMT3A reads and connects histone H3K36me2 to DNA methylation.

The author would like to add the below information in this correction. A similar study from Chao Lu group was published online on 5 September 2019 in Nature, entitled "The histone mark H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape" (Weinberg et al., 2019). Although both the studies reported the preferential recognition of H3K36me2 by DNMT3A PWWP, ours in addition uncovered a stimulation function by such interaction on the activity of DNMT3A. On the disease connections, we used a NSD2 gain-of-function model which led to the discovery of potential therapeutic implication of DNA inhibitors in the related cancers, while the other study only used NSD1 and DNMT3A loss-of-function models.

Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute.

Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by its comprehensive pipeline, which contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control (QC), normalization, batch effect removal, copy-number bias correction, gene hit identification and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires ~3 h on a desktop computer running Linux or Mac OS with R support.

Guide Positioning Sequencing identifies aberrant DNA methylation patterns that alter cell identity and tumor-immune surveillance networks.

Aberrant DNA methylation is a distinguishing feature of cancer. Yet, how methylation affects immune surveillance and tumor metastasis remains ambiguous. We introduce a novel method, Guide Positioning Sequencing (GPS), for precisely detecting whole-genome DNA methylation with cytosine coverage as high as 96% and unbiased coverage of GC-rich and repetitive regions. Systematic comparisons of GPS with whole-genome bisulfite sequencing (WGBS) found that methylation difference between gene body and promoter is an effective predictor of gene expression with a correlation coefficient of 0.67 (GPS) versus 0.33 (WGBS). Moreover, Methylation Boundary Shift (MBS) in promoters or enhancers is capable of modulating expression of genes associated with immunity and tumor metabolism. Furthermore, aberrant DNA methylation results in tissue-specific enhancer switching, which is responsible for altering cell identity during liver cancer development. Altogether, we demonstrate that GPS is a powerful tool with improved accuracy and efficiency over WGBS in simultaneously detecting genome-wide DNA methylation and genomic variation. Using GPS, we show that aberrant DNA methylation is associated with altering cell identity and immune surveillance networks, which may contribute to tumorigenesis and metastasis.

Glucose-regulated phosphorylation of TET2 by AMPK reveals a pathway linking diabetes to cancer.

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications1,2. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer3-5. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel 'phospho-switch' that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.

A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands.

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs' role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.

Suppression of Enhancer Overactivation by a RACK7-Histone Demethylase Complex.

Regulation of enhancer activity is important for controlling gene expression programs. Here, we report that a biochemical complex containing a potential chromatin reader, RACK7, and the histone lysine 4 tri-methyl (H3K4me3)-specific demethylase KDM5C occupies many active enhancers, including almost all super-enhancers. Loss of RACK7 or KDM5C results in overactivation of enhancers, characterized by the deposition of H3K4me3 and H3K27Ac, together with increased transcription of eRNAs and nearby genes. Furthermore, loss of RACK7 or KDM5C leads to de-repression of S100A oncogenes and various cancer-related phenotypes. Our findings reveal a RACK7/KDM5C-regulated, dynamic interchange between histone H3K4me1 and H3K4me3 at active enhancers, representing an additional layer of regulation of enhancer activity. We propose that RACK7/KDM5C functions as an enhancer "brake" to ensure appropriate enhancer activity, which, when compromised, could contribute to tumorigenesis.

MBD3L2 promotes Tet2 enzymatic activity for mediating 5-methylcytosine oxidation.

Ten-eleven translocation (Tet) proteins are key players involved in the dynamic regulation of cytosine methylation and demethylation. Inactivating mutations of Tet2 are frequently found in human malignancies, highlighting the essential role of Tet2 in cellular transformation. However, the factors that control Tet enzymatic activity remain largely unknown. Here, we found that methyl-CpG-binding domain protein 3 (MBD3) and its homolog MBD3-like 2 (MBD3L2) can specifically modulate the enzymatic activity of Tet2 protein, but not Tet1 and Tet3 proteins, in converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Moreover, MBD3L2 is more effective than MBD3 in promoting Tet2 enzymatic activity through strengthening the binding affinity between Tet2 and the methylated DNA target. Further analysis revealed pronounced decreases in 5mC levels at MBD3L2 and Tet2 co-occupied genomic regions, most of which are promoter elements associated with either cancer-related genes or genes involved in the regulation of cellular metabolic processes. Our data add new insights into the regulation of Tet2 activity by MBD3 and MBD3L2, and into how that affects Tet2-mediated modulation of its target genes in cancer development. Thus, they have important applications in understanding how dysregulation of Tet2 might contribute to human malignancy.