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.

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.

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.

Association between adenosine triphosphate luminometry of feeding equipment and environmental and health parameters of preweaning 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 preweaning 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 preweaning 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 cutoff 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 Merrick's nipples. An association was seen between the proportion of preweaning calves suffering from diarrhea on the farm and the contamination of AMF based on ATP luminometry (logistic regression estimate = 0.52). For other feeding equipment, milk replacer, and water, no significant associations were found. This study showed that ATP luminometry measurements of feeding equipment from preweaning 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 preweaning calves and the contamination of AMF based on ATP luminometry, showing the potential clinical importance of this on-farm hygiene assessment tool.

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.

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.

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.

Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity.

Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation.

During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins.

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.

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.

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.

HBO1 HAT complexes target chromatin throughout gene coding regions via multiple PHD finger interactions with histone H3 tail.

The HBO1 HAT protein is the major source of histone H4 acetylation in vivo and has been shown to play critical roles in gene regulation and DNA replication. A distinctive characteristic of HBO1 HAT complexes is the presence of three PHD finger domains in two different subunits: tumor suppressor proteins ING4/5 and JADE1/2/3. Biochemical and functional analyses indicate that these domains interact with histone H3 N-terminal tail region, but with a different specificity toward its methylation status. Their combinatorial action is essential in regulating chromatin binding and substrate specificity of HBO1 complexes, as well as cell growth. Importantly, localization analyses on the human genome indicate that HBO1 complexes are enriched throughout the coding regions of genes, supporting a role in transcription elongation. These results underline the importance and versatility of PHD finger domains in regulating chromatin association and histone modification crosstalk within a single protein complex.

Conditional inactivation of Upstream Binding Factor reveals its epigenetic functions and the existence of a somatic nucleolar precursor body.

Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs).

Eaf1 Links the NuA4 Histone Acetyltransferase Complex to Htz1 Incorporation and Regulation of Purine Biosynthesis.

Proper modulation of promoter chromatin architecture is crucial for gene regulation in order to precisely and efficiently orchestrate various cellular activities. Previous studies have identified the stimulatory effect of the histone-modifying complex NuA4 on the incorporation of the histone variant H2A.Z (Htz1) at the PHO5 promoter (A. Auger, L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Côté, Mol Cell Biol 28:2257-2270, 2008, http://dx.doi.org/10.1128/MCB.01755-07). In vitro studies with a reconstituted system also indicated an intriguing cross talk between NuA4 and the H2A.Z-loading complex, SWR-C (M. Altaf, A. Auger, J. Monnet-Saksouk, J. Brodeur, S. Piquet, M. Cramet, N. Bouchard, N. Lacoste, R. T. Utley, L. Gaudreau, J. Côté, J Biol Chem 285:15966-15977, 2010, http://dx.doi.org/10.1074/jbc.M110.117069). In this work, we investigated the role of the NuA4 scaffold subunit Eaf1 in global gene expression and genome-wide incorporation of Htz1. We found that loss of Eaf1 affects Htz1 levels mostly at the promoters that are normally highly enriched in the histone variant. Analysis of eaf1 mutant cells by expression array unveiled a relationship between NuA4 and the gene network implicated in the purine biosynthesis pathway, as EAF1 deletion cripples induction of several ADE genes. NuA4 directly interacts with Bas1 activation domain, a key transcription factor of adenine genes. Chromatin immunoprecipitation (ChIP) experiments demonstrate that nucleosomes on the inactive ADE17 promoter are acetylated already by NuA4 and enriched in Htz1. Upon derepression, these poised nucleosomes respond rapidly to activate ADE gene expression in a mechanism likely reminiscent of the PHO5 promoter, leading to nucleosome disassembly. These detailed molecular events depict a specific case of cross talk between NuA4-dependent acetylation and incorporation of histone variant Htz1, presetting the chromatin structure over ADE promoters for subsequent chromatin remodeling and activated transcription.

Redirecting splicing with bifunctional oligonucleotides.

Ectopic modulators of alternative splicing are important tools to study the function of splice variants and for correcting mis-splicing events that cause human diseases. Such modulators can be bifunctional oligonucleotides made of an antisense portion that determines target specificity, and a non-hybridizing tail that recruits proteins or RNA/protein complexes that affect splice site selection (TOSS and TOES, respectively, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use of TOSS and TOES has been restricted to a handful of targets. To generalize the applicability and demonstrate the robustness of TOSS, we have tested this approach on more than 50 alternative splicing events. Moreover, we have developed an algorithm that can design active TOSS with a success rate of 80%. To produce bifunctional oligonucleotides capable of stimulating splicing, we built on the observation that binding sites for TDP-43 can stimulate splicing and improve U1 snRNP binding when inserted downstream from 5' splice sites. A TOES designed to recruit TDP-43 improved exon 7 inclusion in SMN2. Overall, our study shows that bifunctional oligonucleotides can redirect splicing on a variety of genes, justifying their inclusion in the molecular arsenal that aims to alter the production of splice variants.

Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.

In most organisms, the primary function of homologous recombination (HR) is to allow genome protection by the faithful repair of DNA double-strand breaks. The vital step of HR is the search for sequence homology, mediated by the RAD51 recombinase, which is stimulated further by proteins mediators such as the tumor suppressor BRCA2. The biochemical interplay between RAD51 and BRCA2 is unknown in Leishmania or Trypanosoma. Here we show that the Leishmania infantum BRCA2 protein possesses several critical features important for the regulation of DNA recombination at the genetic and biochemical level. A BRCA2 null mutant, generated by gene disruption, displayed genomic instability and gene-targeting defects. Furthermore, cytological studies show that LiRAD51 can no longer localize to the nucleus in this mutant. The Leishmania RAD51 and BRCA2 interact together and the purified proteins bind single-strand DNA. Remarkably, LiBRCA2 is a recombination mediator that stimulates the invasion of a resected DNA double-strand break in an undamaged template by LiRAD51 to form a D-loop structure. Collectively, our data show that LiBRCA2 and LiRAD51 promote HR at the genetic and biochemical level in L. infantum, the causative agent of visceral leishmaniasis.

Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress.

Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.

Structure-based protein and small molecule generation using EGNN and diffusion models: A comprehensive review.

Recent breakthroughs in deep learning have revolutionized protein sequence and structure prediction. These advancements are built on decades of protein design efforts, and are overcoming traditional time and cost limitations. Diffusion models, at the forefront of these innovations, significantly enhance design efficiency by automating knowledge acquisition. In the field of de novo protein design, the goal is to create entirely novel proteins with predetermined structures. Given the arbitrary positions of proteins in 3-D space, graph representations and their properties are widely used in protein generation studies. A critical requirement in protein modelling is maintaining spatial relationships under transformations (rotations, translations, and reflections). This property, known as equivariance, ensures that predicted protein characteristics adapt seamlessly to changes in orientation or position. Equivariant graph neural networks offer a solution to this challenge. By incorporating equivariant graph neural networks to learn the score of the probability density function in diffusion models, one can generate proteins with robust 3-D structural representations. This review examines the latest deep learning advancements, specifically focusing on frameworks that combine diffusion models with equivariant graph neural networks for protein generation.