In vitiligo, chronic loss of melanocytes and consequent absence of melanin from the epidermis presents a challenge for long-term tissue maintenance. The stable vitiligo patches are known to attain an irreversible depigmented state. However, the molecular and cellular processes resulting in this remodeled tissue homeostasis is unclear. To investigate the complex interplay of inductive signals and cell intrinsic factors that support the new acquired state, we compared the matched lesional and non-lesional epidermis obtained from stable non-segmental vitiligo subjects. Hierarchical clustering of genome-wide expression of transcripts surprisingly segregated lesional and non-lesional samples in two distinct clades, despite the apparent heterogeneity in the lesions of different vitiligo subjects. Pathway enrichment showed the expected downregulation of melanogenic pathway and a significant downregulation of cornification and keratinocyte differentiation processes. These perturbations could indeed be recapitulated in the lesional epidermal tissue, including blunting of rete-ridges, thickening of stratum corneum and increase in the size of corneocytes. In addition, we identify marked increase in the putrescine levels due to the elevated expression of spermine/spermidine acetyl transferase. Our study provides insights into the intrinsic self-renewing ability of damaged lesional tissue to restore epidermal functionality in vitiligo.

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.

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.

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.

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.

22 differentiation, epigenetics and zebrafish 23 24 Running title: pH regulates melanocyte maturation 25 pH regulates melanocyte maturation 2 Abstract 26 Tanning response and melanocyte differentiation are mediated by the central transcription factor MITF. 27Enigmatically, these involve rapid and selective induction of melanocyte maturation genes, while 28 concomitantly maintaining the expression of other effectors. In this study using cell-based and zebrafish 29 model systems, we elucidate a pH mediated feed-forward mechanism of epigenetic regulation that enables 30 selective amplification of melanocyte maturation program. We demonstrate that MITF activation directly 31 elevates the expression of Carbonic Anhydrase 14 (Ca14) enzyme. Nuclear localized Ca14 increases the 32 intracellular pH, resulting in the activation of histone acetyl transferase activity of p300/CBP. In turn 33 enhanced H3K27 histone acetylation marks of select differentiation genes facilitates their amplified 34 expression by MITF. CRISPR-mediated targeted missense mutation of CA14 in zebrafish results in 35 immature acidic melanocytes with decreased pigmentation, establishing the centrality of this mechanism 36 in rapidly activating melanocyte differentiation. Thereby we reveal a novel epigenetic control through pH 37 modulation that reinforces a deterministic cell fate by altering chromatin dynamics. 38 39 pH regulates melanocyte maturation 102 pH regulates melanocyte maturation 6 Results 103 Alkaline intracellular pH induces pigmentation by enhancing the melanogenesis gene expression 104During the culture of B16 cells in DMEM medium CO 2 -HCO 3 buffering system maintains media pH. 105Hence we resorted to establish the pH-pigmentation link by modulating the prevailing CO 2 levels. We set 106 up progressive pigmentation using the B16 cells as described earlier (Natarajan et al, 2014). In this set up 107 the cells progressively activate the melanogenesis gene expression program and increase pigmentation 108 over a course of 8 to 12 days. To alter the pH, 10% CO 2 levels were tested. We measured the extracellular 109 pH (pH e ) using the standard pH electrode under controlled conditions of temperature and CO 2 saturation. 110While the cells grown in 5% CO 2 showed an increase in pH e from around 7.4 to 7.8 on day 4 after 111 induction of the pigmentation program, the 10% CO 2 sustained a constant pH of around 7.4 112( Supplementary Fig 1A). This trend was reflected in the intracellular pH (pH i ), which is close to 7.9 on 113 day 4 under the routine 5% CO 2 condition. However, under the 10% CO 2 the pH remained close to 7.0, 114 similar to the day 0 where the cells are depigmented (Fig 1A). When the cells were assessed for the 115 cumulative accumulation of melanin on day 8, under the 10% CO 2 condition we observed depigmented 116 cells (Fig 1B). Melanin content assay performed using synthetic melanin standard confirmed that the 117 level of melanin is significantly low (Supplementary Fig S1B). Further electron microscopic evaluation 118 of day...

Running Title: H2A.Z.2 dictates melanocyte fate Summary statementHere we report mechanism behind the critical role of Histone variant H2A.Z.2 in fate allocation and subsequent commitment of melanocyte identity, from a multipotent state. 2 AbstractIn the neural crest lineage, progressive fate-restriction and stem cell assignment are critical for both development and regeneration. While the fate-commitment events have distinct transcriptional footprints, fate-biasing is often transitory and metastable, and is thought to be moulded by epigenetic programs. Hence molecular basis of specification is difficult to define. In this study, we establish a role of a histone variant H2a.z.2 in specification of melanocyte lineage from multipotent neural crest cells.Silencing of H2a.z.2 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 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 a histone based specification code upstream to the core gene regulatory network in the neural crest lineage of melanocytes. This epigenetic code renders a poised state to the promoter of key determinant and enhances activation by external instructive signals thereby establishing melanocyte fate identity.