Prenatal ß-catenin/Brn2/Tbr2 transcriptional cascade regulates adult social and stereotypic behaviors.

Social interaction is a fundamental behavior in all animal species, but the developmental timing of the social neural circuit formation and the cellular and molecular mechanisms governing its formation are poorly understood. We generated a mouse model with mutations in two Disheveled genes, Dvl1 and Dvl3, that displays adult social and repetitive behavioral abnormalities associated with transient embryonic brain enlargement during deep layer cortical neuron formation. These phenotypes were mediated by the embryonic expansion of basal neural progenitor cells (NPCs) via deregulation of a ß-catenin/Brn2/Tbr2 transcriptional cascade. Transient pharmacological activation of the canonical Wnt pathway during this period of early corticogenesis rescued the ß-catenin/Brn2/Tbr2 transcriptional cascade and the embryonic brain phenotypes. Remarkably, this embryonic treatment prevented adult behavioral deficits and partially rescued abnormal brain structure in Dvl mutant mice. Our findings define a mechanism that links fetal brain development and adult behavior, demonstrating a fetal origin for social and repetitive behavior deficits seen in disorders such as autism.

PI3K/AKT signaling determines a dynamic switch between distinct KSRP functions favoring skeletal myogenesis.

Skeletal myogenesis is orchestrated by distinct regulatory signaling pathways, including PI3K/AKT, that ultimately control muscle gene expression. Recently discovered myogenic micro-RNAs (miRNAs) are deeply implicated in muscle biology. Processing of miRNAs from their primary transcripts is emerging as a major step in the control of miRNA levels and might be well suited to be regulated by extracellular signals. Here we report that the RNA binding protein KSRP is required for the correct processing of primary myogenic miRNAs upon PI3K/AKT activation in myoblasts C2C12 and in the course of injury-induced muscle regeneration, as revealed by Ksrp knock-out mice analysis. PI3K/AKT activation regulates in opposite ways two distinct KSRP functions inhibiting its ability to promote decay of myogenin mRNA and activating its ability to favor maturation of myogenic miRNAs. This dynamic regulatory switch eventually contributes to the activation of the myogenic program.

Akt2-mediated phosphorylation of Pitx2 controls Ccnd1 mRNA decay during muscle cell differentiation.

Paired-like homeodomain 2 (Pitx2), first identified as the gene responsible for the Axenfeld-Rieger syndrome, encodes a protein factor that, controlling cell proliferation in a tissue-specific manner, has a crucial role in morphogenesis. During embryonic development, Pitx2 exerts a role in the expansion of muscle progenitors and is expressed at all stages of myogenic progression. In this study, we show that Pitx2 is phosphorylated by the protein kinase Akt2 and is necessary to ensure proper C2C12 myoblast proliferation and differentiation. Pitx2 associates with a ribonucleoprotein complex that includes the mRNA stabilizing factor HuR and sustains Ccnd1 (also known as Cyclin D1) expression, thereby prolonging its mRNA half-life. When the differentiation program is initiated, phosphorylation by Akt2 impairs the ability of Pitx2 to associate with the Ccnd1 mRNA-stabilizing complex that includes HuR and, as a consequence, Ccnd1 mRNA half-life is shortened. We propose that unphosphorylated Pitx2 is required to favor HuR-mediated Ccnd1 mRNA stabilization, thus sustaining myoblast proliferation. Upon Akt2-phosphorylation, the complex Pitx2/HuR/Ccnd1 mRNA dissociates and Ccnd1 mRNA is destabilized. These events contribute to the switch of C2C12 cells from a proliferating to a differentiating phenotype.

Temporal regulation of a paired-like homeodomain repressor/TLE corepressor complex and a related activator is required for pituitary organogenesis.

