Dot1 promotes H2B ubiquitination by a methyltransferase-independent mechanism.

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.

Clinical practicality and patient performance for surface-guided automated VMAT gating for DIBH breast cancer radiotherapy.

BACKGROUND AND PURPOSE: To evaluate the performance of automated surface-guided gating for left-sided breast cancer with DIBH and VMAT. MATERIALS AND METHODS: Patients treated in the first year after introduction of DIBH with VMAT were retrospectively considered for analysis. With automated surface-guided gating the beam automatically switches on/off, if the surface region of interest moved in/out the gating tolerance (±3 mm, ±3°). Patients were coached to hold their breath as long as comfortably possible. Depending on the patient's preference, patients received audio instructions during treatment delivery. Real-time positional variations of the breast/chest wall surface with respect to the reference surface were collected, for all three orthogonal directions. The durations and number of DIBHs needed to complete dose delivery, and DIBH position variations were determined. To evaluate an optimal gating window threshold, smaller tolerances of ±2.5 mm, ±2.0 mm, and ±1.5 mm were simulated. RESULTS: 525 fractions from 33 patients showed that median DIBH duration was 51 s (range: 30-121 s), and median 4 DIBHs per fraction were needed to complete VMAT dose delivery. Median intra-DIBH stability and intrafractional DIBH reproducibility approximated 1.0 mm in each direction. No large differences were found between patients who preferred to perform the DIBH procedure with (n = 21) and without audio-coaching (n = 12). Simulations demonstrated that gating window tolerances could be reduced from ±3.0 mm to ±2.0 mm, without affecting beam-on status. CONCLUSION: Independent of the use of audio-coaching, this study demonstrates that automated surface-guided gating with DIBH and VMAT proved highly efficient. Patients' DIBH performance far exceeded our expectations compared to earlier experiences and literature. Furthermore, gating window tolerances could be reduced.

Corrigendum to "Evaluation of image-guided and surface-guided radiotherapy for breast cancer patients treated in deep inspiration breath-hold: A single institution experience" [Tech. Innov. Patient Support Radiat. Oncol. 21 (2022) 51-57].

[This corrects the article DOI: 10.1016/j.tipsro.2022.02.001.].

Evaluation of image-guided and surface-guided radiotherapy for breast cancer patients treated in deep inspiration breath-hold: A single institution experience.

INTRODUCTION: Nowadays, deep inspiratory breath-hold is a common technique to reduce heart dose in left-sided breast radiotherapy. This study evaluates the evolution of the breath-hold technique in our institute, from portal imaging during dose delivery to continuous monitoring with surface-guided radiotherapy (SGRT). MATERIALS AND METHODS: Setup data and portal imaging results were analyzed for 98 patients treated before 2014, and SGRT data for 228 patients treated between 2018 and 2020. For the pre-SGRT group, systematic and random setup errors were calculated for different correction protocols. Residual errors and reproducibility of breath-holds were evaluated for both groups. The benefit of using SGRT for initial positioning was evaluated for another cohort of 47 patients. RESULTS: Online correction reduced the population mean error from 3.9 mm (no corrections) to 1.4 mm. Despite online setup correction, deviations greater than 3 mm were observed in about 10% and 20% of the treatment beams in ventral-dorsal and cranial-caudal directions, respectively. However, these percentages were much smaller than with offline protocols or no corrections. Mean absolute differences between breath-holds within a fraction were smaller in the SGRT-group (1.69 mm) than in the pre-SGRT-group (2.10 mm), and further improved with addition of visual feedback (1.30 mm). SGRT for positioning did not improve setup accuracy, but slightly reduced the time for imaging and setup correction, allowing completion within 3.5 min for 95% of fractions. CONCLUSION: For accurate radiotherapy breast treatments using deep inspiration breath-hold, daily imaging and correction is required. SGRT provides accurate information on patient positioning during treatment and improves patient compliance with visual feedback.

Dot1 histone methyltransferases share a distributive mechanism but have highly diverged catalytic properties.

The conserved histone methyltransferase Dot1 establishes an H3K79 methylation pattern consisting of mono-, di- and trimethylation states on histone H3 via a distributive mechanism. This mechanism has been shown to be important for the regulation of the different H3K79 methylation states in yeast. Dot1 enzymes in yeast, Trypanosoma brucei (TbDot1A and TbDot1B, which methylate H3K76) and human (hDot1L) generate very divergent methylation patterns. To understand how these species-specific methylation patterns are generated, the methylation output of the Dot1 enzymes was compared by expressing them in yeast at various expression levels. Computational simulations based on these data showed that the Dot1 enzymes have highly distinct catalytic properties, but share a distributive mechanism. The mechanism of methylation and the distinct rate constants have implications for the regulation of H3K79/K76 methylation. A mathematical model of H3K76 methylation during the trypanosome cell cycle suggests that temporally-regulated consecutive action of TbDot1A and TbDot1B is required for the observed regulation of H3K76 methylation states.