Association between ATP luminometry of feeding equipment and environmental and health parameters of preweaned calves on dairy farms.

The objective of this study was to examine the influence of different environmental factors on ATP luminometry measurements of feeding equipment and to investigate associations with health of preweaned calves and the levels of ATP identified through luminometry. On 50 commercial dairy farms in Quebec, Canada, ATP luminometry measurements (in relative light units (RLU)) were obtained using the direct swabbing technique with Hygiena UltraSnap swabs and a liquid rinsing technique with the same swab for automatic milk feeders (AMF), bottles, buckets, esophageal tube feeders (ET), milk replacer, nipples and water. During this visit, environmental factors (including temperature, air draft, humidity, ammonia, and bacterial count) were collected and a clinical examination (including respiratory score and fecal score) was performed for all preweaned calves present at the farm. This process was repeated 4 times in a year, leading to collection of luminometer results, environmental parameters, and overall health of calves for each season per farm. Overall, a difference in luminometer results was seen between the different periods sampled for all feeding equipment (except the ET), milk replacer and water, showing higher RLU values in spring and summer and lower values in autumn and winter. When comparing RLU measurements with environmental factors, only a low to negligible correlation could be found. When feeding equipment was classified as not contaminated or contaminated based on previously described cut-off values, a good agreement within a farm for the different seasons was noticed only for nipples (Gwet's agreement AC1 = 0.64), with a poor to moderate agreement for other feeding equipment. Regarding the different models of nipples, 'Peach Teat' nipples showed higher RLU values compared with 'Merricks' nipples models. An association was seen between the proportion of preweaned calves suffering from diarrhea on the farm and the contamination of AMF based on ATP luminometry (logistic regression estimate = 0.52, P = 0.04). For other feeding equipment, milk replacer and water, no significant associations were found. This study showed that ATP luminometry measurements of feeding equipment from preweaned calves are susceptible to seasonality and type of nipple. Thus, these factors should be taken into consideration when interpreting results. Additionally, an association could be made between diarrhea in preweaned calves and the contamination of AMF based on ATP luminometry, showing the potential clinical importance of this on-farm hygiene assessment tool.

Annealed fractional Lévy-Ito diffusion models for protein generation.

Protein generation has numerous applications in designing therapeutic antibodies and creating new drugs. Still, it is a demanding task due to the inherent complexities of protein structures and the limitations of current generative models. Proteins possess intricate geometry, and sampling their conformational space is challenging due to its high dimensionality. This paper introduces novel Markovian and non-Markovian generative diffusion models based on fractional stochastic differential equations and the Lévy distribution, allowing for a more effective exploration of the conformational space. The approach is applied to a dataset of 40,000 proteins and evaluated in terms of Fréchet distance, fidelity, and diversity, outperforming the state-of-the-art by 25.4%, 35.8%, and 11.8%, respectively.

Comprehensive transcriptomic analysis of long non-coding RNAs in bovine ovarian follicles and early embryos.

Long non-coding RNAs (lncRNAs) have been the subject of numerous studies over the past decade. First thought to come from aberrant transcriptional events, lncRNAs are now considered a crucial component of the genome with roles in multiple cellular functions. However, the functional annotation and characterization of bovine lncRNAs during early development remain limited. In this comprehensive analysis, we review lncRNAs expression in bovine ovarian follicles and early embryos, based on a unique database comprising 468 microarray hybridizations from a single platform designed to target 7,724 lncRNA transcripts, of which 5,272 are intergenic (lincRNA), 958 are intronic, and 1,524 are antisense (lncNAT). Compared to translated mRNA, lncRNAs have been shown to be more tissue-specific and expressed in low copy numbers. This analysis revealed that protein-coding genes and lncRNAs are both expressed more in oocytes. Differences between the oocyte and the 2-cell embryo are also more apparent in terms of lncRNAs than mRNAs. Co-expression network analysis using WGCNA generated 25 modules with differing proportions of lncRNAs. The modules exhibiting a higher proportion of lncRNAs were found to be associated with fewer annotated mRNAs and housekeeping functions. Functional annotation of co-expressed mRNAs allowed attribution of lncRNAs to a wide array of key cellular events such as meiosis, translation initiation, immune response, and mitochondrial related functions. We thus provide evidence that lncRNAs play diverse physiological roles that are tissue-specific and associated with key cellular functions alongside mRNAs in bovine ovarian follicles and early embryos. This contributes to add lncRNAs as active molecules in the complex regulatory networks driving folliculogenesis, oogenesis and early embryogenesis all of which are necessary for reproductive success.

