F-box and WD repeat domain-containing 7 (FBXW7) is a tumor suppressor that targets various oncoproteins for degradation, but its role in modulating cancer-associated fibroblasts (CAFs) in the tumor microenvironment remains elusive. Here, we report that FBXW7 expression is gradually downregulated in CAFs during the progression of human pancreatic and lung cancers. Mechanically, FBXW7 inhibits histone lysine methyltransferase 2 (KMT2) methyltransferase activity via retinoblastoma binding protein 5 (RbBP5) binding, whereas FBXW7 depletion abrogates the binding to activate KMT2, leading to increased H3K4 methylations and global upregulation of gene expression. Activation of the interleukin-17 (IL-17) signaling pathway triggers the secretion of cytokines and chemokines to promote migration, invasion, and sphere formation of lung cancer cells. Coinjection of Fbxw7-depleted mouse embryonic fibroblasts with cancer cells enhances in vivo tumor growth, demonstrating a paracrine effect. Hypoxia downregulates CAF FBXW7 via ETS proto-oncogene 1 (ETS1) to increase H3K4 methylation, whereas conditioned media from hypoxia-exposed CAFs promotes migration and invasion of pancreatic cancer cells, highlighting FBXW7's tumor-suppressing role through KMT2 inactivation.

The APOBEC3 family of polynucleotide cytidine deaminases has diverse roles as viral restriction factors and oncogenic mutators. These enzymes convert cytidine to uridine in single-stranded (ss)DNA, inducing genomic mutations that promote drug resistance and tumor heterogeneity. Of the seven human APOBEC3 members, APOBEC3A (A3A) and APOBEC3B (A3B) are most implicated in driving pro-tumorigenic mutations. How these enzymes engage and selectively deaminate ssDNA over RNA is not well understood. We previously conducted molecular dynamics (MD) simulations that support the role of sugar conformation as a key molecular determinant in nucleic acid recognition by A3B. We hypothesize that A3A and A3B selectively deaminate substrates in the 2 '-endo (DNA) conformation and show reduced activity for 3 '-endo (RNA) conformation substrates. Consequently, we have characterized A3A- and A3B-binding and deaminase activity with chimeric oligonucleotides containing cytidine analogues that promote either the 2 '-endo or 3 '-endo conformation. Using fluorescence polarization and gel-based deamination assays, we determined that sugar conformation preferentially impacts the ability of these enzymes to deaminate substrates and less so binding to substrates. Using MD simulations, we identify specific active site interactions that promote selectivity based on the 2 '-endo conformation. These findings help inform the biological functions of A3A and A3B in providing antiviral innate immunity and pathogenic functions in cancer.

Epigenetic reader domains regulate chromatin structure and modulate gene expression through the recognition of post-translational modifications on histones. Recently, reader domains have also been found to harbor double-stranded (ds) DNA-binding activity, which is as functionally critical as histone association. Here, we explore the dsDNA recognition of the N-terminal bromodomain of the bromodomain and extra-terminal (BET) protein, BRD4. Using protein-observed 19F NMR, 1H-15N HSQC NMR, electrophoretic mobility shift assays (EMSA), and competitive-inhibition assays, we establish the binding surface of dsDNA and find it to be largely overlapping with the acetylated histone (KAc)-binding site. Rather than engaging in electrostatic contacts, we find dsDNA to interact competitively within the KAc-binding pocket. These interactions are distinct from the highly homologous BET bromodomain, BRDT. Nine additional bromodomains have also been characterized for interacting with dsDNA, including tandem BET bromodomains. Together, these studies help establish a binding model for dsDNA interactions with BRD4 bromodomains and elucidate the chromatin-recognition mechanisms of the BRD4 protein for regulating gene expression.