Understanding the functional significance of the coordinate expression of specific corepressors and DNA-binding transcription factors remains a critical question in mammalian development. During the development of the pituitary gland, two highly related paired-like homeodomain factors, a repressor, Hesx1/Rpx and an activator, Prop-1, are expressed in sequential, overlapping temporal patterns. Here we show that while the repressive actions of Hesx1/Rpx may be required for initial pituitary organ commitment, progression beyond the appearance of the first pituitary (POMC) lineage requires both loss of Hesx1 expression and the actions of Prop-1. Although Hesx1 recruits both the Groucho-related corepressor TLE1 and the N-CoR/Sin3/HDAC complex on distinct domains, the repressor functions of Hesx1 in vivo prove to require the specific recruitment of TLE1, which exhibits a spatial and temporal pattern of coexpression during pituitary organogenesis. Furthermore, Hesx1-mediated repression coordinates a negative feedback loop with FGF8/FGF10 signaling in the ventral diencephalon, required to prevent induction of multiple pituitary glands from oral ectoderm. Our data suggest that the opposing actions of two structurally-related DNA-binding paired-like homeodomain transcription factors, binding to similar cognate elements, coordinate pituitary organogenesis by reciprocally repressing and activating target genes in a temporally specific fashion, on the basis of the actions of a critical, coexpressed TLE corepressor.

Regulation of neuronal traits by a novel transcriptional complex.

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Tbx19, a tissue-selective regulator of POMC gene expression.

Pituitary cell types arise in a temporally and spatially specific fashion, in response to combinatorial actions of transcription factors induced by transient signaling gradients. The critical transcriptional determinants of the two pituitary cell types that express the pro-opiomelanocortin (POMC) gene, the anterior lobe corticotropes, producing adrenocorticotropin, and the intermediate lobe melanotropes, producing melanocyte-stimulating hormone (MSH alpha), have remained unknown. Here, we report that a member of the T-box gene family, Tbx19, which is expressed only in the rostral ventral diencephalon and pituitary gland, commencing on e11.5, marks pituitary cells that will subsequently express the POMC gene and is capable of altering progression of ventral cell types and inducing adrenocorticotropin in rostral tip cells. It is suggested that Tbx19, depending on the presence of synergizing transcription factors, can activate POMC gene expression and repress the alpha glycoprotein subunit and thyroid-stimulating hormone beta promoters.

Defect of histone acetyltransferase activity of the nuclear transcriptional coactivator CBP in Rubinstein-Taybi syndrome.

CREB-binding protein (CBP) is a transcriptional coactivator that has intrinsic histone acetyltransferase (HAT) activity. CBP is the causative gene of Rubinstein-Taybi syndrome (RTS). To investigate the relationships between CBP HAT activity and RTS, we analyzed 16 RTS patients. A microdeletion was identified in one patient by fluorescent in situ hybridization analysis. Heteroallelic mutations were identified in five patients by reverse transcriptase-polymerase chain reaction-single-strand conformation polymorphism analysis and sequencing. These included a 2 bp deletion between nucleotides 4319 and 4320, an 11 bp deletion between nucleotides 4898 and 4908, a 14 bp insertion (CCTCGGTCCTGCAC) between nucleotides 5212 and 5213, a 2 bp deletion between nucleotides 5222 and 5223, and a missense mutation from guanine (G) to cytosine (C) at nucleotide 4951 that changed codon 1378 from CGG (arginine) to CCG (proline). The identical missense mutation was introduced into the recombinant mouse CBP. It abolished the HAT activity of CBP and the ability of CBP to transactivate cyclic AMP-response element binding protein (CREB), in HAT assays and in microinjection experiments, respectively. These results suggest that the loss of the HAT activity of CBP may cause RTS, as the first example of a defect of HAT activity in a human disease. Our findings raise the possibility that treatment of RTS patients with histone deacetylase inhibitors might have beneficial effects.

Signaling and transcriptional mechanisms in pituitary development.

During the development of the pituitary gland, distinct hormone-producing cell types arise from a common population of ectodermal progenitors, providing an instructive model system for elucidating the molecular mechanisms of patterning and cell type specification in mammalian organogenesis. Recent studies have established that the development of the pituitary occurs through multiple sequential steps, allowing the coordinate control of the commitment, early patterning, proliferation, and positional determination of pituitary cell lineages in response to extrinsic and intrinsic signals. The early phases of pituitary development appear to be mediated through the activities of multiple signaling gradients emanating from key organizing centers that give rise to temporally and spatially distinct patterns of transcription factor expression. The induction of these transcriptional mediators in turn acts to positionally organize specific pituitary cell lineages within an apparently uniform field of ectodermal progenitors. Ultimately, pituitary cell types have proven to be both specified and maintained through the combinatorial interactions of a series of cell-type-restricted transcription factors that dictate the cell autonomous programs of differentiation in response to the transient signaling events.

