E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis Perrine Goguet-Rubioa,b,c,d,e,1, Berfin Seyrana,b,c,d,e,1, Laurie Gaytea,b,c,d,e, Florence Bernexa,c,f, Anne Suttera,b,c,d,e, Hélène Delpecha,b,c,d,e,g, Laetitia Karine Linaresa,b,c,d,e, Romain Riscala,b,c,d,e, Cendrine Repondh, Geneviève Rodiera,b,c,d,e,g, Olivier Kirshg,i,JawidaTouhamic,g,JeanNoela,b,c,d,f, Charles Vincenta,b,c,d,NellyPirota,b,c,d,f, Guillaume Pavlovicj, Yann Heraultj, Marc Sitbonc,g,LucPellerinh, Claude Sardeta,b,c,d,e,g, Matthieu Lacroixa,b,c,d,e,2,3, and Laurent Le Cama,b,c,d,e,2,3 aInstitut de Recherche en Cancérologie de Montpellier, Montpellier F-34298, France; bINSERM U1194, Montpellier F-34298, France; cUniversity of Montpellier, Montpellier F-34090, France; dInstitut Régional du Cancer de Montpellier, Montpellier F-34298, France; eEquipe labellisée Ligue Contre le Cancer, 75013 Paris, France; fRéseau d’Histologie Expérimentale de Montpellier, BioCampus, CNRS-UMS3426, F-34094 Montpellier, France; gInstitut de Génétique Moléculaire de Montpellier, CNRS-UMR5535, Montpellier 34293, France; hDepartment of Physiology, University of Lausanne, 1005 Lausanne, Switzerland; iEpigenetics and Cell Fate, University Paris Diderot, Sorbonne Paris Cite, UMR7216 CNRS, Paris 75013, France; and jInstitut de la Clinique de la Souris-Mouse Clinical Institute, PHENOMIN, CNRS-UMR7104, INSERM U964, Université de Strasbourg, Illkirch, France Edited by Steven L. McKnight, The University of Texas Southwestern Medical Center, Dallas, TX, and approved August 5, 2016 (received for review February 18, 2016) The multifunctional protein E4 transcription factor 1 (E4F1) is an does not impair maintenance of basal keratinocytes (6). Al- essential regulator of epidermal stem cell (ESC) maintenance. Here, though these results suggest that basal keratinocytes display a we found that E4F1 transcriptionally regulates a metabolic pro- metabolic status that is different from their differentiated counter- gram involved in pyruvate metabolism that is required to maintain parts, further studies are warranted to decipher the poorly un- skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in derstood role of metabolism in the regulation of epidermal deregulated expression of dihydrolipoamide acetyltransferase (Dlat), cell fate. a gene encoding the E2 subunit of the mitochondrial pyruvate de- We previously identified the multifunctional protein E4 tran- hydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) kera- scription factor 1 (E4F1) as an essential regulator of skin homeo- tinocytes exhibited impaired PDH activity and a redirection of the stasis and ESC maintenance (7). E4F1 was originally identified as a PHYSIOLOGY glycolytic flux toward lactate production. The metabolic reprogram- cellular target of the E1A viral oncoprotein (8, 9). Since then, E4f1 ming of KO keratinocytes associated with remodeling of their several laboratories have shown that E4F1 directly interacts with microenvironment and alterations of the basement membrane, led several oncogenes and tumor suppressors, including p53, BMI1, to ESC mislocalization and exhaustion of the ESC pool. ShRNA-medi- RB, RASSF1A, SMAD4, or HMGA2 proteins (10–16). Consistent ated depletion of Dlat in primary keratinocytes recapitulated defects with its implication in different oncogenic pathways, E4F1 acts as observed upon E4f1 inactivation, including increased lactate secre- a survival factor in cancer cells (17, 18). Moreover, characterization tion, enhanced activity of extracellular matrix remodeling enzymes, E4f1 E4f1 and impaired clonogenic potential. Altogether, our data reveal a of knock-out (KO) mice showed that is an essential central role for Dlat in the metabolic program regulated by E4F1 in gene in embryonic stem cells and during early embryogenesis (19). basal keratinocytes and illustrate the importance of PDH activity in Using E4f1 conditional KO mice, we previously reported that E4f1 skin homeostasis. Significance E4F1 | PDH | pyruvate | skin | stem cell We found that the multifunctional protein E4 transcription enewal and wound healing of the epidermis rely on a pool of factor 1 (E4F1) transcriptionally regulates a metabolic program Repidermal stem cells (ESC) located in the basal layer of the involved in pyruvate metabolism that is required to maintain interfollicular epithelium (IFE) and in the bulge region of the skin homeostasis. E4F1 deficiency in skin resulted in deregu- dihydrolipoamide acetlytransferase Dlat hair follicle (HF). In the IFE, these long-lived ESC give rise to lated expression of ( ), progenitors with increased proliferative capacities that differen- a gene encoding the E2 subunit of the mitochondrial pyruvate E4f1 tiate into keratinocytes as they migrate upward into suprabasal dehydrogenase (PDH) complex. Accordingly, knock-out layers. Numerous studies have addressed the role of several key (KO) keratinocytes exhibited impaired PDH activity and a signaling pathways, such as those implicating bone morphoge- metabolic reprogramming