Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca2+-release channels with each subunit containing a binding site for IP3 in the N-terminus. We provide evidence that four IP3 molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca2+ release suggested that IP3Rs are maximally occupied by IP3 before substantial Ca2+ release occurs. We showed that ligand binding–deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP3R subunits, and confirmed the same effect in an IP3R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP3Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP3Rs with four ligand-binding sites exhibited Ca2+ release and electrophysiological properties of native IP3Rs. However, IP3 failed to activate IP3Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP3Rs containing two monomers of IP3R2short, an IP3 binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca2+ release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca2+ release.

Lysine-specific demethylase 1 (LSD1) functions as a transcriptional coregulator by modulating histone methylation. Its role in neural stem cells has not been studied. We show here for the first time that LSD1 serves as a key regulator of neural stem cell proliferation. Inhibition of LSD1 activity or knockdown of LSD1 expression led to dramatically reduced neural stem cell proliferation. LSD1 is recruited by nuclear receptor TLX, an essential neural stem cell regulator, to the promoters of TLX target genes to repress the expression of these genes, which are known regulators of cell proliferation. The importance of LSD1 function in neural stem cells was further supported by the observation that intracranial viral transduction of the LSD1 small interfering RNA (siRNA) or intraperitoneal injection of the LSD1 inhibitors pargyline and tranylcypromine led to dramatically reduced neural progenitor proliferation in the hippocampal dentate gyri of wild-type adult mouse brains. However, knockout of TLX expression abolished the inhibitory effect of pargyline and tranylcypromine on neural progenitor proliferation, suggesting that TLX is critical for the LSD1 inhibitor effect. These findings revealed a novel role for LSD1 in neural stem cell proliferation and uncovered a mechanism for neural stem cell proliferation through recruitment of LSD1 to modulate TLX activity.TLX is an orphan nuclear receptor that plays an important role in vertebrate brain functions (12,14,27,28). We have shown that TLX is an essential regulator of neural stem cell maintenance and self-renewal in both embryonic and adult brains (8,14,18,30). TLX acts by controlling the expression of a network of target genes to establish the undifferentiated and self-renewable state of neural stem cells. Elucidating molecular mechanisms underlying TLX regulation would be a significant advance in understanding neural stem cell self-renewal and neurogenesis. The transcription action of nuclear receptors is modulated by an extensive set of nuclear receptor cofactors (4, 10, 13). The identification and characterization of the coregulator complexes are essential for understanding the mechanistic basis of nuclear receptor-regulated events. Identifying TLX transcriptional coregulators in neural stem cells would represent a major step in uncovering TLX-mediated transcriptional regulation.Histone modifications, such as acetylation, phosphorylation, and methylation, are switches that alter chromatin structure to form a binding platform for downstream "effector" proteins to allow transcriptional activation or repression (24). Each modification can affect chromatin architecture, yet the sum of these modifications may be the ultimate determinant of the chromatin state that regulates gene transcription (5, 17). Histone methylation has been linked to transcriptional activation and repression (29). Whether methylation leads to transcriptional activation or repression is influenced by a variety of factors, including the types of histone, the lysine acceptor, the histone location, an...

Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP 3 receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts. This role is independent of calcium fluxes. We also show that, while each isoform can support contacts, type 2 IP3R is the most effective in delivering calcium to the mitochondria. Thus, these studies reveal a non-canonical, structural role for the IP3Rs and direct attention towards the type 2 IP3R that was previously neglected in the context of ER-mitochondrial calcium signaling.

Gillespie syndrome (GS) is a rare variant form of aniridia characterized by non-progressive cerebellar ataxia, intellectual disability, and iris hypoplasia. Unlike the more common dominant and sporadic forms of aniridia, there has been no significant association with PAX6 mutations in individuals with GS and the mode of inheritance of the disease had long been regarded as uncertain. Using a combination of trio-based whole-exome sequencing and Sanger sequencing in five simplex GS-affected families, we found homozygous or compound heterozygous truncating mutations (c.4672C>T [p.Gln1558(∗)], c.2182C>T [p.Arg728(∗)], c.6366+3A>T [p.Gly2102Valfs5(∗)], and c.6664+5G>T [p.Ala2221Valfs23(∗)]) and de novo heterozygous mutations (c.7687_7689del [p.Lys2563del] and c.7659T>G [p.Phe2553Leu]) in the inositol 1,4,5-trisphosphate receptor type 1 gene (ITPR1). ITPR1 encodes one of the three members of the IP3-receptors family that form Ca(2+) release channels localized predominantly in membranes of endoplasmic reticulum Ca(2+) stores. The truncation mutants, which encompass the IP3-binding domain and varying lengths of the modulatory domain, did not form functional channels when produced in a heterologous cell system. Furthermore, ITPR1 p.Lys2563del mutant did not form IP3-induced Ca(2+) channels but exerted a negative effect when co-produced with wild-type ITPR1 channel activity. In total, these results demonstrate biallelic and monoallelic ITPR1 mutations as the underlying genetic defects for Gillespie syndrome, further extending the spectrum of ITPR1-related diseases.

276, 3123-3129). In the current study, we sought to define the mechanism behind this adaptive response. We show that GnRH induces a rapid and dramatic increase in InsP 3 receptor polyubiquitination and that proteasome inhibitors block InsP 3 receptor down-regulation and cause the accumulation of polyubiquitinated receptors. Thus, the ubiquitin/proteasome pathway is active in ␣T3-1 cells, and GnRH regulates the levels of InsP 3 receptors via this mechanism. Given these findings and further characterization of this system, we also examined the possibility that ␣T3-1 cells could be used to examine the ubiquitination of exogenous InsP 3 receptors introduced by cDNA transfection. This was found to be the case, since exogenous wild-type InsP 3 receptors, but not bindingdefective mutant receptors, were polyubiquitinated in a GnRH-dependent manner, and agents that inhibited the polyubiquitination of endogenous receptors also inhibited the polyubiquitination of exogenous receptors. Further, we used this system to determine whether phosphorylation was involved in triggering InsP 3 receptor polyubiquitination. This was not the case, since mutation of serine residues 1588 and 1755 (the predominant phosphorylation sites in the type I receptor) did not inhibit polyubiquitination. In total, these data show that the ubiquitin/proteasome pathway is active in anterior pituitary cells, that this pathway targets both endogenous and exogenous InsP 3 receptors in GnRH-stimulated ␣T3-1 cells, and that, in contrast to the situation for many other substrates, phosphorylation does not trigger InsP 3 receptor polyubiquitination.