Search

Abstract Background Methylation of histone H3 lysine 79 (H3K79) by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome. Results Targeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1. Conclusions Targeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.

Objectives: In order to recognise and facilitate the development of excellent medical doctors (physicians and residents), it is important to first identify the characteristics of excellence. Failure to recognising excellence causes loss of talent, loss of role models and it lowers work ethos. This causes less than excellent patient care and lack of commitment to improve the healthcare system., Design: Systematic review performed according to the Association for Medical Education in Europe guideline., Information Sources: We searched Medline, Embase, Psycinfo, ERIC and CINAHL until 14 March 2022., Eligibility Criteria: We included original studies describing characteristics of excellent medical doctors, using a broad approach as to what is considered excellence. Assuming that excellence will be viewed differently depending on the interplay, and that different perspectives (peers, supervisors and patients) will add to a complete picture of the excellent medical doctor, we did not limit this review to a specific perspective., Data Extraction and Synthesis: Data extraction and quality assessment were performed independently by two researchers. We used the Quality Assessment Tool for Different Designs for quality assessment., Results: Eleven articles were eligible and described the characteristics from different perspectives: (1) physicians on physicians, (2) physicians on residents, (3) patients on physicians and (4) mixed group (diverse sample of participants on physicians). The included studies showed a wide range of characteristics, which could be grouped into competencies (communication, professionalism and knowledge), motivation (directed to learning and to patient care) and personality (flexibility, empathy)., Conclusions: In order to define excellence of medical doctors three clusters seem important: competence, motivation and personality. This is in line with Renzulli's model of gifted behaviour. Our work adds to this model by specifying the content of these clusters, and as such provides a basis for definition and recognition of medical excellence., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To identify risk factors for failed induction in nulliparous women., Material and Methods: A retrospective cohort study of nulliparous women admitted for induction of labour (IOL). Identification of risk factors for failed IOL by comparing clinical characteristics of patients with a failed IOL defined as birth by caesarean section (LSCS) with those achieving vaginal birth., Results: During a 12 month episode, 400 nulliparous women had an IOL; of these 168 (42%) failed to deliver vaginally. Independent antenatal risk factors for failed IOL were higher maternal age (OR = 1.052 per additional year), being shorter (OR = 1.112 per cm less maternal height) and a lower cervical dilatation score (OR = 1.411 per lower cervical dilatation score). A longer active phase (OR = 1.004 per additional minute) was the only independent intrapartum risk factor for having a LSCS., Conclusions: Maternal age, height and cervical dilatation are independent antepartum risk factors, while duration of active phase is the single independent intrapartum risk factor for a failed IOL. Increased maternal BMI was less of a risk factor than anticipated with increased risk for failed IOL, as independent risk factor, more or less restricted to the morbidly obese women.

Post-translational modifications of histone proteins have a crucial role in regulating gene expression. If efficiently re-established after chromosome duplication, histone modifications could help propagate gene expression patterns in dividing cells by epigenetic mechanisms. We used an integrated approach to investigate the dynamics of the conserved methylation of histone H3 Lys 79 (H3K79) by Dot1. Our results show that methylation of H3K79 progressively changes after histone deposition, which is incompatible with a rapid copy mechanism. Instead, methylation accumulates on ageing histones, providing the cell with a timer mechanism to directly couple cell-cycle length to changes in chromatin modification on the nucleosome core.

In the eukaryotic cell nucleus, the DNA is packaged in a structure called chromatin. The fundamental building block of chromatin is the nucleosome, which is composed of DNA wrapped around an octamer of four distinct histone proteins. Post-translational modifications (PTMs) of histone proteins can affect chromatin structure and function and thereby play critical roles in regulating gene expression. Most histone PTMs are found in unstructured histone tails that protrude from the nucleosome core. As a consequence, (synthetic) peptide truncations of these tails provide convenient substrates for the analysis of histone binding proteins and modifying enzymes. Modifications located on residues that reside in the nucleosome core are more difficult to study because short peptides do not recapitulate this defined structured state well. Methylation of histone H3 on Lys79 (H3K79), mediated by the Dot1 enzyme, is an example of such a core PTM. This modification, which is highly conserved, is linked to human leukemia, and pharmacological modulation of Dot1 activity could be a strategy to treat leukemia. Here we review the available and emerging genetic, biochemical, and chemical methods that together are starting to reveal the function and regulation of this and other histone modifications on the nucleosome core., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Dot1 is a highly conserved methyltransferase that modifies histone H3 on the nucleosome core surface. In contrast to yeast, flies, and humans where a single Dot1 enzyme is responsible for all methylation of H3 lysine 79 (H3K79), African trypanosomes express two DOT1 proteins that methylate histone H3K76 (corresponding to H3K79 in other organisms) in a cell-cycle-regulated manner. Whereas DOT1A is essential for normal cell cycle progression, DOT1B is involved in differentiation and control of antigenic variation of this protozoan parasite. Analysis of DOT1A and DOT1B in trypanosomes or in vitro, to understand how H3K76 methylation is controlled during the cell cycle, is complicated by the lack of genetic tools and biochemical assays. To eliminate these problems, we developed a heterologous expression system in yeast. Whereas Trypanosoma brucei DOT1A predominantly dimethylated H3K79, DOT1B trimethylated H3K79 even in the absence of dimethylation by DOT1A. Furthermore, DOT1A activity was selectively reduced by eliminating ubiquitylation of H2B. The tail of histone H4 was not required for activity of DOT1A or DOT1B. These findings in yeast provide new insights into possible mechanisms of regulation of H3K76 methylation in Trypanosoma brucei.

