Regional specific differentiation of integumentary organs: SATB2 is involved in α- and ß-keratin gene cluster switching in the chicken.

BACKGROUND: Animals develop skin regional specificities to best adapt to their environments. Birds are excellent models in which to study the epigenetic mechanisms that facilitate these adaptions. Patients suffering from SATB2 mutations exhibit multiple defects including ectodermal dysplasia-like changes. The preferential expression of SATB2, a chromatin regulator, in feather-forming compared to scale-forming regions, suggests it functions in regional specification of chicken skin appendages by acting on either differentiation or morphogenesis. RESULTS: Retrovirus mediated SATB2 misexpression in developing feathers, beaks, and claws causes epidermal differentiation abnormalities (e.g. knobs, plaques) with few organ morphology alterations. Chicken ß-keratins are encoded in 5 sub-clusters (Claw, Feather, Feather-like, Scale, and Keratinocyte) on Chromosome 25 and a large Feather keratin cluster on Chromosome 27. Type I and II α-keratin clusters are located on Chromosomes 27 and 33, respectively. Transcriptome analyses showed these keratins (1) are often tuned up or down collectively as a sub-cluster, and (2) these changes occur in a temporo-spatial specific manner. CONCLUSIONS: These results suggest an organizing role of SATB2 in cluster-level gene co-regulation during skin regional specification.

Regional Specific Differentiation of Integumentary Organs: Regulation of Gene Clusters within the Avian Epidermal Differentiation Complex and Impacts of SATB2 Overexpression.

The epidermal differentiation complex (EDC) encodes a group of unique proteins expressed in late epidermal differentiation. The EDC gave integuments new physicochemical properties and is critical in evolution. Recently, we showed ß-keratins, members of the EDC, undergo gene cluster switching with overexpression of SATB2 (Special AT-rich binding protein-2), considered a chromatin regulator. We wondered whether this unique regulatory mechanism is specific to ß-keratins or may be derived from and common to EDC members. Here we explore (1) the systematic expression patterns of non-ß-keratin EDC genes and their preferential expression in different skin appendages during development, (2) whether the expression of non-ß-keratin EDC sub-clusters are also regulated in clusters by SATB2. We analyzed bulk RNA-seq and ChIP-seq data and also evaluated the disrupted expression patterns caused by overexpressing SATB2. The results show that the expression of whole EDDA and EDQM sub-clusters are possibly mediated by enhancers in E14-feathers. Overexpressing SATB2 down-regulates the enriched EDCRP sub-cluster in feathers and the EDCH sub-cluster in beaks. These results reveal the potential of complex epigenetic regulation activities within the avian EDC, implying transcriptional regulation of EDC members acting at the gene and/or gene cluster level in a temporal and skin regional-specific fashion, which may contribute to the evolution of diverse avian integuments.

Symmetry breaking of tissue mechanics in wound induced hair follicle regeneration of laboratory and spiny mice.

Tissue regeneration is a process that recapitulates and restores organ structure and function. Although previous studies have demonstrated wound-induced hair neogenesis (WIHN) in laboratory mice (Mus), the regeneration is limited to the center of the wound unlike those observed in African spiny (Acomys) mice. Tissue mechanics have been implicated as an integral part of tissue morphogenesis. Here, we use the WIHN model to investigate the mechanical and molecular responses of laboratory and African spiny mice, and report these models demonstrate opposing trends in spatiotemporal morphogenetic field formation with association to wound stiffness landscapes. Transcriptome analysis and K14-Cre-Twist1 transgenic mice show the Twist1 pathway acts as a mediator for both epidermal-dermal interactions and a competence factor for periodic patterning, differing from those used in development. We propose a Turing model based on tissue stiffness that supports a two-scale tissue mechanics process: (1) establishing a morphogenetic field within the wound bed (mm scale) and (2) symmetry breaking of the epidermis and forming periodically arranged hair primordia within the morphogenetic field (µm scale). Thus, we delineate distinct chemo-mechanical events in building a Turing morphogenesis-competent field during WIHN of laboratory and African spiny mice and identify its evo-devo advantages with perspectives for regenerative medicine.

Making region-specific integumentary organs in birds: evolution and modifications.