Hygiene management practices and adenosine triphosphate luminometry of feeding equipment in preweaning calves on dairy farms in Quebec, Canada.

The objective of this study was to describe the cleaning practices currently used for preweaning calves on dairy farms in Quebec, Canada. In addition, contamination of feeding equipment for preweaning calves was described using ATP (expressed as relative light units, RLU), visual assessment, and bacteriological analysis. A questionnaire was administered on 50 commercial dairy farms in Quebec, Canada, regarding the self-reported cleaning protocol used for feeding equipment of preweaning calves. During the visit, a visual score was given to the feeding equipment available at the farm. Afterward, ATP luminometry measurements were obtained using Hygiene UltraSnap and MicroSnap swabs (Hygiene, Camarillo, CA), and the liquid rinsing technique for buckets, nipples, bottles, esophageal tube feeders (ET), the tube of automatic milk feeders (AMF), water samples, and milk replacer. An additional direct swabbing technique was performed on buckets and nipples. The fluid retrieved from the liquid rinsing technique was also used to determine the total bacterial count (TBC) and total coliform count. Based on the bacteriological analysis, optimal RLU cutoff values to determine contamination were obtained. The median (interquartile range) luminometer measurements using the UltraSnap and direct technique for buckets and nipples were 2,082 (348-7,410) and 3,462 (462-7,518) RLU, respectively; and, using the liquid technique for bottles, ET, AMF, water, and milk replacer were 43 (4-974), 15 (4-121), 301 (137-1,323), 190 (71-358), and 94 (38-218) RLU, respectively. Overall, for all equipment and both techniques used, higher RLU values were seen in UltraSnap samples compared with MicroSnap samples. Additionally, for buckets and nipples, higher RLU values were obtained for the direct swabbing method compared with the liquid sampling method for both swabs used. No differences in the level of contamination were seen between the different feeding equipment used within a farm. Overall, a higher correlation with bacteriological results was noticed for ATP luminometry compared with the visual score, with a high correlation for nipples and bottles using the UltraSnap and liquid technique. Based on the classification of "contaminated" (TBC ≥100,000 cfu/mL) or "not contaminated" (TBC <100,000 cfu/mL), optimal ATP luminometer cutoff values for buckets, nipples, bottles, AMF, water, and milk replacer were 798, 388, 469, 282, 1,432, and 93 RLU, respectively. No clear association was found between ATP measurements and the self-reported cleaning protocol. This study gave new insights into the current cleaning procedures and contamination of feeding equipment for preweaning calves on dairy farms in Quebec. In addition, ATP luminometry cutoff values could help benchmark farms regarding cleaning practices and provide customized advice, improving the overall hygiene management, and thus the health, of preweaning calves on dairy farms.

Standardization and validation of ATP luminometry as a diagnostic tool to assess the cleanliness of feeding equipment in preweaning calves.

The objective of this cross-sectional study was to standardize a reliable and repeatable swabbing technique using ATP luminometry (light emission proportional to the amount of ATP with result provided in relative light units [RLU]) to describe the cleanliness of various feeding equipment used for preweaning calves in dairy farms. A total of 7 Québec commercial dairy herds were selected conveniently. Following visual hygiene scoring, the cleanliness of every available piece of feeding equipment was assessed using direct surface swabbing for buckets and nipples with Hygiena UltraSnap swabs. A liquid rinsing technique was used for esophageal feeders, bottles, and automatic milk feeders (AMF) with UltraSnap, AquaSnap, and MicroSnap swabs. To validate direct swabbing technique of buckets, a stage within and between operators was realized, as well as a conventional bacterial culture. A total of 519 swab samples were obtained from 201 pieces of equipment. The median (interquartile range) contamination in RLU for a bottle, esophageal feeder, AMF, bucket and nipple was 2 (1;6), 2 (0;12), 52 (19;269), 886 (128;7,230) and 899 (142;6,928), respectively. The direct swabbing technique, which consists in swabbing directly the surface of an equipment, showed excellent correlation for intrarater reliability (intraclass correlation (ICC) = 0.93; 95% CI: 0.88-0.96). The interoperator (2 sessions with 3 different operators) reliability also showed high correlation (ICC = 0.88; 95% CI: 0.78-0.94 for the first session, and ICC = 0.89; 95% CI: 0.79-0.95 for the second session). Luminometer values were positively associated with the visual score of esophageal feeders, AMF and buckets. A positive correlation between bacterial culture and direct swabbing of buckets was also found for the UltraSnap (rs = 0.653; 95% CI: 0.283-0.873; P = 0.0003) and MicroSnap (rs = 0.569, 95% CI: 0.309-0.765; P = 0.002). This study describes a standardized and practical on-farm swabbing technique for assessing the hygienic status of feeding equipment by luminometry, which can be integrated in the investigation of preweaning dairy calves problems.

