Distinct melanocyte subpopulations defined by stochastic expression of proliferation or maturation programs enable a rapid and sustainable pigmentation response.

The ultraviolet (UV) radiation triggers a pigmentation response in human skin, wherein, melanocytes rapidly activate divergent maturation and proliferation programs. Using single-cell sequencing, we demonstrate that these 2 programs are segregated in distinct subpopulations in melanocytes of human and zebrafish skin. The coexistence of these 2 cell states in cultured melanocytes suggests possible cell autonomy. Luria-Delbrück fluctuation test reveals that the initial establishment of these states is stochastic. Tracking of pigmenting cells ascertains that the stochastically acquired state is faithfully propagated in the progeny. A systemic approach combining single-cell multi-omics (RNA+ATAC) coupled to enhancer mapping with H3K27 acetylation successfully identified state-specific transcriptional networks. This comprehensive analysis led to the construction of a gene regulatory network (GRN) that under the influence of noise, establishes a bistable system of pigmentation and proliferation at the population level. This GRN recapitulates melanocyte behaviour in response to external cues that reinforce either of the states. Our work highlights that inherent stochasticity within melanocytes establishes dedicated states, and the mature state is sustained by selective enhancers mark through histone acetylation. While the initial cue triggers a proliferation response, the continued signal activates and maintains the pigmenting subpopulation via epigenetic imprinting. Thereby our study provides the basis of coexistence of distinct populations which ensures effective pigmentation response while preserving the self-renewal capacity.

Temporal analysis of melanogenesis identifies fatty acid metabolism as key skin pigment regulator.

Therapeutic methods to modulate skin pigmentation has important implications for skin cancer prevention and for treating cutaneous hyperpigmentary conditions. Towards defining new potential targets, we followed temporal dynamics of melanogenesis using a cell-autonomous pigmentation model. Our study elucidates 3 dominant phases of synchronized metabolic and transcriptional reprogramming. The melanogenic trigger is associated with high MITF levels along with rapid uptake of glucose. The transition to pigmented state is accompanied by increased glucose channelisation to anabolic pathways that support melanosome biogenesis. SREBF1-mediated up-regulation of fatty acid synthesis results in a transient accumulation of lipid droplets and enhancement of fatty acids oxidation through mitochondrial respiration. While this heightened bioenergetic activity is important to sustain melanogenesis, it impairs mitochondria lately, shifting the metabolism towards glycolysis. This recovery phase is accompanied by activation of the NRF2 detoxication pathway. Finally, we show that inhibitors of lipid metabolism can resolve hyperpigmentary conditions in a guinea pig UV-tanning model. Our study reveals rewiring of the metabolic circuit during melanogenesis, and fatty acid metabolism as a potential therapeutic target in a variety of cutaneous diseases manifesting hyperpigmentary phenotype.

Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes.

Melanin protects skin cells from ultraviolet radiation-induced DNA damage. However, intermediates of eumelanin are highly reactive quinones that are potentially genotoxic. In this study, we systematically investigate the effect of sustained elevation of melanogenesis and map the consequent cellular repair response of melanocytes. Pigmentation increases γH2AX foci, DNA abasic sites, causes replication stress and invokes translesion polymerase Polκ in primary human melanocytes, as well as mouse melanoma cells. Confirming the causal link, CRISPR-based genetic ablation of tyrosinase results in depigmented cells with low Polκ levels. During pigmentation, Polκ activates replication stress response and keeps a check on uncontrolled proliferation of cells harboring melanin-damaged DNA. The mutational landscape observed in human melanoma could in part explain the error-prone bypass of DNA lesions by Polκ, whose absence would lead to genome instability. Thereby, translesion polymerase Polκ is a critical response of pigmenting melanocytes to combat melanin-induced DNA alterations. Our study illuminates the dark side of melanin and identifies (eu)melanogenesis as a key missing link between tanning response and mutagenesis, mediated via the necessary evil translesion polymerase, Polκ.

