GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development.

Ectodermal dysplasias including skin abnormalities and cleft lip/palate result from improper surface ectoderm (SE) patterning. However, the connection between SE gene regulatory networks and disease remains poorly understood. Here, we dissect human SE differentiation with multiomics and establish GRHL2 as a key mediator of early SE commitment, which acts by skewing cell fate away from the neural lineage. GRHL2 and master SE regulator AP2a balance early cell fate output, with GRHL2 facilitating AP2a binding to SE loci. In turn, AP2a restricts GRHL2 DNA binding away from de novo chromatin contacts. Integration of these regulatory sites with ectodermal dysplasia-associated genomic variants annotated within the Biomedical Data Commons identifies 55 loci previously implicated in craniofacial disorders. These include ABCA4/ARHGAP29 and NOG regulatory regions where disease-linked variants directly affect GRHL2/AP2a binding and gene transcription. These studies elucidate the logic underlying SE commitment and deepen our understanding of human oligogenic disease pathogenesis.

Translational control of a human CDKN1A mRNA splice variant regulates the fate of UVB-irradiated human keratinocytes.

In response to sublethal ultraviolet B (UVB) irradiation, human keratinocytes transiently block progression of the cell cycle to allow ample time for DNA repair and cell fate determination. These cellular activities are important for avoiding the initiation of carcinogenesis in skin. Central to these processes is the repression of initiation of mRNA translation through GCN2 phosphorylation of eIF2α (eIF2α-P). Concurrent with reduced global protein synthesis, eIF2α-P and the accompanying integrated stress response (ISR) selectively enhance translation of mRNAs involved in stress adaptation. In this study, we elucidated a mechanism for eIF2α-P cytoprotection in response to UVB in human keratinocytes. Loss of eIF2α-P induced by UVB diminished G1 arrest, DNA repair, and cellular senescence coincident with enhanced cell death in human keratinocytes. Genome-wide analysis of translation revealed that the mechanism for these critical adaptive responses by eIF2α-P involved induced expression of CDKN1A encoding the p21 (CIP1/WAF1) protein. We further show that human CDKN1A mRNA splice variant 4 is preferentially translated following stress-induced eIF2α-P by a mechanism mediated in part by upstream ORFs situated in the 5'-leader of CDKN1A mRNA. We conclude that eIF2α-P is cytoprotective in response to UVB by a mechanism featuring translation of a specific splice variant of CDKN1A that facilitates G1 arrest and subsequent DNA repair.

Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2.

Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. Although transcriptional regulation has proven to be important for keratinocyte differentiation, little is known about the role of translational control. A key mechanism for modulating translation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2). A family of different eIF2α kinases function in the integrative stress response to inhibit general protein synthesis coincident with preferential translation of select mRNAs that participate in stress alleviation. Here we demonstrate that translational control through eIF2α phosphorylation is required for normal keratinocyte differentiation. Analyses of polysome profiles revealed that key differentiation genes, including involucrin, are bound to heavy polysomes during differentiation, despite decreased general protein synthesis. Induced eIF2α phosphorylation by the general control nonderepressible 2 (GCN2) protein kinase facilitated translational control and differentiation-specific protein expression during keratinocyte differentiation. Furthermore, loss of GCN2 thwarted translational control, normal epidermal differentiation, and differentiation gene expression in organotypic skin culture. These findings underscore a previously unknown function for GCN2 phosphorylation of eIF2α and translational control in the formation of an intact human epidermis.

Insulin-like growth factor-1 receptor regulates repair of ultraviolet B-induced DNA damage in human keratinocytes in vivo.

The activation status of the insulin-like growth factor-1 receptor (IGF-1R) regulates the cellular response of keratinocytes to ultraviolet B (UVB) exposure, both in vitro and in vivo. Geriatric skin is deficient in IGF-1 expression resulting in an aberrant IGF-1R-dependent UVB response which contributes to the development of aging-associated squamous cell carcinoma. Furthermore, our lab and others have reported that geriatric keratinocytes repair UVB-induced DNA damage less efficiently than young adult keratinocytes. Here, we show that IGF-1R activation influences DNA damage repair in UVB-irradiated keratinocytes. Specifically, in the absence of IGF-1R activation, the rate of DNA damage repair following UVB-irradiation was significantly slowed (using immortalized human keratinocytes) or inhibited (using primary human keratinocytes). Furthermore, inhibition of IGF-1R activity in human skin, using either ex vivo explant cultures or in vivo xenograft models, suppressed DNA damage repair. Primary keratinocytes with an inactivated IGF-1R also exhibited lower steady-state levels of nucleotide excision repair mRNAs. These results suggest that deficient UVB-induced DNA repair in geriatric keratinocytes is due in part to silenced IGF-1R activation in geriatric skin and provide a mechanism for how the IGF-1 pathway plays a role in the initiation of squamous cell carcinoma in geriatric patients.

Translational Repression Protects Human Keratinocytes from UVB-Induced Apoptosis through a Discordant eIF2 Kinase Stress Response.

This study delineates the mechanisms by which UVB regulates protein synthesis in human keratinocytes and the importance of translational control in cell survival. Translation initiation is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2-P) that causes decreased global protein synthesis coincident with enhanced translation of selected stress-related transcripts, such as activating transcription factor 4 (ATF4). ATF4 is a transcriptional activator of the integrated stress response (ISR) that has cytoprotective functions as well as apoptotic signals through the downstream transcriptional regulator C/EBP homologous protein (CHOP; GADD153/DDIT3). We determined that UVB irradiation is a potent inducer of eIF2-P in keratinocytes, leading to decreased levels of translation initiation. However, expression of ATF4 or CHOP was not induced by UVB as compared with traditional ISR activators. The rationale for this discordant response is that ATF4 mRNA is reduced by UVB, and despite its ability to be preferentially translated, there are diminished levels of available transcript. Forced expression of ATF4 and CHOP protein before UVB irradiation significantly enhanced apoptosis, suggesting that this portion of the ISR is deleterious in keratinocytes following UVB. Inhibition of eIF2-P and translational control reduced viability following UVB that was alleviated by cycloheximide (CHX), indicating that translation repression through eIF2-P is central to keratinocyte survival.