CBX4 plays a bidirectional role in transcriptional regulation and lung adenocarcinoma progression.

Lung adenocarcinoma (LUAD) remains a leading cause of cancer-related mortality worldwide. Understanding the dysregulated epigenetics governing LUAD progression is pivotal for identifying therapeutic targets. CBX4, a chromobox protein, is reported to be upregulated in LUAD. This study highlights the dual impact of CBX4 on LUAD proliferation and metastasis through a series of rigorous in vitro and in vivo experiments. Further investigation into the underlying mechanism through high-throughput ChIP-seq and RNA-seq reveals that CBX4 functions in promoting LUAD proliferation via upregulating PHGDH expression and subsequent serine biosynthesis, while concurrently suppressing LUAD metastasis by inhibiting ZEB2 transcription. CBX4 facilitates PHGDH transcription through the interaction with GCN5, inducing heightened histone acetylation on the PHGDH promoter. Simultaneously, the inhibition of ZEB2 transcription involves CBX4-mediated recruitment of canonical PRC1 (cPRC1), establishing H2K119ub on the ZEB2 promoter. These findings underscore CBX4's pivotal role as a regulator of LUAD progression, emphasizing its diverse transcriptional regulatory functions contingent upon interactions with specific epigenetic partners. Understanding the nuanced interplay between CBX4 and epigenetic factors sheds light on potential therapeutic avenues in LUAD.

InPACT: a computational method for accurate characterization of intronic polyadenylation from RNA sequencing data.

Alternative polyadenylation can occur in introns, termed intronic polyadenylation (IPA), has been implicated in diverse biological processes and diseases, as it can produce noncoding transcripts or transcripts with truncated coding regions. However, a reliable method is required to accurately characterize IPA. Here, we propose a computational method called InPACT, which allows for the precise characterization of IPA from conventional RNA-seq data. InPACT successfully identifies numerous previously unannotated IPA transcripts in human cells, many of which are translated, as evidenced by ribosome profiling data. We have demonstrated that InPACT outperforms other methods in terms of IPA identification and quantification. Moreover, InPACT applied to monocyte activation reveals temporally coordinated IPA events. Further application on single-cell RNA-seq data of human fetal bone marrow reveals the expression of several IPA isoforms in a context-specific manner. Therefore, InPACT represents a powerful tool for the accurate characterization of IPA from RNA-seq data.

A CRISPR-Cas9 library screening identifies CARM1 as a critical inhibitor of ferroptosis in hepatocellular carcinoma cells.

Ferroptosis is an iron-catalyzed form of regulated cell death that results from the accumulation of lipid peroxidation products and reactive oxygen species to a lethal content. However, the transcriptional regulation of ferroptosis is not well understood. Sorafenib, a standard drug for hepatocellular carcinoma (HCC), induces ferroptosis in HCC cells. In this study, we conducted a CRISPR-Cas9 library screening targeting epigenetic factors and identified coactivator-associated arginine methyltransferase 1 (CARM1) as a critical inhibitor of ferroptosis. CARM1 depletion intensified Sorafenib-induced ferroptosis, resulting in decreased cell viability, reduced cellular glutathione level, increased lipid peroxidation, and altered mitochondrial crista structure. Additionally, we investigated a CARM1 inhibitor (CARM1i) as a potential ferroptosis inducer. Combining the CARM1i with Sorafenib enhanced the induction of ferroptosis. Notably, both CARM1 knockdown and CARM1i showed cooperative effects with Sorafenib in inhibiting HCC growth in mice. The underlying mechanism involves CARM1-catalyzed H3R26me2a on the promoter of glutathione peroxidase 4, leading to its transcriptional activation and subsequent ferroptosis inhibition. Furthermore, Sorafenib treatment induced the transcription of CARM1 through the MDM2-p53 axis. In summary, our findings establish CARM1 as a critical ferroptosis inhibitor and highlight the potential of CARM1is as novel ferroptosis inducers, providing promising therapeutic strategies for HCC treatment.

CBX8 promotes lung adenocarcinoma growth and metastasis through transcriptional repression of CDKN2C and SCEL.

