Deconvolving the recognition of DNA shape from sequence.

Protein-DNA binding is mediated by the recognition of the chemical signatures of the DNA bases and the 3D shape of the DNA molecule. Because DNA shape is a consequence of sequence, it is difficult to dissociate these modes of recognition. Here, we tease them apart in the context of Hox-DNA binding by mutating residues that, in a co-crystal structure, only recognize DNA shape. Complexes made with these mutants lose the preference to bind sequences with specific DNA shape features. Introducing shape-recognizing residues from one Hox protein to another swapped binding specificities in vitro and gene regulation in vivo. Statistical machine learning revealed that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence, and feature selection identified shape features important for recognition. Thus, shape readout is a direct and independent component of binding site selection by Hox proteins.

Low affinity binding site clusters confer hox specificity and regulatory robustness.

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression.

Context-Dependent Gene Regulation by Homeodomain Transcription Factor Complexes Revealed by Shape-Readout Deficient Proteins.

Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.

Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins.

Members of transcription factor families typically have similar DNA binding specificities yet execute unique functions in vivo. Transcription factors often bind DNA as multiprotein complexes, raising the possibility that complex formation might modify their DNA binding specificities. To test this hypothesis, we developed an experimental and computational platform, SELEX-seq, that can be used to determine the relative affinities to any DNA sequence for any transcription factor complex. Applying this method to all eight Drosophila Hox proteins, we show that they obtain novel recognition properties when they bind DNA with the dimeric cofactor Extradenticle-Homothorax (Exd). Exd-Hox specificities group into three main classes that obey Hox gene collinearity rules and DNA structure predictions suggest that anterior and posterior Hox proteins prefer DNA sequences with distinct minor groove topographies. Together, these data suggest that emergent DNA recognition properties revealed by interactions with cofactors contribute to transcription factor specificities in vivo.

Dense and pleiotropic regulatory information in a developmental enhancer.

Changes in gene regulation underlie much of phenotypic evolution1. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations2. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

A combinatorial regulatory signature controls terminal differentiation of the dopaminergic nervous system in C. elegans.

Terminal differentiation programs in the nervous system are encoded by cis-regulatory elements that control the expression of terminal features of individual neuron types. We decoded the regulatory information that controls the expression of five enzymes and transporters that define the terminal identity of all eight dopaminergic neurons in the nervous system of the Caenorhabditis elegans hermaphrodite. We show that the tightly coordinated, robust expression of these dopaminergic enzymes and transporters ("dopamine pathway") is ensured through a combinatorial cis-regulatory signature that is shared by all dopamine pathway genes. This signature is composed of an Ets domain-binding site, recognized by the previously described AST-1 Ets domain factor, and two distinct types of homeodomain-binding sites that act in a partially redundant manner. Through genetic screens, we identified the sole C. elegans Distalless/Dlx ortholog, ceh-43, as a factor that acts through one of the homeodomain sites to control both induction and maintenance of terminal dopaminergic fate. The second type of homeodomain site is a Pbx-type site, which is recognized in a partially redundant and neuron subtype-specific manner by two Pbx factors, ceh-20 and ceh-40, revealing novel roles of Pbx factors in the context of terminal neuron differentiation. Taken together, we revealed a specific regulatory signature and cognate, terminal selector-type transcription factors that define the entire dopaminergic nervous system of an animal. Dopaminergic neurons in the mouse olfactory bulb express a similar combinatorial transcription factor collective of Ets/Dlx/Pbx factors, suggesting deep phylogenetic conservation of dopaminergic regulatory programs.

Factors influencing life-space mobility change after total knee arthroplasty in patients with severe knee osteoarthritis.

