A fear conditioned cue orchestrates a suite of behaviors in rats.

Pavlovian fear conditioning has been extensively used to study the behavioral and neural basis of defensive systems. In a typical procedure, a cue is paired with foot shock, and subsequent cue presentation elicits freezing, a behavior theoretically linked to predator detection. Studies have since shown a fear conditioned cue can elicit locomotion, a behavior that - in addition to jumping, and rearing - is theoretically linked to imminent or occurring predation. A criticism of studies observing fear conditioned cue-elicited locomotion is that responding is non-associative. We gave rats Pavlovian fear discrimination over a baseline of reward seeking. TTL-triggered cameras captured 5 behavior frames/s around cue presentation. Experiment 1 examined the emergence of danger-specific behaviors over fear acquisition. Experiment 2 examined the expression of danger-specific behaviors in fear extinction. In total, we scored 112,000 frames for nine discrete behavior categories. Temporal ethograms show that during acquisition, a fear conditioned cue suppresses reward seeking and elicits freezing, but also elicits locomotion, jumping, and rearing - all of which are maximal when foot shock is imminent. During extinction, a fear conditioned cue most prominently suppresses reward seeking, and elicits locomotion that is timed to shock delivery. The independent expression of these behaviors in both experiments reveals a fear conditioned cue to orchestrate a temporally organized suite of behaviors.

Knowing that an animal is fearful is crucial for many psychology and neuroscience studies. For instance, this knowledge allows researchers to examine the brain pathways involved in processing and responding to fear. Typically, researchers consider that a rodent is experiencing fear if it 'freezes' ­ a response which, in the wild, helps to evade detection by predators. In Pavlovian fear conditioning experiments, for example, rats and mice freeze when exposed to a stimulus (often a specific sound) previously associated with unpleasant sensations. However, rodents can also respond more actively to threats, for instance by running or jumping away. It remains unclear whether the 'fearful stimuli' used in Pavlovian approaches specifically elicits only freezing, or other fear-related behaviors as well. To investigate this, Chu et al. used high-speed cameras to record rats' responses to a sound cue they had 'learned' to associate with a mild foot shock. In addition to freezing, the animals ran, jumped, stood on their hind legs and stopped their usual reward-seeking behavior in response to the cue. Crucially, these reactions were absent when the rats were exposed to sound cues not associated with pain. Overall, these experiments demonstrate that Pavlovian conditioning can elicit a full range of fear-related behaviors beyond freezing. Understanding the neural activity behind these diverse responses could lead to more targeted therapies and interventions addressing the various ways stress and anxiety manifest in people.

Physics-Informed Generative Model for Drug-like Molecule Conformers.

We present a diffusion-based generative model for conformer generation. Our model is focused on the reproduction of the bonded structure and is constructed from the associated terms traditionally found in classical force fields to ensure a physically relevant representation. Techniques in deep learning are used to infer atom typing and geometric parameters from a training set. Conformer sampling is achieved by taking advantage of recent advancements in diffusion-based generation. By training on large, synthetic data sets of diverse, drug-like molecules optimized with the semiempirical GFN2-xTB method, high accuracy is achieved for bonded parameters, exceeding that of conventional, knowledge-based methods. Results are also compared to experimental structures from the Protein Databank and the Cambridge Structural Database.

Comparison of outcomes after appendectomy in First Nations and non-First Nations patients in Northern Alberta.