Dysregulation of polycomb group (PcG) proteins that mediate epigenetic gene silencing contributes to tumorigenesis. As core components of the polycomb repressive complex 1 (PRC1), chromobox (CBX) proteins recognize H3K27me3 to recruit PRC1 to maintain a repressive transcriptional state. However, the individual biological functions of these CBX proteins in tumorigenesis warrant in-depth investigation. In this study, we analyzed the mRNA expression of CBX family genes across multiple cancers using The Cancer Genome Atlas data and found different expression patterns of the five CBX genes in different types of cancer. This analyses together with the result of immunohistochemistry indicated that CBX8 expression was significantly higher in lung adenocarcinoma (LUAD) tissues compared to adjacent nontumor tissues. Overexpression approaches demonstrated that CBX8 facilitated LUAD cell proliferation and migration in vitro. Consistently, CBX8 knockdown reduced LUAD cell proliferation and migration in both cell culture and mouse models. RNA sequencing combined with real-time RT-PCR assays revealed CDKN2C and SCEL as target genes of CBX8. Furthermore, chromatin immunoprecipitation assays indicated that CBX8 directly bound to the promoters of CDKN2C and SCEL to establish H2AK119ub. CBX8 depletion reduced the enrichment of H2AK119ub on CDKN2C and SCEL promoters. Moreover, depletion of CDKN2C and SCEL restored the repressed growth and invasion ability of LUAD cells caused by CBX8 knockdown. These findings demonstrate that CBX8 promotes LUAD growth and metastasis through the transcriptional repression of CDKN2C and SCEL. Our study uncovers the oncogenic role of CBX8 in LUAD progression and provides a new target for the diagnosis and therapy of LUAD.

HDAC6 deacetylates IDH1 to promote the homeostasis of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are cells mainly present in the bone marrow and capable of forming mature blood cells. However, the epigenetic mechanisms governing the homeostasis of HSPCs remain elusive. Here, we demonstrate an important role for histone deacetylase 6 (HDAC6) in regulating this process. Our data show that the percentage of HSPCs in Hdac6 knockout mice is lower than in wild-type mice due to decreased HSPC proliferation. HDAC6 interacts with isocitrate dehydrogenase 1 (IDH1) and deacetylates IDH1 at lysine 233. The deacetylation of IDH1 inhibits its catalytic activity and thereby decreases the 5-hydroxymethylcytosine level of ten-eleven translocation 2 (TET2) target genes, changing gene expression patterns to promote the proliferation of HSPCs. These findings uncover a role for HDAC6 and IDH1 in regulating the homeostasis of HSPCs and may have implications for the treatment of hematological diseases.

Leveraging aptamers for targeted protein degradation.

Targeted protein degradation (TPD) technologies, particularly proteolysis-targeting chimeras (PROTACs), have emerged as a significant advancement in drug discovery. However, several hurdles - such as the difficulty of identifying suitable ligands for traditionally undruggable proteins, poor solubility and impermeability, nonspecific biodistribution, and on-target off-tissue toxicity - present challenges to their clinical applications. Aptamers are promising ligands for broad-ranging molecular recognition. Utilizing aptamers in TPD has shown potential advantages in overcoming these challenges. Here, we provide an overview of recent developments in aptamer-based TPD, emphasizing their potential to achieve targeted delivery and their promise for the spatiotemporal degradation of undruggable proteins. We also discuss the challenges and future directions of aptamer-based TPD with the goal of facilitating their clinical applications.

A C-terminal glutamine recognition mechanism revealed by E3 ligase TRIM7 structures.

The E3 ligase TRIM7 has emerged as a critical player in viral infection and pathogenesis. However, the mechanism governing the TRIM7-substrate association remains to be defined. Here we report the crystal structures of TRIM7 in complex with 2C peptides of human enterovirus. Structure-guided studies reveal the C-terminal glutamine residue of 2C as the primary determinant for TRIM7 binding. Leveraged by this finding, we identify norovirus and SARS-CoV-2 proteins, and physiological proteins, as new TRIM7 substrates. Crystal structures of TRIM7 in complex with multiple peptides derived from SARS-CoV-2 proteins display the same glutamine-end recognition mode. Furthermore, TRIM7 could trigger the ubiquitination and degradation of these substrates, possibly representing a new Gln/C-degron pathway. Together, these findings unveil a common recognition mode by TRIM7, providing the foundation for further mechanistic characterization of antiviral and cellular functions of TRIM7.

CBX8 Together with SET Facilitates Ovarian Carcinoma Growth and Metastasis by Suppressing the Transcription of SUSD2.

