The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs) with latrophilins (LPHNs/ADGRLs) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. Here, we report the 2.9 Å resolution cryo-EM structure of the TEN2-LPHN3 complex, and describe the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity.

SummaryIt is clear that neural differentiation from human pluripotent stem cells generates cells that are developmentally immature. Here, we show that the let-7 plays a functional role in the developmental decision making of human neural progenitors, controlling whether these cells make neurons or glia. Through gain- and loss-of-function studies on both tissue and pluripotent derived cells, our data show that let-7 specifically regulates decision making in this context by regulation of a key chromatin-associated protein, HMGA2. Furthermore, we provide evidence that the let-7/HMGA2 circuit acts on HES5, a NOTCH effector and well-established node that regulates fate decisions in the nervous system. These data link the let-7 circuit to NOTCH signaling and suggest that this interaction serves to regulate human developmental progression.

Abstractp97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.

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SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a corepressor of the glucocorticoid receptor, constitutive androstane receptor, and hepatocyte nuclear factor 4␣. Here we show that SMILE also represses estrogen receptor-related receptor ␥ (ERR␥) transactivation. Knockdown of SMILE gene expression increases ERR␥ activity. SMILE directly interacts with ERR␥ in vitro and in vivo. Domain mapping analysis showed that SMILE binds to the AF2 domain of ERR␥. SMILE represses ERR␥ transactivation partially through competition with coactivators PGC-1␣, PGC-1␤, and GRIP1. Interestingly, the repression of SMILE on ERR␥ is released by SIRT1 inhibitors, a catalytically inactive SIRT1 mutant, and SIRT1 small interfering RNA but not by histone protein deacetylase inhibitor. In vivo glutathione S-transferase pulldown and coimmunoprecipitation assays validated that SMILE physically interacts with SIRT1. Furthermore, the ERR␥ inverse agonist GSK5182 enhances the interaction of SMILE with ERR␥ and SMILE-mediated repression. Knockdown of SMILE or SIRT1 blocks the repressive effect of GSK5182. Moreover, chromatin immunoprecipitation assays revealed that GSK5182 augments the association of SMILE and SIRT1 on the promoter of the ERR␥ target PDK4. GSK5182 and adenoviral overexpression of SMILE cooperate to repress ERR␥-induced PDK4 gene expression, and this repression is released by overexpression of a catalytically defective SIRT1 mutant. Finally, we demonstrated that ERR␥ regulates SMILE gene expression, which in turn inhibits ERR␥. Overall, these findings implicate SMILE as a novel corepressor of ERR␥ and recruitment of SIRT1 as a novel repressive mechanism for SMILE and ERR␥ inverse agonist.

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is an atypical member of the nuclear receptor family and acts as a corepressor of a number of nuclear receptors. HNF4␣ (hepatocyte nuclear factor 4␣) is a liver-enriched transcription factor that controls the expression of a variety of genes involved in cholesterol, fatty acid, and glucose metabolism. Here we show that DAX-1 inhibits transcriptional activity of HNF4␣ and modulates hepatic gluconeogenic gene expression. Hepatic DAX-1 expression is increased by insulin and SIK1 (salt-inducible kinase 1), whereas it is decreased in high fat diet-fed and diabetic mice. Coimmunoprecipitation assay from mouse liver samples depicts that endogenous DAX-1 interacts with HNF4␣ in vivo. In vivo chromatin immunoprecipitation assay affirms that the recruitment of DAX-1 on the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is inversely correlated with the recruitment of PGC-1␣ and HNF4␣ under fasting and refeeding, showing that DAX-1 could compete with the coactivator PGC-1␣ for binding to HNF4␣. Adenovirus-mediated expression of DAX-1 decreased both HNF4␣-and forskolin-mediated gluconeogenic gene expressions. In addition, knockdown of DAX-1 partially reverses the insulin-mediated inhibition of gluconeogenic gene expression in primary hepatocytes. Finally, DAX-1 inhibits PEPCK and glucose-6-phosphatase gene expression and significantly lowers fasting blood glucose level in high fat diet-fed mice, suggesting that DAX-1 can modulate hepatic gluconeogenesis in vivo. Overall, this study demonstrates that DAX-1 acts as a corepressor of HNF4␣ to negatively regulate hepatic gluconeogenic gene expression in liver.The nuclear receptor superfamily comprises a diverse group of transcription factors including conventional receptors with known ligands and orphan nuclear receptors without ligands (1, 2). Among nuclear receptors, DAX-1 belongs to a group of atypical nuclear receptors that do not possess classical DNA binding domains and is closely related to the same family member SHP (small heterodimer partner) (3-5). DAX-1 was identified through a screening for genes linked to adrenal hypoplasia critical, a disease that affects the normal development of the adrenal cortex and is often associated with hypogonadism (6). DAX-1 has a putative ligand binding domain (LBD) 4 in the C terminus, although no ligand has been identified yet (3), whereas N-terminal region contains a unique repeat domain that is involved in single-stranded DNA and RNA binding and protein-protein interactions (7,8). This N-terminal region comprises three LXXLL motif-like sequences that are necessary for the interaction with estrogen receptors (ERs) (9). It is well established that DAX-1 functions as a coregulatory protein rather than a transcriptional factor, since it inhibits the transcriptional activity of other nuclear receptors, such as steroidogenic factor 1 (10), ER (9), androgen receptor (11), progesterone receptor (12), liver receptor homolog-1 ...

SummaryHypoxia augments human embryonic stem cell (hESC) self-renewal via hypoxia-inducible factor 2α-activated OCT4 transcription. Hypoxia also increases the efficiency of reprogramming differentiated cells to a pluripotent-like state. Combined, these findings suggest that low O2 tension would impair the purposeful differentiation of pluripotent stem cells. Here, we show that low O2 tension and hypoxia-inducible factor (HIF) activity instead promote appropriate hESC differentiation. Through gain- and loss-of-function studies, we implicate O2 tension as a modifier of a key cell fate decision, namely whether neural progenitors differentiate toward neurons or glia. Furthermore, our data show that even transient changes in O2 concentration can affect cell fate through HIF by regulating the activity of MYC, a regulator of LIN28/let-7 that is critical for fate decisions in the neural lineage. We also identify key small molecules that can take advantage of this pathway to quickly and efficiently promote the development of mature cell types.

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