Cryo-EM structures of the KMT2A/MLL1 core complex bound on nucleosome core particles (NCPs) suggest unusual rotational dynamics of the MLL1 complex approaching its physiological substrate. However, the functional implication of such dynamics remains unclear. Here, we show that the MLL1 core complex also shows high rotational dynamics bound on the NCP carrying the catalytically inert histone H3 lysine 4 to methionine (K4M) mutation. There are two major binding modes of the MLL1 complex on the NCPK4M. Importantly, disruption of only one of the binding modes compromised the overall MLL1 activity in an NCP-specific manner. We propose that the MLL1 core complex probably exists in an equilibrium of poised and active binding modes. The high rotational dynamics of the MLL1 complex on the NCP is a feature that can be exploited for loci-specific regulation of H3K4 methylation in higher eukaryotes.

Here we solve the single particle cryoEM structure for the MLL1 complex with nucleosome core particle (NCP) carrying histone H3 lysine 4 to methionine mutation. The MLL1 complex displays significant rotational dynamics on the NCP, a feature distinct from the yeast SET1 complex. We identified two major binding modes of the MLL1 complex on the NCP. Both binding modes anchor on the NCP through ASH2L, but they differ drastically with regard to where the MLL1 SET domain and RbBP5 bind. We show that one of the binding modes is catalytically inactive since disrupting interactions unique to this binding mode does not affect overall MLL1 activity in an NCP-specific manner. Interestingly, the inactive binding mode is in a configuration similar to that of the ySET1- NCP complex, which is intrinsically inactive on an unmodified NCP. The high rotational dynamics of the MLL1 complex as well as distinction between MLL and yeast SET1 complexes may reflect the necessity for loci-specific regulation of H3K4 methylation states in higher eukaryotes.

Recent cryo-EM structures show the highly dynamic nature of the MLL1-NCP (nucleosome core particle) interaction. Functional implication and regulation of such dynamics remain unclear. Here we show that DPY30 and the intrinsically disordered regions (IDRs) of ASH2L work together in restricting the rotational dynamics of the MLL1 complex on the NCP. We show that DPY30 binding to ASH2L leads to stabilization and integration of ASH2L IDRs into the MLL1 complex and establishes new ASH2L-NCP contacts. The significance of ASH2L-DPY30 interactions is demonstrated by requirement of both ASH2L IDRs and DPY30 for dramatic increase of processivity and activity of the MLL1 complex. This DPY30 and ASH2L-IDR dependent regulation is NCP-specific and applies to all members of the MLL/SET1 family of enzymes. We further show that DPY30 is causal for de novo establishment of H3K4me3 in ESCs. Our study provides a paradigm of how H3K4me3 is regulated on chromatin and how H3K4me3 heterogeneity can be modulated by ASH2L IDR interacting proteins.

In eukaryotes, histone H3K4 methylation by the MLL/SET1 family histone methyltransferases is enriched at transcription regulatory elements including gene promoters and enhancers. The level of H3K4 methylation is highly correlated with transcription activation and is one of the most frequently used histone post-translational modifications to predict transcriptional outcome. Recently, it has been shown that rearrangement of the cellular landscape of H3K4 mono-methylation at distal enhancers precedes cell fate transition and is used for identification of novel regulatory elements for development and disease progression. Similarly, broad H3K4 tri-methylation regions have also been used to predict intrinsic tumor suppression properties of regulator regions in a variety of cellular models. Understanding the regulation for how H3K4 methylation is deposited and regulated is of paramount importance. In this review, we will discuss new findings on how the MLL/SET1 family enzymes are regulated on chromatin and their potential functional and regulatory implications. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.

Heterotrichous ciliates play an important role in aquatic ecosystem energy flow processes and many are model organisms for research in cytology, regenerative biology, and toxicology. In the present study, we combine both morphological and molecular data to infer phylogenetic relationships at family-genus level and propose new evolutionary hypotheses for the class Heterotrichea. The main results include: (1) 96 new ribosomal DNA sequences from 36 populations, representing eight families and 13 genera, including three poorly annotated genera,Folliculinopsis, AmpullofolliculinaandLinostomella;(2) the earliest-branching families are Spirostomidae in single-gene trees and Peritromidae in the concatenated tree, but the family Peritromidae probably represents the basal lineage based on its possession of many "primitive" morphological characters; (3) some findings in molecular trees are not supported by morphological evidence, such as the family Blepharismidae is one of the most recent branches and the relationship between Fabreidae and Folliculinidae is very close; (4) the systematic positions of Condylostomatidae, Climacostomidae, and Gruberiidae remain uncertain based either on morphological or molecular data; and (5) the monophyly of each genus included in the present study is supported by the molecular phylogenetic trees, except forBlepharismain the SSU rDNA tree andFolliculinain the ITS1-5.8S-ITS2 tree.