BACKGROUND: Internationally, Indigenous Peoples experience worse surgical outcomes than non-Indigenous patients, but equity of surgical care is less well studied in Canada. This study compares outcomes after appendectomy in First Nations and non-First Nations patients. METHODS: In this population-based study, we reviewed administrative data of patients who underwent appendectomy between Apr. 1, 2004, and Mar. 31, 2017, in Northern Alberta. Demographic variables and characteristics of surgical care for First Nations and non-First Nations patients were collected. We identified adverse outcomes by the presence of predefined administrative codes. We identified variables related to a complex postoperative course (at least 1 of wound dehiscence, surgical site infection, abscess, bowel obstruction, pneumonia, deep vein thrombosis, sepsis, emergency department visit, readmission or death within 30 d after appendectomy) through a logistic regression model, and those related to longer length of stay using a Cox proportional hazards model. RESULTS: A total of 28 453 patients met the selection criteria, of whom 1737 (6.1%) had First Nations status. Compared to non-First Nations patients, First Nations patients were younger, lived farther away from the hospital of their appendectomy, were in lower socioeconomic quintiles, and had higher rates of obesity and diabetes (all p < 0.001). After adjustment for age, sex, distance to hospital, socioeconomic deprivation and comorbidities, First Nations status remained independently associated with higher rates of adverse outcomes (odds ratio 1.548, 95% confidence interval [CI] 1.384-1.733) and longer lengths of stay (hazard ratio 0.877, 95% CI 0.832-0.924). CONCLUSION: Although rurality, comorbidities and socioeconomic status contributed to worse outcomes after appendectomy for First Nations patients, First Nations status remained independently associated with worse surgical outcomes. Surgical care, an integral component of health care delivery, must be improved for First Nations patients in order to achieve equitable health care.

Analysis of the complex between MBD2 and the histone deacetylase core of NuRD reveals key interactions critical for gene silencing.

The nucleosome remodeling and deacetylase (NuRD) complex modifies nucleosome positioning and chromatin compaction to regulate gene expression. The methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) play a critical role in complex formation; however, the molecular details of how they interact with other NuRD components have yet to be fully elucidated. We previously showed that an intrinsically disordered region (IDR) of MBD2 is necessary and sufficient to bind to the histone deacetylase core of NuRD. Building on that work, we have measured the inherent structural propensity of the MBD2-IDR using solvent and site-specific paramagnetic relaxation enhancement measurements. We then used the AlphaFold2 machine learning software to generate a model of the complex between MBD2 and the histone deacetylase core of NuRD. This model is remarkably consistent with our previous studies, including the current paramagnetic relaxation enhancement data. The latter suggests that the free MBD2-IDR samples conformations similar to the bound structure. We tested this model of the complex extensively by mutating key contact residues and measuring binding using an intracellular bioluminescent resonance energy transfer assay. Furthermore, we identified protein contacts that, when mutated, disrupted gene silencing by NuRD in a cell model of fetal hemoglobin regulation. Hence, this work provides insights into the formation of NuRD and highlights critical binding pockets that may be targeted to block gene silencing for therapy. Importantly, we show that AlphaFold2 can generate a credible model of a large complex that involves an IDR that folds upon binding.

MBD2a-NuRD binds to the methylated γ-globin gene promoter and uniquely forms a complex required for silencing of HbF expression.

During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2-NuRD. In this report, we present direct evidence that MBD2-NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a-NuRD, and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine-rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine-binding domain (MBD) of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings support a unified model that integrates the respective roles of BCL11A, MBD2a-NuRD, PRMT5, and DNA methylation in HbF silencing.

DNALI1 interacts with the MEIG1/PACRG complex within the manchette and is required for proper sperm flagellum assembly in mice.

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.

Evaluation of acyllysine isostere interactions with the aromatic pocket of the AF9 YEATS domain.

Amide-π interactions, in which an amide interacts with an aromatic group, are ubiquitous in biology, yet remain understudied relative to other noncovalent interactions. Recently, we demonstrated that an electrostatically tunable amide-π interaction is key to recognition of histone acyllysine by the AF9 YEATS domain, a reader protein which has emerged as a therapeutic target due to its dysregulation in cancer. Amide isosteres are commonly employed in drug discovery, often to prevent degradation by proteases, and have proven valuable in achieving selectivity when targeting epigenetic proteins. However, like amide-π interactions, interactions of amide isosteres with aromatic rings have not been thoroughly studied despite widespread use. Herein, we evaluate the recognition of a series of amide isosteres by the AF9 YEATS domain using genetic code expansion to evaluate the amide isostere-π interaction. We show that compared to the amide-π interaction with the native ligand, each isostere exhibits similar electrostatic tunability with an aromatic residue in the binding pocket, demonstrating that the isosteres maintain similar interactions with the aromatic residue. We identify a urea-containing ligand that binds with enhanced affinity for the AF9 YEATS domain, offering a promising starting point for inhibitor development. Furthermore, we demonstrate that carbamate and urea isosteres of crotonyllysine are resistant to enzymatic removal by SIRT1, a protein that cleaves acyl post-translational modifications, further indicating the potential of amide isosteres in YEATS domain inhibitor development. These results also provide experimental precedent for interactions of these common drug discovery moieties with aromatic rings that can inform computational methods.

