ZFP207 sustains pluripotency by coordinating OCT4 stability, alternative splicing and RNA export.

The pluripotent state is not solely governed by the action of the core transcription factors OCT4, SOX2, and NANOG, but also by a series of co-transcriptional and post-transcriptional events, including alternative splicing (AS) and the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs. Zinc Finger Protein 207 (ZFP207) is an essential transcription factor for mammalian embryonic development. Here, we employ multiple functional analyses to characterize its role in mouse embryonic stem cells (ESCs). We find that ZFP207 plays a pivotal role in ESC maintenance, and silencing of Zfp207 leads to severe neuroectodermal differentiation defects. In striking contrast to human ESCs, mouse ZFP207 does not transcriptionally regulate neuronal and stem cell-related genes but exerts its effects by controlling AS networks and by acting as an RBP. Our study expands the role of ZFP207 in maintaining ESC identity, and underscores the functional versatility of ZFP207 in regulating neural fate commitment.

MODOMICS: a database of RNA modification pathways. 2021 update.

The MODOMICS database has been, since 2006, a manually curated and centralized resource, storing and distributing comprehensive information about modified ribonucleosides. Originally, it only contained data on the chemical structures of modified ribonucleosides, their biosynthetic pathways, the location of modified residues in RNA sequences, and RNA-modifying enzymes. Over the years, prompted by the accumulation of new knowledge and new types of data, it has been updated with new information and functionalities. In this new release, we have created a catalog of RNA modifications linked to human diseases, e.g., due to mutations in genes encoding modification enzymes. MODOMICS has been linked extensively to RCSB Protein Data Bank, and sequences of experimentally determined RNA structures with modified residues have been added. This expansion was accompanied by including nucleotide 5'-monophosphate residues. We redesigned the web interface and upgraded the database backend. In addition, a search engine for chemically similar modified residues has been included that can be queried by SMILES codes or by drawing chemical molecules. Finally, previously available datasets of modified residues, biosynthetic pathways, and RNA-modifying enzymes have been updated. Overall, we provide users with a new, enhanced, and restyled tool for research on RNA modification. MODOMICS is available at https://iimcb.genesilico.pl/modomics/.

A mark of disease: how mRNA modifications shape genetic and acquired pathologies.

RNA modifications have recently emerged as a widespread and complex facet of gene expression regulation. Counting more than 170 distinct chemical modifications with far-reaching implications for RNA fate, they are collectively referred to as the epitranscriptome. These modifications can occur in all RNA species, including messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). In mRNAs the deposition, removal, and recognition of chemical marks by writers, erasers and readers influence their structure, localization, stability, and translation. In turn, this modulates key molecular and cellular processes such as RNA metabolism, cell cycle, apoptosis, and others. Unsurprisingly, given their relevance for cellular and organismal functions, alterations of epitranscriptomic marks have been observed in a broad range of human diseases, including cancer, neurological and metabolic disorders. Here, we will review the major types of mRNA modifications and editing processes in conjunction with the enzymes involved in their metabolism and describe their impact on human diseases. We present the current knowledge in an updated catalog. We will also discuss the emerging evidence on the crosstalk of epitranscriptomic marks and what this interplay could imply for the dynamics of mRNA modifications. Understanding how this complex regulatory layer can affect the course of human pathologies will ultimately lead to its exploitation toward novel epitranscriptomic therapeutic strategies.

ZNF217/ZFP217 Meets Chromatin and RNA.

The Kruppel-like transcription factor zinc finger protein (ZNF)217 (mouse homolog ZFP217) contributes to tumorigenesis by dysregulating gene expression programs. The newly discovered molecular function of ZFP217 in controlling N6-methyladenosine (m6A) deposition in embryonic stem cells (ESCs) sheds new light on the role of this transcription factor in tumor development.

The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer.

We observed overexpression and increased intra-nuclear accumulation of the PRMT5/WDR77 in breast cancer cell lines relative to immortalized breast epithelial cells. Utilizing mass spectrometry and biochemistry approaches we identified the Zn-finger protein ZNF326, as a novel interaction partner and substrate of the nuclear PRMT5/WDR77 complex. ZNF326 is symmetrically dimethylated at arginine 175 (R175) and this modification is lost in a PRMT5 and WDR77-dependent manner. Loss of PRMT5 or WDR77 in MDA-MB-231 cells leads to defects in alternative splicing, including inclusion of A-T rich exons in target genes, a phenomenon that has previously been observed upon loss of ZNF326. We observed that the alternatively spliced transcripts of a subset of these genes, involved in proliferation and tumor cell migration like REPIN1/AP4, ST3GAL6, TRNAU1AP and PFKM are degraded upon loss of PRMT5. In summary, we have identified a novel mechanism through which the PRMT5/WDR77 complex maintains the balance between splicing and mRNA stability through methylation of ZNF326.