Kupyaphores are zinc homeostatic metallophores required for colonization of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) endures a combination of metal scarcity and toxicity throughout the human infection cycle, contributing to complex clinical manifestations. Pathogens counteract this paradoxical dysmetallostasis by producing specialized metal trafficking systems. Capture of extracellular metal by siderophores is a widely accepted mode of iron acquisition, and Mtb iron-chelating siderophores, mycobactin, have been known since 1965. Currently, it is not known whether Mtb produces zinc scavenging molecules. Here, we characterize low-molecular-weight zinc-binding compounds secreted and imported by Mtb for zinc acquisition. These molecules, termed kupyaphores, are produced by a 10.8 kbp biosynthetic cluster and consists of a dipeptide core of ornithine and phenylalaninol, where amino groups are acylated with isonitrile-containing fatty acyl chains. Kupyaphores are stringently regulated and support Mtb survival under both nutritional deprivation and intoxication conditions. A kupyaphore-deficient Mtb strain is unable to mobilize sufficient zinc and shows reduced fitness upon infection. We observed early induction of kupyaphores in Mtb-infected mice lungs after infection, and these metabolites disappeared after 2 wk. Furthermore, we identify an Mtb-encoded isonitrile hydratase, which can possibly mediate intracellular zinc release through covalent modification of the isonitrile group of kupyaphores. Mtb clinical strains also produce kupyaphores during early passages. Our study thus uncovers a previously unknown zinc acquisition strategy of Mtb that could modulate host-pathogen interactions and disease outcome.

Histone variant dictates fate biasing of neural crest cells to melanocyte lineage.

In the neural crest lineage, progressive fate restriction and stem cell assignment are crucial for both development and regeneration. Whereas fate commitment events have distinct transcriptional footprints, fate biasing is often transitory and metastable, and is thought to be moulded by epigenetic programmes. Therefore, the molecular basis of specification is difficult to define. In this study, we established a role for a histone variant, H2a.z.2, in specification of the melanocyte lineage from multipotent neural crest cells. H2a.z.2 silencing reduces the number of melanocyte precursors in developing zebrafish embryos and from mouse embryonic stem cells in vitro We demonstrate that this histone variant occupies nucleosomes in the promoter of the key melanocyte determinant mitf, and enhances its induction. CRISPR/Cas9-based targeted mutagenesis of this gene in zebrafish drastically reduces adult melanocytes, as well as their regeneration. Thereby, our study establishes the role of a histone variant upstream of the core gene regulatory network in the neural crest lineage. This epigenetic mark is a key determinant of cell fate and facilitates gene activation by external instructive signals, thereby establishing melanocyte fate identity.

Polyketide Quinones Are Alternate Intermediate Electron Carriers during Mycobacterial Respiration in Oxygen-Deficient Niches.

Mycobacterium tuberculosis (Mtb) adaptation to hypoxia is considered crucial to its prolonged latent persistence in humans. Mtb lesions are known to contain physiologically heterogeneous microenvironments that bring about differential responses from bacteria. Here we exploit metabolic variability within biofilm cells to identify alternate respiratory polyketide quinones (PkQs) from both Mycobacterium smegmatis (Msmeg) and Mtb. PkQs are specifically expressed in biofilms and other oxygen-deficient niches to maintain cellular bioenergetics. Under such conditions, these metabolites function as mobile electron carriers in the respiratory electron transport chain. In the absence of PkQs, mycobacteria escape from the hypoxic core of biofilms and prefer oxygen-rich conditions. Unlike the ubiquitous isoprenoid pathway for the biosynthesis of respiratory quinones, PkQs are produced by type III polyketide synthases using fatty acyl-CoA precursors. The biosynthetic pathway is conserved in several other bacterial genomes, and our study reveals a redox-balancing chemicocellular process in microbial physiology.

STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis.

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions form functionally active microdomains that connect intracellular and extracellular environments. While the key role of these interfaces in maintenance of intracellular Ca2+ levels has been uncovered in recent years, the functional significance of ER-PM junctions in non-excitable cells has remained unclear. Here, we show that the ER calcium sensor protein STIM1 (stromal interaction molecule 1) interacts with the plasma membrane-localized adenylyl cyclase 6 (ADCY6) to govern melanogenesis. The physiological stimulus α-melanocyte-stimulating hormone (αMSH) depletes ER Ca2+ stores, thus recruiting STIM1 to ER-PM junctions, which in turn activates ADCY6. Using zebrafish as a model system, we further established STIM1's significance in regulating pigmentation in vivo STIM1 domain deletion studies reveal the importance of Ser/Pro-rich C-terminal region in this interaction. This mechanism of cAMP generation creates a positive feedback loop, controlling the output of the classical αMSH-cAMP-MITF axis in melanocytes. Our study thus delineates a signaling module that couples two fundamental secondary messengers to drive pigmentation. Given the central role of calcium and cAMP signaling pathways, this module may be operative during various other physiological processes and pathological conditions.

pH-controlled histone acetylation amplifies melanocyte differentiation downstream of MITF.

