Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution

5-Hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC) catalyzed by ten-eleven translocation enzymes, plays an important role in many biological processes as an epigenetic mediator. Prior studies have shown that 5hmC can be selectively labeled with chemically modified glucose moieties and enriched using click chemistry with biotin affinity approaches. Besides, DNA deaminases of the AID/APOBEC family can discriminate modified 5hmC bases from cytosine (C) or 5mC. Herein, we developed a method based on embryonic stem cell (ESC) whole-genome analysis, which could enrich 5hmC-containing DNA by selective chemical labeling and locate 5hmC sites at single-base resolution with enzyme-based deamination. The combination experimental design is an extension of previous methods, and we hope that this cost-effective single-base resolution 5hmC sequencing method could be used to promote the mechanism and diagnosis research of 5hmC.

Pan-cancer landscape of AID-related mutations, composite mutations and its potential role in the ICI response

Activation-induced cytidine deaminase, AICDA or AID, is a driver of somatic hypermutation and class-switch recombination in immunoglobulins. In addition, this deaminase belonging to the APOBEC family, may have off-target effects genome-wide, but its effects at pan-cancer level are not well elucidated. Here, we used different pan-cancer datasets, totaling more than 50,000 samples analyzed by whole-genome, whole-exome or targeted sequencing. AID synergizes initial hotspot mutations by a second composite mutation. Analysis of 2.5 million cells, normal and oncogenic, revealed AICDA expression activation after oncogenic transformation and cell cycle regulation loss. AID mutational load was found to be independently associated with favorable outcome in immune-checkpoint inhibitors (ICI) treated patients across cancers after analyzing 2,000 samples. Finally, we found that AID related neoepitopes, resulting from mutations at more frequent hotspots if compared to other mutational signatures, enhance CXCL13/CCR5 expression, immunogenicity and T-cell exhaustion, which may increase ICI sensitivity.

Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution

5-Hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC) catalyzed by ten-eleven translocation (TET) enzymes, plays an important role in many biological processes as an epigenetic mediator. Prior studies have shown that 5hmC can be selectively labeled with chemically-modified glucose moieties and enriched using click chemistry with biotin affinity approaches. Besides, DNA deaminases of the AID/APOBEC family can discriminate modified 5hmC bases from cytosine (C)or 5-methylcytosine (5mC). Herein, we developed a method based on ESC whole-genome analysis which could enrich 5hmC-containing DNA by selective chemical labeling and locate 5hmC sites at single-base resolution with enzyme APOBEC-based deamination. The combination experimental design is an extension of previous methods, and we hope that this cost-effective single base resolution 5hmC sequencing method could be used to promote the mechanism and diagnosis research of 5hmC.

HPV Meets APOBEC: New Players in Head and Neck Cancer

Besides smoking and alcohol, human papillomavirus (HPV) is a factor promoting head and neck squamous cell carcinoma (HNSCC). In some human tumors, including HNSCC, a number of mutations are caused by aberrantly activated DNA-modifying enzymes, such as the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) family of cytidine deaminases. As the enzymatic activity of APOBEC proteins contributes to the innate immune response to viruses, including HPV, the role of APOBEC proteins in HPV-driven head and neck carcinogenesis has recently gained increasing attention. Ongoing research efforts take the cue from two key observations: (1) APOBEC expression depends on HPV infection status in HNSCC; and (2) APOBEC activity plays a major role in HPV-positive HNSCC mutagenesis. This review focuses on recent advances on the role of APOBEC proteins in HPV-positive vs. HPV-negative HNSCC.

Parallelized Latent Dirichlet Allocation Provides a Novel Interpretability of Mutation Signatures in Cancer Genomes

Mutation signatures are defined as the distribution of specific mutations such as activity of AID/APOBEC family proteins. Previous studies have reported numerous signatures, using matrix factorization methods for mutation catalogs. Different mutation signatures are active in different tumor types; hence, signature activity varies greatly among tumor types and becomes sparse. Because of this, many previous methods require dividing mutation catalogs for each tumor type. Here, we propose parallelized latent Dirichlet allocation (PLDA), a novel Bayesian model to simultaneously predict mutation signatures with all mutation catalogs. PLDA is an extended model of latent Dirichlet allocation (LDA), which is one of the methods used for signature prediction. It has parallelized hyperparameters of Dirichlet distributions for LDA, and they represent the sparsity of signature activities for each tumor type, thus facilitating simultaneous analyses. First, we conducted a simulation experiment to compare PLDA with previous methods (including SigProfiler and SignatureAnalyzer) using artificial data and confirmed that PLDA could predict signature structures as accurately as previous methods without searching for the optimal hyperparameters. Next, we applied PLDA to PCAWG (Pan-Cancer Analysis of Whole Genomes) mutation catalogs and obtained a signature set different from the one predicted by SigProfiler. Further, we have shown that the mutation spectrum represented by the predicted signature with PLDA provides a novel interpretability through post-analyses.