Discovery of novel N-acylpyrazoles as potent and selective thrombin inhibitors.

Direct oral anticoagulants (DOACs), which includes thrombin and factor Xa inhibitors, have emerged as the preferred therapeutics for thrombotic disorders, penetrating a market previously dominated by warfarin and heparin. This article describes the discovery and profiling of a novel series of N-acylpyrazoles, which act as selective, covalent, reversible, non-competitive inhibitors of thrombin. We describe in vitro stability issues associated with this chemotype and, importantly, demonstrate that N-acylpyrazoles successfully act in vivo as anticoagulants in basic thrombotic animal models. Crucially, this anticoagulant nature is unaccompanied by the higher bleeding risk profile that has become an undesirable characteristic of the DTIs and factor Xa inhibitors. We propose that the N-acylpyrazole chemotype shows intriguing promise as next-generation oral anticoagulants.

Densely methylated DNA traps Methyl-CpG-binding domain protein 2 but permits free diffusion by Methyl-CpG-binding domain protein 3.

The methyl-CpG-binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status.

The Ancient and Evolved Mouse Sperm-Associated Antigen 6 Genes Have Different Biologic Functions In Vivo.

Sperm-associated antigen 6 (SPAG6) is the mammalian orthologue of Chlamydomonas PF16, an axonemal central pair protein involved in flagellar motility. In mice, two Spag6 genes have been identified. The ancestral gene, on mouse chromosome 2, is named Spag6. A related gene originally called Spag6, localized on mouse chromosome 16, evolved from the ancient Spag6 gene. It has been renamed Spag6-like (Spag6l). Spag6 encodes a 1.6 kb transcript consisting of 11 exons, while Spag6l encodes a 2.4 kb transcript which contains an additional non-coding exon in the 3'-end as well as the 11 exons found in Spag6. The two Spag6 genes share high similarities in their nucleotide and amino acid sequences. Unlike Spag6l mRNA, which is widely expressed, Spag6 mRNA expression is limited to a smaller number of tissues, including the testis and brain. In transfected mammalian cells, SPAG6/GFP is localized on microtubules, a similar localization as SPAG6L. A global Spag6l knockout mouse model was generated previously. In addition to a role in modulating the ciliary beat, SPAG6L has many unexpected functions, including roles in the regulation of ciliogenesis/spermatogenesis, hearing, and the immunological synapse, among others. To investigate the role of the ancient Spag6 gene, we phenotyped global Spag6 knockout mice. All homozygous mutant mice were grossly normal, and fertility was not affected in both males and females. The homozygous males had normal sperm parameters, including sperm number, motility, and morphology. Examination of testis histology revealed normal spermatogenesis. Testicular protein expression levels of selected SPAG6L binding partners, including SPAG16L, were not changed in the Spag6 knockout mice, even though the SPAG16L level was significantly reduced in the Spag6l knockout mice. Structural analysis of the two SPAG6 proteins shows that both adopt very similar folds, with differences in a few amino acids, many of which are solvent-exposed. These differences endow the two proteins with different functional characteristics, even though both have eight armadillo repeats that mediate protein-protein interaction. Our studies suggest that SPAG6 and SPAG6L have different functions in vivo, with the evolved SPAG6L protein being more important. Since the two proteins have some overlapping binding partners, SPAG6 could have functions that are yet to be identified.

Post-discharge after surgery Virtual Care with Remote Automated Monitoring-1 (PVC-RAM-1) technology versus standard care: randomised controlled trial.