ProtInteract: A deep learning framework for predicting protein-protein interactions.

Proteins mainly perform their functions by interacting with other proteins. Protein-protein interactions underpin various biological activities such as metabolic cycles, signal transduction, and immune response. However, due to the sheer number of proteins, experimental methods for finding interacting and non-interacting protein pairs are time-consuming and costly. We therefore developed the ProtInteract framework to predict protein-protein interaction. ProtInteract comprises two components: first, a novel autoencoder architecture that encodes each protein's primary structure to a lower-dimensional vector while preserving its underlying sequence attributes. This leads to faster training of the second network, a deep convolutional neural network (CNN) that receives encoded proteins and predicts their interaction under three different scenarios. In each scenario, the deep CNN predicts the class of a given encoded protein pair. Each class indicates different ranges of confidence scores corresponding to the probability of whether a predicted interaction occurs or not. The proposed framework features significantly low computational complexity and relatively fast response. The contributions of this work are twofold. First, ProtInteract assimilates the protein's primary structure into a pseudo-time series. Therefore, we leverage the nature of the time series of proteins and their physicochemical properties to encode a protein's amino acid sequence into a lower-dimensional vector space. This approach enables extracting highly informative sequence attributes while reducing computational complexity. Second, the ProtInteract framework utilises this information to identify protein interactions with other proteins based on its amino acid configuration. Our results suggest that the proposed framework performs with high accuracy and efficiency in predicting protein-protein interactions.

Deformable Protein Shape Classification Based on Deep Learning, and the Fractional Fokker-Planck and Kähler-Dirac Equations.

The classification of deformable protein shapes, based solely on their macromolecular surfaces, is a challenging problem in protein-protein interaction prediction and protein design. Shape classification is made difficult by the fact that proteins are dynamic, flexible entities with high geometrical complexity. In this paper, we introduce a novel description for such deformable shapes. This description is based on the bifractional Fokker-Planck and Dirac-Kähler equations. These equations analyse and probe protein shapes in terms of a scalar, vectorial and non-commuting quaternionic field, allowing for a more comprehensive description of the protein shapes. An underlying non-Markovian Lévy random walk establishes geometrical relationships between distant regions while recalling previous analyses. Classification is performed with a multiobjective deep hierarchical pyramidal neural network, thus performing a multilevel analysis of the description. Our approach is applied to the SHREC'19 dataset for deformable protein shapes classification and to the SHREC'16 dataset for deformable partial shapes classification, demonstrating the effectiveness and generality of our approach.

Protein-protein interaction prediction with deep learning: A comprehensive review.

Most proteins perform their biological function by interacting with themselves or other molecules. Thus, one may obtain biological insights into protein functions, disease prevalence, and therapy development by identifying protein-protein interactions (PPI). However, finding the interacting and non-interacting protein pairs through experimental approaches is labour-intensive and time-consuming, owing to the variety of proteins. Hence, protein-protein interaction and protein-ligand binding problems have drawn attention in the fields of bioinformatics and computer-aided drug discovery. Deep learning methods paved the way for scientists to predict the 3-D structure of proteins from genomes, predict the functions and attributes of a protein, and modify and design new proteins to provide desired functions. This review focuses on recent deep learning methods applied to problems including predicting protein functions, protein-protein interaction and their sites, protein-ligand binding, and protein design.

Recurrent chromosomal translocations in sarcomas create a megacomplex that mislocalizes NuA4/TIP60 to Polycomb target loci.

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.

Masking terminal neo-epitopes of linear peptides through glycosylation favours immune responses towards core epitopes producing parental protein bound antibodies.