CHD6 regulates the topological arrangement of the CFTR locus.

The control of transcription is regulated through the well-coordinated spatial and temporal interactions between distal genomic regulatory elements required for specialized cell-type and developmental gene expression programs. With recent findings CFTR has served as a model to understand the principles that govern genome-wide and topological organization of distal intra-chromosomal contacts as it relates to transcriptional control. This is due to the extensive characterization of the DNase hypersensitivity sites, modification of chromatin, transcription factor binding sites and the arrangement of these sites in CFTR consistent with the restrictive expression in epithelial cell types. Here, we identified CHD6 from a screen among several chromatin-remodeling proteins as a putative epigenetic modulator of CFTR expression. Moreover, our findings of CTCF interactions with CHD6 are consistent with the role described previously for CTCF in CFTR regulation. Our results now reveal that the CHD6 protein lies within the infrastructure of multiple transcriptional complexes, such as the FACT, PBAF, PAF1C, Mediator, SMC/Cohesion and MLL complexes. This model underlies the fundamental role CHD6 facilitates by tethering cis-acting regulatory elements of CFTR in proximity to these multi-subunit transcriptional protein complexes. Finally, we indicate that CHD6 structurally coordinates a three-dimensional stricture between intragenic elements of CFTR bound by several cell-type specific transcription factors, such as CDX2, SOX18, HNF4α and HNF1α. Therefore, our results reveal new insights into the epigenetic regulation of CFTR expression, whereas the manipulation of CFTR gene topology could be considered for treating specific indications of cystic fibrosis and/or pancreatitis.

Interplay between Homeobox proteins and Polycomb repressive complexes in p16INK4a regulation.

The INK4/ARF locus regulates senescence and is frequently altered in cancer. In normal cells, the INK4/ARF locus is found silenced by Polycomb repressive complexes (PRCs). Which are the mechanisms responsible for the recruitment of PRCs to INK4/ARF and their other target genes remains unclear. In a genetic screen for transcription factors regulating senescence, we identified the homeodomain-containing protein HLX1 (H2.0-like homeobox 1). Expression of HLX1 extends cellular lifespan and blunts oncogene-induced senescence. Using quantitative proteomics, we identified p16(INK4a) as the key target mediating the effects of HLX1 in senescence. HLX1 represses p16(INK4a) transcription by recruiting PRCs and HDAC1. This mechanism has broader implications, as HLX1 also regulates a subset of PRC targets besides p16(INK4a). Finally, sampling members of the Homeobox family, we identified multiple genes with ability to repress p16(INK4a). Among them, we found HOXA9 (Homeobox A9), a putative oncogene in leukaemia, which also recruits PRCs and HDAC1 to regulate p16(INK4a). Our results reveal an unexpected and conserved interplay between homeodomain-containing proteins and PRCs with implications in senescence, development and cancer.

Long Non-coding RNA ANRIL and Polycomb in Human Cancers and Cardiovascular Disease.

The long non-coding RNA CDKN2B-AS1, commonly referred to as the A ntisense N on-coding R NA in the I NK4 L ocus (ANRIL), is a 3.8-kb-long RNA transcribed from the short arm of human chromosome 9 on p21.3 that overlaps a critical region encompassing three major tumor suppressor loci juxtaposed to the INK4b-ARF-INK4a gene cluster and the methyl-thioadenosine phosphorylase (MTAP) gene. Genome-wide association studies have identified this region with a remarkable and growing number of disease-associated DNA alterations and single nucleotide polymorphisms, which corresponds to increased susceptibility to human disease. Recent attention has been devoted on whether these alterations in the ANRIL sequence affect its expression levels and/or its splicing transcript variation, and in consequence, global cellular homeostasis. Moreover, recent evidence postulates that ANRIL not only can regulate their immediate genomic neighbors in cis, but also has the capacity to regulate additional loci in trans. This action would further increase the complexity for mechanisms imposed through ANRIL and furthering the scope of this lncRNA in disease pathogenesis. In this chapter, we summarize the most recent findings on the investigation of ANRIL and provide a perspective on the biological and clinical significance of ANRIL as a putative biomarker, specifically, its potential role in directing cellular fates leading to cancer and cardiovascular disease.