OBJECTIVE: To determine if virtual care with remote automated monitoring (RAM) technology versus standard care increases days alive at home among adults discharged after non-elective surgery during the covid-19 pandemic. DESIGN: Multicentre randomised controlled trial. SETTING: 8 acute care hospitals in Canada. PARTICIPANTS: 905 adults (≥40 years) who resided in areas with mobile phone coverage and were to be discharged from hospital after non-elective surgery were randomised either to virtual care and RAM (n=451) or to standard care (n=454). 903 participants (99.8%) completed the 31 day follow-up. INTERVENTION: Participants in the experimental group received a tablet computer and RAM technology that measured blood pressure, heart rate, respiratory rate, oxygen saturation, temperature, and body weight. For 30 days the participants took daily biophysical measurements and photographs of their wound and interacted with nurses virtually. Participants in the standard care group received post-hospital discharge management according to the centre's usual care. Patients, healthcare providers, and data collectors were aware of patients' group allocations. Outcome adjudicators were blinded to group allocation. MAIN OUTCOME MEASURES: The primary outcome was days alive at home during 31 days of follow-up. The 12 secondary outcomes included acute hospital care, detection and correction of drug errors, and pain at 7, 15, and 30 days after randomisation. RESULTS: All 905 participants (mean age 63.1 years) were analysed in the groups to which they were randomised. Days alive at home during 31 days of follow-up were 29.7 in the virtual care group and 29.5 in the standard care group: relative risk 1.01 (95% confidence interval 0.99 to 1.02); absolute difference 0.2% (95% confidence interval -0.5% to 0.9%). 99 participants (22.0%) in the virtual care group and 124 (27.3%) in the standard care group required acute hospital care: relative risk 0.80 (0.64 to 1.01); absolute difference 5.3% (-0.3% to 10.9%). More participants in the virtual care group than standard care group had a drug error detected (134 (29.7%) v 25 (5.5%); absolute difference 24.2%, 19.5% to 28.9%) and a drug error corrected (absolute difference 24.4%, 19.9% to 28.9%). Fewer participants in the virtual care group than standard care group reported pain at 7, 15, and 30 days after randomisation: absolute differences 13.9% (7.4% to 20.4%), 11.9% (5.1% to 18.7%), and 9.6% (2.9% to 16.3%), respectively. Beneficial effects proved substantially larger in centres with a higher rate of care escalation. CONCLUSION: Virtual care with RAM shows promise in improving outcomes important to patients and to optimal health system function. TRIAL REGISTRATION: ClinicalTrials.gov NCT04344665.

Post Discharge after Surgery Virtual Care with Remote Automated Monitoring Technology (PVC-RAM): protocol for a randomized controlled trial.

BACKGROUND: After nonelective (i.e., semiurgent, urgent and emergent) surgeries, patients discharged from hospitals are at risk of readmissions, emergency department visits or death. During the coronavirus disease 2019 (COVID-19) pandemic, we are undertaking the Post Discharge after Surgery Virtual Care with Remote Automated Monitoring Technology (PVC-RAM) trial to determine if virtual care with remote automated monitoring (RAM) compared with standard care will increase the number of days adult patients remain alive at home after being discharged following nonelective surgery. METHODS: We are conducting a randomized controlled trial in which 900 adults who are being discharged after nonelective surgery from 8 Canadian hospitals are randomly assigned to receive virtual care with RAM or standard care. Outcome adjudicators are masked to group allocations. Patients in the experimental group learn how to use the study's tablet computer and RAM technology, which will measure their vital signs. For 30 days, patients take daily biophysical measurements and complete a recovery survey. Patients interact with nurses via the cellular modem-enabled tablet, who escalate care to preassigned and available physicians if RAM measurements exceed predetermined thresholds, patients report symptoms, a medication error is identified or the nurses have concerns they cannot resolve. The primary outcome is number of days alive at home during the 30 days after randomization. INTERPRETATION: This trial will inform management of patients after discharge following surgery in the COVID-19 pandemic and offer insights for management of patients who undergo nonelective surgery in a nonpandemic setting. Knowledge dissemination will be supported through an online multimedia resource centre, policy briefs, presentations, peer-reviewed journal publications and media engagement. TRIAL REGISTRATION: ClinicalTrials.gov, no. NCT04344665.

Gene expression profile of epithelial-mesenchymal transition mediators in papillary thyroid cancer.

PURPOSE: Platelet derived growth receptor alpha (PDGFRA) promotes the epithelial-mesenchymal transition (EMT) in thyroid follicular cells and is linked to lymphatic metastases in papillary thyroid cancer (PTC). We probed the regulatory network of genes linked to PDGFRA and EMT, comparing matched patient primary tumor and metastatic specimens, as well as engineered cell lines and ex vivo primary cultures with and without PDGFRA. METHODS: Freshly isolated thyroid tumors with or without metastases, with matching neighboring benign or normal tissue, was isolated for comparative transcriptional analysis using a TaqMan Low Density array (TLDA) assay with genes representing important markers of EMT, cellular adhesion, apoptosis, differentiation, senescence, and signal transduction pathways in thyroid cancer. Transfected primary cultures and immortalized cell lines were also analyzed with respect to PDGFRA expression and cell phenotype. RESULTS: We reveal the consistent upregulation of serine protease DPP4 and structural protein SPP1 with the progression of PTC to metastatic disease, as well as with PDGFRA expression. Conversely, epithelial integrity gene TFF3 and transcription factor SOX10 were strongly down-regulated. This gene network also includes important mediators of EMT including DSG1, MMP3, MMP9, and BECN. We observed similar genomic changes in ex vivo normal thyroid cells transfected with PDGFRA that also exhibited a partially dedifferentiated phenotype. In particular, we observed lamellopodia with induction of PDGFRA and illustrate that DPP4 and SPP1 were upregulated in this process, with decreased TFF3 and SOX10 as seen in tissue specimens. PDGFRA did decrease nuclear protein levels of differentiation factor TTF1, but not the transcription of TTF1 and PAX8. CONCLUSIONS: We demonstrate that PDGFRA activates EMT pathways and decreases expression of genes favoring epithelial integrity, pushing follicular cells toward a dedifferentiated phenotype. SPP1 and DPP4, previously linked with adverse outcomes in thyroid cancer, appear to be regulated by PDGFRA. PDGFRA expression promotes metastatic disease through multiple EMT levers that favor formation of an invasive phenotype and increased metalloproteinase expression.

The Nucleosome Remodeling and Deacetylase Complex Has an Asymmetric, Dynamic, and Modular Architecture.

The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.

VE-1902-A direct thrombin inhibitor with reversible covalent mechanism of action shows efficacy with reduced bleeding in rodent models of thrombosis.

INTRODUCTION: High incidence of bleeding events remains a key risk for patients taking anticoagulants, especially those in need of long-term combination therapy with antiplatelet agents. As a consequence, patients may not receive clinically indicated combination antithrombotic therapy. Here, we report on VE-1902, a member of a novel class of precision oral anticoagulants (PROACs) that combines effective anticoagulation with reduced bleeding in preclinical testing. METHODS AND RESULTS: Acting through covalent, reversible active-site modification of thrombin similar to a previously described molecule [1], VE-1902 shows potency and selectivity for thrombin inhibition in human plasma comparable to clinically relevant direct thrombin inhibitors (DTI) such as argatroban and dabigatran (thrombin generation assay ETP EC50 = 1.3 µM compared to 0.36 µM and 0.31 µM for argatroban and dabigatran; >100-fold selectivity against related serine proteases). Unlike the current anticoagulants, VE-1902 does not significantly inhibit thrombin-mediated platelet activation in in vivo models of thrombosis. In the thrombin generation assay, the compound inhibits thrombin formation without significantly delaying the initiation phase of the clotting cascade. These features are possibly responsible for the observed reduced bleeding in tail bleeding and saphenous vein bleeding models. Consistent with this novel pharmacological profile, VE-1902 shows efficacious anticoagulation in several fibrin-driven animal models of thrombosis (arteriovenous shunt, venous stasis thrombosis, and thrombin-induced thromboembolism models), whereas it does not significantly prevent arterial occlusion in the platelet dependent FeCl3 model. CONCLUSIONS: By leaving platelet activation following vascular injury mostly unaffected, VE-1902, and the PROACs more generally, represent a new generation of precision anticoagulants with reduced bleeding risk.

DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices.

BACKGROUND: MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA-protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins. RESULTS: We demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), we determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops. CONCLUSIONS: Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosine-binding proteins can vary drastically between strong compaction of DNA and no discernable large-scale impact of protein binding. We demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping.

Thermodynamic consequences of Tyr to Trp mutations in the cation-π-mediated binding of trimethyllysine by the HP1 chromodomain.

Evolution has converged on cation-π interactions for recognition of quaternary alkyl ammonium groups such as trimethyllysine (Kme3). While computational modelling indicates that Trp provides the strongest cation-π interaction of the native aromatic amino acids, there is limited corroborative data from measurements within proteins. Herein we investigate a Tyr to Trp mutation in the binding pocket of the HP1 chromodomain, a reader protein that recognizes Kme3. Binding studies demonstrate that the Trp-mediated cation-π interaction is about -5 kcal mol-1 stronger, and the Y24W crystal structure shows that the mutation is not perturbing. Quantum mechanical calculations indicate that greater enthalpic binding is predominantly due to increased cation-π interactions. NMR studies indicate that differences in the unbound state of the Y24W mutation lead to enthalpy-entropy compensation. These results provide direct experimental quantification of Trp versus Tyr in a cation-π interaction and afford insight into the conservation of aromatic cage residues in Kme3 reader domains.

The Methyl-CpG-Binding Domain 2 and 3 Proteins and Formation of the Nucleosome Remodeling and Deacetylase Complex.

The Nucleosome Remodeling and Deacetylase (NuRD) complex uniquely combines both deacetylase and remodeling enzymatic activities in a single macromolecular complex. The methyl-CpG-binding domain 2 and 3 (MBD2 and MBD3) proteins provide a critical structural link between the deacetylase and remodeling components, while MBD2 endows the complex with the ability to selectively recognize methylated DNA. Hence, NuRD combines three major arms of epigenetic gene regulation. Research over the past few decades has revealed much of the structural basis driving formation of this complex and started to uncover the functional roles of NuRD in epigenetic gene regulation. However, we have yet to fully understand the molecular and biophysical basis for methylation-dependent chromatin remodeling and transcription regulation by NuRD. In this review, we discuss the structural information currently available for the complex, the role MBD2 and MBD3 play in forming and recruiting the complex to methylated DNA, and the biological functions of NuRD.

Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells.

As high fetal hemoglobin levels ameliorate the underlying pathophysiological defects in sickle cell anemia and beta (ß)-thalassemia, understanding the mechanisms that enforce silencing of fetal hemoglobin postnatally offers the promise of effective molecular therapy. Depletion of the Nucleosome Remodeling and Deacetylase complex member MBD2 causes a 10-20-fold increase in γ-globin gene expression in adult ß-globin locus yeast artificial chromosome transgenic mice. To determine the effect of MBD2 depletion in human erythroid cells, genome editing technology was utilized to knockout MBD2 in Human Umbilical cord Derived Erythroid Progenitor-2 cells resulting in γ/γ+ß mRNA levels of approximately 50% and approximately 40% fetal hemoglobin by high performance liquid chromatography. In contrast, MBD3 knockout had no appreciable effect on γ-globin expression. Knockdown of MBD2 in primary adult erythroid cells consistently increased γ/γ+ß mRNA ratios by approximately 10-fold resulting in approximately 30-40% γ/γ+ß mRNA levels and a corresponding increase in γ-globin protein. MBD2 exerts its repressive effects through recruitment of the chromatin remodeler CHD4 via a coiled-coil domain, and the histone deacetylase core complex via an intrinsically disordered region. Enforced expression of wild-type MBD2 in MBD2 knockout cells caused a 5-fold decrease in γ-globin mRNA while neither the coiled-coil mutant nor the intrinsically disordered region mutant MBD2 proteins had an inhibitory effect. Co-immunoprecipitation assays showed that the coiled-coil and intrinsically disorder region mutations disrupt complex formation by dissociating the CHD4 and the histone deacetylase core complex components, respectively. These results establish the MBD2 Nucleosome Remodeling and Deacetylase complex as a major silencer of fetal hemoglobin in human erythroid cells and point to the coiled-coil and intrinsically disordered region of MBD2 as potential therapeutic targets.