associated with remodeling of their netic proteins, TGF-β, Notch, Sonic Hedgehog, or Wnt in skin microenvironment and alterations of the basement membrane, homeostasis, and how they control ESC maintenance (1–3). The leading to epidermal stem cell mislocalization and exhaus- role of these pathways in regulating stemness has been attributed tion of the epidermal stem cell pool. Our data reveal a cen- Dlat to the regulation of cell proliferation, cell death, cellular senes- tral role for in the metabolic program regulated by E4F1 cence, cell adhesion, or differentiation. Although previous data in skin and illustrate the importance of PDH activity in skin indicate that some of these stem cell regulators also control energy homeostasis. metabolism in the hematopoietic or neuronal lineages (4), very few Author contributions: M.S., L.P., C.S., M.L., and L.L.C. designed research; P.G.-R., B.S., L.G., studies have yet addressed their metabolic functions in keratino- A.S., H.D., L.K.L., R.R., C.R., G.R., O.K., J.T., J.N., C.V., N.P., and M.L. performed research; cytes. In addition, the potential role of specific metabolic regulators G.P., Y.H., and M.S. contributed new reagents/analytic tools; F.B., M.L., and L.L.C. analyzed in the control of skin homeostasis remains poorly documented. data; and M.L. and L.L.C. wrote the paper. Nevertheless, previous observations indicate that deregulation The authors declare no conflict of interest. of the nutrient-sensing mammalian target of rapamycin path- This article is a PNAS Direct Submission. way in basal keratinocytes occurs as a consequence of pro- 1P.G.-R. and B.S. contributed equally to this work. longed Wnt signaling, leading to the progressive exhaustion of 2M.L. and L.L.C. contributed equally to this work. HF bulge stem cells (5). Recent data also indicate that genetic 3To whom correspondence may be addressed. Email: [email protected] or inactivation in mouse epidermis of mitochondrial transcription [email protected]. factor A (Tfam), a gene involved in mitochondrial DNA replication This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and transcription, impinges on keratinocyte differentiation but 1073/pnas.1602751113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1602751113 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 inactivation in the epidermis results in ESC defects through a mechanism that involves, at least partly, the deregulation of the Bmi1–Arf–p53 pathway (7). Here, we show evidence supporting a major role for E4F1 in pyruvate metabolism that governs ESC maintenance and skin homeostasis. Results E4f1 Inactivation in Basal but Not Suprabasal Adult Keratinocytes Leads to Epidermal Defects and Exhaustion of the ESC Pool. Using E4f1 whole-body conditional KO mice (E4f1KO; RERT), we previously identified an essential role for E4f1 in adult skin ho- meostasis (7). In this genetically engineered mouse model, E4f1 inactivation was achieved in the entire skin of adult animals, including the dermal compartment. To assess the cell of origin of these skin defects, we generated new mouse strains by crossing E4f1 conditional KO mice to transgenic animals expressing the tamoxifen (tam) -inducible CreER recombinase under the control of the keratin 14 (K14) or keratin 10 (K10) promoters [hereafter referred to as E4f1(K14)KO and E4f1(K10)KO strains], allowing Fig. 1. E4F1 deficiency in basal keratinocytes leads to skin defects and ex- acute inactivation of E4f1 in adult keratinocytes of the basal or haustion of the ESC pool. (A) Microphotographs of (HES)-stained skin sec- spinous layers, respectively (20). tions prepared from E4F1(K14)KO mice or E4F1(K14)CTR littermates, 1, 2, or Molecular and histological analyses of adult back skin of 8- to 5 wk after tam administration. Dashed lines indicate the separation between 12-wk-old E4f1(K10)KO animals confirmed that topical skin the epidermis and the dermis. (Scale bars, 100 μm.) (B) Whole mounts of tail applications of tam activated the Cre recombinase in suprabasal epidermis prepared from adult E4F1(K14)KO and CTR mice, 5 wk after tam but not in basal keratinocytes (Fig. S1). Neither histological al- application, stained with K15 antibody and DAPI. Brackets: bulge area (BG) of the HF. (Scale bar, 100 μm.) (C) Number of follicular stem cells (FSC) in back terations nor differences in the expression pattern of the basal- skin epidermis prepared from the same mice as in B. FACS-analysis of α6/CD34 cell specific K14 marker and of the differentiation markers K10 CD34high FSC in back skin epidermis prepared from the same mice as in B (mean ± + and involucrin were identified in skin samples harvested up to SEM; n = 10). (D) Number of label-retaining (EdU ) interfollicular stem cells (LRCs) 4 mo upon regular tam administration (Fig. S2). In sharp contrast, detected by immunofluorescence (IF) on back-skin sections prepared from adult inactivation of E4f1 in adult basal keratinocytes of E4f1(K14)KO E4F1(K14)KO mice or E4F1(K14)CTR littemates, 5 wk after tam application.