Dot1 is a conserved histone methyltransferase that methylates histone H3 on lysine 79. We previously observed that in Saccharomyces cerevisiae, a single DOT1 gene encodes two Dot1 protein species. Here, we show that the relative abundance of the two isoforms changed under nutrient-limiting conditions. A mutagenesis approach showed that the two Dot1 isoforms are produced from two alternative translation start sites as a result of leaky scanning by the ribosome. The leaky scanning was not affected by the 5'- or 3'-untranslated regions of DOT1, indicating that translation initiation is determined by the DOT1 coding sequence. Construction of yeast strains expressing either one of the isoforms showed that both were sufficient for Dot1's role in global H3K79 methylation and telomeric gene silencing. However, the absence of the long isoform of Dot1 altered the resistance of yeast cells to the chitin-binding drug Calcofluor White, suggesting that the two Dot1 isoforms have a differential function in cell wall biogenesis.

Dot1 methylates histone H3 lysine 79 (H3K79) on the nucleosome core and is involved in Sir protein-mediated silencing. Previous studies suggested that H3K79 methylation within euchromatin prevents nonspecific binding of the Sir proteins, which in turn facilitates binding of the Sir proteins in unmethylated silent chromatin. However, the mechanism by which the Sir protein binding is influenced by this modification is unclear. We performed genome-wide synthetic genetic array (SGA) analysis and identified interactions of DOT1 with SIR1 and POL32. The synthetic growth defects found by SGA analysis were attributed to the loss of mating type identity caused by a synthetic silencing defect. By using epistasis analysis, DOT1, SIR1, and POL32 could be placed in different pathways of silencing. Dot1 shared its silencing phenotypes with the NatA N-terminal acetyltransferase complex and the conserved N-terminal bromo adjacent homology (BAH) domain of Sir3 (a substrate of NatA). We classified all of these as affecting a common silencing process, and we show that mutations in this process lead to nonspecific binding of Sir3 to chromatin. Our results suggest that the BAH domain of Sir3 binds to histone H3K79 and that acetylation of the BAH domain is required for the binding specificity of Sir3 for nucleosomes unmethylated at H3K79.

Whereas mono-, di- and trimethylation states of lysines on histones typically have specific functions, no specific functions have been attributed so far to the different methylation states of histone H3 Lysine 79 (H3K79) generated by Dot1. Here we show that Dot1, in contrast to other known histone methyltransferases, introduces multiple methyl groups via a nonprocessive mechanism. The kinetic mechanism implies that the H3K79 methylation states cannot be generated independently, suggesting functional redundancy. Indeed, gene silencing in yeast, which is dependent on Dot1, relied on global H3K79 methylation levels and not on one specific methylation state. Furthermore, our findings suggest that histone H2B ubiquitination affects H3K79 trimethylation by enhancing synthesis of all H3K79 methylation states. Our results suggest that multiple methylation of H3K79 leads to a binary code, which is expected to limit the possibilities for regulation by putative demethylases or binding proteins.

Bites by Loxosceles spiders can produce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, hemolysis, and persistent inflammation. The causative factor is a sphingomyelinase D (SMaseD) that cleaves sphingomyelin into choline and ceramide 1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. However, the molecular basis for SMaseD toxicity is not well understood, which hampers effective therapy. Here we show that the spider and bacterial SMases D hydrolyze albumin-bound lysophosphatidylcholine (LPC), but not sphingosylphosphorylcholine, with K(m) values ( approximately 20-40 microm) well below the normal LPC levels in blood. Thus, toxic SMases D have intrinsic lysophospholipase D activity toward LPC. LPC hydrolysis yields the lipid mediator lysophosphatidic acid (LPA), a known inducer of platelet aggregation, endothelial hyperpermeability, and pro-inflammatory responses. Introduction of LPA(1) receptor cDNA into LPA receptor-negative cells renders non-susceptible cells susceptible to SmaseD, but only in LPC-containing media. Degradation of circulating LPC to LPA with consequent activation of LPA receptors may have a previously unappreciated role in the pathophysiology of secreted SMases D.