Glycosylation of hydrophobic peptides at one terminus effectively increases their water-solubility, and conjugation through the opposing end to a carrier protein, renders them more immunogenic. Moreover, the glycosylation minimizes antibody responses to potentially deleterious, non-productive terminal neo-epitope regions of the peptides, and consequently shifts peptide immunogenicity towards the core amino acid residues. As proof of concept, glycopeptide-protein conjugates related to influenza hemagglutinin (HA), neuraminidase (NA), and the dimerization loop region of human epidermal growth factor receptor 2 (Her2), demonstrated a favorable production of core peptide specific antibodies as determined by ELISA studies. Furthermore, glycosylated Her2 peptide conjugate antisera were also shown to recognize full length Her2 protein by ELISA and at the cell surface through flow cytometry analysis. In contrast, unmasked peptide conjugates generated significant antibody populations that were specific to the terminal neo-epitope of the peptide immunogen that are notably absent in parental proteins. Antibodies generated in this manner to peptides in the dimerization loop of Her2 are also functional as demonstrated by the growth inhibition of Her2 expressing SKBR3 carcinoma cells. This method provides a technique to tailor-make epitope-specific antibodies that may facilitate vaccine, therapeutic and diagnostic antibody development.

Engineered signaling centers for the spatially controlled patterning of human pluripotent stem cells.

Signaling centers, localized groups of cells that secrete morphogens, play a key role in early development and organogenesis by orchestrating spatial cell fate patterning. Here we present a microfluidic approach that exposes human pluripotent stem cell (hPSC) colonies to spatiotemporally controlled morphogen gradients generated from artificial signaling centers. In response to a localized source of bone morphogenetic protein 4 (BMP4), hPSC colonies reproducibly break their intrinsic radial symmetry to produce distinct, axially arranged differentiation domains. Counteracting sources of the BMP antagonist NOGGIN enhance this spatial control of cell fate patterning. We also show how morphogen concentration and cell density affect the BMP response and germ layer patterning. These results demonstrate that the intrinsic capacity of stem cells for self-organization can be extrinsically controlled through the use of engineered signaling centers.

Low-dimensional Dynamics of Two Coupled Biological Oscillators.

The circadian clock and the cell cycle are two biological oscillatory processes that coexist within individual cells. These two oscillators were found to interact, which can lead to their synchronization. Here, we develop a method to identify a low-dimensional stochastic model of the coupled system directly from time-lapse imaging in single cells. In particular, we infer the coupling and non-linear dynamics of the two oscillators from thousands of mouse and human single-cell fluorescence microscopy traces. This coupling predicts multiple phase-locked states showing different degrees of robustness against molecular fluctuations inherent to cellular-scale biological oscillators. For the 1:1 state, the predicted phase-shifts upon period perturbations were validated experimentally. Moreover, the phase-locked states are temperature-independent and evolutionarily conserved from mouse to human, hinting at a common underlying dynamical mechanism. Finally, we detect a signature of the coupled dynamics in a physiological context, explaining why tissues with different proliferation states exhibited shifted circadian clock phases.

Single Live Cell Monitoring of Protein Turnover Reveals Intercellular Variability and Cell-Cycle Dependence of Degradation Rates.

Cells need to reliably control their proteome composition to maintain homeostasis and regulate growth. How protein synthesis and degradation interplay to control protein expression levels remains unclear. Here, we combined a tandem fluorescent timer and pulse-chase protein labeling to disentangle how protein synthesis and degradation control protein homeostasis in single live mouse embryonic stem cells. We discovered substantial cell-cycle dependence in protein synthesis rates and stabilization of a large number of proteins around cytokinesis. Protein degradation rates were highly variable between cells, co-varied within individual cells for different proteins, and were positively correlated with synthesis rates. This suggests variability in proteasome activity as an important source of global extrinsic noise in gene expression. Our approach paves the way toward understanding the complex interplay of synthesis and degradation processes in determining protein levels of individual mammalian cells.

Casein kinase 2 mediated phosphorylation of Spt6 modulates histone dynamics and regulates spurious transcription.