[Purpose] The purpose of this study was to identify the factors influencing change in life-space mobility after total knee arthroplasty (TKA) in patients with severe knee osteoarthritis (knee OA). [Participants and Methods] Overall, 58 primary unilateral TKA recipients (9 males and 49 females; age ± SD 74.6 ± 6.5 years) were enrolled. We evaluated Life-Space Assessment (LSA) scores, knee extensor strength, Timed Up and Go test (TUG), one-leg standing time (OLS), Western Ontario and McMaster Universities osteoarthritis Index, and physical activity self-efficacy (SE) before surgery and at 3 months post-operation. [Results] Life space mobility significantly expanded 3 months after surgery compared with preoperative baseline. Preoperatively, walking SE and knee extensor muscle strength on the operative side were found to have strong correlation with LSA scores, while stairs SE and knee extensor muscle strength of the operative side were correlated at 3 months post-operation. [Conclusion] These findings suggest that to expand the life-space mobility of TKA recipients, it is important to enhance self-efficacy for general physical activity in addition to strengthening the quadriceps muscles.

Quantitative modeling of transcription factor binding specificities using DNA shape.

DNA binding specificities of transcription factors (TFs) are a key component of gene regulatory processes. Underlying mechanisms that explain the highly specific binding of TFs to their genomic target sites are poorly understood. A better understanding of TF-DNA binding requires the ability to quantitatively model TF binding to accessible DNA as its basic step, before additional in vivo components can be considered. Traditionally, these models were built based on nucleotide sequence. Here, we integrated 3D DNA shape information derived with a high-throughput approach into the modeling of TF binding specificities. Using support vector regression, we trained quantitative models of TF binding specificity based on protein binding microarray (PBM) data for 68 mammalian TFs. The evaluation of our models included cross-validation on specific PBM array designs, testing across different PBM array designs, and using PBM-trained models to predict relative binding affinities derived from in vitro selection combined with deep sequencing (SELEX-seq). Our results showed that shape-augmented models compared favorably to sequence-based models. Although both k-mer and DNA shape features can encode interdependencies between nucleotide positions of the binding site, using DNA shape features reduced the dimensionality of the feature space. In addition, analyzing the feature weights of DNA shape-augmented models uncovered TF family-specific structural readout mechanisms that were not revealed by the DNA sequence. As such, this work combines knowledge from structural biology and genomics, and suggests a new path toward understanding TF binding and genome function.

The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types.

Transcription factors that drive neuron type-specific terminal differentiation programs in the developing nervous system are often expressed in several distinct neuronal cell types, but to what extent they have similar or distinct activities in individual neuronal cell types is generally not well explored. We investigate this problem using, as a starting point, the C. elegans LIM homeodomain transcription factor ttx-3, which acts as a terminal selector to drive the terminal differentiation program of the cholinergic AIY interneuron class. Using a panel of different terminal differentiation markers, including neurotransmitter synthesizing enzymes, neurotransmitter receptors and neuropeptides, we show that ttx-3 also controls the terminal differentiation program of two additional, distinct neuron types, namely the cholinergic AIA interneurons and the serotonergic NSM neurons. We show that the type of differentiation program that is controlled by ttx-3 in different neuron types is specified by a distinct set of collaborating transcription factors. One of the collaborating transcription factors is the POU homeobox gene unc-86, which collaborates with ttx-3 to determine the identity of the serotonergic NSM neurons. unc-86 in turn operates independently of ttx-3 in the anterior ganglion where it collaborates with the ARID-type transcription factor cfi-1 to determine the cholinergic identity of the IL2 sensory and URA motor neurons. In conclusion, transcription factors operate as terminal selectors in distinct combinations in different neuron types, defining neuron type-specific identity features.

Syntaxin13 expression is regulated by mammalian target of rapamycin (mTOR) in injured neurons to promote axon regeneration.

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.

Nail lesions in palmoplantar pustulosis and pustulotic arthro-osteitis impairs patients' quality of life: Suggesting new assessment tool of PPP nail lesions.

