Impaired differentiation is a hallmark of myeloid malignancies1,2. Therapies that enable cells to circumvent the differentiation block, such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), are by and large curative in acute promyelocytic leukaemia3, but whether 'differentiation therapy' is a generalizable therapeutic approach for acute myeloid leukaemia (AML) and beyond remains incompletely understood. Here we demonstrate that simultaneous inhibition of the histone demethylase LSD1 (LSD1i) and the WNT pathway antagonist GSK3 kinase4 (GSK3i) robustly promotes therapeutic differentiation of established AML cell lines and primary human AML cells, as well as reducing tumour burden and significantly extending survival in a patient-derived xenograft mouse model. Mechanistically, this combination promotes differentiation by activating genes in the type I interferon pathway via inducing expression of transcription factors such as IRF7 (LSD1i) and the co-activator β-catenin (GSK3i), and their selective co-occupancy at targets such as STAT1, which is necessary for combination-induced differentiation. Combination treatment also suppresses the canonical, pro-oncogenic WNT pathway and cell cycle genes. Analysis of datasets from patients with AML suggests a correlation between the combination-induced transcription signature and better prognosis, highlighting clinical potential of this strategy. Collectively, this combination strategy rewires transcriptional programs to suppress stemness and to promote differentiation, which may have important therapeutic implications for AML and WNT-driven cancers beyond AML.

A hallmark of acute myeloid leukemia (AML) is the inability of self-renewing malignant cells to mature into a non-dividing terminally differentiated state. This differentiation block has been linked to dysregulation of multiple cellular processes, including transcriptional, chromatin, and metabolic regulation. The transcription factor HOXA9 and the histone demethylase LSD1 are examples of such regulators that promote differentiation blockade in AML. To identify metabolic targets that interact with LSD1 inhibition to promote myeloid maturation, we screened a small molecule library to identify druggable substrates. We found that differentiation caused by LSD1 inhibition is enhanced by combined perturbation of purine nucleotide salvage and de novo lipogenesis pathways, and identified multiple lines of evidence to support the specificity of these pathways and suggest a potential basis of how perturbation of these pathways may interact synergistically to promote myeloid differentiation. In sum, these findings suggest potential drug combination strategies in the treatment of AML.

Recent studies reported that METTL4 regulates DNA 6mA in vivo and therefore is a candidate DNA m6A methyltransfease. However, the enzymatic activity of METTL4 in vitro has not been demonstrated in part due to the difficulties of obtaining well-folded proteins. Here we show that mettl4 is a major methyltransfase responsible for m6A methylation of U2 snRNA both in vitro and in vivo in fly, and identify adenosine at 29th position as the site of m6A methylation. This study answered a long-standing question regarding the enzymatic activity of METTL4, and thus paved the way for further investigating the functions of METTL4 in different biological settings.

The essential human enzyme lysine specific demethylase 1 (LSD1) silences genes by demethylating mono- and dimethylated lysine 4 in histone H3 (H3K4me1/2). Studies of the minimal requirements for LSD1 activity are complicated by the heterogeneity of histone modification states in cells. We overcame this challenge by generating homogeneous mononucleosome substrates containing semisynthetic H3K4me2. Biophysical and biochemical assays with full-length LSD1 revealed its ability to bind and demethylate nucleosomes. Consistent with a requirement for nucleosome binding prior to demethylation, a competing nucleosome-binding peptide from the high-mobility group protein effectively inhibited LSD1 activity. Thus, our studies provide the first glimpse of nucleosome demethylation by LSD1 in the absence of other scaffolding proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SummarymRNA modifications play an important role in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5’ ends of mRNAs. Furthermore, PCIF1 catalyzes only 5’ m6Am methylation of capped mRNAs, but not internal m6A methylationin vitroandin vivo. Our global mRNA methylation analysis revealed that there is no crosstalk between m6Am and m6A mRNA methylation events, suggesting that m6Am is functionally distinct from m6A. Importantly, our data indicate that m6Am negatively impacts translation of methylated mRNAs by antagonizing cap binding protein eIF4E. Together, we identify the first and only human mRNA m6Am methyltransferase and demonstrate a novel mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation in eukaryotes.HighlightsPCIF1 is an evolutionarily conserved mRNA m6Am methyltransferaseLoss of PCIF1 leads to a complete loss of m6Am, whereas m6A level and distribution are not affectedPCIF1 mediated m6Am does not affect RNA Pol II transcription or mRNA stabilitym6Am-Exo-Seq is a robust methodology that enables global m6Am mappingm6Am suppresses cap dependent translation

Acute myeloid leukemia (AML) is one of the most lethal blood cancers, accounting for close to a quarter of a million annual deaths worldwide. Even though genetically heterogeneous, all AMLs are characterized by two interrelated features—blocked differentiation and high proliferative capacity. Despite significant progress in our understanding of the molecular and genetic basis of AML, the treatment of AMLs with chemotherapeutic regimens has remained largely unchanged in the past 30 years. In this review, we will consider the role of two cellular processes, metabolism and epigenetics, in the development and progression of AML and highlight the studies that suggest an interconnection of therapeutic importance between the two. Large-scale whole-exome sequencing of AML patients has revealed the presence of mutations, translocations or duplications in several epigenetic effectors such as DNMT3, MLL, ASXL1, and TET2, often times co-occuring with mutations in metabolic enzymes such as IDH1 and IDH2. These mutations often result in impaired enzymatic activity which leads to an altered epigenetic landscape through dysregulation of chromatin modifications such as DNA methylation, histone acetylation and methylation. We will discuss the role of enzymes that are responsible for establishing these modifications, namely histone acetyl transferases (HAT), histone methyl transferases (HMT), demethylases (KDMs), and deacetylases (HDAC), and also highlight the merits and demerits of using inhibitors that target these enzymes. Furthermore, we will tie in the metabolic regulation of co-factors such as acetyl-CoA, SAM, and α-ketoglutarate that are utilized by these enzymes and examine the role of metabolic inhibitors as a treatment option for AML. In doing so, we hope to stimulate interest in this topic and help generate a rationale for the consideration of the combinatorial use of metabolic and epigenetic inhibitors for the treatment of AML.

RING1 is an E3-ubiquitin ligase that is involved in epigenetic control of transcription during development. It is a component of the polycomb repressive complex 1, and its role in that complex is to ubiquitylate histone H2A. In a 13-year-old girl with syndromic neurodevelopmental disabilities, we identified a de novo mutation, RING1 p.R95Q, which alters a conserved arginine residue in the catalytic RING domain. In vitro assays demonstrated that the mutant RING1 retains capacity to catalyze ubiquitin chain formation, but is defective in its ability to ubiquitylate histone H2A in nucleosomes. Consistent with this in vitro effect, cells of the patient showed decreased monoubiquitylation of histone H2A. We modeled the mutant RING1 in Caenorhabditis elegans by editing the comparable amino acid change into spat-3, the suggested RING1 ortholog. Animals with either the missense mutation or complete knockout of spat-3 were defective in monoubiquitylation of histone H2A and had defects in neuronal migration and axon guidance. Relevant to our patient, animals heterozygous for either the missense or knockout allele also showed neuronal defects. Our results support three conclusions: mutation of RING1 is the likely cause of a human neurodevelopmental syndrome, mutation of RING1 can disrupt histone H2A ubiquitylation without disrupting RING1 catalytic activity, and the comparable mutation in C. elegans spat-3 both recapitulates the effects on histone H2A ubiquitylation and leads to neurodevelopmental abnormalities. This role for RING1 adds to our understanding of the importance of aberrant epigenetic effects as causes of human neurodevelopmental disorders.

The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family.

Missense mutations that disrupt the RING domain of the tumor suppressor gene BRCA1 lead to increased risk of breast and ovarian cancer. The BRCA1 RING domain is a ubiquitin ligase, whose structure and function rely critically on forming a heterodimer with BARD1, which also harbors a RING domain. The function of the BARD1 RING domain is unknown. In families severely affected with breast cancer, we identified inherited BARD1 missense mutations Cys53Trp, Cys71Tyr, and Cys83Arg that alter three zincbinding residues of the BARD1 RING domain. Each of these mutant BARD1 proteins retained the ability to form heterodimeric complexes with BRCA1 to make an active ubiquitin ligase, but the mutant BRCA1/BARD1 complexes were deficient in binding to nucleosomes and in ubiquitylating histone H2A. The BARD1 mutations also caused loss of transcriptional repression of BRCA1-regulated estrogen metabolism genes CYP1A1 and CYP3A4; breast epithelial cells edited to create heterozygous loss of BARD1 showed significantly higher expression of CYP1A1 and CYP3A4. Reintroduction of wild-type BARD1 into these cells restored CYP1A1 and CYP3A4 transcription to normal levels, but introduction of the cancer-predisposing BARD1 RING mutants failed to do so. These results indicate that an intact BARD1 RING domain is critical to BRCA1/BARD1 binding to nucleosomes and hence to ubiquitylation of histone H2A and also critical to transcriptional repression of BRCA1-regulated genes active in estrogen metabolism.

Lysine-specific demethylase 1 (LSD1) downregulates eukaryotic gene activity by demethylating mono- and dimethylated Lys4 in histone H3. Elucidating the biochemical crosstalk of LSD1 with histone post-translational modifications (PTMs) is essential for developing LSD1-targeted therapeutics in human cancers. We interrogated the small ubiquitin-like modifier (SUMO)-driven regulation of LSD1 activity with semisynthetic nucleosomes containing site-specifically methylated and sumoylated histones. We discovered that nucleosomes containing sumoylated histone H4 (suH4), a modification associated with gene repression, stimulate LSD1 activity by a mechanism dependent upon the SUMO-interaction motif in CoREST. Furthermore, the stimulatory effect of suH4 was spatially limited and did not extend to the demethylation of adjacent nonsumoylated nucleosomes. Thus, we have identified histone modification by SUMO as the first PTM that stimulates intranucleosomal demethylation by the developmentally critical LSD1-CoREST complex.

The density and diversity of posttranslational modifications (PTMs) observed in histone proteins typically limit their purification to homogeneity from biological sources. Access to quantities of uniformly modified histones is, however, critical for investigating the downstream effects of histone PTMs on chromatin-templated processes. Therefore, a number of semisynthetic methodologies have been developed to generate histones bearing precisely defined PTMs or close analogs thereof. In this chapter, we present two optimized and rapid strategies for generating functional analogs of site-specifically acetylated and sumoylated histones. First, we describe a convergent strategy to site-specifically attach the small ubiquitin-like modifier-3 (SUMO-3) protein to the site of Lys12 in histone H4 by means of a disulfide linkage. We then describe the generation of thialysine analogs of histone H3 acetylated at Lys14 or Lys56, using thiol-ene coupling chemistry. Both strategies afford multimilligram quantities of uniformly modified histones that are easily incorporated into mononucleosomes and nucleosome arrays for biophysical and biochemical investigations. These methods are readily extendable to any desired sites in the four core nucleosomal histones and their variant forms.

Investigating the Effect of Histone H4 Sumoylation on Chromatin Structure and Function

Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical and biochemical investigations aimed at deconvoluting the myriad biological roles for Ub. Current chemical strategies for protein ubiquitylation, however, employ temporary ligation auxiliaries that are removed under harsh denaturing conditions and have limited applicability. We report an unprecedented aromatic thiol-mediated N-O bond cleavage and its application towards native chemical ubiquitylation with the ligation auxiliary 2-aminooxyethanethiol. Our interrogation of the reaction mechanism suggests a disulfide radical anion as the active species capable of cleaving the N-O bond. The successful semisynthesis of full-length histone H2B modified by the small ubiquitin-like modifier-3 (SUMO-3) protein further demonstrates the generalizability and compatibility of our strategy with folded proteins.

In order to fit a large amount of genetic material in the small volume of the cell nucleus, eukaryotic DNA is packaged as repeating units of mononucleosomes. Each mononucleosome consists of about 147 bp of DNA wrapped tightly around a protein spool comprised of the four core histones H2A, H2B, H3, and H4. Reversible posttranslational modifications (PTMs) of histones play critical roles in regulating gene function and their dysregulation underlies several acute human diseases. Therefore, there is an urgent need to elucidate the mechanistic roles for histone PTMs and to identify the biochemical relationships (crosstalk) between various PTMs. This chapter highlights chemical and genetic tools that have yielded access to homogeneously site-specifically modified histones for mechanistic investigations. Results from the biochemical and biophysical characterization of mononucleosomes and nucleosomal arrays reconstituted with precisely modified histones are discussed, and the extension of current technologies to probe the crosstalk between histone PTMs at a system-wide level is presented.

Background: Human histone H4 is post-translationally modified at Lys-12 by the small ubiquitin-like modifier protein (SUMO-3). Results: Chemical sumoylation at H4 Lys-12 revealed the inhibition of chromatin compaction and oligomerization by SUMO-3. Conclusion: Sumoylation changes chromatin structure by inhibiting long-range internucleosomal interactions and decreasing the affinity between adjacent nucleosomes. Significance: Learning how sumoylation changes the structure of chromatin suggests that it may mediate gene repression without chromatin compaction.The structure of eukaryotic chromatin directly influences gene function, and is regulated by chemical modifications of the core histone proteins. Modification of the human histone H4 N-terminal tail region by the small ubiquitin-like modifier protein, SUMO-3, is associated with transcription repression. However, the direct effect of sumoylation on chromatin structure and function remains unknown. Therefore, we employed a disulfide-directed strategy to generate H4 homogenously and site-specifically sumoylated at Lys-12 (suH4(ss)). Chromatin compaction and oligomerization assays with nucleosomal arrays containing suH4(ss) established that SUMO-3 inhibits array folding and higher order oligomerization, which underlie chromatin fiber formation. Moreover, the effect of sumoylation differed from that of acetylation, and could be recapitulated with the structurally similar protein ubiquitin. Mechanistic studies at the level of single nucleosomes revealed that, unlike acetylation, the effect of SUMO-3 arises from the attenuation of long-range internucleosomal interactions more than from the destabilization of a compacted dinucleosome state. Altogether, our results present the first insight on the direct structural effects of histone H4 sumoylation and reveal a novel mechanism by which SUMO-3 inhibits chromatin compaction.

The prokaryotic ubiquitin-like protein (Pup)-based proteasomal system in the pathogen Mycobacterium tuberculosis (Mtb) is essential for its survival in a mammalian host. The Pup ligase enzyme, PafA, conjugates Pup to a suite of proteins targeted for proteasomal degradation and is necessary for persistent infection by Mtb. We report the design and application of fluorescent probes for use in elucidating the mechanisms of Pup and substrate recognition by PafA. Our studies revealed that the C-terminal 26 amino acid sequence of Pup is the minimal ligase recognition motif in Mtb. Specific hydrophobic residues within this sequence that are known to be important for the interactions of Pup with proteasomes are also critical for the activation of Pup by PafA.

The process of neurodegeneration in Parkinson's Disease is intimately associated with the aggregation of the protein α-synuclein into toxic oligomers and fibrils. Interestingly, many of these protein aggregates are found to be post-translationally modified by ubiquitin at several different lysine residues. However, the inability to generate homogeneously ubiquitin modified α-synuclein at each site has prevented the understanding of the specific biochemical consequences. We have used protein semisynthesis to generate nine site-specifically ubiquitin modified α-synuclein derivatives and have demonstrated that different ubiquitination sites have differential effects on α-synuclein aggregation.