Birds are the most diversified terrestrial vertebrates due to highly diverse integumentary organs that enable robust adaptability to various eco-spaces. Here we show that this complexity is built upon multi-level regional specifications. Across-the-body (macro-) specification includes the evolution of beaks and feathers as new integumentary organs that are formed with regional specificity. Within-an-organ (micro-) specification involves further modifications of organ shapes. We review recent progress in elucidating the molecular mechanisms underlying feather diversification as an example. (1) ß-Keratin gene clusters are regulated by typical enhancers or high order chromatin looping to achieve macro- and micro-level regional specification, respectively. (2) Multi-level symmetry-breaking of feather branches confers new functional forms. (3) Complex color patterns are produced by combinations of macro-patterning and micro-patterning processes. The integration of these findings provides new insights toward the principle of making a robustly adaptive bio-interface.

Folding Keratin Gene Clusters during Skin Regional Specification.

Regional specification is critical for skin development, regeneration, and evolution. The contribution of epigenetics in this process remains unknown. Here, using avian epidermis, we find two major strategies regulate ß-keratin gene clusters. (1) Over the body, macro-regional specificities (scales, feathers, claws, etc.) established by typical enhancers control five subclusters located within the epidermal differentiation complex on chromosome 25; (2) within a feather, micro-regional specificities are orchestrated by temporospatial chromatin looping of the feather ß-keratin gene cluster on chromosome 27. Analyses suggest a three-factor model for regional specification: competence factors (e.g., AP1) make chromatin accessible, regional specifiers (e.g., Zic1) target specific genome regions, and chromatin regulators (e.g., CTCF and SATBs) establish looping configurations. Gene perturbations disrupt morphogenesis and histo-differentiation. This chicken skin paradigm advances our understanding of how regulation of big gene clusters can set up a two-dimensional body surface map.

Instructive role of melanocytes during pigment pattern formation of the avian skin.

Animal skin pigment patterns are excellent models to study the mechanism of biological self-organization. Theoretical approaches developed mathematical models of pigment patterning and molecular genetics have brought progress; however, the responsible cellular mechanism is not fully understood. One long unsolved controversy is whether the patterning information is autonomously determined by melanocytes or nonautonomously determined from the environment. Here, we transplanted purified melanocytes and demonstrated that melanocytes could form periodic pigment patterns cell autonomously. Results of heterospecific transplantation among quail strains are consistent with this finding. Further, we observe that developing melanocytes directly connect with each other via filopodia to form a network in culture and in vivo. This melanocyte network is reminiscent of zebrafish pigment cell networks, where connexin is implicated in stripe formation via genetic studies. Indeed, we found connexin40 (cx40) present on developing melanocytes in birds. Stripe patterns can form in quail skin explant cultures. Several calcium channel modulators can enhance or suppress pigmentation globally, but a gap junction inhibitor can change stripe patterning. Most interestingly, in ovo, misexpression of dominant negative cx40 expands the black region, while overexpression of cx40 expands the yellow region. Subsequently, melanocytes instruct adjacent dermal cells to express agouti signaling protein (ASIP), the regulatory factor for pigment switching, which promotes pheomelanin production. Thus, we demonstrate Japanese quail melanocytes have an autonomous periodic patterning role during body pigment stripe formation. We also show dermal agouti stripes and how the coupling of melanocytes with dermal cells may confer stable and distinct pigment stripe patterns.

STAT3 signalling pathway is implicated in keloid pathogenesis by preliminary transcriptome and open chromatin analyses.

