Measuring the nanoscale molecular environment dictating the transcriptional state of the Epidermal Differentiation Complex

PROJECT SUMMARY In the stratified epithelia, each layer of cells exhibits a distinct and tightly regulated chromatin organization and gene expression profile. During epidermal differentiation mechanical signals from the extracellular matrix and linker of the nuclear-envelope linker of the cytoskeleton (LINC) are propagated to the nucleus, correlating with a change in chromatin organization and expression. These events coincide with the marked transcriptional activation of a 3.1 Mb chromosomal region known as the epidermal differentiation complex (EDC), which contains genes that are a hallmark feature of differentiation. However, the mechanism by which mechanical force impacts or regulates gene activation and silencing remains unclear. In this proposal, I will use the EDC as a framework by which to investigate the mechanism by which direct force and/or nuclear lamina associated proteins are necessary and sufficient to inhibit or activate gene expression in epidermal differentiation. Activation of the EDC is characterized by a change in nuclear localization, chromatin organization, and shift in associated proteins. However, the spatial organization of these partners remains unresolved. I will apply a new single-molecule superresolution technique that will simultaneously image EDC-associated proteins, histone modification, and the EDC at nanometer scales (<25nm) (Aim1). By doing this I will be able to measure the spatial distance between individual targets to create a map of the nuclear environment in mouse keratinocytes in which the EDC is either in the active or repressed chromatin state. To investigate the mechanism by which nuclear localization influences gene expression of the EDC, I will apply a synthetic inducible tethering system by which EDC localization can be biophysically manipulated (Aim 2). In wild-type mouse keratinocytes it has been shown that the EDC occupies different nuclear localizations in the active and repressed state but the mechanism that regulates this remains unknown. I will evaluate in wild- type keratinocytes and keratinocytes in which LINC-complex dependent tension has been ablated and EDC expression is active, if tethering/localization to the nuclear periphery is sufficient and necessary in inhibiting expression. The completion of these aims will uncover the relationship between chromatin organization and gene expression in the context of epidermal differentiation. Thus far, this structure and function relationship has been correlative; by applying and developing new technology I have the unique ability to reveal mechanistic insight into the nanoscale molecular environment that dictates the transcriptional state of the epidermal differentiation complex in normal and pathogenic states.