METTL3 as a master regulator of translation in cancer: mechanisms and implications.

Translational regulation is an important step in the control of gene expression. In cancer cells, the orchestration of both global control of protein synthesis and selective translation of specific mRNAs promote tumor cell survival, angiogenesis, transformation, invasion and metastasis. N6-methyladenosine (m6A), the most prevalent mRNA modification in higher eukaryotes, impacts protein translation. Over the past decade, the development of m6A mapping tools has facilitated comprehensive functional investigations, revealing the involvement of this chemical mark, together with its writer METTL3, in promoting the translation of both oncogenes and tumor suppressor transcripts, with the impact being context-dependent. This review aims to consolidate our current understanding of how m6A and METTL3 shape translation regulation in the realm of cancer biology. In addition, it delves into the role of cytoplasmic METTL3 in protein synthesis, operating independently of its catalytic activity. Ultimately, our goal is to provide critical insights into the interplay between m6A, METTL3 and translational regulation in cancer, offering a deeper comprehension of the mechanisms sustaining tumorigenesis.

The scaffolding function of LSD1 controls DNA methylation in mouse ESCs.

Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.

Quantitative trait mapping reveals a regulatory axis involving peroxisome proliferator-activated receptors, PRDM16, transforming growth factor-ß2 and FLT3 in hematopoiesis.

Hematopoiesis is the process whereby BM HSCs renew to maintain their number or to differentiate into committed progenitors to generate all blood cells. One approach to gain mechanistic insight into this complex process is the investigation of quantitative genetic variation in hematopoietic function among inbred mouse strains. We previously showed that TGF-ß2 is a genetically determined positive regulator of hematopoiesis. In the presence of unknown nonprotein serum factors TGF-ß2, but not TGF-ß1 or -ß3, enhances progenitor proliferation in vitro, an effect that is subject to mouse strain-dependent variation mapping to a locus on chr.4, Tb2r1. TGF-ß2-deficient mice show hematopoietic defects, demonstrating the physiologic role of this cytokine. Here, we show that TGF-ß2 specifically and predominantly cell autonomously enhances signaling by FLT3 in vitro and in vivo. A coding polymorphism in Prdm16 (PR-domain-containing 16) underlies Tb2r1 and differentially regulates transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ), identifying lipid PPAR ligands as the serum factors required for regulation of FLT3 signaling by TGF-ß2. We furthermore show that PPARγ agonists play a FLT3-dependent role in stress responses of progenitor cells. These observations identify a novel regulatory axis that includes PPARs, Prdm16, and TGF-ß2 in hematopoiesis.

Prdm16 is a physiologic regulator of hematopoietic stem cells.

Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation.

METTL3 regulates breast cancer-associated alternative splicing switches.

Alternative splicing (AS) enables differential inclusion of exons from a given transcript, thereby contributing to the transcriptome and proteome diversity. Aberrant AS patterns play major roles in the development of different pathologies, including breast cancer. N6-methyladenosine (m6A), the most abundant internal modification of eukaryotic mRNA, influences tumor progression and metastasis of breast cancer, and it has been recently linked to AS regulation. Here, we identify a specific AS signature associated with breast tumorigenesis in vitro. We characterize for the first time the role of METTL3 in modulating breast cancer-associated AS programs, expanding the role of the m6A-methyltransferase in tumorigenesis. Specifically, we find that both m6A deposition in splice site boundaries and in splicing and transcription factor transcripts, such as MYC, direct AS switches of specific breast cancer-associated transcripts. Finally, we show that five of the AS events validated in vitro are associated with a poor overall survival rate for patients with breast cancer, suggesting the use of these AS events as a novel potential prognostic biomarker.

Rewired m6A epitranscriptomic networks link mutant p53 to neoplastic transformation.

