We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.

Docosahexaenoic acid (DHA, 22:6n-3) is an omega-3 fatty acid essential for proper brain development. N-docosahexaenoylethanolamine (synaptamide), an endogenous metabolite of DHA, potently promotes neurogenesis, neuritogenesis and synaptogenesis; however, the underlying molecular mechanism is not known. Here, we demonstrate orphan G-protein coupled receptor 110 (GPR110, ADGRF1) as the synaptamide receptor, mediating synaptamide-induced bioactivity in a cAMP-dependent manner. Mass spectrometry-based proteomic characterization and cellular fluorescence tracing with chemical analogues of synaptamide reveal specific binding of GPR110 to synaptamide, which triggers cAMP production with low nM potency. Disruption of this binding or GPR110 gene knockout abolishes while GPR110 overexpression enhances synaptamide-induced bioactivity. GPR110 is highly expressed in fetal brains but rapidly decreases after birth. GPR110 knockout mice show significant deficits in object recognition and spatial memory. GPR110 deorphanized as a functional synaptamide receptor provides a novel target for neurodevelopmental control and new insight into mechanisms by which DHA promotes brain development and function.

Background: Myocilin, a secreted glaucoma-associated protein, is detected in ocular and non-ocular tissues. Results: Myocilin is expressed in mesenchymal stem cells (MSCs) and stimulates their differentiation into osteoblasts. Conclusion: Myocilin-stimulated osteogenic differentiation of MSCs is associated with activation of MAP kinase signaling pathways. Significance: Modulation of myocilin activity could potentially be targeted to improve the bone-regenerative potential of MSCs.

The MYOCILIN gene encodes a secreted glycoprotein which is highly expressed in eye drainage structures. Mutations in this gene may lead to juvenile open-angle glaucoma and adult onset primary open-angle glaucoma, one of the leading causes of irreversible blindness in the world. Functions of wild-type myocilin are still unclear. We have recently demonstrated that myocilin is a modulator of Wnt signaling and may affect actin cytoskeleton organization. Here we report that myocilin and its naturally occurring proteolytic fragments, similar to Wnt3a, are able to stimulate trabecular meshwork, NIH3T3 and FHL124 cell migration with the N-terminal proteolytic fragment of myocilin lacking the olfactomedin domain producing the highest stimulatory effect. Stimulation of cell migration occurs through activation of the integrin-focal adhesion kinase (FAK)-serine/threonine kinase (AKT) signaling pathway. Inhibition of FAK by siRNA reduced the stimulatory action of myocilin by 3 fold. Activation of several components of this signaling pathway was also demonstrated in the eyes of transgenic mice expressing elevated levels of myocilin in the eye drainage structures. These data extend the similarities between actions of myocilin and Wnt proteins acting through a β-catenin-independent mechanism. The modification of the migratory ability of cells by myocilin may play a role in normal functioning of the eye anterior segment and its pathology including glaucoma.