Polycomb group proteins are often dysregulated in cancer, leading to disruption of epigenetic landscapes and acquisition of cancer hallmarks. Chromobox 8 (CBX8) is a core component of canonical polycomb repressive complex 1; however, its role in transcriptional regulation and in ovarian carcinoma progression has not been extensively investigated. In this study, we find that CBX8 is upregulated in ovarian cancer. Overexpression and knockdown approaches show that CBX8 facilitates the growth and migration of CAOV3, A2780, and SKOV3 cells in vitro. Consistently, depletion of CBX8 suppresses the growth and metastasis of ovarian carcinoma in vivo. Mechanistically, RNA-sequencing assays together with functional rescue experiments identify a tumor suppressor, SUSD2, as the functional target of CBX8 in ovarian carcinoma cells. Significantly, FLAG affinity coupled with mass spectrometry discovers that CBX8 interacts with a subunit of inhibitor of acetyltransferases (INHAT), SET, which also promotes the growth and migration of A2780 cells. CBX8 and SET cobind to the promoter of SUSD2 to establish H2AK119ub1 and prevent the acetylation of histone H3, resulting in transcriptional suppression of SUSD2. IMPLICATIONS: Our study uncovers a novel mechanism CBX8 explores to execute gene repression, and provides new therapeutic targets for ovarian carcinoma.

Integrated analysis of genes encoding ATP-dependent chromatin remodellers identifies CHD7 as a potential target for colorectal cancer therapy.

BACKGROUND: Genes participating in chromatin organization and regulation are frequently mutated or dysregulated in cancers. ATP-dependent chromatin remodelers (ATPCRs) play a key role in organizing genomic DNA within chromatin, therefore regulating gene expression. The oncogenic role of ATPCRs and the mechanism involved remains unclear. METHODS: We analyzed the genomic and transcriptional aberrations of the genes encoding ATPCRs in The Cancer Genome Atlas (TCGA) cohort. A series of cellular experiments and mouse tumor-bearing experiments were conducted to reveal the regulatory function of CHD7 on the growth of colorectal cancer cells. RNA-seq and ATAC-seq approaches together with ChIP assays were performed to elucidate the downstream targets and the molecular mechanisms. RESULTS: Our data showed that many ATPCRs represented a high frequency of somatic copy number alterations, widespread somatic mutations, remarkable expression abnormalities, and significant correlation with overall survival, suggesting several somatic driver candidates including chromodomain helicase DNA-binding protein 7 (CHD7) in colorectal cancer. We experimentally demonstrated that CHD7 promotes the growth of colorectal cancer cells in vitro and in vivo. CHD7 can bind to the promoters of target genes to maintain chromatin accessibility and facilitate transcription. We found that CHD7 knockdown downregulates AK4 expression and activates AMPK phosphorylation, thereby promoting the phosphorylation and stability of p53 and leading to the inhibition of the colorectal cancer growth. Our muti-omics analyses of ATPCRs across large-scale cancer specimens identified potential therapeutic targets and our experimental studies revealed a novel CHD7-AK4-AMPK-p53 axis that plays an oncogenic role in colorectal cancer.

CBX2 and EZH2 cooperatively promote the growth and metastasis of lung adenocarcinoma.

The disruption of epigenetic regulation is common in tumors; the abnormal expression of epigenetic factors leads to cancer occurrence and development. In this study, to investigate the potential function of histone methylation regulators in lung adenocarcinoma (LUAD), we performed differential expression analysis using RNA-seq data downloaded from The Cancer Genome Atlas (TCGA) database, and identified CBX2 and EZH2 as obviously upregulated histone methylation regulators. CBX2 knockdown significantly inhibited LUAD cell growth and metastasis in vitro and in vivo. The combined high expression of CBX2 and EZH2 was an indicator of poor prognosis in LUAD. The inhibition of both CBX2 and EZH2 exerted cooperative suppressive effects on the growth and metastasis of LUAD cells. Mechanistically, we revealed that CBX2 and EZH2 downregulated several PPAR signaling pathway genes and tumor suppressor genes through binding to their promoter cooperatively or separately. Furthermore, knockdown of CBX2 improved the therapeutic efficiency of EZH2 inhibitor on A549 cells. Our study reveals the cooperative oncogenic role of CBX2 and EZH2 in promoting LUAD progression, thereby providing potential targets for LUAD diagnosis and therapy.

Ultrafast and scalable variant annotation and prioritization with big functional genomics data.

