Date of Award
Doctor of Philosophy (PhD)
The epidermis covers the surface of the skin and provides a functional barrier across the entire body. Epidermal cells or keratinocytes proliferate in the innermost basal layer and migrate upwards into the suprabasal spinous and granular layers as they differentiate, and finally into the terminally differentiated outermost stratum corneum. Keratinocytes undergoing terminal differentiation are marked by tissue-specific concomitant expression of genes encoded in the Epidermal Differentiation Complex (EDC) locus. The EDC genes are organized into four gene families - S100, Sprr, Lce, and Flg-like, which are coordinately expressed upon activation of the terminal differentiation program in keratinocytes. The molecular mechanisms that govern the activation of the EDC during epidermal differentiation are poorly understood. The synteny and colinearity of the locus across multiple mammalian species and the coordinate expression of EDC genes upon keratinocyte differentiation suggest molecular mechanisms operating at the chromatin level. I hypothesize coordinate activation of the EDC by an enhancer regulatory element. Enhancers are non-coding regulatory DNA sequences that upon binding specific transcription factors, are able to increase expression of a proximal or distal target gene. Previous work in our lab identified an epidermal-specific enhancer, CNE 923, that was active in in cell-based luciferase assays and transgenic mice. Here, I examine the function of the 923 enhancer for epidermal differentiation. Using an independent transgenic mouse line, I identified spatiotemporal sensitivity of the 923 enhancer that correlated with the patterning of epidermal barrier formation during mouse embryonic development. To determine if 923 formed chromatin interactions with the EDC gene promoters, I performed chromosome conformation capture (3C) assays in proliferating and differentiated primary mouse keratinocytes. The 3C studies identified physiologically sensitive chromatin interactions between 923 and EDC gene promoters. The data supports a dynamic EDC chromatin topology during keratinocyte differentiation. A requirement for c-Jun/AP-1 in relation to 923-mediated EDC chromatin remodeling for normal EDC gene expression during keratinocyte differentiation was further determined by chromatin immunoprecipitation, 3C, and RNA-seq upon pharmacological inhibition of AP-1 binding. To further determine the function of 923 in vivo, I generated a series of mutation alleles using CRISPR/Cas9 genome editing in mice. Cas9 nuclease activity targeted to the flanking ends of the 923 enhancer in mouse zygotes by a pair of guide RNAs, coupled with homologous recombination-mediated loxP insertions, generated 1 floxed (923flox), 2 independent deletions (923delA, 923delB), and 1 partial deletion (923pdel) alleles for the 923 enhancer. My results from the 923 knockout mice identified decreased expression of nearby Smcp, Lce6a, and involucrin gene expression, decreased distal Crnn and Lce gene family members, and a correlative increase in expression of Sprr gene family members. To identify the chromatin interactions for the 923 enhancer on a genome-wide scale, I performed high-throughput circular chromosome conformation capture (4C-seq) assays with respect to the 923 enhancer and an additional Flg promoter viewpoint in proliferating and differentiated keratinocytes and P5424 T-cells. My results revealed 923 enhancer-mediated chromatin interactions indicative of a topologically associated domain encompassing the EDC. However, an enrichment of 923 mediated chromatin interactions within the EDC, were identified in keratinocytes relative to the T-cells, specifically between the 923 enhancer and the Sprr and Lce gene families, and with non-coding regions in the gene desert between the S100 and Sprr gene families. Of note was a 923 interaction with another putative enhancer near Crct1, enriched specifically in proliferating keratinocytes, and suggesting cross-talk between enhancers. Keratinocyte-specific trans-interactions identified by MACS and GREAT algorithms included genes important for epidermal function including Trp63, an important regulator of keratinocyte differentiation. Together, my 4C-seq identifies unique chromatin architectures of the EDC in keratinocytes and T cells, including keratinocyte-specific enhancer-enhancer crosstalk in cis and interactions between transcriptionally active loci in trans. My studies identify, for the first time, a link between the 923 enhancer and proximal (Ivl, Smcp, Lce6a) and distal genes (Crnn, distal Lce family), the loss of which coincides with upregulation of other epidermal differentiation genes (Sprr family) to maintain skin barrier function. Together, my work has identified 923 as an epidermal-specific enhancer that participates in a chromatin looping network to co-regulate expression of genes important for epidermal development, as a mechanism for maintaining skin barrier integrity.
Chair and Committee
Cristina de Guzman Strong
Shiming Chen, John Edwards, Eugene Oltz, Gary Stormo
Oh, Inez, "The Role of Epidermal Enhancer 923 in the Chromatin Architecture and Transcriptional Regulation of the Epidermal Differentiation Complex" (2016). Arts & Sciences Electronic Theses and Dissertations. 880.