The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14’s codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. A proteomics screen identified 14-3-3 as K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14 C373A keratinocytes. Residue C373 in K14, which is conserved in a subset of keratins, is revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in epidermis.

In the fruit fly Drosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (FruM) alters the presence, anatomy, and/or connectivity of >60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of FruM in sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. Here, we take a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of FruM. We identify 436 high-confidence genomic regions differentially accessible in male fruitless neurons, validate candidate regions as bona-fide, differentially regulated enhancers, and describe the particular cell types in which these enhancers are active. We find that individual enhancers are not activated universally but are dedicated to specific fru+ cell types. Aside from fru itself, genes are not dedicated to or common across the fru circuit; rather, FruM appears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, we find FruM motifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that FruM acts cell-type-specifically to decommission regulatory elements in male fruitless neurons.

Chromatin insulators are DNA-protein complexes that regulate chromatin structure and gene expression in a wide range of organisms. These complexes also harbor enhancer blocking and barrier activities. Increasing evidence suggests that RNA molecules are integral components of insulator complexes. However, how these RNA molecules are involved in insulator function remains unclear. The Drosophila RNA-binding protein Shep associates with the gypsy insulator complex and inhibits insulator activities. By mutating key residues in the RRM domains, we generated a Shep mutant protein incapable of RNA-binding, and this mutant lost the ability to inhibit barrier activity. In addition, we found that one of many wildtype Shep isoforms but not RRM mutant Shep was sufficient to repress enhancer blocking activities. Finally, wildtype Shep rescued synthetic lethality of shep , mod(mdg4) double-mutants and developmental defects of shep mutant neurons, whereas mutant Shep failed to do so. These results indicate that the RNA-binding ability of Shep is essential for its ability to antagonize insulator activities and promote neuronal maturation. Our findings suggest that regulation of insulator function by RNA-binding proteins relies on RNA-mediated interactions.