CK2 is an essential protein kinase implicated in various cellular processes. In this study, we address a potential role of this kinase in chromatin modulations associated with transcription. We found that CK2 depletion from yeast cells leads to replication-independent increase of histone H3K56 acetylation and global activation of H3 turnover in coding regions. This suggests a positive role of CK2 in maintenance/recycling of the histone H3/H4 tetramers during transcription. Interestingly, strand-specific RNA-seq analyses show that CK2 inhibits global cryptic promoters driving both sense and antisense transcription. This further indicates a role of CK2 in the modulation of chromatin during transcription. Next, we showed that CK2 interacts with the major histone chaperone Spt6, and phosphorylates it in vivo and in vitro. CK2 phosphorylation of Spt6 is required for its cellular levels, for the suppression of histone H3 turnover and for the inhibition of spurious transcription. Finally, we showed that CK2 and Spt6 phosphorylation sites are important to various transcriptional responses suggesting that cryptic intragenic and antisense transcript production are associated with a defective adaptation to environmental cues. Altogether, our data indicate that CK2 mediated phosphorylation of Spt6 regulates chromatin dynamics associated with transcription, and prevents aberrant transcription.

Serum vitamin C levels modulate the lifespan and endoplasmic reticulum stress response pathways in mice synthesizing a nonfunctional mutant WRN protein.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase (WRN). Mice lacking part of the helicase domain of the WRN ortholog exhibit several phenotypic features of WS. In this study, we generated a Wrn mutant line that, like humans, relies entirely on dietary sources of vitamin C (ascorbate) to survive, by crossing them to mice that lack the gulonolactone oxidase enzyme required for ascorbate synthesis. In the presence of 0.01% ascorbate (w/v) in drinking water, double-mutant mice exhibited a severe reduction in lifespan, small size, sterility, osteopenia, and metabolic profiles different from wild-type (WT) mice. Although increasing the dose of ascorbate to 0.4% improved dramatically the phenotypes of double-mutant mice, the metabolic and cytokine profiles were different from age-matched WT mice. Finally, double-mutant mice treated with 0.01% ascorbate revealed a permanent activation of all the 3 branches of the ER stress response pathways due to a severe chronic oxidative stress in the ER compartment. In addition, markers associated with the ubiquitin-proteasome-dependent ER-associated degradation pathway were increased. Augmenting the dose of ascorbate reversed the activation of this pathway to WT levels rendering this pathway a potential therapeutic target in WS.-Aumailley, L., Dubois, M. J., Brennan, T. A., Garand, C., Paquet, E. R., Pignolo, R. J., Marette, A., Lebel, M. Serum vitamin C levels modulate the lifespan and endoplasmic reticulum stress response pathways in mice synthesizing a nonfunctional mutant WRN protein.

Assessment of early response biomarkers in relation to long-term survival in patients with HER2-negative breast cancer receiving neoadjuvant chemotherapy plus bevacizumab: Results from the Phase II PROMIX trial.

Pathologic complete response (pCR) is a predictor for favorable outcome after neoadjuvant treatment in early breast cancer. Modulation of gene expression may also provide early readouts of biological activity and prognosis, offering the possibility for timely response-guided treatment adjustment. The role of early transcriptional changes in predicting response to neoadjuvant chemotherapy plus bevacizumab was investigated. One-hundred-and-fifty patients with large, operable and locally advanced HER2-negative breast cancer received epirubicin and docetaxel, with the addition of bevacizumab. Patients underwent tumor biopsies at baseline, after Cycle 2 and at the time of surgery. The primary end point, pCR, and its relation with the secondary endpoints event-free survival (EFS), overall survival (OS) and gene expression profiles, are reported. The pCR rate was 13% (95% CI 8.6-20.2), with significantly more pCRs among triple-negative [28% (95% CI 14.8-45.4)] than among hormone receptor positive (HR+) tumors [9% (95% CI 4.6-16.3); (OR = 3.9 [CI = 1.5-10.3])]. pCR rates were not associated with EFS or OS. PAM50 subtypes significantly changed after Cycle 2 (p = 0.03) and an index of absolute changes in PAM50 correlations between these time-points was associated with EFS [HR = 0.62 (CI = 0.3-1.1)]. In univariable analyses, signatures for angiogenesis, proliferation, estrogen receptor signaling, invasion and metastasis, and immune response, measured after Cycle 2, were associated with pCR in HR+ tumors. Evaluation of changes in molecular subtypes and other signatures early in the course of neoadjuvant treatment may be predictive of pCR and EFS. These factors may help guide further treatment and should be considered when designing neoadjuvant trials.

Generation of monoclonal pan-hemagglutinin antibodies for the quantification of multiple strains of influenza.