The advances of large-scale genomics studies have enabled compilation of cell type-specific, genome-wide DNA functional elements at high resolution. With the growing volume of functional annotation data and sequencing variants, existing variant annotation algorithms lack the efficiency and scalability to process big genomic data, particularly when annotating whole-genome sequencing variants against a huge database with billions of genomic features. Here, we develop VarNote to rapidly annotate genome-scale variants in large and complex functional annotation resources. Equipped with a novel index system and a parallel random-sweep searching algorithm, VarNote shows substantial performance improvements (two to three orders of magnitude) over existing algorithms at different scales. It supports both region-based and allele-specific annotations and introduces advanced functions for the flexible extraction of annotations. By integrating massive base-wise and context-dependent annotations in the VarNote framework, we introduce three efficient and accurate pipelines to prioritize the causal regulatory variants for common diseases, Mendelian disorders, and cancers.

JMJD6 modulates DNA damage response through downregulating H4K16ac independently of its enzymatic activity.

The initiation and transduction of DNA damage response (DDR) occur in the context of chromatin, and modifications as well as the structure of chromatin are crucial for DDR signaling. How the profound chromatin alterations are confined to DNA lesions by epigenetic factors remains largely unclear. Here, we discover that JMJD6, a Jumonji C domain-containing protein, is recruited to DNA double-strand breaks (DSBs) after microirradiation. JMJD6 controls the spreading of histone ubiquitination, as well as the subsequent accumulation of repair proteins and transcriptional silencing around DSBs, but does not regulate the initial DNA damage sensing. Furthermore, JMJD6 deficiency results in promotion of the efficiency of nonhomologous end joining (NHEJ) and homologous recombination (HR), rapid cell-cycle checkpoint recovery, and enhanced survival after irradiation. Regarding the mechanism involved, we demonstrate that JMJD6, independently of its catalytic activity, interacts with SIRT1 and recruits it to chromatin to downregulate H4K16ac around DSBs. Our study reveals JMJD6 as a modulator of the epigenome around DNA lesions, and adds to the understanding of the role of epigenetic factors in DNA damage response.

GWAS4D: multidimensional analysis of context-specific regulatory variant for human complex diseases and traits.

Genome-wide association studies have generated over thousands of susceptibility loci for many human complex traits, and yet for most of these associations the true causal variants remain unknown. Tissue/cell type-specific prediction and prioritization of non-coding regulatory variants will facilitate the identification of causal variants and underlying pathogenic mechanisms for particular complex diseases and traits. By leveraging recent large-scale functional genomics/epigenomics data, we develop an intuitive web server, GWAS4D (http://mulinlab.tmu.edu.cn/gwas4d or http://mulinlab.org/gwas4d), that systematically evaluates GWAS signals and identifies context-specific regulatory variants. The updated web server includes six major features: (i) updates the regulatory variant prioritization method with our new algorithm; (ii) incorporates 127 tissue/cell type-specific epigenomes data; (iii) integrates motifs of 1480 transcriptional regulators from 13 public resources; (iv) uniformly processes Hi-C data and generates significant interactions at 5 kb resolution across 60 tissues/cell types; (v) adds comprehensive non-coding variant functional annotations; (vi) equips a highly interactive visualization function for SNP-target interaction. Using a GWAS fine-mapped set for 161 coronary artery disease risk loci, we demonstrate that GWAS4D is able to efficiently prioritize disease-causal regulatory variants.

Exploring genetic associations with ceRNA regulation in the human genome.

Competing endogenous RNAs (ceRNAs) are RNA molecules that sequester shared microRNAs (miRNAs) thereby affecting the expression of other targets of the miRNAs. Whether genetic variants in ceRNA can affect its biological function and disease development is still an open question. Here we identified a large number of genetic variants that are associated with ceRNA's function using Geuvaids RNA-seq data for 462 individuals from the 1000 Genomes Project. We call these loci competing endogenous RNA expression quantitative trait loci or 'cerQTL', and found that a large number of them were unexplored in conventional eQTL mapping. We identified many cerQTLs that have undergone recent positive selection in different human populations, and showed that single nucleotide polymorphisms in gene 3΄UTRs at the miRNA seed binding regions can simultaneously regulate gene expression changes in both cis and trans by the ceRNA mechanism. We also discovered that cerQTLs are significantly enriched in traits/diseases associated variants reported from genome-wide association studies in the miRNA binding sites, suggesting that disease susceptibilities could be attributed to ceRNA regulation. Further in vitro functional experiments demonstrated that a cerQTL rs11540855 can regulate ceRNA function. These results provide a comprehensive catalog of functional non-coding regulatory variants that may be responsible for ceRNA crosstalk at the post-transcriptional level.