N6-methyladenosine (m6A), one of the most prevalent mRNA modifications in eukaryotes, plays a critical role in modulating both biological and pathological processes. However, it is unknown whether mutant p53 neomorphic oncogenic functions exploit dysregulation of m6A epitranscriptomic networks. Here, we investigate Li-Fraumeni syndrome (LFS)-associated neoplastic transformation driven by mutant p53 in iPSC-derived astrocytes, the cell-of-origin of gliomas. We find that mutant p53 but not wild-type (WT) p53 physically interacts with SVIL to recruit the H3K4me3 methyltransferase MLL1 to activate the expression of m6A reader YTHDF2, culminating in an oncogenic phenotype. Aberrant YTHDF2 upregulation markedly hampers expression of multiple m6A-marked tumor-suppressing transcripts, including CDKN2B and SPOCK2, and induces oncogenic reprogramming. Mutant p53 neoplastic behaviors are significantly impaired by genetic depletion of YTHDF2 or by pharmacological inhibition using MLL1 complex inhibitors. Our study reveals how mutant p53 hijacks epigenetic and epitranscriptomic machinery to initiate gliomagenesis and suggests potential treatment strategies for LFS gliomas.

m6A RNA Immunoprecipitation Followed by High-Throughput Sequencing to Map N6-Methyladenosine.

N6-methyladenosine (m6A) is the most abundant internal modification on messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) in eukaryotes. It influences gene expression by regulating RNA processing, nuclear export, mRNA decay, and translation. Hence, m6A controls fundamental cellular processes, and dysregulated deposition of m6A has been acknowledged to play a role in a broad range of human diseases, including cancer. m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq or m6A-seq) is a powerful technique to map m6A in a transcriptome-wide level. After immunoprecipitation of fragmented polyadenylated (poly(A)+) rich RNA by using specific anti-m6A antibodies, both the immunoprecipitated RNA fragments together with the input control are subjected to massively parallel sequencing. The generation of such comprehensive methylation profiles of signal enrichment relative to input control is necessary in order to better comprehend the pathogenesis behind aberrant m6A deposition.

Transcriptional regulation of the human acetoacetyl-CoA synthetase gene by PPARgamma.

In the cytosol of lipogenic tissue, ketone bodies are activated by AACS (acetoacetyl-CoA synthetase) and incorporated into cholesterol and fatty acids. AACS gene expression is particularly abundant in white adipose tissue, as it is induced during adipocyte differentiation. In order to elucidate the mechanism controlling the gene expression of human AACS and to clarify its physiological role, we isolated the human promoter, characterized the elements required to initiate transcription and analysed the expression of the gene in response to PPARgamma (peroxisome-proliferator-activated receptor gamma), an inducer of adipogenesis. We show that the human AACS promoter is a PPARgamma target gene and that this nuclear receptor is recruited to the AACS promoter by direct interaction with Sp1 (stimulating protein-1).

Regulatory roles of RNA modifications in breast cancer.

Collectively referred to as the epitranscriptome, RNA modifications play important roles in gene expression control regulating relevant cellular processes. In the last few decades, growing numbers of RNA modifications have been identified not only in abundant ribosomal (rRNA) and transfer RNA (tRNA) but also in messenger RNA (mRNA). In addition, many writers, erasers and readers that dynamically regulate the chemical marks have also been characterized. Correct deposition of RNA modifications is prerequisite for cellular homeostasis, and its alteration results in aberrant transcriptional programs that dictate human disease, including breast cancer, the most frequent female malignancy, and the leading cause of cancer-related death in women. In this review, we emphasize the major RNA modifications that are present in tRNA, rRNA and mRNA. We have categorized breast cancer-associated chemical marks and summarize their contribution to breast tumorigenesis. In addition, we describe less abundant tRNA modifications with related pathways implicated in breast cancer. Finally, we discuss current limitations and perspectives on epitranscriptomics for use in therapeutic strategies against breast and other cancers.

LSD1: Expanding Functions in Stem Cells and Differentiation.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC) provide a powerful model system to uncover fundamental mechanisms that control cellular identity during mammalian development. Histone methylation governs gene expression programs that play a key role in the regulation of the balance between self-renewal and differentiation of ESCs. Lysine-specific demethylase 1 (LSD1, also known as KDM1A), the first identified histone lysine demethylase, demethylates H3K4me1/2 and H3K9me1/2 at target loci in a context-dependent manner. Moreover, it has also been shown to demethylate non-histone substrates playing a central role in the regulation of numerous cellular processes. In this review, we summarize current knowledge about LSD1 and the molecular mechanism by which LSD1 influences the stem cells state, including the regulatory circuitry underlying self-renewal and pluripotency.

FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell.

Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N6,2'-O-dimethyladenosine (m6Am) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.