Tanning response and melanocyte differentiation are mediated by the central transcription factor MITF. This involves the rapid and selective induction of melanocyte maturation genes, while concomitantly the expression of other effector genes is maintained. In this study, using cell-based and zebrafish model systems, we report on a pH-mediated feed-forward mechanism of epigenetic regulation that enables selective amplification of the melanocyte maturation program. We demonstrate that MITF activation directly elevates the expression of the enzyme carbonic anhydrase 14 (CA14). Nuclear localization of CA14 leads to an increase of the intracellular pH, resulting in the activation of the histone acetyl transferase p300/CBP. In turn, enhanced H3K27 histone acetylation at selected differentiation genes facilitates their amplified expression via MITF. CRISPR-mediated targeted missense mutation of CA14 in zebrafish results in the formation of immature acidic melanocytes with decreased pigmentation, establishing a central role for this mechanism during melanocyte differentiation in vivo. Thus, we describe an epigenetic control system via pH modulation that reinforces cell fate determination by altering chromatin dynamics.

Myg1 exonuclease couples the nuclear and mitochondrial translational programs through RNA processing.

Semi-autonomous functioning of mitochondria in eukaryotic cell necessitates coordination with nucleus. Several RNA species fine-tune mitochondrial processes by synchronizing with the nuclear program, however the involved components remain enigmatic. In this study, we identify a widely conserved dually localized protein Myg1, and establish its role as a 3'-5' RNA exonuclease. We employ mouse melanoma cells, and knockout of the Myg1 ortholog in Saccharomyces cerevisiae with complementation using human Myg1 to decipher the conserved role of Myg1 in selective RNA processing. Localization of Myg1 to nucleolus and mitochondrial matrix was studied through imaging and confirmed by sub-cellular fractionation studies. We developed Silexoseqencing, a methodology to map the RNAse trail at single-nucleotide resolution, and identified in situ cleavage by Myg1 on specific transcripts in the two organelles. In nucleolus, Myg1 processes pre-ribosomal RNA involved in ribosome assembly and alters cytoplasmic translation. In mitochondrial matrix, Myg1 processes 3'-termini of the mito-ribosomal and messenger RNAs and controls translation of mitochondrial proteins. We provide a molecular link to the possible involvement of Myg1 in chronic depigmenting disorder vitiligo. Our study identifies a key component involved in regulating spatially segregated organellar RNA processing and establishes the evolutionarily conserved ribonuclease as a coordinator of nucleo-mitochondrial crosstalk.

Non-invasive Oil-Based Method to Increase Topical Delivery of Nucleic Acids to Skin.

Topical delivery of nucleic acids to skin has huge prospects in developing therapeutic interventions for cutaneous disorders. In spite of initial success, clinical translation is vastly impeded by the constraints of bioavailability as well as stability in metabolically active environment of skin. Various physical and chemical methods used to overcome these limitations involve invasive procedures or compounds that compromise skin integrity. Hence, there is an increasing demand for developing safe skin penetration enhancers for efficient nucleic acid delivery to skin. Here, we demonstrate that pretreatment of skin with silicone oil can increase the transfection efficiency of non-covalently associated peptide-plasmid DNA nanocomplexes in skin ex vivo and in vivo. The method does not compromise skin integrity, as indicated by microscopic evaluation of cellular differentiation, tissue architecture, enzyme activity assessment, dye penetration tests using Franz assay, and cytotoxicity and immunogenicity analyses. Stability of nanocomplexes is not hampered on pretreatment, thereby avoiding nuclease-mediated degradation. The mechanistic insights through Fourier transform infrared (FTIR) spectroscopy reveal some alterations in the skin hydration status owing to possible occlusion effects of the enhancer. Overall, we describe a topical, non-invasive, efficient, and safe method that can be used to increase the penetration and delivery of plasmid DNA to skin for possible therapeutic applications.

IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation.