FOXK2 Elicits Massive Transcription Repression and Suppresses the Hypoxic Response and Breast Cancer Carcinogenesis.

Although clinically associated with severe developmental defects, the biological function of FOXK2 remains poorly explored. Here we report that FOXK2 interacts with transcription corepressor complexes NCoR/SMRT, SIN3A, NuRD, and REST/CoREST to repress a cohort of genes including HIF1ß and EZH2 and to regulate several signaling pathways including the hypoxic response. We show that FOXK2 inhibits the proliferation and invasion of breast cancer cells and suppresses the growth and metastasis of breast cancer. Interestingly, FOXK2 is transactivated by ERα and transrepressed via reciprocal successive feedback by HIF1ß/EZH2. Significantly, the expression of FOXK2 is progressively lost during breast cancer progression, and low FOXK2 expression is strongly correlated with higher histologic grades, positive lymph nodes, and ERα-/PR-/HER2- status, all indicators of poor prognosis.

DOT1L promotes angiogenesis through cooperative regulation of VEGFR2 with ETS-1.

Histone methyltransferase DOT1L is implicated in various biological processes including cell proliferation, differentiation and embryogenesis. Gene ablation of Dot1l results in embryonic lethality and cardiovascular defects including decreased vasculature. However, how DOT1L might contribute to the development of vasculature is not clear. Here, we report that DOT1L is required for angiogenesis. We demonstrated that silencing of DOT1L in human umbilical vein endothelial cells (HUVECs) leads to decreased cell viability, migration, tube formation, and capillary sprout formation in vitro, as well as reduced formation of functional vascular networks in matrigel plugs in vivo. Genome-wide analysis of DOT1L targets via H3K79me2 ChIP-seq annotation in HUVECs identified a number of genes including VEGFR2 that are critically involved in angiogenesis. We showed that DOT1L cooperates with transcription factor ETS-1 to stimulate the expression of VEGFR2, thereby activating ERK1/2 and AKT signaling pathways and promoting angiogenesis. Our study revealed a mechanistic role for DOT1L in the promotion of angiogenesis, adding to the understanding of the biological function of this histone methyltransferase.

Ubiquitin ligase RNF20/40 facilitates spindle assembly and promotes breast carcinogenesis through stabilizing motor protein Eg5.

Whether transcriptional regulators are functionally involved in mitosis is a fundamental question in cell biology. Here we report that the RNF20/40 complex, a major ubiquitin ligase catalysing histone H2B monoubiquitination, interacts with the motor protein Eg5 during mitosis and participates in spindle assembly. We show that the RNF20/40 complex monoubiquitinates and stabilizes Eg5. Loss of RNF20/40 results in spindle assembly defects, cell cycle arrest and apoptosis. Consistently, depletion of either RNF20/40 or Eg5 suppresses breast cancer in vivo. Significantly, RNF20/40 and Eg5 are concurrently upregulated in human breast carcinomas and high Eg5 expression is associated with poorer overall survival of patients with luminal A, or B, breast cancer. Our study uncovers an important spindle assembly role of the RNF20/40 complex, and implicates the RNF20/40-Eg5 axis in breast carcinogenesis, supporting the pursuit of these proteins as potential targets for breast cancer therapeutic interventions.

Dysfunction of the Reciprocal Feedback Loop between GATA3- and ZEB2-Nucleated Repression Programs Contributes to Breast Cancer Metastasis.

How loss-of-function of GATA3 contributes to the development of breast cancer is poorly understood. Here, we report that GATA3 nucleates a transcription repression program composed of G9A and MTA3-, but not MTA1- or MTA2-, constituted NuRD complex. Genome-wide analysis of the GATA3/G9A/NuRD(MTA3) targets identified a cohort of genes including ZEB2 that are critically involved in epithelial-to-mesenchymal transition and cell invasion. We demonstrate that the GATA3/G9A/NuRD(MTA3) complex inhibits the invasive potential of breast cancer cells in vitro and suppresses breast cancer metastasis in vivo. Strikingly, the expression of GATA3, G9A, and MTA3 is concurrently downregulated during breast cancer progression, leading to an elevated expression of ZEB2, which, in turn, represses the expression of G9A and MTA3 through the recruitment of G9A/NuRD(MTA1).