APOBEC2 binds Chromatin and Represses Transcription during Myoblast Differentiation

ABSTRACTAPOBEC2 is a member of the prolific activation induced cytidine deaminase/ apolipoprotein B editing complex (AID/APOBEC) family of DNA or RNA editors. This family of nucleic acid editors has diverse molecular roles ranging from antibody diversification to RNA transcript editing. However, even though APOBEC2 is an evolutionarily conserved zinc-dependent cytidine deaminase, it neither has an established molecular substrate nor function. In this work, we use the C2C12 skeletal myoblast differentiation model to confirm that APOBEC2 is upregulated during differentiation and is critical to proper differentiation. Furthermore, we show that APOBEC2 has none of the attributed molecular functions of the AID/APOBEC family, such as mRNA editing, DNA demethylation, and DNA mutation. Unexpectedly, we reveal that APOBEC2 binds chromatin at promoter regions of actively transcribed genes, and binding correlates with transcriptional repression of non-myogenesis related gene pathways. APOBEC2 occupied regions co-occur with sequence motifs for several transcription factors such as Specificity Protein/Krüppel-like Factor (SP/KLF), and we demonstrate in vitro that APOBEC2 binds directly and co-operatively to double stranded DNA containing a SP1 binding site. Finally, protein-proximity data show that APOBEC2 directly interacts with histone deacetylase (HDAC) transcriptional co-repressor complexes. Taken together, these data suggest a role for APOBEC2 as a transcriptional repressor for muscle differentiation, a novel role that is unique among AID/APOBEC family members.

Association between APOBEC3H-Mediated Demethylation and Immune Landscape in Head and Neck Squamous Carcinoma

Immunotherapy has been demonstrated as a promising strategy in controlling head and neck squamous cell carcinoma (HNSC). The AID/APOBEC family is well characterized as DNA mutator and considered to play critical roles in immune responses in HNSC. However, the expression pattern and deamination-dependent demethylation roles of AID/APOBECs in HNSC are unclear. In this study, the RNA-seq and DNA methylation profiles of HNSC from TCGA database and cell-based experiments were applied to analyze the relationships between AID/APOBEC expression levels, patients’ clinical outcomes, methylation alterations, and immune responses. Here, we found that APOBEC3H was abnormally upregulated in HNSC patients. HPV+ patients tended to have higher APOBEC3H levels than HPV- patients. Remarkably, patients with high APOBEC3H levels showed a favorable overall survival. Furthermore, tumors with high APOBEC3H levels exhibited a genome-wide DNA hypomethylation pattern. APOBEC3H was identified to demethylate and upregulate CXCL10 and improve CD8+ T cell tumor infiltration in the tumor microenvironment. Collectively, APOBEC3H plays critical roles in CD8+ T cell immune infiltration and activation in HNSC, which may be a potential biomarker for oncoimmunotherapy in HNSC.

A PRESPECTIVE STUDY ON APOBEC PROTEIN FAMILY

Apobec is an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like" is a family of deaminases. It has two types of APOBEC enzymes n-terminal of apobec enzyme and C-terminal of APOBEC enzyme. N-terminal domain is catalytic domain and C-terminal domain is a pseudo catalytic domain. Pathogen and cellular genome undergo mutation by human DNA cytosine to uracil deaminases. Three subtypes of APOBEC3D. APOBEC3F, APOBEC3G and APOBEC3H restrict human deficiency virus-1. Two APOBEC enzymes are the sources of somatic mutagenesis in cancer cells that drive tumor evolution and manifest clinically as theraphy resistance. This review of the APOBEC family will focus on an open question in regulation, namely what role the interactions of these proteins with RNA have in editing substrate recognition or allosteric regulation of DNA mutagenic and host-defense activities.