Modification of representational difference analysis applied to the isolation of forskolin-regulated genes from Schwann cells.

Many aspects of the response of Schwann cells to axonal cues can be induced in vitro by the adenylyl cyclase activator forskolin, yet the role of cAMP signaling in regulating Schwann cell differentiation remains unclear. To define better the relationship between cAMP signaling and Schwann cell differentiation, we used a modification of cDNA representational difference analysis (RDA) that permits the analysis of small amounts of mRNA and identified additional genes that are differentially expressed by forskolin-treated and untreated Schwann cells. The genes that we have identified, including MKP3, a regulator of ERK signaling, and the sphingosine-1-phosphate receptor edg3/lp(B3), may play important roles in mediating Schwann cell differentiation.

POU domain factors in the neuroendocrine system: lessons from developmental biology provide insights into human disease.

POU domain factors are transcriptional regulators characterized by a highly conserved DNA-binding domain referred to as the POU domain. The structure of the POU domain has been solved, facilitating the understanding of how these proteins bind to DNA and regulate transcription via complex protein-protein interactions. Several members of the POU domain family have been implicated in the control of development and function of the neuroendocrine system. Such roles have been most clearly established for Pit-1, which is required for formation of somatotropes, lactotropes, and thyrotropes in the anterior pituitary gland, and for Brn-2, which is critical for formation of magnocellular and parvocellular neurons in the paraventricular and supraoptic nuclei of the hypothalamus. While genetic evidence is lacking, molecular biology experiments have implicated several other POU factors in the regulation of gene expression in the hypothalamus and pituitary gland. Pit-1 mutations in humans cause combined pituitary hormone deficiency similar to that found in mice deleted for the Pit-1 gene, providing a striking example of how basic developmental biology studies have provided important insights into human disease.

Hedgehog signaling is required for pituitary gland development.

Pituitary gland development serves as an excellent model system in which to study the emergence of distinct cell types from a common primordium in mammalian organogenesis. We have investigated the role of the morphogen Sonic hedgehog (SHH) in outgrowth and differentiation of the pituitary gland using loss- and gain-of-function studies in transgenic mice. Shh is expressed throughout the ventral diencephalon and the oral ectoderm, but its expression is subsequently absent from the nascent Rathke's pouch as soon as it becomes morphologically visible, creating a Shh boundary within the oral epithelium. We used oral ectoderm/Rathke's pouch-specific 5' regulatory sequences (Pitx1(HS)) from the bicoid related pituitary homeobox gene (Pitx1) to target overexpression of the Hedgehog inhibitor Hip (Huntingtin interacting protein) to block Hedgehog signaling, finding that SHH is required for proliferation of the pituitary gland. In addition, we provide evidence that Hedgehog signaling, acting at the Shh boundary within the oral ectoderm, may exert a role in differentiation of ventral cell types (gonadotropes and thyrotropes) by inducing Bmp2 expression in Rathke's pouch, which subsequently regulates expression of ventral transcription factors, particularly Gata2. Furthermore, our data suggest that Hedgehog signaling, together with FGF8/10 signaling, synergizes to regulate expression of the LIM homeobox gene Lhx3, which has been proved to be essential for initial pituitary gland formation. Thus, SHH appears to exert effects on both proliferation and cell-type determination in pituitary gland development.

Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification.

Reciprocal gene activation and restriction during cell type differentiation from a common lineage is a hallmark of mammalian organogenesis. A key question, then, is whether a critical transcriptional activator of cell type-specific gene targets can also restrict expression of the same genes in other cell types. Here, we show that whereas the pituitary-specific POU domain factor Pit-1 activates growth hormone gene expression in one cell type, the somatotrope, it restricts its expression from a second cell type, the lactotrope. This distinction depends on a two-base pair spacing in accommodation of the bipartite POU domains on a conserved growth hormone promoter site. The allosteric effect on Pit-1, in combination with other DNA binding factors, results in the recruitment of a corepressor complex, including nuclear receptor corepressor N-CoR, which, unexpectedly, is required for active long-term repression of the growth hormone gene in lactotropes.