Vaccination is the most effective course of action to prevent influenza. About 150 million doses of influenza vaccines were distributed for the 2015-2016 season in the USA alone according to the Centers for Disease Control and Prevention. Vaccine dosage is calculated based on the concentration of hemagglutinin (HA), the main surface glycoprotein expressed by influenza which varies from strain to strain. Therefore yearly-updated strain-specific antibodies and calibrating antigens are required. Preparing these quantification reagents can take up to three months and significantly slows down the release of new vaccine lots. Therefore, to circumvent the need for strain-specific sera, two anti-HA monoclonal antibodies (mAbs) against a highly conserved sequence have been produced by immunizing mice with a novel peptide-conjugate. Immunoblots demonstrate that 40 strains of influenza encompassing HA subtypes H1 to H13, as well as B strains from the Yamagata and Victoria lineage were detected when the two mAbs are combined to from a pan-HA mAb cocktail. Quantification using this pan-HA mAbs cocktail was achieved in a dot blot assay and results correlated with concentrations measured in a hemagglutination assay with a coefficient of correlation of 0.80. A competitive ELISA was also optimised with purified viral-like particles. Regardless of the quantification method used, pan-HA antibodies can be employed to accelerate process development when strain-specific antibodies are not available, and represent a valuable tool in case of pandemics. These antibodies were also expressed in CHO cells to facilitate large-scale production using bioreactor technologies which might be required to meet industrial needs for quantification reagents. Finally, a simulation model was created to predict the binding affinity of the two anti-HA antibodies to the amino acids composing the highly conserved epitope; different probabilities of interaction between a given amino acid and the antibodies might explain the affinity of each antibody against different influenza strains.

A new promoter element associated with daily time keeping in Drosophila.

Circadian clocks are autonomous daily timekeeping mechanisms that allow organisms to adapt to environmental rhythms as well as temporally organize biological functions. Clock-controlled timekeeping involves extensive regulation of rhythmic gene expression. To date, relatively few clock-associated promoter elements have been identified and characterized. In an unbiased search of core clock gene promoters from 12 species of Drosophila, we discovered a 29-bp consensus sequence that we designated as the Clock-Associated Transcriptional Activation Cassette or 'CATAC'. To experimentally address the spatiotemporal expression information associated with this element, we generated constructs with four separate native CATAC elements upstream of a basal promoter driving expression of either the yeast Gal4 or firefly luciferase reporter genes. Reporter assays showed that presence of wild-type, but not mutated CATAC elements, imparted increased expression levels as well as rhythmic regulation. Part of the CATAC consensus sequence resembles the E-box binding site for the core circadian transcription factor CLOCK/CYCLE (CLK/CYC), and CATAC-mediated expression rhythms are lost in the presence of null mutations in either cyc or the gene encoding the CLK/CYC inhibitor, period (per). Nevertheless, our results indicate that CATAC's enhancer function persists in the absence of CLK/CYC. Thus, CATAC represents a novel cis-regulatory element encoding clock-controlled regulation.

Combined Action of Histone Reader Modules Regulates NuA4 Local Acetyltransferase Function but Not Its Recruitment on the Genome.

Recognition of histone marks by reader modules is thought to be at the heart of epigenetic mechanisms. These protein domains are considered to function by targeting regulators to chromosomal loci carrying specific histone modifications. This is important for proper gene regulation as well as propagation of epigenetic information. The NuA4 acetyltransferase complex contains two of these reader modules, an H3K4me3-specific plant homeodomain (PHD) within the Yng2 subunit and an H3K36me2/3-specific chromodomain in the Eaf3 subunit. While each domain showed a close functional interaction with the respective histone mark that it recognizes, at the biochemical level, genetic level (as assessed with epistatic miniarray profile screens), and phenotypic level, cells with the combined loss of both readers showed greatly enhanced phenotypes. Chromatin immunoprecipitation coupled with next-generation sequencing experiments demonstrated that the Yng2 PHD specifically directs H4 acetylation near the transcription start site of highly expressed genes, while Eaf3 is important downstream on the body of the genes. Strikingly, the recruitment of the NuA4 complex to these loci was not significantly affected. Furthermore, RNA polymerase II occupancy was decreased only under conditions where both PHD and chromodomains were lost, generally in the second half of the gene coding regions. Altogether, these results argue that methylated histone reader modules in NuA4 are not responsible for its recruitment to the promoter or coding regions but, rather, are required to orient its acetyltransferase catalytic site to the methylated histone 3-bearing nucleosomes in the surrounding chromatin, cooperating to allow proper transition from transcription initiation to elongation.

The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation.

The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin.