METTL8 links mt-tRNA m3C modification to the HIF1α/RTK/Akt axis to sustain GBM stemness and tumorigenicity.

Epitranscriptomic RNA modifications are crucial for the maintenance of glioma stem cells (GSCs), the most malignant cells in glioblastoma (GBM). 3-methylcytosine (m3C) is a new epitranscriptomic mark on RNAs and METTL8 represents an m3C writer that is dysregulated in cancer. Although METTL8 has an established function in mitochondrial tRNA (mt-tRNA) m3C modification, alternative splicing of METTL8 can also generate isoforms that localize to the nucleolus where they may regulate R-loop formation. The molecular basis for METTL8 dysregulation in GBM, and which METTL8 isoform(s) may influence GBM cell fate and malignancy remain elusive. Here, we investigated the role of METTL8 in regulating GBM stemness and tumorigenicity. In GSC, METTL8 is exclusively localized to the mitochondrial matrix where it installs m3C on mt-tRNAThr/Ser(UCN) for mitochondrial translation and respiration. High expression of METTL8 in GBM is attributed to histone variant H2AZ-mediated chromatin accessibility of HIF1α and portends inferior glioma patient outcome. METTL8 depletion impairs the ability of GSC to self-renew and differentiate, thus retarding tumor growth in an intracranial GBM xenograft model. Interestingly, METTL8 depletion decreases protein levels of HIF1α, which serves as a transcription factor for several receptor tyrosine kinase (RTK) genes, in GSC. Accordingly, METTL8 loss inactivates the RTK/Akt axis leading to heightened sensitivity to Akt inhibitor treatment. These mechanistic findings, along with the intimate link between METTL8 levels and the HIF1α/RTK/Akt axis in glioma patients, guided us to propose a HIF1α/Akt inhibitor combination which potently compromises GSC proliferation/self-renewal in vitro. Thus, METTL8 represents a new GBM dependency that is therapeutically targetable.

PHF2 regulates genome topology and DNA replication in neural stem cells via cohesin.

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs), which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here, we discover that the histone demethylase PHF2 associates with RAD21, a core subunit of cohesin, to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably, the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient, active replication origins, and can act as boundaries to separate adjacent domains. Accordingly, PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore, we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation, and that PHF2's histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops, where cohesin represses dormant replication origins directly or indirectly, to sustain DNA replication in NSC.

Telomere shortening induces aging-associated phenotypes in hiPSC-derived neurons and astrocytes.

Telomere shortening is a well-established hallmark of cellular aging. Telomerase reverse transcriptase (TERT) plays a crucial role in maintaining the length of telomeres, which are specialised protective caps at the end of chromosomes. The lack of in vitro aging models, particularly for the central nervous system (CNS), has impeded progress in understanding aging and age-associated neurodegenerative diseases. In this study, we aimed to explore the possibility of inducing aging-associated features in cell types of the CNS using hiPSC (human induced pluripotent stem cell) technology. To achieve this, we utilised CRISPR/Cas9 to generate hiPSCs with a loss of telomerase function and shortened telomeres. Through directed differentiation, we generated motor neurons and astrocytes to investigate whether telomere shortening could lead to age-associated phenotypes. Our findings revealed that shortened telomeres induced age-associated characteristics in both motor neurons and astrocytes including increased cellular senescence, heightened inflammation, and elevated DNA damage. We also observed cell-type specific age-related morphology changes. Additionally, our study highlighted the fundamental role of TERT and telomere shortening in neural progenitor cell (NPC) proliferation and neuronal differentiation. This study serves as a proof of concept that telomere shortening can effectively induce aging-associated phenotypes, thereby providing a valuable tool to investigate age-related decline and neurodegenerative diseases.

Transcriptional repression by a secondary DNA binding surface of DNA topoisomerase I safeguards against hypertranscription.

Regulation of global transcription output is important for normal development and disease, but little is known about the mechanisms involved. DNA topoisomerase I (TOP1) is an enzyme well-known for its role in relieving DNA supercoils for enabling transcription. Here, we report a non-enzymatic function of TOP1 that downregulates RNA synthesis. This function is dependent on specific DNA-interacting residues located on a conserved protein surface. A loss-of-function knock-in mutation on this surface, R548Q, is sufficient to cause hypertranscription and alter differentiation outcomes in mouse embryonic stem cells (mESCs). Hypertranscription in mESCs is accompanied by reduced TOP1 chromatin binding and change in genomic supercoiling. Notably, the mutation does not impact TOP1 enzymatic activity; rather, it diminishes TOP1-DNA binding and formation of compact protein-DNA structures. Thus, TOP1 exhibits opposing influences on transcription through distinct activities which are likely to be coordinated. This highlights TOP1 as a safeguard of appropriate total transcription levels in cells.

CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established DNA damage response protein, has been implicated in mitotic checkpoint regulation but its exact role remains poorly understood. Here, RNF8 proximity proteomics uncovered a role of RNF8-MAD2 in generating the mitotic checkpoint signal. Specifically, RNF8 competes with a small pool of p31comet for binding to the closed conformer of MAD2 via its RING domain, while CAMK2D serves as a molecular scaffold to concentrate the RNF8-MAD2 complex via transient/weak interactions between its p-Thr287 and RNF8's FHA domain. Accordingly, RNF8 overexpression impairs glioma stem cell (GSC) mitotic progression in a FHA- and RING-dependent manner. Importantly, low RNF8 expression correlates with inferior glioma outcome and RNF8 overexpression impedes GSC tumorigenicity. Last, we identify PLK1 inhibitor that mimics RNF8 overexpression using a chemical biology approach, and demonstrate a PLK1/HSP90 inhibitor combination that synergistically reduces GSC proliferation and stemness. Thus, our study has unveiled a previously unrecognized CAMK2D-RNF8-MAD2 complex in regulating mitotic checkpoint with relevance to gliomas, which is therapeutically targetable.

E2F and STAT3 provide transcriptional synergy for histone variant H2AZ activation to sustain glioblastoma chromatin accessibility and tumorigenicity.

The histone variant H2AZ is overexpressed in diverse cancer types where it facilitates the accessibility of transcriptional regulators to the promoters of cell cycle genes. However, the molecular basis for its dysregulation in cancer remains unknown. Here, we report that glioblastomas (GBM) and glioma stem cells (GSCs) preferentially overexpress H2AZ for their proliferation, stemness and tumorigenicity. Chromatin accessibility analysis of H2AZ2 depleted GSC revealed that E2F1 occupies the enhancer region within H2AZ2 gene promoter, thereby activating H2AZ2 transcription. Exploration of other H2AZ2 transcriptional activators using a customized "anti-H2AZ2" query signature for connectivity map analysis identified STAT3. Co-targeting E2F and STAT3 synergistically reduced the levels of H2AZ, histone 3 lysine 27 acetylation (H3K27ac) and cell cycle gene transcription, indicating that E2F1 and STAT3 synergize to activate H2AZ gene transcription in GSCs. Remarkably, an E2F/STAT3 inhibitor combination durably suppresses GSC tumorigenicity in an orthotopic GBM xenograft model. In glioma patients, high STAT3 signaling is associated with high E2F1 and H2AZ2 expression. Thus, GBM has uniquely opted the use of E2F1- and STAT3-containing "enhanceosomes" that integrate multiple signaling pathways to achieve H2AZ gene activation, supporting a translational path for the E2F/STAT3 inhibitor combination to be applied in GBM treatment.

A chemical biology approach reveals a dependency of glioblastoma on biotin distribution.

Glioblastoma (GBM) is a uniformly lethal disease driven by glioma stem cells (GSCs). Here, we use a chemical biology approach to unveil previously unknown GBM dependencies. By studying sulconazole (SN) with anti-GSC properties, we find that SN disrupts biotin distribution to the carboxylases and histones. Transcriptomic and metabolomic analyses of SN-treated GSCs reveal metabolic alterations that are characteristic of biotin-deficient cells, including intracellular cholesterol depletion, impairment of oxidative phosphorylation, and energetic crisis. Furthermore, SN treatment reduces histone biotinylation, histone acetylation, and expression of superenhancer-associated GSC critical genes, which are also observed when biotin distribution is genetically disrupted by holocarboxylase synthetase (HLCS) depletion. HLCS silencing impaired GSC tumorigenicity in an orthotopic xenograft brain tumor model. In GBM, high HLCS expression robustly indicates a poor prognosis. Thus, the dependency of GBM on biotin distribution suggests that the rational cotargeting of biotin-dependent metabolism and epigenetic pathways may be explored for GSC eradication.

Tumor-adjacent tissue co-expression profile analysis reveals pro-oncogenic ribosomal gene signature for prognosis of resectable hepatocellular carcinoma.

Currently, molecular markers are not used when determining the prognosis and treatment strategy for patients with hepatocellular carcinoma (HCC). In the present study, we proposed that the identification of common pro-oncogenic pathways in primary tumors (PT) and adjacent non-malignant tissues (AT) typically used to predict HCC patient risks may result in HCC biomarker discovery. We examined the genome-wide mRNA expression profiles of paired PT and AT samples from 321 HCC patients. The workflow integrated differentially expressed gene selection, gene ontology enrichment, computational classification, survival predictions, image analysis and experimental validation methods. We developed a 24-ribosomal gene-based HCC classifier (RGC), which is prognostically significant in both PT and AT. The RGC gene overexpression in PT was associated with a poor prognosis in the training (hazard ratio = 8.2, P = 9.4 × 10-6 ) and cross-cohort validation (hazard ratio = 2.63, P = 0.004) datasets. The multivariate survival analysis demonstrated the significant and independent prognostic value of the RGC. The RGC displayed a significant prognostic value in AT of the training (hazard ratio = 5.0, P = 0.03) and cross-validation (hazard ratio = 1.9, P = 0.03) HCC groups, confirming the accuracy and robustness of the RGC. Our experimental and bioinformatics analyses suggested a key role for c-MYC in the pro-oncogenic pattern of ribosomal biogenesis co-regulation in PT and AT. Microarray, quantitative RT-PCR and quantitative immunohistochemical studies of the PT showed that DKK1 in PT is the perspective biomarker for poor HCC outcomes. The common co-transcriptional pattern of ribosome biogenesis genes in PT and AT from HCC patients suggests a new scalable prognostic system, as supported by the model of tumor-like metabolic redirection/assimilation in non-malignant AT. The RGC, comprising 24 ribosomal genes, is introduced as a robust and reproducible prognostic model for stratifying HCC patient risks. The adjacent non-malignant liver tissue alone, or in combination with HCC tissue biopsy, could be an important target for developing predictive and monitoring strategies, as well as evidence-based therapeutic interventions, that aim to reduce the risk of post-surgery relapse in HCC patients.

Sense-antisense gene-pairs in breast cancer and associated pathological pathways.

More than 30% of human protein-coding genes form hereditary complex genome architectures composed of sense-antisense (SA) gene pairs (SAGPs) transcribing their RNAs from both strands of a given locus. Such architectures represent important novel components of genome complexity contributing to gene expression deregulation in cancer cells. Therefore, the architectures might be involved in cancer pathways and, in turn, be used for novel drug targets discovery. However, the global roles of SAGPs in cancer pathways has not been studied. Here we investigated SAGPs associated with breast cancer (BC)-related pathways using systems biology, prognostic survival and experimental methods. Gene expression analysis identified 73 BC-relevant SAGPs that are highly correlated in BC. Survival modelling and metadata analysis of the 1161 BC patients allowed us to develop a novel patient prognostic grouping method selecting the 12 survival-significant SAGPs. The qRT-PCR-validated 12-SAGP prognostic signature reproducibly stratified BC patients into low- and high-risk prognostic subgroups. The 1381 SAGP-defined differentially expressed genes common across three studied cohorts were identified. The functional enrichment analysis of these genes revealed the GABPA gene network, including BC-relevant SAGPs, specific gene sets involved in cell cycle, spliceosomal and proteasomal pathways. The co-regulatory function of GABPA in BC cells was supported using siRNA knockdown studies. Thus, we demonstrated SAGPs as the synergistically functional genome architectures interconnected with cancer-related pathways and associated with BC patient clinical outcomes. Taken together, SAGPs represent an important component of genome complexity which can be used to identify novel aspects of coordinated pathological gene networks in cancers.

Contrasting expression patterns of coding and noncoding parts of the human genome upon oxidative stress.

Oxidative stress (OS) is caused by an imbalance between pro- and anti-oxidant reactions leading to accumulation of reactive oxygen species within cells. We here investigate the effect of OS on the transcriptome of human fibroblasts. OS causes a rapid and transient global induction of transcription characterized by pausing of RNA polymerase II (PolII) in both directions, at specific promoters, within 30 minutes of the OS response. In contrast to protein-coding genes, which are commonly down-regulated, this novel divergent, PolII pausing-phenomenon leads to the generation of thousands of long noncoding RNAs (lncRNAs) with promoter-associated antisense lncRNAs transcripts (si-paancRNAs) representing the major group of stress-induced transcripts. OS causes transient dynamics of si-lncRNAs in nucleus and cytosol, leading to their accumulation at polysomes, in contrast to mRNAs, which get depleted from polysomes. We propose that si-lncRNAs represent a novel component of the transcriptional stress that is known to determine the outcome of immediate-early and later cellular stress responses and we provide insights on the fate of those novel mature lncRNA transcripts by showing that their association with polysomal complexes is significantly increased in OS.

Complex sense-antisense architecture of TNFAIP1/POLDIP2 on 17q11.2 represents a novel transcriptional structural-functional gene module involved in breast cancer progression.

BACKGROUND: A sense-antisense gene pair (SAGP) is a gene pair where two oppositely transcribed genes share a common nucleotide sequence region. In eukaryotic genomes, SAGPs can be organized in complex sense-antisense architectures (CSAGAs) in which at least one sense gene shares loci with two or more antisense partners. As shown in several case studies, SAGPs may be involved in cancers, neurological diseases and complex syndromes. However, CSAGAs have not yet been characterized in the context of human disease or cancer. RESULTS: We characterize five genes (TMEM97, IFT20, TNFAIP1, POLDIP2 and TMEM199) organized in a CSAGA on 17q11.2 (we term this the TNFAIP1/POLDIP2 CSAGA) and demonstrate their strong and reproducible co-regulatory transcription pattern in breast cancer tumours. Genes of the TNFAIP1/POLDIP2 CSAGA are located inside the smallest region of recurrent amplification on 17q11.2 and their expression profile correlates with the DNA copy number of the region. Survival analysis of a group of 410 breast cancer patients revealed significant survival-associated individual genes and gene pairs in the TNFAIP1/POLDIP2 CSAGA. Moreover, several of the gene pairs associated with survival, demonstrated synergistic effects. Expression of genes-members of the TNFAIP1/POLDIP2 CSAGA also strongly correlated with expression of genes of ERBB2 core region of recurrent amplification on 17q12. We clearly demonstrate that the observed co-regulatory transcription profile of the TNFAIP1/POLDIP2 CSAGA is maintained not only by a DNA amplification mechanism, but also by chromatin remodelling and local transcription activation. CONCLUSION: We have identified a novel TNFAIP1/POLDIP2 CSAGA and characterized its co-regulatory transcription profile in cancerous breast tissues. We suggest that the TNFAIP1/POLDIP2 CSAGA represents a clinically significant transcriptional structural-functional gene module associated with amplification of the genomic region on 17q11.2 and correlated with expression ERBB2 amplicon core genes in breast cancer. Co-expression pattern of this module correlates with histological grades and a poor prognosis in breast cancer when over-expressed. TNFAIP1/POLDIP2 CSAGA maps the risks of breast cancer relapse onto the complex genomic locus on 17q11.2.

Integrative analysis of the human cis-antisense gene pairs, miRNAs and their transcription regulation patterns.

Cis-antisense gene pairs (CASGPs) can transcribe mRNAs from an opposite strand of a given locus. To classify and understand diverse CASGP phenomena in the human we compiled a genome-wide catalog of CASGPs and integrated these sequences with microarray, SAGE and miRNA data. Using the concept of overlapping regions and clustering of SA transcripts by chromosome coordinates, we identified up to 9000 overlapping antisense loci. Four thousand three hundred and seventy-four of these CASGPs form 1759 complex gene architectures. We found that approximately 35% (6347/18160) of RefSeq genes are overlapped with the antisense transcripts. About 30% of Affymetrix U133 microarray initial sequences map transcripts of approximately 35% CASGPs and reveal mostly concordant expression in CASGPs. We found strong significant overrepresentation of human miRNA genes in loci of CASGPs. We developed a data-driven model of cross-talk between co-expressed CASGPs and DICER1-mediated miRNA pathway in normal spermatogenesis and in severe teratozoospermia. Specifically, we revealed complex SA structural-functional gene module composing the protein-coding genes, WDR6, DALRD3, NDUFAF3 and ncRNA precursors, mir-425 and mir-191, which could provide downregulation of ncRNA pathway via direct targeting DICER1 and basonuclin 2 transcripts by mir-425 and mir-191 in normal spermatogenesis, but this mechanism is switched off in severe teratozoospermia. The database is available from http://globalisland.bii.a-star.edu.sg/ approximately jiangtao/sas/index3.php?link =about.