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

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

In eukaryotes, histone H3K4 methylation by the MLL/SET1 family histone methyltransferases is enriched at transcription regulatory elements including gene promoters and enhancers. The level of H3K4 methylation is highly correlated with transcription activation and is one of the most frequently used histone post-translational modifications to predict transcriptional outcome. Recently, it has been shown that rearrangement of the cellular landscape of H3K4 mono-methylation at distal enhancers precedes cell fate transition and is used for identification of novel regulatory elements for development and disease progression. Similarly, broad H3K4 tri-methylation regions have also been used to predict intrinsic tumor suppression properties of regulator regions in a variety of cellular models. Understanding the regulation for how H3K4 methylation is deposited and regulated is of paramount importance. In this review, we will discuss new findings on how the MLL/SET1 family enzymes are regulated on chromatin and their potential functional and regulatory implications. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.

Mixed Lineage Leukemia (MLL) family histone methyltransferases are the key enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanism by which the MLL complexes recognize histone H3K4 within the nucleosome core particle (NCP) remains unclear. Here, we report the single-particle cryo-electron microscopy (cryo-EM) structure of the human MLL1 core complex bound to the NCP. The MLL1 core complex anchors on the NCP through RbBP5 and ASH2L, which interacts extensively with nucleosomal DNA as well as the surface close to histone H4 N-terminal tail. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, allowing symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.

Mixed lineage leukemia (MLL) family histone methyltransferases are enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanisms whereby the MLL complexes recognize histone H3K4 within nucleosome core particles (NCPs) remain unclear. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the NCP-bound human MLL1 core complex. We show that the MLL1 core complex anchors to the NCP via the conserved RbBP5 and ASH2L, which interact extensively with nucleosomal DNA and the surface close to the N-terminal tail of histone H4. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1 SET domain at the nucleosome dyad, thereby facilitating symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.

As regulators of transcription, epigenetic proteins that interpret post-translational modifications to N-terminal histone tails are essential for maintaining cellular homeostasis. When dysregulated, "reader" proteins become drivers of disease. In the case of bromodomains, which recognize N-ε-acetylated lysine, selective inhibition of individual bromodomain-and-extra-terminal (BET)-family bromodomains has proven challenging. We describe the >55-fold N-terminal-BET bromodomain selectivity of 1,4,5-trisubstituted-imidazole dual kinase-bromodomain inhibitors. Selectivity for the BRD4 N-terminal bromodomain (BRD4(1)) over its second bromodomain (BRD4(2)) arises from the displacement of ordered waters and the conformational flexibility of lysine-141 in BRD4(1). Cellular efficacy was demonstrated via reduction of c-Myc expression, inhibition of NF-κB signaling, and suppression of IL-8 production through potential synergistic inhibition of BRD4(1) and p38α. These dual inhibitors provide a new scaffold for domain-selective inhibition of BRD4, the aberrant function of which plays a key role in cancer and inflammatory signaling.

Several chemical probes have been developed for use in fluorescence polarization screening assays to aid in drug discovery for the bromodomain and extra-terminal domain (BET) proteins. However, few of those have been characterized in the literature. We have designed, synthesized, and thoroughly characterized a novel fluorescence polarization pan-BET chemical probe suitable for high-throughput screening, structure-activity relationships, and hit-to-lead potency and selectivity assays to identify and characterize BET bromodomain inhibitors.

APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2'-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease. We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay. Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains. Structure-activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity. Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure-activity data. An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement. Given these findings, novel and easily synthesized inhibitors are being introduced to the growing field of bromodomain inhibitor development.