BACKGROUND: Palmoplantar pustulosis (PPP) sometimes presents with nail lesions, which affect the patients' quality of life (QOL). However, little is known about nail lesions in PPP, and there is currently no established method for assessing them. OBJECTIVE: This study aimed to analyze the impact of PPP-related nail lesions with the patients' QOL. In addition, we considered whether they might constitute a risk factor of pustulotic arthro-osteitis (PAO).' METHODS: A total of 178 patients with PPP were enrolled. Among the 178 patients, 66 patients participated in the following quality of life questionnaires; GHQ28, DLQI, and Skindex-16. The severity of the nail lesions was classified according to the Nail Psoriasis Severity Index (NAPSI), and the types of nail lesion were investigated. RESULTS: The DLQI, Skindex-16 and PPPASI scores were significantly higher in patients with nail lesions than in those without them. Indentions, transverse ridging, and nail thickening were relatively common in PPP. Nail lesions were unrelated to the presence of PAO complications, but leukonychia and discoloration were likely to be related to PAO lesion site. CONCLUSIONS: The study demonstrated that the presence of nail lesions is associated with a decreased QOL regardless of the severity of the skin lesions. The nail lesions were not a risk factor of PAO, but a predictor of skin lesion severity and PAO lesion site. Given this association, indention, transverse ridging, and thickening of the nail, currently not included in the NAPSI, should be added as an assessment item in the evaluation of PPP nail lesions.

Mammalian target of rapamycin (mTOR) activation increases axonal growth capacity of injured peripheral nerves.

Unlike neurons in the central nervous system (CNS), injured neurons in the peripheral nervous system (PNS) can regenerate their axons and reinnervate their targets. However, functional recovery in the PNS often remains suboptimal, especially in cases of severe damage. The lack of regenerative ability of CNS neurons has been linked to down-regulation of the mTOR (mammalian target of rapamycin) pathway. We report here that PNS dorsal root ganglial neurons (DRGs) activate mTOR following damage and that this activity enhances axonal growth capacity. Furthermore, genetic up-regulation of mTOR activity by deletion of tuberous sclerosis complex 2 (TSC2) in DRGs is sufficient to enhance axonal growth capacity in vitro and in vivo. We further show that mTOR activity is linked to the expression of GAP-43, a crucial component of axonal outgrowth. However, although TSC2 deletion in DRGs facilitates axonal regrowth, it leads to defects in target innervation. Thus, whereas manipulation of mTOR activity could provide new strategies to stimulate nerve regeneration in the PNS, fine control of mTOR activity is required for proper target innervation.

Clinical background of patients with psoriasiform skin lesions due to tumor necrosis factor antagonist administration at a single center.

Paradoxical reaction (PR) occurs when a drug elicits a reaction contrary to what was expected. To clarify the clinical features and genetic background of individuals susceptible to PR, we analyzed the clinical course of patients in whom psoriatic eruptions worsened or newly developed during tumor necrosis factor (TNF) antagonist administration and the role of focal infections and genetic variations. Of 125 patients who received TNF antagonist therapy for psoriasis, acrodermatitis continua of Hallopeau (ACH), generalized pustular psoriasis (GPP), or palmoplantar pustular psoriasis (PPP), eight patients with PR were surveyed at our hospital Dermatology Department between 2010 and 2021. A survey was also done on six patients who received TNF antagonist therapy for Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, and hidradenitis suppurativa and were referred to our department due to PR. Additionally, Sanger sequencing analysis was performed for all exons and flanking introns of IL36RN (interleukin 36 receptor antagonist), CARD14 (caspase recruitment domain-containing protein 14), and AP1S3 (adaptor-related protein complex 1 subunit sigma 3). The clinical assessment of the 14 patients demonstrated an average age at PR onset of 48.4 years, a male : female ratio of 5:9, and a mean administration period until onset of 9.2 months. The clinical types of PR were plaque psoriasis, PPP, GPP, pustulosis, acne, ACH, hair loss, and exacerbation of arthralgia. Histopathology revealed psoriasiform dermatitis in three patients. One patient continued TNF antagonist therapy. All of the patients with psoriasis and GPP had dental infections, suggesting that focal infection may be a risk factor of the development of PR following TNF antagonist therapy. Gene analysis demonstrated CARD14 gene variants associated with RA, CD, AS, or PPP in four patients. In addition, all of the patients with ACH and PPP experienced PR, suggesting that these diseases may predispose patients to PR to TNF antagonist therapy.

