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-trisubstitutedimidazole 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.

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.

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 MLL1SET 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.

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.

AimTo evaluate and compare the feedback of final year undergraduate dental students in eight Malaysian dental schools on the application of a new system for classifying root canal morphology in teaching and clinical practice.MethodsOne PowerPoint presentation describing two classification systems for root canal morphology (Oral Surgery Oral Medicine Oral Pathology, 1974 38, 456 and its supplemental configurations, International Endodontic Journal 2017, 50, 761) was delivered to final year undergraduate dental students in eight dental schools in Malaysia by two presenters (each presented to four schools). To examine students’ feedback on the utility of each system, printed questionnaires consisting of six questions (five multiple choice questions and one open‐ended question) were distributed and collected after the lecture. The questionnaire was designed to compare the classification systems in terms of accuracy, practicability, understanding of root canal morphology and recommendation for use in pre‐clinical and clinical courses. The exact test was used for statistical analysis, with the level of significance set at 0.05 (P = 0.05).ResultsA total of 382 (out of 447) students participated giving a response rate of 86%. More than 90% of students reported that the new system was more accurate and more practical compared with the Vertucci system (P < 0.001). Overall, 97% of students reported the new system helped their understanding of root and canal morphology compared with the Vertucci classification (P < 0.001). Over 97% of students recommended the use of the new system in teaching, pre‐clinical courses and clinical practice (P < 0.001). Except for two schools, no significant difference was detected between the responses of students for all questions at the different schools (P > 0.05). The students’ responses for all questions were almost similar for both presenters (P > 0.05).ConclusionsThe new system of International Endodontic Journal 2017, 50, 761 for classifying root and canal morphology was favoured by final year undergraduate dental students in Malaysia. The new system has the potential to be included in the undergraduate endodontic curriculum for teaching courses related to root and canal morphology.

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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.

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, Ampullofolliculina and Linostomella; (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 for Blepharisma in the SSU rDNA tree and Folliculina in the ITS1-5.8S-ITS2 tree.