Selective Recruitment Research Articles

Impact of the EndoPredict genomic assay on treatment decisions for oestrogen receptor-positive early breast cancer patients: benefits of physician selective testing.

Genomic tests improve accuracy of risk prediction for early breast cancers but these are expensive. This study evaluated the clinical utility of EndoPredict®, in terms of impact on adjuvant therapy recommendations and identification of parameters to guide selective application. Patients with ER-positive, HER2-negative, and early-stage invasive breast cancer were tested with EndoPredict®. Two cohorts were recruited: one consecutively and another at clinical team discretion. Systemic treatment recommendations were recorded before and after EndoPredict® results were revealed to the multidisciplinary team. 233 patients were recruited across five sites: 123 consecutive and 110 at clinical team discretion. In the consecutive cohort 50.6% (62/123) cases were classified high risk of recurrence by EndoPredict®, compared with 62.7% (69/110) in the selective cohort. A change in treatment recommendation was significantly more likely (p < 0.0001) in the selective cohort (43/110, 39.1%) compared to the consecutive group (11/123, 8.9%). The strongest driver of selective recruitment was intermediate grade histology, whilst logistic regression modelling demonstrated that nodal status (p < 0.001), proliferative rate (p = 0.001), and progesterone receptor positivity (p < 0.001) were the strongest discriminators of risk. Whilst molecular risk can be predicted by traditional variables in a high proportion of cases, EndoPredict® had a greater impact on treatment decisions in those cases selected for testing at team discretion. This is indicative of the robust ability of the clinical team to identify cases most likely to benefit from testing, underscoring the value of genomic tests in the oncologists' tool kit.

Clinicians' Views of Patient-initiated Follow-up in Head and Neck Cancer: a Qualitative Study to Inform the PETNECK2 Trial

AimsCurrent follow-up for head and neck cancer (HNC) is ineffective, expensive and fails to address patients' needs. The PETNECK2 trial will compare a new model of patient-initiated follow-up (PIFU) with routine scheduled follow-up. This article reports UK clinicians' views about HNC follow-up and PIFU, to inform the trial design. Materials and methodsOnline focus groups with surgeons (ear, nose and throat/maxillofacial), oncologists, clinical nurse specialists and allied health professionals. Clinicians were recruited from professional bodies, mailing lists and personal contacts. Focus groups explored views on current follow-up and acceptability of the proposed PIFU intervention and randomised controlled trial design (presented by the study co-chief investigator), preferences, margins of equipoise, potential organisational barriers and thoughts about the content and format of PIFU. Data were interpreted using inductive thematic analysis. ResultsEight focus groups with 34 clinicians were conducted. Clinicians highlighted already known limitations with HNC follow-up – lack of flexibility to address the wide-ranging needs of HNC patients, expense and lack of evidence – and agreed that follow-up needs to change. They were enthusiastic about the PETNECK2 trial to develop and evaluate PIFU but had concerns that PIFU may not suit disengaged patients and may aggravate patient anxiety/fear of recurrence and delay detection of recurrence. Anticipated issues with implementation included ensuring a reliable route back to clinic and workload burden on nurses and allied health professionals. ConclusionsClinicians supported the evaluation of PIFU but voiced concerns about barriers to help-seeking. An emphasis on patient engagement, psychosocial issues, symptom reporting and reliable, quick routes back to clinic will be important. Certain patient groups may be less suited to PIFU, which will be evaluated in the trial. Early, meaningful, ongoing engagement with clinical teams and managers around the trial rationale and recruitment process will be important to discourage selective recruitment and address risk-averse behaviour and potential workload burden.

Liver ischaemia-reperfusion injury: a new understanding of the role of innate immunity.

Liver ischaemia-reperfusion injury (LIRI), a local sterile inflammatory response driven by innate immunity, is one of the primary causes of early organ dysfunction and failure after liver transplantation. Cellular damage resulting from LIRI is an important risk factor not only for graft dysfunction but also for acute and even chronic rejection and exacerbates the shortage of donor organs for life-saving liver transplantation. Hepatocytes, liver sinusoidal endothelial cells and Kupffer cells, along with extrahepatic monocyte-derived macrophages, neutrophils and platelets, are all involved in LIRI. However, the mechanisms underlying the responses of these cells in the acute phase of LIRI and how these responses are orchestrated to control and resolve inflammation and achieve homeostatic tissue repair are not well understood. Technological advances allow the tracking of cells to better appreciate the role of hepatic macrophages and platelets (such as their origin and immunomodulatory and tissue-remodelling functions) and hepatic neutrophils (such as their selective recruitment, anti-inflammatory and tissue-repairing functions, and formation of extracellular traps and reverse migration) in LIRI. In this Review, we summarize the role of macrophages, platelets and neutrophils in LIRI, highlight unanswered questions, and discuss prospects for innovative therapeutic regimens against LIRI in transplant recipients.

Building a Mosque's Competitive Advantage through the Management of Imams of Mosque

Like business enterprises, mosques and other nonprofit organizations also need to have a vital source of competitive advantage to increase their worshippers' uniqueness and attractiveness. If it can be adequately managed, imam masjid can be one of the uniqueness and continuing excellence for the mosque. One of the best practices in managing mosque’ imams as a competitive advantage is Masjid Raya Hubbul Wathan Islamic Center in West Nusa Tenggara. This research examines differentiation strategies in managing imams at Masjid Raya Hubbul Wathan Islamic Center in West Nusa Tenggara and how imams in the mosque can be a source of sustainable excellence and not only temporary. This research was conducted using qualitative methods with a case study approach. The collected data is analyzed descriptively based on the theory of competitive advantage, differentiation, and the VRIO framework. Among the critical findings in this study is the differentiation strategy in the management of imams at Masjid Raya Hubbul Wathan Islamic Center in West Nusa Tenggara carried out by: (1) the existence of a measurable and selective imam recruitment process; (2) the availability of competent priests with extensive and varied numbers; (3) involvement of international priests; and (4) the existence of a system of appreciation, improvement, empowerment of good mosque imams.

CD271 antibody-functionalized microspheres capable of selective recruitment of reparative endogenous stem cells for in situ bone regeneration

In the strategy of in situ bone regeneration, it used to be difficult to specifically recruit bone marrow mesenchymal stem cells (BM-MSCs) by a single marker. Recently, CD271 has been considered to be one of the most specific markers to isolate BM-MSCs; however, the effectiveness of CD271 antibodies in recruiting BM-MSCs has not been explored yet. In this study, we developed novel CD271 antibody-functionalized chitosan (CS) microspheres with the aid of polydopamine (PDA) coating to recruit endogenous BM-MSCs for in situ bone regeneration. The CS microspheres were sequentially modified with PDA and CD271 antibody through dopamine self-polymerization and bioconjugation, respectively. In vitro studies showed that the CD271 antibody-functionalized microspheres selectively captured significantly more BM-MSCs from a fluorescently labeled heterotypic cell population than non-functionalized controls. In addition, the PDA coating was critical for supporting stable adhesion and proliferation of the captured BM-MSCs. Effective early recruitment of CD271+ stem cells by the functionalized microspheres at bone defect site of SD rat was observed by the CD271/DAPI immunofluorescence staining, which led to significantly enhanced new bone formation in rat femoral condyle defect over long term. Together, findings from this study have demonstrated, for the first time, that the CD271 antibody-functionalized CS microspheres are promising for in situ bone regeneration.

Microglia-specific overexpression of \u03b1-synuclein leads to severe dopaminergic neurodegeneration by phagocytic exhaustion and oxidative toxicity

Recent findings in human samples and animal models support the involvement of inflammation in the development of Parkinson’s disease. Nevertheless, it is currently unknown whether microglial activation constitutes a primary event in neurodegeneration. We generated a new mouse model by lentiviral-mediated selective α-synuclein (αSYN) accumulation in microglial cells. Surprisingly, these mice developed progressive degeneration of dopaminergic (DA) neurons without endogenous αSYN aggregation. Transcriptomics and functional assessment revealed that αSYN-accumulating microglial cells developed a strong reactive state with phagocytic exhaustion and excessive production of oxidative and proinflammatory molecules. This inflammatory state created a molecular feed-forward vicious cycle between microglia and IFNγ-secreting immune cells infiltrating the brain parenchyma. Pharmacological inhibition of oxidative and nitrosative molecule production was sufficient to attenuate neurodegeneration. These results suggest that αSYN accumulation in microglia induces selective DA neuronal degeneration by promoting phagocytic exhaustion, an excessively toxic environment and the selective recruitment of peripheral immune cells.

The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious.

Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infection induces autophagy that enhances virus replication and vesicle release to evade immune system surveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells and showed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobility group box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradient centrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in the cytoplasm of the infected cells were detected in the purified double-membrane autophagosome by immunogold labeling under transmission electron microscopy. In DENV infected cells, the levels of capsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramatic accumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked by chloroquine, indicating that these proteins are not regulated by autophagic degradation machinery. We further demonstrated that purified autophagosomes were infectious when co-cultured with uninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negative-strand and full-length genomic RNAs, but no viral particles. It is possible that the infectivity of the autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2 promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the first to report that DENV2-induced double-membrane autophagosomes containing viral proteins and full-length RNAs are infectious and not undergoing autophagic degradation. Our novel finding warrants further validation of whether these intracellular vesicles undergo exocytosis to become infectious autophagic vesicles.

Competition-based screening helps to secure the evolutionary stability of a defensive microbiome

BackgroundThe cuticular microbiomes of Acromyrmex leaf-cutting ants pose a conundrum in microbiome biology because they are freely colonisable, and yet the prevalence of the vertically transmitted bacteria Pseudonocardia, which contributes to the control of Escovopsis fungus garden disease, is never compromised by the secondary acquisition of other bacterial strains. Game theory suggests that competition-based screening can allow the selective recruitment of antibiotic-producing bacteria from the environment, by providing abundant resources to foment interference competition between bacterial species and by using Pseudonocardia to bias the outcome of competition in favour of antibiotic producers.ResultsHere, we use RNA-stable isotope probing (RNA-SIP) to confirm that Acromyrmex ants can maintain a range of microbial symbionts on their cuticle by supplying public resources. We then used RNA sequencing, bioassays, and competition experiments to show that vertically transmitted Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of other microbes, ultimately biassing the bacterial competition to allow the selective establishment of secondary antibiotic-producing strains while excluding non-antibiotic-producing strains that would parasitise the symbiosis.ConclusionsOur findings are consistent with the hypothesis that competition-based screening is a plausible mechanism for maintaining the integrity of the co-adapted mutualism between the leaf-cutting ant farming symbiosis and its defensive microbiome. Our results have broader implications for explaining the stability of other complex symbioses involving horizontal acquisition.

Histone H4 lysine 20 mono-methylation directly facilitates chromatin openness and promotes transcription of housekeeping genes

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.

Selective recruitment for pollen and nectar sources in honeybees.

Honeybees (Apis mellifera) use cues and signals to recruit nestmates to profitable food sources. Here, we investigated whether the type of resource advertised within the colony (i.e. pollen or nectar) correlates with the choices of recruits at the feeding site. We observed that pollen recruits preferred to collect pollen once arrived for the first time at the feeding site, while nectar recruits preferred to forage sucrose solutions. Bees recruited by foragers carrying both resources showed intermediate preferences. Studying the plasticity of this response, we found that nectar recruits have a low probability of switching to pollen collection, yet pollen recruits were likely to switch to sucrose solution of increasing concentrations. Our results show that cues associated with the advertised resource type correlate with the foraging tendency of recruits for pollen and sucrose solution, a feature that would guarantee an efficient resource collection.

Interracial contact differentially shapes brain networks involved in social and non-social judgments from faces: a combination of univariate and multivariate approaches.

The present work explores the relationship between interracial contact and the neural substrates of explicit social and non-social judgments about both racial ingroup and outgroup targets. Convergent evidence from univariate and multivariate partial least squares (PLS) analyses reveals that contact shapes the recruitment of brain regions involved in social cognition similarly for both ingroup and outgroup targets. Results support the hypothesis that increased contact is associated with generalized changes in social cognition toward both ingroup and outgroup faces. Specifically, regardless of target race, low- and average-contact perceivers showed the typically observed increased recruitment of temporoparietal junction and dorsomedial prefrontal cortex during social compared to perceptual judgments. However, high-contact perceivers did not show selective recruitment of these brain regions for social judgments. Complimenting univariate results, multivariate PLS analyses reveal that greater perceiver contact leads to reduced co-activation in networks of brain regions associated with face processing (e.g. fusiform gyrus) and salience detection (e.g. anterior cingulate cortex and insula). Across univariate and multivariate analyses, we found no evidence that contact differentially impacted cross-race face perception. Instead, when performing either a social or a novel perceptual task, interracial contact appears to broadly shape how perceivers engage with all faces.

Recent Advances in Synergy Among SNARE Proteins and Multi-Subunit Tethering Complexes (MTCs) in Vesicle Trafficking and Plant Development

Plants comprise an expanded endomembrane system, and transport within the network requires well-organized and accurate vesicle transport. Tethering complexes facilitate the early, exact contact among donor and acceptor membranes, operate to bring vesicles into a closer proximity for trans-SNARE complex assembly docking; these are classified as either long coiled-coil proteins or multi-subunit tethering complexes (MTCs). Numerous MTCs that function at different membrane trafficking steps have been recognized, where they function as significant interfaces between SNARE proteins, Rabs, and phosphoinositides. SNARE proteins assemble into complexes that catalyze the fusion between a donor and a target membrane. Studies of the diverse SNARE proteins provided further valuable information about vacuole biogenesis and vacuolar trafficking pathways related to cell-type specificity, plant development, growth, and the plant developed a specific traffic route to overcome environmental stress. In conclusion, tethers' selective recruitment during membrane fusion is controlled via diverse small GTPases, such as those in the RAB family. The MTCs promote SNARE complex assembly by direct interactions of MTC subunits with Q-SNAREs. A subset of MTC subunits exploits structurally similar CATCHR domains to mediate inter-subunit interactions as well as SNARE protein interactions. MTCs are subdivided into CATCHR (complexes associated with tethering containing helical rods: Dsl1, COG, GARP, EARP, and exocyst) and non‐CATCHR (TRAPP I, II and III, HOPS and CORVET) complexes based on the structure of their subunits. This review summarized new information about SNARE proteins and tethering complexes, highlight new insights about their function, and discuss current debates and future perspectives.

Anti-tumor immunity in mismatch repair-deficient colorectal cancers requires type I IFN-driven CCL5 and CXCL10.

Colorectal cancers (CRCs) deficient in DNA mismatch repair (dMMR) contain abundant CD8+ tumor-infiltrating lymphocytes (TILs) responding to the abundant neoantigens from their unstable genomes. Priming of such tumor-targeted TILs first requires recruitment of CD8+ T cells into the tumors, implying that this is an essential prerequisite of successful dMMR anti-tumor immunity. We have discovered that selective recruitment and activation of systemic CD8+ T cells into dMMR CRCs strictly depend on overexpression of CCL5 and CXCL10 due to endogenous activation of cGAS/STING and type I IFN signaling by damaged DNA. TIL infiltration into orthotopic dMMR CRCs is neoantigen-independent and followed by induction of a resident memory-like phenotype key to the anti-tumor response. CCL5 and CXCL10 could be up-regulated by common chemotherapies in all CRCs, indicating that facilitating CD8+ T cell recruitment underlies their efficacy. Induction of CCL5 and CXCL10 thus represents a tractable therapeutic strategy to induce TIL recruitment into CRCs, where local priming can be maximized even in neoantigen-poor CRCs.

TRIMs: selective recruitment at different steps of the NF-κB pathway-determinant of activation or resolution of inflammation.

TNF-α-induced NF-κB pathway is an essential component of innate and adaptive immune pathway, and it is tightly regulated by various post-translational modifications including ubiquitination. Oscillations in NF-κB activation and temporal gene expression are emerging as critical determinants of inflammatory response, however, the regulators of unique outcomes in different patho-physiological conditions are not well understood. Tripartite Motif-containing proteins (TRIMs) are RING domain-containing E3 ligases involved in the regulation of cellular homeostasis, metabolism, cell death, inflammation, and host defence. Emerging reports suggest that TRIMs are recruited at different steps of TNF-α-induced NF-κB pathway and modulate via their E3 ligase activity. TRIMs show synergy and antagonism in the regulation of the NF-κB pathway and also regulate it in a feedback manner. TRIMs also regulate pattern recognition receptors (PRRs) mediated inflammatory pathways and may have evolved to directly regulate a specific arm of immune signalling. The review emphasizes TRIM-mediated ubiquitination and modulation of TNF-α-regulated temporal and NF-κB signaling and its possible impact on unique transcriptional and functional outcomes.

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

Harnessing self-labeling enzymes for selective and concurrent A-to-I and C-to-U RNA base editing

The SNAP-ADAR tool enables precise and efficient A-to-I RNA editing in a guideRNA-dependent manner by applying the self-labeling SNAP-tag enzyme to generate RNA-guided editases in cell culture. Here, we extend this platform by combining the SNAP-tagged tool with further effectors steered by the orthogonal HALO-tag. Due to their small size (ca. 2 kb), both effectors are readily integrated into one genomic locus. We demonstrate selective and concurrent recruitment of ADAR1 and ADAR2 deaminase activity for optimal editing with extended substrate scope and moderate global off-target effects. Furthermore, we combine the recruitment of ADAR1 and APOBEC1 deaminase activity to achieve selective and concurrent A-to-I and C-to-U RNA base editing of endogenous transcripts inside living cells, again with moderate global off-target effects. The platform should be readily transferable to further epitranscriptomic writers and erasers to manipulate epitranscriptomic marks in a programmable way with high molecular precision.

A Cluster of Enhancer Modules Directs Differential Myc Expression Along the Normal and Leukemic Hematopoietic Stem Cell Hierarchies

Large enhancer regions, known as “super-enhancers”, have been described as critical regulatory units governing cell identity, but their actual contribution to stem cell-driven hierarchies remains poorly understood. The transcription factor MYC has critical roles in the regulation of hematopoietic stem cells (HSCs) and progenitors as well as in hematopoietic malignancies.Here we show that a region previously identified as a “super-enhancer”, located 1.7 Mb telomeric from Myc plays an essential role in regulating Myc expression throughout hematopoiesis. Deletion of this “super enhancer” in mice leads to a complete loss of Myc expression in HSC/progenitors, causing an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, similarly to the conditional deletion of the Myc gene in HSCs. This “Blood ENhancer Cluster” (BENC), which is functionally conserved in mice and humans, is comprised of distinct modules with selective activity and recruitment of such transcription factors as GFI1b, RUNX1 and MYB in different hematopoietic cell types. Analysis of mice carrying deletions of individual enhancer modules revealed a modular and combinatorial organization of BENC controlling Myc expression levels throughout most of the hematopoietic hierarchy. Additionally, we show that individual enhancer modules are activated at distinct stages during B cell development.In a malignant setting, we demonstrate that BENC is essential for the maintenance of MLL-AF9 leukemia in mice. Furthermore, in human acute myeloid leukemia, BENC modules show an increased accessibility in primary leukemic stem cells compared to blasts. The accessibility of the module directing Myc expression in normal HSCs correlates with Myc expression and a favorable patient outcome. Altogether, these data indicate a major role of BENC in regulating MYC levels across both normal hematopoietic and leukemic hierarchies. The composition of BENC may serve as a blueprint for other enhancer clusters operating across cellular hierarchies in other tissue types. DisclosuresNo relevant conflicts of interest to declare.

α-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics.

Microtubules are non-covalent polymers of αβ-tubulin dimers. Posttranslational processing of the intrinsically disordered C-terminal α-tubulin tail produces detyrosinated and Δ2-tubulin. Although these are widely employed as proxies for stable cellular microtubules, their effect (and of the α-tail) on microtubule dynamics remains uncharacterized. Using recombinant, engineered human tubulins, we now find that neither detyrosinated nor Δ2-tubulin affect microtubule dynamics, while the α-tubulin tail is an inhibitor of microtubule growth. Consistent with the latter, molecular dynamics simulations show the α-tubulin tail transiently occluding the longitudinal microtubule polymerization interface. The marked differential invivo stabilities of the modified microtubule subpopulations, therefore, must result exclusively from selective effector recruitment. We find that tyrosination quantitatively tunes CLIP-170 density at the growing plus end and that CLIP170 and EB1 synergize to selectively upregulate the dynamicity of tyrosinated microtubules. Modification-dependent recruitment of regulators thereby results in microtubule subpopulations with distinct dynamics, a tenet of the tubulin code hypothesis.

T3-Dependent Transcriptional Programming by TRβ in Thyroid Cells Requires SWI/SNF Chromatin Remodelers

Abstract Transcriptional regulation in response to thyroid hormone (T3) is a dynamic and cell-type specific process that maintains cellular homeostasis and identity. A bimodal switch model, where T3 binding alters the co-regulator profile of a constitutively DNA-bound thyroid hormone receptor (TR) to affect downstream gene expression, is widely used to describe the interaction between TRβ and chromatin. To test this model on a genome-wide scale, we used an integrated genomics approach to profile and characterize the cistrome of TRβ by CUT&amp;RUN, map changes in chromatin accessibility by ATAC-seq, and capture the transcriptomic changes in response to T3 by RNA-seq in the normal thyroid cell line, Nthy-ORI. Our CUT&amp;RUN data demonstrated that T3 binding causes significant shifts in TRβ genomic occupancy; these shifts are associated with differential chromatin accessibility. Most of the T3-induced differentially expressed genes have a TRβ binding site associated with the proximal protomer region within one kilobase of the transcriptional start site, suggesting that these are direct TRβ regulatory target genes. Remarkably, the majority of TRβ binding sites were found in transgenic regions and distal regulatory elements. In order to identify the co-regulatory proteins that are required for execution of a T3-dependent transcriptional program in our thyroid cells, we used a TRβ-miniTurboID fusion construct to perform a proximity ligation assay followed by mass spectrometry. We identified 1,138 nuclear proteins that interact with TRβ. Of these proteins, 75 interact preferentially in the presence of T3 and 68 in the absence of T3. All of the core SWI/SNF complex subunits from both of the major subtypes (BAF and PBAF) were identified as TRβ. Interestingly, we found that the PBAF-specific subunit, PBRM1, was significantly enriched in the presence of T3. BAF complex-specific subunits, such as ARID1A, were also present in our dataset. To test whether TRβ could differentially recruit BAF and PBAF complexes to its binding sites, we performed CUT&amp;RUN targeting BRG1, PBRM, and ARID1A to determine the degree of co-occupancy with TRβ. Based on our comprehensive genomic and proteomic analyses, we propose a new model for selective recruitment of BAF and PBAF SWI/SNF complexes to TRβ binding sites for differential functions in regulating chromatin accessibility.

The N2A region of titin has a unique structural configuration.

The N2A segment of titin is a main signaling hub in the sarcomeric I-band that recruits various signaling factors and processing enzymes. It has also been proposed to play a role in force production through its Ca2+-regulated association with actin. However, the molecular basis by which N2A performs these functions selectively within the repetitive and extensive titin chain remains poorly understood. Here, we analyze the structure of N2A components and their association with F-actin. Specifically, we characterized the structure of its Ig domains by elucidating the atomic structure of the I81-I83 tandem using x-ray crystallography and computing a homology model for I80. Structural data revealed these domains to present heterogeneous and divergent Ig folds, where I81 and I83 have unique loop structures. Notably, the I81-I83 tandem has a distinct rotational chain arrangement that confers it a unique multi-domain topography. However, we could not identify specific Ca2+-binding sites in these Ig domains, nor evidence of the association of titin N2A components with F-actin in transfected C2C12 myoblasts or C2C12-derived myotubes. In addition, F-actin cosedimentation assays failed to reveal binding to N2A. We conclude that N2A has a unique architecture that predictably supports its selective recruitment of binding partners in signaling, but that its mechanical role through interaction with F-actin awaits validation.