Dietary habits in Japanese patients with palmoplantar pustulosis.

Palmoplantar pustulosis (PPP) is a chronic dermatitis characterized by sterile intra-epidermal pustules associated with erythema and scales on the palms and soles. Tumor necrosis factor (TNF)-α/interleukin (IL)-23/IL-17 inflammatory pathway may be involved in the pathogenesis of PPP, and the skin lesions manifest the enhanced expression of IL-8 in keratinocytes and increased levels of antimicrobial peptide cathelicidin, leucine leucine-37 in vesicles/pustules. Some PPP patients are associated with arthro-osteitis, called pustulotic arthro-osteitis (PAO). Dietary habits may modulate the pathogenesis of PPP, however, have not been investigated in PPP patients. We evaluated dietary habits in adult Japanese PPP patients, using a validated, brief-type self-administered diet history questionnaire, and compared their results to those of age- and sex-matched healthy controls. The results in PPP patients with PAO were compared to those in the patients without. Japanese PPP patients showed higher body mass indices (BMIs), higher intakes of pulses and sugar/sweeteners, and lower intake of vitamin A, compared to those of healthy controls. The bivariate and multivariable logistic regression analysis showed that PPP was associated with high BMI, high intake of pulses, and low intake of vitamin A. The sodium intake and BMI were positively correlated with palmoplantar pustulosis area and severity index (PPPASI). The linear multivariate regression analysis revealed that sodium intake and BMI were predictors of PPPASI. The age and sodium intake in the patients with PAO were lower than those in the patients without. The bivariate and multivariable logistic regression analysis showed that PAO was negatively associated with age and sodium intake. This is the first study showing the dietary habits in patients with PPP. Further studies should clarify if the dietary intervention to correct the BMI and sodium intake will alter the progress of PPP.

Sunday driver interacts with two distinct classes of axonal organelles.

The extreme polarized morphology of neurons poses a challenging problem for intracellular trafficking pathways. The distant synaptic terminals must communicate via axonal transport with the cell soma for neuronal survival, function, and repair. Multiple classes of organelles transported along axons may establish and maintain the polarized morphology of neurons, as well as control signaling and neuronal responses to extracellular cues such as neurotrophic or stress factors. We reported previously that the motor-binding protein Sunday Driver (syd), also known as JIP3 or JSAP1, links vesicular axonal transport to injury signaling. To better understand syd function in axonal transport and in the response of neurons to injury, we developed a purification strategy based on anti-syd antibodies conjugated to magnetic beads to identify syd-associated axonal vesicles. Electron microscopy analyses revealed two classes of syd-associated vesicles of distinct morphology. To identify the molecular anatomy of syd vesicles, we determined their protein composition by mass spectrometry. Gene Ontology analyses of each vesicle protein content revealed their unique identity and indicated that one class of syd vesicles belongs to the endocytic pathway, whereas another may belong to an anterogradely transported vesicle pool. To validate these findings, we examined the transport and localization of components of syd vesicles within axons of mouse sciatic nerve. Together, our results lead us to propose that endocytic syd vesicles function in part to carry injury signals back to the cell body, whereas anterograde syd vesicles may play a role in axonal outgrowth and guidance.

Nerve injury signaling.

Although neurons within the peripheral nervous system (PNS) have a remarkable ability to repair themselves after injury, neurons within the central nervous system (CNS) do not spontaneously regenerate. This problem has remained recalcitrant despite a century of research on the reaction of axons to injury. The balance between inhibitory cues present in the environment and the intrinsic growth capacity of the injured neuron determines the extent of axonal regeneration following injury. The cell body of an injured neuron must receive accurate and timely information about the site and extent of axonal damage in order to increase its intrinsic growth capacity and successfully regenerate. One of the mechanisms contributing to this process is retrograde transport of injury signals. For example, molecules activated at the injury site convey information to the cell body leading to the expression of regeneration-associated genes and increased growth capacity of the neuron. Here we discuss recent studies that have begun to dissect the injury-signaling pathways involved in stimulating the intrinsic growth capacity of injured neurons.