Cellular homeostasis is an outcome of complex interacting processes with nonlinear feedbacks that can span distinct spatial and temporal dimensions. Skin tanning is one such dynamic response that maintains genome integrity of epidermal cells. Although pathways underlying hyperpigmentation cascade are recognized, negative feedback regulatory loops that can dampen the activated melanogenesis process are not completely understood. In this study, we delineate a regulatory role of IFN-γ in skin pigmentation biology. We show that IFN-γ signaling impedes maturation of the key organelle melanosome by concerted regulation of several pigmentation genes. Withdrawal of IFN-γ signal spontaneously restores normal cellular programming. This effect in melanocytes is mediated by IFN regulatory factor-1 and is not dependent on the central regulator microphthalmia-associated transcription factor. Chronic IFN-γ signaling shows a clear hypopigmentation phenotype in both mouse and human skin. Interestingly, IFN-γ KO mice display a delayed recovery response to restore basal state of epidermal pigmentation after UV-induced tanning. Together, our studies delineate a new spatiotemporal role of the IFN-γ signaling network in skin pigmentation homeostasis, which could have implications in various cutaneous depigmentary and malignant disorders.

Population diversity and adaptive evolution in keratinization genes: impact of environment in shaping skin phenotypes.

Several studies have demonstrated the role of climatic factors in shaping skin phenotypes, particularly pigmentation. Keratinization is another well-designed feature of human skin, which is involved in modulating transepidermal water loss (TEWL). Although this physiological process is closely linked to climate, presently it is not clear whether genetic diversity is observed in keratinization and whether this process also responds to the environmental pressure. To address this, we adopted a multipronged approach, which involved analysis of 1) copy number variations in diverse Indian and HapMap populations from varied geographical regions; 2) genetic association with geoclimatic parameters in 61 populations of dbCLINE database in a set of 549 genes from four processes namely keratinization, pigmentation, epidermal differentiation, and housekeeping functions; 3) sequence divergence in 4,316 orthologous promoters and corresponding exonic regions of human and chimpanzee with macaque as outgroup, and 4) protein sequence divergence (Ka/Ks) across nine vertebrate classes, which differ in their extent of TEWL. Our analyses demonstrate that keratinization and epidermal differentiation genes are under accelerated evolution in the human lineage, relative to pigmentation and housekeeping genes. We show that this entire pathway may have been driven by environmental selection pressure through concordant functional polymorphisms across several genes involved in skin keratinization. Remarkably, this underappreciated function of skin may be a crucial determinant of adaptation to diverse environmental pressures across world populations.

Multifaceted pathways protect human skin from UV radiation.

The recurrent interaction of skin with sunlight is an intrinsic constituent of human life, and exhibits both beneficial and detrimental effects. The apparent robust architectural framework of skin conceals remarkable mechanisms that operate at the interface between the surface and environment. In this Review, we discuss three distinct protective mechanisms and response pathways that safeguard skin from deleterious effects of ultraviolet (UV) radiation. The unique stratified epithelial architecture of human skin along with the antioxidant-response pathways constitutes the important defense mechanisms against UV radiation. The intricate pigmentary system and its intersection with the immune-system cytokine axis delicately balance tissue homeostasis. We discuss the relationship among these networks in the context of an unusual depigmenting disorder, vitiligo. The elaborate tunable mechanisms, elegant multilayered architecture and evolutionary selection pressures involved in skin and sunlight interaction makes this a compelling model to understand biological complexity.

Bioinspired Functionalized Melanin Nanovariants with a Range of Properties Provide Effective Color Matched Photoprotection in Skin.

Melanin and related polydopamine hold great promise; however, restricted fine-tunabilility limits their usefulness in biocompatible applications. In the present study, by taking a biomimetic approach, we synthesize peptide-derived melanin with a range of physicochemical properties. Characterization of these melanin polymers indicates that they exist as nanorange materials with distinct size distribution, shapes, and surface charges. These variants demonstrate similar absorption spectra but have different optical properties that correlate with particle size. Our approach enables incorporation of chemical groups to create functionalized polyvalent organic nanomaterials and enables customization of melanin. Further, we establish that these synthetic variants are efficiently taken up by the skin keratinocytes, display appreciable photoprotection with minimal cytotoxicity, and thereby function as effective color matched photoprotective agents. In effect we demonstrate that an array of functionalized melanins with distinct properties could be synthesized using bioinspired green chemistry, and these are of immense utility in generating customized melanin/polydopamine like materials.

Development of pathophysiologically relevant models of sickle cell disease and ß-thalassemia for therapeutic studies.

Ex vivo cellular system that accurately replicates sickle cell disease and ß-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and ß-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications.

Respiratory Quinone Switches from Menaquinone to Polyketide Quinone during the Development Cycle in Streptomyces sp. Strain MNU77.

