BAP1 regulation of the key adaptor protein NCoR1 is critical for γ-globin gene repression.

Human globin gene production transcriptionally "switches" from fetal to adult synthesis shortly after birth and is controlled by macromolecular complexes that enhance or suppress transcription by elements scattered throughout the locus. The DRED (direct repeat erythroid-definitive) repressor is recruited to the ε-globin and γ-globin promoters by the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2) to engender their silencing in adult erythroid cells. Here we found that nuclear receptor corepressor-1 (NCoR1) is a critical component of DRED that acts as a scaffold to unite the DNA-binding and epigenetic enzyme components (e.

Targeted inhibition of the Shroom3-Rho kinase protein-protein interaction circumvents Nogo66 to promote axon outgrowth.

Background: Inhibitory molecules in the adult central nervous system, including NogoA, impede neural repair by blocking axon outgrowth. The actin-myosin regulatory protein Shroom3 directly interacts with Rho kinase and conveys axon outgrowth inhibitory signals from Nogo66, a C-terminal inhibitory domain of NogoA. The purpose of this study was to identify small molecules that block the Shroom3-Rho kinase protein-protein interaction as a means to modulate NogoA signaling and, in the longer term, enhance axon outgrowth during neural repair.

POSH is an intracellular signal transducer for the axon outgrowth inhibitor Nogo66.

Myelin-derived inhibitors limit axon outgrowth and plasticity during development and in the adult mammalian CNS. Nogo66, a functional domain of the myelin-derived inhibitor NogoA, signals through the PirB receptor to inhibit axon outgrowth. The signaling pathway mobilized by Nogo66 engagement of PirB is not well understood.

The scaffold protein POSH regulates axon outgrowth.

How scaffold proteins integrate signaling pathways with cytoskeletal components to drive axon outgrowth is not well understood. We report here that the multidomain scaffold protein Plenty of SH3s (POSH) regulates axon outgrowth. Reduction of POSH function by RNA interference (RNAi) enhances axon outgrowth in differentiating mouse primary cortical neurons and in neurons derived from mouse P19 cells, suggesting POSH negatively regulates axon outgrowth.

UNC-51-like kinase regulation of fibroblast growth factor receptor substrate 2/3.

UNC-51-like kinases (ULK) are members of an evolutionarily conserved sub-family of ubiquitously expressed serine/threonine-specific protein kinases. Here we report that fibroblast growth factor receptor substrate (FRS) 2/3 are novel ULK2 carboxy-terminal domain interacting proteins. FRS2/3 are homologs that function as adaptor proteins to mediate signaling of multiple receptor tyrosine kinases.

Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155.

Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells.

Use of short hairpin RNA expression vectors to study mammalian neural development.

The use of RNA interference (RNAi) in mammalian cells has become a powerful tool for the analysis of gene function. Here we discuss the use of DNA vectors to produce short hairpin RNAs (shRNAs) and inhibit gene expression in mammalian neural progenitors and neurons. Protocols are presented for introducing shRNA vectors into mouse P19 cells differentiated as neurons in vitro and for electroporation of shRNA vectors into primary neural progenitors from the embryonic mouse dorsal telencephalon (prospective cerebral cortex).

Akt2 negatively regulates assembly of the POSH-MLK-JNK signaling complex.

We demonstrate that POSH, a scaffold for the JNK signaling pathway, binds to Akt2. A POSH mutant that is unable to bind Akt2 (POSH W489A) exhibits enhanced-binding to MLK3, and this increase in binding is accompanied by increased activation of the JNK signaling pathway. In addition, we show that the association of MLK3 with POSH is increased upon inhibition of the endogenous phosphatidylinositol 3-kinase/Akt signaling pathway.

Akt negatively regulates translation of the ternary complex factor Elk-1.

Cross-talk between signaling pathways plays an important role in regulation of cell growth, differentiation, survival, and death. Here, we show that Akt regulates the Elk-1 transcription factor, independent of its negative regulation of Raf kinases. Using a constitutively active Mek1 to bypass the regulation of Raf by Akt, we find that the Elk-1 and Sap1a proteins are dramatically decreased in the presence of activated Akt.

Fgd1, the Cdc42 GEF responsible for Faciogenital Dysplasia, directly interacts with cortactin and mAbp1 to modulate cell shape.

FGD1 mutations result in Faciogenital Dysplasia (FGDY), an X-linked human disease that affects skeletal formation and embryonic morphogenesis. FGD1 and Fgd1, the mouse FGD1 ortholog, encode guanine nucleotide exchange factors (GEF) that specifically activate Cdc42, a Rho GTPase that controls the organization of the actin cytoskeleton. To further understand FGD1/Fgd1 signaling and begin to elucidate the molecular pathophysiology of FGDY, we demonstrate that Fgd1 directly interacts with cortactin and mouse actin-binding protein 1 (mAbp1), actin-binding proteins that regulate actin polymerization through the Arp2/3 complex.

Akt regulates basic helix-loop-helix transcription factor-coactivator complex formation and activity during neuronal differentiation.

Neural basic helix-loop-helix (bHLH) transcription factors regulate neurogenesis in vertebrates. Signaling by peptide growth factors also plays critical roles in regulating neuronal differentiation and survival. Many peptide growth factors activate phosphatidylinositol 3-kinase (PI3K) and subsequently the Akt kinases, raising the possibility that Akt may impact bHLH protein function during neurogenesis.

Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors.

Short interfering RNAs (siRNAs) can mediate sequence-specific inhibition of gene expression in mammalian cells. We and others have recently developed expression vector-based systems for synthesizing siRNAs or hairpin siRNAs in mammalian cells. Expression vector-based RNA interference (RNAi) effectively suppresses expression of target genes and is likely to be a powerful tool for analysis of gene function.