Arne Klungland

Glioblastomas are the most prevalent and aggressive malignant brain tumors, characterized by hypertranscription and dependence on neurodevelopmental transcription factors. The transcriptional cycle is regulated by phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII) by transcriptional cyclin-dependent kinases (tCDKs), including CDK7, CDK9, CDK12, and CDK13. Here we find that glioblastoma stem cells (GSCs) are selectively sensitive to CDK12/CDK13 inhibition, whereas CDK7 and CDK9 inhibition cause non-specific cytotoxicity. This selective targeting halts GSC and organoid proliferation, curtails GSC invasion and suppresses tumor growth in a xenograft mouse model. In GSCs, CDK12/CDK13 inhibition leads to a rapid and genome-wide loss of serine-2 phosphorylation (pSer2) of the RNAPII CTD, abolishing transcriptional elongation and a transcriptional program sustained by key neurodevelopmental transcription factors. CDK12/CDK13 inhibition unexpectedly arrests DNA replication and replication fork progression in a manner distinct from the effect of inhibiting other tCDKs. This dramatic arrest precedes DNA damage response activation and cell cycle arrest, directly linking RNAPII elongation to replication fork dynamics and revealing a previously unrecognized dependence of DNA replication on CDK12/CDK13-RNAPII regulation.

Epigenomes of mammalian oocytes and embryos undergo major transitions essential for successful development. Here, we provide genome-wide maps of histone variant H2A.Z during twelve stages of mouse oogenesis and preimplantation embryo development and relate it to histone marks and genomic features. This revealed that major waves of H2A.Z incorporation occur early in growing oocytes, forming distinct patterns of maternal, embryonic, and persistent H2A.Z enrichment. Late maternal enrichment is inherited by the zygote and precedes reduced formation of lamina associated domains and early replication in the maternal genome of 2-cell embryos. Persistent H2A.Z enrichment is strongly associated with CpG islands and H3K4me3 near transcription start sites of active genes, but thousands of maternal and embryonic H2A.Z incorporation sites exist elsewhere, frequently at transposable elements. The persisting H2A.Z enrichments across related developmental stages enable preservation of epigenetic information despite major concurrent changes in H3K4me3, H3K27me3, and DNA methylation. Altogether, this advances our understanding of how histone variants contribute to epigenetic reprogramming during mammalian oogenesis and early development.

The dentate gyrus (DG), a crucial region of the hippocampus responsible for learning, spatial encoding, and memory formation, undergoes its main development and maturation after birth. Despite its importance, the regulatory mechanisms underlying postnatal DG development remain poorly understood. This study is aimed to investigate the role of H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 27 trimethylation (H3K27me3) in the development and function of the postnatal DG. We show robust enrichment of H3K4me3 in the subgranular zone (SGZ), a primary neurogenic region, while high levels of H3K27me3 were mainly presented in granule cell layer. Enhanced H3K4me3 level facilitated proliferation and development of neonatal mouse neural stem cells (NSCs), promoted differentiation towards GABA neurons, as well as improved mouse spatial learning and memory. Enhancing H3K27me3 level exerts the opposite function, additionally promoting NSCs entry into a quiescent-like state. During the neuronal differentiation of NSCs, the integration of RNA-Seq and ChIP-Seq datasets reveals that H3K4me3 and H3K27me3 co-regulate the expression of genes essential for neural development, such as Gli1, through the formation of bivalent domains. Manipulation activation of the Shh/Gli1 pathway abolishes the effect of alterations in the levels of H3K4me3 and H3K27me3 in NSCs. Based on these findings, we propose that H3K4me3 and H3K27me3 serve as molecular “switches” to dynamically regulate NSCs proliferation and differentiation and in turn, influence the postnatal developmental progression of DG, additionally to provide potential therapeutic targets for treating diseases associated with abnormal hippocampal development. During dentate gyrus development in neonatal mice, the active transcription mark H3K4me3 and the repressive mark H3K27me3 are co-localized at the promoter regions of essential neurodevelopmental genes, and thus forming bivalent chromatin domains in neural stem cells. These domains serve as a “molecular switch” that regulates the dynamic processes of cell proliferation and differentiation. The enhanced ratio of H3K4me3 to H3K27me3 markedly upregulates the expression related genes, thereby promoting cell proliferation and neuronal differentiation, ultimately leading to improved spatial learning and memory. Conversely, decreasing this ratio has the opposite effect.