Keloids are wounding-induced fibroproliferative human tumor-like skin scars of complex genetic makeup and poorly defined pathogenesis. To reveal dynamic epigenetic and transcriptome changes of keloid fibroblasts, we performed RNA-seq and ATAC-seq analysis on an early passage keloid fibroblast cell strain and its paired normal control fibroblasts. This keloid strain produced keloid-like scars in a plasma clot-based skin equivalent humanized keloid animal model. RNA-seq analysis reveals gene ontology terms including hepatic fibrosis, Wnt-ß-catenin, TGF-ß, regulation of epithelial-mesenchymal transition (EMT), STAT3 and adherens junction. ATAC-seq analysis suggests STAT3 signalling is the most significantly enriched gene ontology term in keloid fibroblasts, followed by Wnt signalling (Wnt5) and regulation of the EMT pathway. Immunohistochemistry confirms that STAT3 (Tyr705 phospho-STAT3) is activated and ß-catenin is up-regulated in the dermis of keloid clinical specimens and keloid skin equivalent implants from the humanized mouse model. A non-linear dose-response of cucurbitacin I, a selective JAK2/STAT3 inhibitor, in collagen type I expression of keloid-derived plasma clot-based skin equivalents implicates a likely role of STAT3 signalling in keloid pathogenesis. This work also demonstrates the utility of the recently established humanized keloid mouse model in exploring the mechanism of keloid formation.

Transcriptome analyses of reprogrammed feather / scale chimeric explants revealed co-expressed epithelial gene networks during organ specification.

BACKGROUND: The molecular mechanism controlling regional specific skin appendage phenotypes is a fundamental question that remains unresolved. We recently identified feather and scale primordium associated genes and with functional studies, proposed five major modules are involved in scale-to-feather conversion and their integration is essential to form today's feathers. Yet, how the molecular networks are wired and integrated at the genomic level is still unknown. RESULTS: Here, we combine classical recombination experiments and systems biology technology to explore the molecular mechanism controlling cell fate specification. In the chimeric explant, dermal fate is more stable, while epidermal fate is reprogrammed to be similar to the original appendage type of the mesenchyme. We analyze transcriptome changes in both scale-to-feather and feather-to-scale transition in the epidermis. We found a highly interconnected regulatory gene network controlling skin appendage types. These gene networks are organized around two molecular hubs, ß-catenin and retinoic acid (RA), which can bind to regulatory elements controlling downstream gene expression, leading to scale or feather fates. ATAC sequencing analyses revealed about 1000 altered widely distributed chromatin open sites. We find that perturbation of a key gene alters the expression of many other co-expressed genes in the same module. CONCLUSIONS: Our findings suggest that these feather / scale fate specification genes form an interconnected network and rewiring of the gene network can lead to changes of appendage phenotypes, acting similarly to endogenous reprogramming at the tissue level. This work shows that key hub molecules, ß-catenin and retinoic acid, regulate scale / feather fate specification gene networks, opening up new possibilities to understand the switches controlling organ phenotypes in a two component (epithelial and mesenchyme) system.

SUMO5, a Novel Poly-SUMO Isoform, Regulates PML Nuclear Bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) are PML-based nuclear structures that regulate various cellular processes. SUMOylation, the process of covalently conjugating small ubiquitin-like modifiers (SUMOs), is required for both the formation and the disruption of PML-NBs. However, detailed mechanisms of how SUMOylation regulates these processes remain unknown. Here we report that SUMO5, a novel SUMO variant, mediates the growth and disruption of PML-NBs. PolySUMO5 conjugation of PML at lysine 160 facilitates recruitment of PML-NB components, which enlarges PML-NBs. SUMO5 also increases polySUMO2/3 conjugation of PML, resulting in RNF4-mediated disruption of PML-NBs. The acute promyelocytic leukemia oncoprotein PML-RARα blocks SUMO5 conjugation of PML, causing cytoplasmic displacement of PML and disruption of PML-NBs. Our work not only identifies a new member of the SUMO family but also reveals the mechanistic basis of the PML-NB life cycle in human cells.

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation.

Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein-protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.

FKBPs in chromatin modification and cancer.

FK506-binding proteins (FKBPs) are intracellular receptors for FK506 and rapamycin, immunosuppressants that have recently been utilized as anticancer drugs. In the cytoplasm, FKBPs and these drugs modulate signal transduction pathways. However, recent reports reveal novel functions of FKBPs in the nucleus, which include regulation of transcription factors, histone chaperone activity, and modifications of chromatin structure. These activities are known to affect gene expression, DNA repair, and DNA replication. Therefore, elucidation of the nuclear functions of FKBPs will help researchers and clinicians better understand how immunosuppressants work as anticancer drugs, which might in turn lead to novel designs of cancer therapy.