Type III polyketide synthases (PKSs) found across Streptomyces species are primarily known for synthesis of a vast repertoire of clinically and industrially relevant secondary metabolites. However, our understanding of the functional relevance of these bioactive metabolites in Streptomyces physiology is still limited. Recently, a role of type III PKS harboring gene cluster in producing alternate electron carrier, polyketide quinone (PkQ) was established in a related member of the Actinobacteria, Mycobacteria, highlighting the critical role these secondary metabolites play in primary cellular metabolism of the producer organism. Here, we report the developmental stage-specific transcriptional regulation of homologous type III PKS containing gene cluster in freshwater Streptomyces sp. strain MNU77. Gene expression analysis revealed the type III PKS gene cluster to be stringently regulated, with significant upregulation observed during the dormant sporulation stage of Streptomyces sp. MNU77. In contrast, the expression levels of only known electron carrier, menaquinone biosynthetic genes were interestingly found to be downregulated. Our liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of a metabolite extract from the Streptomyces sp. MNU77 spores also showed 10 times more metabolic abundance of PkQs than menaquinones. Furthermore, through heterologous complementation studies, we demonstrate that Streptomyces sp. MNU77 type III PKS rescues a respiratory defect of the Mycobacterium smegmatis type III PKS deletion mutant. Together, our studies reveal that freshwater Streptomyces sp. MNU77 robustly produces novel PkQs during the sporulation stage, suggesting utilization of PkQs as alternate electron carriers across Actinobacteria during dormant hypoxic conditions. IMPORTANCE The complex developmental life cycle of Streptomyces sp. mandates efficient cellular respiratory reconfiguration for a smooth transition from aerated nutrient-rich vegetative hyphal growth to the hypoxic-dormant sporulation stage. Polyketide quinones (PkQs) have recently been identified as a class of alternate electron carriers from a related member of the Actinobacteria, Mycobacteria, that facilitates maintenance of membrane potential in oxygen-deficient niches. Our studies with the newly identified freshwater Streptomyces sp. strain MNU77 show conditional transcriptional upregulation and metabolic abundance of PkQs in the spore state of the Streptomyces life cycle. In parallel, the levels of menaquinones, the only known Streptomyces electron carrier, were downregulated, suggesting deployment of PkQs as universal electron carriers in low-oxygen, unfavorable conditions across the Actinobacteria family.

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish.

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis.

The recent discovery of fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis (Mtb) provided a new perspective of fatty acid activation. These proteins convert fatty acids to the corresponding adenylates, which are intermediates of acyl-CoA-synthesizing fatty acyl-CoA ligases (FACLs). Presently, it is not evident how obligate pathogens such as Mtb have evolved such new themes of functional versatility and whether the activation of fatty acids to acyladenylates could indeed be a general mechanism. Here, based on elucidation of the first structure of an FAAL protein and by generating loss-of-function and gain-of-function mutants that interconvert FAAL and FACL activities, we demonstrate that an insertion motif dictates formation of acyladenylate. Because FAALs in Mtb are crucial nodes in the biosynthetic network of virulent lipids, inhibitors directed against these proteins provide a unique multipronged approach to simultaneously disrupting several pathways.

Baseline cell proliferation rates and response to UV differ in lymphoblastoid cell lines derived from healthy individuals of extreme constitution types.

Differences in human phenotypes and susceptibility to complex diseases are an outcome of genetic and environmental interactions. This is evident in diseases that progress through a common set of intermediate patho-endophenotypes. Precision medicine aims to delineate molecular players for individualized and early interventions. Functional studies of lymphoblastoid cell line (LCL) model of phenotypically well-characterized healthy individuals can help deconvolute and validate these molecular mechanisms. In this study, LCLs are developed from eight healthy individuals belonging to three extreme constitution types, deep phenotyped on the basis of Ayurveda. LCLs were characterized by karyotyping and immunophenotyping. Growth characteristics and response to UV were studied in these LCLs. Significant differences in cell proliferation rates were observed between the contrasting groups such that one type (Kapha) proliferates significantly slower than the other two (Vata, Pitta). In response to UV, one of the fast growing groups (Vata) shows higher cell death but recovers its numbers due to an inherent higher rates of proliferation. This study reveals that baseline differences in cell proliferation could be a key to understanding the survivability of cells under UV stress. Variability in baseline cellular phenotypes not only explains the cellular basis of different constitution types but can also help set priors during the design of an individualized therapy with DNA damaging agents. This is the first study of its kind that shows variability of intermediate patho-phenotypes among healthy individuals with potential implications in precision medicine.