Hypoxic Ischemic Encephalopathy (HIE) causes brain damage, ranging from mild to severe, in up to one million newborns worldwide every year. While HIE has been shown to modulate gene expression through epigenetic regulation, studies have primarily focused on limited loci, leaving a gap in genome-wide understanding. Using a neonatal mouse model that recapitulates human HIE pathology, we comprehensively profiled gene expression, DNA secondary structures, and histone modifications in the hippocampi of control, hypoxic, and hypoxic-ischemic mice. Our results demonstrate that both hypoxic and hypoxic-ischemic conditions reshape the hippocampal G-quadruplexes (G4s) landscape during the acute phase, following a strong transcriptional response in the hypoxic-ischemic hippocampi, whereas the hypoxic part displays a very moderate response. Substantial upregulation of stress-responsive genes in hypoxic-ischemic hippocampi correlated with G4 loss and the enrichment of active histone marks (H3K4me3 and H3K27ac) at promoters and CpG islands. Brain-specific enhancers also showed G4s depletion alongside asymmetric H3K27ac enrichment, suggesting a broader role for G4s in regulating mRNA expression and processing. Vulnerable hippocampal neurons (CA1/CA3) displayed pronounced G4 depletion following insult, while resilient neurons maintained low G4 occupancy. Together, these findings highlight the neuroprotective potential of G4s during acute neonatal hypoxia-ischemia, suggesting new directions for biomarker discovery and therapeutic development.

RNA N ⁶ -methyladenosine (m ⁶ A, m6A) modification is a critical regulator for a range of physiological processes. However, the dynamic m ⁶ A profiles within human preimplantation embryos remain uncharacterized. Here, we present the first RNA m ⁶ A landscape of single human oocytes and early embryos. Comparative analyses with mouse data reveal an intriguing divergence during the window of zygotic genome activation. m ⁶ A-modified genes are involved in regulation of gene transcription, while unmodified genes are mainly associated with basic metabolic processes. Maternal decay mRNAs exhibit a propensity for m ⁶ A modifications, and these genes are targeted by miRNAs. m ⁶ A modified genes that are constantly expressed across all stages demonstrate higher translation efficiency. Moreover, we observe frequent m ⁶ A enrichment on stage-specifically expressed retrotransposons, particularly within young subfamilies. m ⁶ A inhibitor leads to m ⁶ A erasure on massive retrotransposons. In summary, this study provides a resource to broaden our understanding about the regulatory roles of m ⁶ A during early human embryo development.

