Giacomo Donati

Diverse epigenetic strategies interact to control epidermal differentiation

It is becoming clear that interconnected functional gene networks, rather than individual genes, govern stem cell self-renewal and differentiation. To identify epigenetic factors that impact on human epidermal stem cells we performed siRNA-based genetic screens for 332 chromatin modifiers. We developed a Bayesian mixture model to predict putative functional interactions between epigenetic modifiers that regulate differentiation. We discovered a network of genetic interactions involving EZH2, UHRF1 (both known to regulate epidermal self-renewal), ING5 (a MORF complex component), BPTF and SMARCA5 (NURF complex components). Genome-wide localization and global mRNA expression analysis revealed that these factors impact two distinct but functionally related gene sets, including integrin extracellular matrix receptors that mediate anchorage of epidermal stem cells to their niche. Using a competitive epidermal reconstitution assay we confirmed that ING5, BPTF, SMARCA5, EZH2 and UHRF1 control differentiation under physiological conditions. Thus, regulation of distinct gene expression programs through the interplay between diverse epigenetic strategies protects epidermal stem cells from differentiation.

Stem cell heterogeneity and plasticity in epithelia.

Epithelia cover the surfaces and line the cavities of the body. Recent studies have highlighted the existence of multiple stem cell compartments within individual epithelia that exhibit striking plasticity in response to tissue damage, transplantation, or tumor development. New knowledge about the composition of the epithelial niche and the transcription factor networks that maintain cell identity has provided new insights into the extrinsic and intrinsic regulation of stem cell behavior. In addition new in vitro tissue substitutes allow better integration of data from human and mouse models.

Epidermal Wnt/β-catenin signaling regulates adipocyte differentiation via secretion of adipogenic factors

Significance The synchronized patterns of hair follicle growth and expansion of the dermal adipocyte layer have long been recognized. Although factors secreted by adipocytes are known to regulate the hair growth cycle, it is unclear whether, conversely, the epidermis can regulate adipogenesis. Our study now demonstrates that activation of epidermal Wnt/β-catenin signaling stimulates adipocyte differentiation in vivo and in vitro. The effect can be mediated by secreted factors, including insulin-like growth factor 2 and bone morphogenetic proteins 2 and 6. It has long been recognized that the hair follicle growth cycle and oscillation in the thickness of the underlying adipocyte layer are synchronized. Although factors secreted by adipocytes are known to regulate the hair growth cycle, it is unclear whether the epidermis can regulate adipogenesis. We show that inhibition of epidermal Wnt/β-catenin signaling reduced adipocyte differentiation in developing and adult mouse dermis. Conversely, ectopic activation of epidermal Wnt signaling promoted adipocyte differentiation and hair growth. When the Wnt pathway was activated in the embryonic epidermis, there was a dramatic and premature increase in adipocytes in the absence of hair follicle formation, demonstrating that Wnt activation, rather than mature hair follicles, is required for adipocyte generation. Epidermal and dermal gene expression profiling identified keratinocyte-derived adipogenic factors that are induced by β-catenin activation. Wnt/β-catenin signaling-dependent secreted factors from keratinocytes promoted adipocyte differentiation in vitro, and we identified ligands for the bone morphogenetic protein and insulin pathways as proadipogenic factors. Our results indicate epidermal Wnt/β-catenin as a critical initiator of a signaling cascade that induces adipogenesis and highlight the role of epidermal Wnt signaling in synchronizing adipocyte differentiation with the hair growth cycle.

Innate sensing of microbial products promotes wound-induced skin cancer

The association between tissue damage, chronic inflammation and cancer is well known. However, the underlying mechanisms are unclear. Here we characterize a mouse model in which constitutive epidermal extracellular-signal-regulated kinase-MAP-kinase signalling results in epidermal inflammation, and skin wounding induces tumours. We show that tumour incidence correlates with wound size and inflammatory infiltrate. Ablation of tumour necrosis factor receptor (TNFR)-1/-2, Myeloid Differentiation primary response gene 88 or Toll-like receptor (TLR)-5, the bacterial flagellin receptor, but not other innate immune sensors, in radiosensitive leukocytes protects against tumour formation. Antibiotic treatment inhibits, whereas injection of flagellin induces, tumours in a TLR-5-dependent manner. TLR-5 is also involved in chemical-induced skin carcinogenesis in wild-type mice. Leukocytic TLR-5 signalling mediates upregulation of the alarmin HMGB1 (High Mobility Group Box 1) in wound-induced papillomas. HMGB1 is elevated in tumours of patients with Recessive Dystrophic Epidermolysis Bullosa, a disease characterized by chronic skin damage. We conclude that in our experimental model the combination of bacteria, chronic inflammation and wounding cooperate to trigger skin cancer.

