The Proteins of mRNA Modification: Writers, Readers, and Erasers.

Over the past decade, mRNA modifications have emerged as important regulators of gene expression control in cells. Fueled in large part by the development of tools for detecting RNA modifications transcriptome wide, researchers have uncovered a diverse epitranscriptome that serves as an additional layer of gene regulation beyond simple RNA sequence. Here, we review the proteins that write, read, and erase these marks, with a particular focus on the most abundant internal modification, N6-methyladenosine (m6A). We first describe the discovery of the key enzymes that deposit and remove m6A and other modifications and discuss how our understanding of these proteins has shaped our views of modification dynamics. We then review current models for the function of m6A reader proteins and how our knowledge of these proteins has evolved. Finally, we highlight important future directions for the field and discuss key questions that remain unanswered.

The transcription regulators ZNF750 and LSD1/KDM1A dampen inflammation on the skin's surface by silencing pattern recognition receptors.

The surface of the skin is continually exposed to pro-inflammatory stimuli; however, it is unclear why it is not constantly inflamed due to this exposure. Here, we showed undifferentiated keratinocytes residing in the deep epidermis could trigger a strong inflammatory response due to their high expression of pattern recognition receptors (PRRs) that detect damage or pathogens. As keratinocytes differentiated, they migrated outward toward the surface of the skin and decreased their PRR expression, which led to dampened immune responses. ZNF750, a transcription factor expressed only in differentiated keratinocytes, recruited the histone demethylase KDM1A/LSD1 to silence genes coding for PRRs (TLR3, IFIH1/MDA5, and DDX58/RIG1). Loss of ZNF750 or KDM1A in human keratinocytes or mice resulted in sustained and excessive inflammation resembling psoriatic skin, which could be restored to homeostatic conditions upon silencing of TLR3. Our findings explain how the skin's surface prevents excessive inflammation through ZNF750- and KDM1A-mediated suppression of PRRs.

Dynamic Control of X Chromosome Conformation and Repression by a Histone H4K20 Demethylase.

Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We have discovered the machinery and mechanism underlying the dynamic enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during C. elegans dosage compensation and demonstrated H4K20me1's pivotal role in regulating higher-order chromosome structure and X-chromosome-wide gene expression. The structure and the activity of the dosage compensation complex (DCC) subunit DPY-21 define a Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminates H4K20me1 enrichment in somatic cells, elevates X-linked gene expression, reduces X chromosome compaction, and disrupts X chromosome conformation by diminishing the formation of topologically associating domains (TADs). Unexpectedly, DPY-21 also associates with autosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Our findings demonstrate the direct link between chromatin modification and higher-order chromosome structure in long-range regulation of gene expression.

Epigenetic modulators provide a path to understanding disease and therapeutic opportunity.

The discovery of epigenetic modulators (writers, erasers, readers, and remodelers) has shed light on previously underappreciated biological mechanisms that promote diseases. With these insights, novel biomarkers and innovative combination therapies can be used to address challenging and difficult to treat disease states. This review highlights key mechanisms that epigenetic writers, erasers, readers, and remodelers control, as well as their connection with disease states and recent advances in associated epigenetic therapies.

ALKBH1-Mediated tRNA Demethylation Regulates Translation.

tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.

RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

Versatile JMJD proteins: juggling histones and much more.

Jumonji C domain-containing (JMJD) proteins are found in bacteria, fungi, animals, and plants. They belong to the 2-oxoglutarate-dependent oxygenase superfamily and are endowed with various enzymatic activities, including demethylation of histones and hydroxylation of non-histone proteins. Many JMJD proteins are involved in the epigenetic control of gene expression, yet they also modulate a myriad other cellular processes. In this review we focus on the 33 human JMJD proteins and their established and controversial catalytic properties, survey their epigenetic and non-epigenetic functions, emphasize their contribution to sex-specific disease differences, and highlight how they sense metabolic changes. All this underlines not only their key roles in development and homeostasis, but also that JMJD proteins are destined to become drug targets in multiple diseases.

Taking Me away: the function of phosphorylation on histone lysine demethylases.

Histone lysine demethylases (KDMs) regulate eukaryotic gene transcription by catalysing the removal of methyl groups from histone proteins. These enzymes are intricately regulated by the kinase signalling system in response to internal and external stimuli. Here, we review the mechanisms by which kinase-mediated phosphorylation influence human histone KDM function. These include the changing of histone KDM subcellular localisation or chromatin binding, the altering of protein half-life, changes to histone KDM complex formation that result in histone demethylation, non-histone demethylation or demethylase-independent effects, and effects on histone KDM complex dissociation. We also explore the structural context of phospho-sites on histone KDMs and evaluate how this relates to function.

Cotranscriptional demethylation induces global loss of H3K4me2 from active genes in Arabidopsis.

Based on studies of animals and yeasts, methylation of histone H3 lysine 4 (H3K4me1/2/3, for mono-, di-, and tri-methylation, respectively) is regarded as the key epigenetic modification of transcriptionally active genes. In plants, however, H3K4me2 correlates negatively with transcription, and the regulatory mechanisms of this counterintuitive H3K4me2 distribution in plants remain largely unexplored. A previous genetic screen for factors regulating plant regeneration identified Arabidopsis LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3), which is a major H3K4me2 demethylase. Here, we show that LDL3-mediated H3K4me2 demethylation depends on the transcription elongation factor Paf1C and phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). In addition, LDL3 binds to phosphorylated RNAPII. These results suggest that LDL3 is recruited to transcribed genes by binding to elongating RNAPII and demethylates H3K4me2 cotranscriptionally. Importantly, the negative correlation between H3K4me2 and transcription is significantly attenuated in the ldl3 mutant, demonstrating the genome-wide impacts of the transcription-driven LDL3 pathway to control H3K4me2 in plants. Our findings implicate H3K4me2 demethylation in plants as chromatin records of transcriptional activity, which ensures robust gene control.

Dynamic multilayered control of m6A RNA demethylase activity.

Similar to DNA and histone, RNA can also be methylated. In its most common form, a N6-methyladenosine (m6A) chemical modification is introduced into nascent messenger ribonucleic acid (mRNA) by a specialized methyltransferase complex and removed by the RNA demethylases, Fat mass and obesity-associated (FTO), and ALKBH5. The fate of m6A-marked mRNA is uniquely diverse, ranging from degradation to stabilization/translation, which has been suggested to be largely dependent on its interaction with the family of YT521-B homology (YTH) domain-containing proteins. Here, we highlight a series of control levers that impinge on the RNA demethylases. We present evidence to indicate that intermediary metabolism and various posttranslation modifications modulate the activity, stability, and the subcellular localization of FTO and ALKBH5, further dispelling the notion that m6A methylation is not a dynamic process. We also discuss how examination of these underappreciated regulatory nodes adds a more nuanced view of the role of FTO and ALKBH5 and should guide their study in cancer and nonmalignant conditions alike.

KDM5-mediated activation of genes required for mitochondrial biology is necessary for viability in Drosophila.

Histone-modifying proteins play important roles in the precise regulation of the transcriptional programs that coordinate development. KDM5 family proteins interact with chromatin through demethylation of H3K4me3 as well as demethylase-independent mechanisms that remain less understood. To gain fundamental insights into the transcriptional activities of KDM5 proteins, we examined the essential roles of the single Drosophila Kdm5 ortholog during development. KDM5 performs crucial functions in the larval neuroendocrine prothoracic gland, providing a model to study its role in regulating key gene expression programs. Integrating genome binding and transcriptomic data, we identify that KDM5 regulates the expression of genes required for the function and maintenance of mitochondria, and we find that loss of KDM5 causes morphological changes to mitochondria. This is key to the developmental functions of KDM5, as expression of the mitochondrial biogenesis transcription factor Ets97D, homolog of GABPα, is able to suppress the altered mitochondrial morphology as well as the lethality of Kdm5 null animals. Together, these data establish KDM5-mediated cellular functions that are important for normal development and could contribute to KDM5-linked disorders when dysregulated.

N6-Methyladenine DNA Modification in the Human Genome.

DNA N6-methyladenine (6mA) modification is the most prevalent DNA modification in prokaryotes, but whether it exists in human cells and whether it plays a role in human diseases remain enigmatic. Here, we showed that 6mA is extensively present in the human genome, and we cataloged 881,240 6mA sites accounting for ∼0.051% of the total adenines. [G/C]AGG[C/T] was the most significantly associated motif with 6mA modification. 6mA sites were enriched in the coding regions and mark actively transcribed genes in human cells. DNA 6mA and N6-demethyladenine modification in the human genome were mediated by methyltransferase N6AMT1 and demethylase ALKBH1, respectively. The abundance of 6mA was significantly lower in cancers, accompanied by decreased N6AMT1 and increased ALKBH1 levels, and downregulation of 6mA modification levels promoted tumorigenesis. Collectively, our results demonstrate that DNA 6mA modification is extensively present in human cells and the decrease of genomic DNA 6mA promotes human tumorigenesis.

The Intellectual Disability Risk Gene Kdm5b Regulates Long-Term Memory Consolidation in the Hippocampus.

The histone lysine demethylase KDM5B is implicated in recessive intellectual disability disorders, and heterozygous, protein-truncating variants in KDM5B are associated with reduced cognitive function in the population. The KDM5 family of lysine demethylases has developmental and homeostatic functions in the brain, some of which appear to be independent of lysine demethylase activity. To determine the functions of KDM5B in hippocampus-dependent learning and memory, we first studied male and female mice homozygous for a Kdm5b Δ ARID allele that lacks demethylase activity. Kdm5b Δ ARID/ Δ ARID mice exhibited hyperactivity and long-term memory deficits in hippocampus-dependent learning tasks. The expression of immediate early, activity-dependent genes was downregulated in these mice and hyperactivated upon a learning stimulus compared with wild-type (WT) mice. A number of other learning-associated genes were also significantly dysregulated in the Kdm5b Δ ARID/ Δ ARID hippocampus. Next, we knocked down Kdm5b specifically in the adult, WT mouse hippocampus with shRNA. Kdm5b knockdown resulted in spontaneous seizures, hyperactivity, and hippocampus-dependent long-term memory and long-term potentiation deficits. These findings identify KDM5B as a critical regulator of gene expression and synaptic plasticity in the adult hippocampus and suggest that at least some of the cognitive phenotypes associated with KDM5B gene variants are caused by direct effects on memory consolidation mechanisms.

Neuronal splicing of the unmethylated histone H3K4 reader, PHF21A, prevents excessive synaptogenesis.

PHF21A is a histone-binding protein that recognizes unmethylated histone H3K4, the reaction product of LSD1 histone demethylase. PHF21A and LSD1 form a complex, and both undergo neuron-specific microexon splicing. The PHF21A neuronal microexon interferes with nucleosome binding, whereas the LSD1 neuronal microexon weakens H3K4 demethylation activity and can alter the substrate specificity to H3K9 or H4K20. However, the temporal expression patterns of PHF21A and LSD1 splicing isoforms during brain development and their biological roles remain unknown. In this work, we report that neuronal PHF21A isoform expression precedes neuronal LSD1 expression during human neuron differentiation and mouse brain development. The asynchronous splicing events resulted in stepwise deactivation of the LSD1-PHF21A complex in reversing H3K4 methylation. An unbiased proteomics survey revealed that the enzymatically inactive LSD1-PHF21A complex interacts with neuron-specific binding partners, including MYT1-family transcription factors and post-transcriptional mRNA processing proteins such as VIRMA. The interaction with the neuron-specific components, however, did not require the PHF21A microexon, indicating that the neuronal proteomic milieu, rather than the microexon-encoded PHF21A segment, is responsible for neuron-specific complex formation. Finally, by using two Phf21a mutant mouse models, we found that Phf21a neuronal splicing prevents excess synapse formation that otherwise would occur when canonical PHF21A is expressed in neurons. These results suggest that the role of the PHF21A microexon is to dampen LSD1-mediated H3K4 demethylation, thereby containing aberrant synaptogenesis.

Upregulation of nuclear protein Hemgn by transcriptional repressor Gfi1 through repressing PU.1 contributes to the anti-apoptotic activity of Gfi1.

Gfi1 is a transcriptional repressor that plays a critical role in hematopoiesis. The repressive activity of Gfi1 is mediated mainly by its SNAG domain that interacts with and thereby recruits the histone demethylase LSD1 to its target genes. An important function of Gfi1 is to protect hematopoietic cells against stress-induced apoptosis, which has been attributed to its participation in the posttranscriptional modifications of p53 protein, leading to suppression of p53 activity. In this study, we show that Gfi1 upregulated the expression of Hemgn, a nuclear protein, through a 16-bp promoter region spanning from +47 to +63 bp relative to the transcription start site (TSS), which was dependent on its interaction with LSD1. We further demonstrate that Gfi1, Ikaros, and PU.1 are bound to this 16-bp region. However, while Ikaros activated Hemgn and collaborated with Gfi1 to augment Hemgn expression, it was not required for Gfi1-mediated Hemgn upregulation. In contrast, PU.1 repressed Hemgn and inhibited Hemgn upregulation by Gfi1. Notably, PU.1 knockdown and deficiency, while augmenting Hemgn expression, abolished Hemgn upregulation by Gfi1. PU.1 (Spi-1) is repressed by Gfi1. We show here that PU.1 repression by Gfi1 preceded and correlated well with Hemgn upregulation. Thus, our data strongly suggest that Gfi1 upregulates Hemgn by repressing PU.1. In addition, we demonstrate that Hemgn upregulation contributed to the anti-apoptotic activity of Gfi1 in a p53-independent manner.

Oxygen-dependent histone lysine demethylase 4 restricts hepatitis B virus replication.

Mammalian cells have evolved strategies to regulate gene expression when oxygen is limited. Hypoxia-inducible factors (HIF) are the major transcriptional regulators of host gene expression. We previously reported that HIFs bind and activate hepatitis B virus (HBV) DNA transcription under low oxygen conditions; however, the global cellular response to low oxygen is mediated by a family of oxygenases that work in concert with HIFs. Recent studies have identified a role for chromatin modifiers in sensing cellular oxygen and orchestrating transcriptional responses, but their role in the HBV life cycle is as yet undefined. We demonstrated that histone lysine demethylase 4 (KDM4) can restrict HBV, and pharmacological or oxygen-mediated inhibition of the demethylase increases viral RNAs derived from both episomal and integrated copies of the viral genome. Sequencing studies demonstrated that KDM4 is a major regulator of the hepatic transcriptome, which defines hepatocellular permissivity to HBV infection. We propose a model where HBV exploits cellular oxygen sensors to replicate and persist in the liver. Understanding oxygen-dependent pathways that regulate HBV infection will facilitate the development of physiologically relevant cell-based models that support efficient HBV replication.

Autoregulatory mechanism of enzyme activity by the nuclear localization signal of lysine-specific demethylase 1.

The N-terminal region of the human lysine-specific demethylase 1 (LSD1) has no predicted structural elements, contains a nuclear localization signal (NLS), undergoes multiple posttranslational modifications (PTMs), and acts as a protein-protein interaction hub. This intrinsically disordered region (IDR) extends from core LSD1 structure, resides atop the catalytic active site, and is known to be dispensable for catalysis. Here, we show differential nucleosome binding between the full-length and an N terminus deleted LSD1 and identify that a conserved NLS and PTM containing element of the N terminus contains an alpha helical structure, and that this conserved element impacts demethylation. Enzyme assays reveal that LSD1's own electropositive NLS amino acids 107 to 120 inhibit demethylation activity on a model histone 3 lysine 4 dimethyl (H3K4me2) peptide (Kiapp âˆ¼ 3.3 µM) and histone 3 lysine 4 dimethyl nucleosome substrates (IC50 ∼ 30.4 µM), likely mimicking the histone H3 tail. Further, when the identical, inhibitory NLS region contains phosphomimetic modifications, inhibition is partially relieved. Based upon these results and biophysical data, a regulatory mechanism for the LSD1-catalyzed demethylation reaction is proposed whereby NLS-mediated autoinhibition can occur through electrostatic interactions, and be partially relieved through phosphorylation that occurs proximal to the NLS. Taken together, the results highlight a dynamic and synergistic role for PTMs, intrinsically disordered regions, and structured regions near LSD1 active site and introduces the notion that phosphorylated mediated NLS regions can function to fine-tune chromatin modifying enzyme activity.

OsJMJ718, a histone demethylase gene, positively regulates seed germination in rice.

Seed vigor has major impact on the rate and uniformity of seedling growth, crop yield, and quality. However, the epigenetic regulatory mechanism of crop seed vigor remains unclear. In this study, a (jumonji C) JmjC gene of the histone lysine demethylase OsJMJ718 was cloned in rice, and its roles in seed germination and its epigenetic regulation mechanism were investigated. OsJMJ718 was located in the nucleus and was engaged in H3K9 methylation. Histochemical GUS staining analysis revealed OsJMJ718 was highly expressed in seed embryos. Abiotic stress strongly induced the OsJMJ718 transcriptional accumulation level. Germination percentage and seedling vigor index of OsJMJ718 knockout lines (OsJMJ718-CR) were lower than those of the wild type (WT). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) of seeds imbibed for 24 h showed an increase in H3K9me3 deposition of thousands of genes in OsJMJ718-CR. ChIP-seq results and transcriptome analysis showed that differentially expressed genes were enriched in ABA and ethylene signal transduction pathways. The content of ABA in OsJMJ718-CR was higher than that in WT seeds. OsJMJ718 overexpression enhanced sensitivity to ABA during germination and early seedling growth. In the seed imbibition stage, ABA and ethylene content diminished and augmented, separately, suggesting that OsJMJ718 may adjust rice seed germination through the ABA and ethylene signal transduction pathways. This study displayed the important function of OsJMJ718 in adjusting rice seed germination and vigor, which will provide an essential reference for practical issues, such as improving rice vigor and promoting direct rice sowing production.

FTO in oral diseases: Functions, mechanisms, and therapeutic potential.

Fat mass and obesity-associated protein (FTO) is the first identified N6-methyladenosine (m6A) demethylase widely distributed in various tissues in adults and children. It plays an essential role in diverse mRNA-associated processes including transcriptional stability, selective splicing, mRNA translocation, and also protein translation. Recently, emerging studies have shown that FTO is involved in the genesis and development of oral diseases. However, the correlation between FTO and oral diseases and its specific regulatory mechanism still needs further study. In this review, we will summarize the discovery, distribution, gene expression, protein structure, biological functions, inhibitors, and quantifying methods of FTO, as well as its regulatory role and mechanism in oral diseases. Notably, FTO genetic variants are strongly associated with periodontal diseases (PDs), temporomandibular joint osteoarthritis (TMJOA), and obstructive sleep apnea (OSA). Besides, the latest studies that describe the relationship between FTO and PDs, head and neck squamous cell carcinoma (HNSCCs), TMJOA, and OSA will be discussed. We elaborate on the regulatory roles of FTO in PDs, HNSCCs, and TMJOA, which are modulated through cell proliferation, cell migration, apoptosis, bone metabolism, and immune response. The review will enrich our understanding of RNA epigenetic modifications in oral diseases and present a solid theoretical foundation for FTO to serve as a novel diagnosis and prognostic biomarker for oral diseases.

Arginine Methylation: The Coming of Age.

Arginine methylation is a common post-translational modification functioning as an epigenetic regulator of transcription and playing key roles in pre-mRNA splicing, DNA damage signaling, mRNA translation, cell signaling, and cell fate decision. Recently, a wealth of studies using transgenic mouse models and selective PRMT inhibitors helped define physiological roles for protein arginine methyltransferases (PRMTs) linking them to diseases such as cancer and metabolic, neurodegenerative, and muscular disorders. This review describes the recent molecular advances that have been uncovered in normal and diseased mammalian cells.