Background: Bladder cancer (BLCA) is a prevalent and life-threatening condition that significantly impacts patients’ quality of life while imposing substantial financial costs on healthcare systems. Advancing our knowledge of the mechanisms underlying tumor development is crucial for improving treatment outcomes. Emerging studies emphasize the critical role of the RNA modification 6-methyladenine (m⁶A) and its associated proteins, methyltransferase-like 3 (METTL3), Vir-like m⁶A methyltransferase associated (VIRMA) (writers), Alkb homolog 5 (ALKBH5) and fat mass and obesity associated protein (FTO) (erasers), in maintaining m⁶A homeostasis. Dysregulation of these enzymes leads to aberrant m⁶A methylation, a hallmark of various cancers, including BLCA. Furthermore, m⁶A modifications influence cisplatin sensitivity, a key drug in muscle-invasive bladder cancer (MIBC) treatment. With this background, we investigated the combined effects of ALKBH5 and FTO knock-down in bladder tumor cell lines. Methods: We first investigated the expression of METTL3, VIRMA, ALKBH5 and FTO in BLCA tissues and human bladder tumor cell lines from urinary cancer cells by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Simultaneous knock-down of the expression of the erasers was then performed to explore their consequences in bladder cells. We then conducted cisplatin and mitomycin (MMC) treatment in knock-down cells to decipher the effect of their reduction. The cell viability was evaluated with cell counting kit-8 (CCK-8) assay after the two treatment regimes. Results: Lower expression of ALKBH5 and FTO was identified in BLCA tissue and bladder tumor cell lines. Notably, this trend was consistent across both low-grade and high-grade tissue samples. Furthermore, lower expression levels of ALKBH5 and FTO were observed in tumor cell lines derived from both men and women compared to the non-tumorigenic SV-HUC1 cell line. In contrast, both tissue and cell line data revealed an increased expression tendency of the m⁶A writers METTL3 and VIRMA. Additionally, knock-down of the two m⁶A erasers was found to enhance tolerance to cisplatin and MMC treatment, resulting in increased resistance to cell death. Conclusions: Our findings reveal that ALKBH5 and FTO are down-regulated in BLCA and their knock-down confers resistance to cisplatin and MMC in vitro. This suggests that m⁶A erasers play a critical role in modulating chemotherapy sensitivity, potentially serving as biomarkers or therapeutic targets for enhancing treatment efficacy in BLCA.

Dentate gyrus (DG) is part of the hippocampus formation and primarily mature after birth. It undergoes rapid early development followed by a protracted developmental process and is crucial for learning and memory. This unique developmental trajectory challenge mechanistic studies and our understanding of its regulatory processes are limited. Our study demonstrates that histone H3K4me3 and H3K27me3 are essential in regulating DG development. H3K4me3, which is enriched in the subgranular zone, promotes the proliferation and differentiation of neural stem cells (NSCs) into GABAergic neurons, thereby enhancing spatial learning and memory in mice. Conversely, enhancing H3K27me3 levels exert the opposite effect, additionally driving NSCs into a quiescent-like state. Furthermore, we show that H3K4me3 and H3K27me3 co-regulate the expression of genes essential for neural development, such as GLI1, through the formation of bivalent domains. These findings indicate that H3K4me3 and H3K27me3 act as molecular “switches” governing DG development.

Current N⁶-methyladenosine (m⁶A) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale m⁶A RNA immunoprecipitation and sequencing (picoMeRIP–seq) for studying m⁶A in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark m⁶A mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos.

Despite the significance of N⁶-methyladenosine (m⁶A) in gene regulation, the requirement for large amounts of RNA has hindered m⁶A profiling in mammalian early embryos. Here we apply low-input methyl RNA immunoprecipitation and sequencing to map m⁶A in mouse oocytes and preimplantation embryos. We define the landscape of m⁶A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m⁶A. In contrast to m⁶A-marked zygotic ally-activated genes, m⁶A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m⁶A. Our results provide a foundation for future studies exploring the regulatory roles of m⁶A in mammalian early embryonic development.

RNA modifications are important regulatory elements of RNA functions. However, most genome-wide mapping of RNA modifications has focused on messenger RNAs and transfer RNAs, but such datasets have been lacking for small RNAs. Here we mapped N¹-methyladenosine (m¹A) in the cellular small RNA space. Benchmarked with synthetic m¹A RNAs, our workflow identified specific groups of m¹A-containing small RNAs, which are otherwise disproportionally under-represented. In particular, 22-nucleotides long 3′ tRNA-fragments are highly enriched for TRMT6/61A-dependent m¹A located within the seed region. TRMT6/61A-dependent m¹A negatively affects gene silencing by tRF-3s. In urothelial carcinoma of the bladder, where TRMT6/61A is over-expressed, higher m¹A modification on tRFs is detected, correlated with a dysregulation of tRF targetome. Lastly, TRMT6/61A regulates tRF-3 targets involved in unfolded protein response. Together, our results reveal a mechanism of regulating gene expression via base modification of small RNA.