Spindle checkpoint deficiency is tolerated by murine epidermal cells but not hair follicle stem cells

The spindle assembly checkpoint (SAC) ensures correct chromosome segregation during mitosis by preventing aneuploidy, an event that is detrimental to the fitness and survival of normal cells but oncogenic in tumor cells. Deletion of SAC genes is incompatible with early mouse development, and RNAi-mediated depletion of SAC components in cultured cells results in rapid death. Here we describe the use of a conditional KO of mouse Mad2, an essential component of the SAC signaling cascade, as a means to selectively induce chromosome instability and aneuploidy in the epidermis of the skin. We observe that SAC inactivation is tolerated by interfollicular epidermal cells but results in depletion of hair follicle bulge stem cells. Eventually, a histologically normal epidermis develops within ∼1 mo after birth, albeit without any hair. Mad2-deficient cells in this epidermis exhibited abnormal transcription of metabolic genes, consistent with aneuploid cell state. Hair follicle bulge stem cells were completely absent, despite the continued presence of rudimentary hair follicles. These data demonstrate that different cell lineages within a single tissue respond differently to chromosome instability: some proliferating cell lineages can survive, but stem cells are highly sensitive.

BLIMP1 is required for postnatal epidermal homeostasis but does not define a sebaceous gland progenitor under steady-state conditions

Summary B-lymphocyte-induced nuclear maturation protein 1 (BLIMP1) was previously reported to define a sebaceous gland (SG) progenitor population in the epidermis. However, the recent identification of multiple stem cell populations in the hair follicle junctional zone has led us to re-evaluate its function. We show, in agreement with previous studies, that BLIMP1 is expressed by postmitotic, terminally differentiated epidermal cells within the SG, interfollicular epidermis, and hair follicle. Epidermal overexpression of c-Myc results in loss of BLIMP1+ cells, an effect modulated by androgen signaling. Epidermal-specific deletion of Blimp1 causes multiple differentiation defects in the epidermis in addition to SG enlargement. In culture, BLIMP1+ sebocytes have no greater clonogenic potential than BLIMP1− sebocytes. Finally, lineage-tracing experiments reveal that, under steady-state conditions, BLIMP1-expressing cells do not divide. Thus, rather than defining a sebocyte progenitor population, BLIMP1 functions in terminally differentiated cells to maintain homeostasis in multiple epidermal compartments.

Wounding induces dedifferentiation of epidermal Gata6 + cells and acquisition of stem cell properties

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6+ cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

Single-cell analysis of CD4+ T-cell differentiation reveals three major cell states and progressive acceleration of proliferation

Differentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood. Here, we interrogate and quantitatively model how proliferation is linked to differentiation in CD4+ T cells. We perform ex vivo single-cell RNA-sequencing of CD4+ T cells during a mouse model of infection that elicits a type 2 immune response and infer that the differentiated, cytokine-producing cells cycle faster than early activated precursor cells. To dissect this phenomenon quantitatively, we determine expression profiles across consecutive generations of differentiated and undifferentiated cells during Th2 polarization in vitro. We predict three discrete cell states, which we verify by single-cell quantitative PCR. Based on these three states, we extract rates of death, division and differentiation with a branching state Markov model to describe the cell population dynamics. From this multi-scale modelling, we infer a significant acceleration in proliferation from the intermediate activated cell state to the mature cytokine-secreting effector state. We confirm this acceleration both by live imaging of single Th2 cells and in an ex vivo Th1 malaria model by single-cell RNA-sequencing. The link between cytokine secretion and proliferation rate holds both in Th1 and Th2 cells in vivo and in vitro, indicating that this is likely a general phenomenon in adaptive immunity.BackgroundDifferentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood. Here, we interrogate and quantitatively model how proliferation is linked to differentiation in CD4+ T cells.ResultsWe perform ex vivo single-cell RNA-sequencing of CD4+ T cells during a mouse model of infection that elicits a type 2 immune response and infer that the differentiated, cytokine-producing cells cycle faster than early activated precursor cells. To dissect this phenomenon quantitatively, we determine expression profiles across consecutive generations of differentiated and undifferentiated cells during Th2 polarization in vitro. We predict three discrete cell states, which we verify by single-cell quantitative PCR. Based on these three states, we extract rates of death, division and differentiation with a branching state Markov model to describe the cell population dynamics. From this multi-scale modelling, we infer a significant acceleration in proliferation from the intermediate activated cell state to the mature cytokine-secreting effector state. We confirm this acceleration both by live imaging of single Th2 cells and in an ex vivo Th1 malaria model by single-cell RNA-sequencing.ConclusionThe link between cytokine secretion and proliferation rate holds both in Th1 and Th2 cells in vivo and in vitro, indicating that this is likely a general phenomenon in adaptive immunity.

Type XVII collagen coordinates proliferation in the interfollicular epidermis

Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin. DOI: http://dx.doi.org/10.7554/eLife.26635.001

A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells

Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2.