Regulation of somatic growth by the p160 coactivator p/CIP.

A family of p160 coactivators was initially identified based on ligand-dependent interactions with nuclear receptors and thought to function, in part, by recruiting CREB-binding protein/p300 to several classes of transcription factors. One of the p160 factors, p/CIP/AIB1, often amplified and overexpressed in breast cancer, also exhibits particularly strong interaction with CREB-binding protein/p300. In this manuscript, we report that p/CIP, which exhibits regulated transfer from cytoplasm to nucleus, is required for normal somatic growth from embryonic day 13.5 through maturity. Our data suggest that a short stature phenotype of p/CIP gene-deleted mice reflect both altered regulation of insulin-like growth factor-1 (IGF-1) gene expression in specific tissues and a cell-autonomous defect of response to IGF-1, including ineffective transcriptional activities by several classes of regulated transcription factors under specific conditions. The actions of p/CIP are therefore required for full expression of a subset of genes critical for regulating physiological patterns of somatic growth in mammals.

Multistep signaling and transcriptional requirements for pituitary organogenesis in vivo.

During development of the mammalian pituitary gland, specific hormone-producing cell types, critical in maintaining homeostasis, emerge in a spatially and temporally specific fashion from an ectodermal primordium. We have investigated the molecular basis of generating diverse cell phenotypes from a common precursor, providing in vivo and in vitro evidence that development of these cell types involves at least four sequential phases of signaling events and the action of a gradient at an ectodermal boundary. In the first phase, we hypothesize that this notochord induces invagination of Rathke's pouch from the oral ectoderm. This is followed by appearance of an ectodermal boundary, formed with exclusion of Shh from the nascent pouch. Next, signals from the ventral diencephalon--expressing BMP4, Wnt5a, FGF10, and FGF8--in concert with Shh represent critical in vivo signals for pituitary determination. Subsequently, a dorsal-ventral BMP2 signal gradient emanates from a ventral pituitary organizing center, forming at the boundary to oral ectoderm region from which Shh expression is selectively excluded. In concert with a dorsal FGF8 signal, this creates opposing gradients that generate overlapping patterns of specific transcription factors that underlie cell lineage specification events. The mechanisms by which these transient gradients of signaling molecules lead to the appearance of four ventral pituitary cell types appear to involve the reciprocal interactions of two transcription factors, Pit-1 and GATA-2, which are epistatic to the remainder of the cell type-specific transcription programs and serve as a molecular memory of the transient signaling events. Unexpectedly, this program includes a DNA-binding-independent function of Pit-1, suppressing the ventral GATA-2-dependent gonadotrope program by inhibiting GATA-2 binding to gonadotrope- but not thyrotrope-specific genes. This indicates that both DNA-binding-dependent and-independent actions of abundant determining factors contribute to generate distinct cell phenotypes. In the fourth phase, temporally specific loss of the BMP2 signal is required to allow terminal differentiation. The consequence of these sequential organ and cellular determination events is that each of the pituitary cell types--gonadotropes, thyrotropes, somatotropes, lactotropes, corticotropes, and melanotropes appears to be determined, in a ventral to dorsal gradient, respectively, apparently based on a combinatorial code of transcription factors induced by the gradient of specific signaling molecules.

Combinatorial roles of the nuclear receptor corepressor in transcription and development.

Transcriptional repression plays crucial roles in diverse aspects of metazoan development, implying critical regulatory roles for corepressors such as N-CoR and SMRT. Altered patterns of transcription in tissues and cells derived from N-CoR gene-deleted mice and the resulting block at specific points in CNS, erythrocyte, and thymocyte development indicated that N-CoR was a required component of short-term active repression by nuclear receptors and MAD and of a subset of long-term repression events mediated by REST/NRSF. Unexpectedly, N-CoR and a specific deacetylase were also required for transcriptional activation of one class of retinoic acid response element. Together, these findings suggest that specific combinations of corepressors and histone deacetylases mediate the gene-specific actions of DNA-bound repressors in development of multiple organ systems.

The histone deacetylase-3 complex contains nuclear receptor corepressors.

Acetylation and deacetylation of nucleosomal histones have profound effects on gene transcription in all eukaryotes. In humans, three highly homologous class I and four class II histone deacetylase (HDAC) enzymes have been identified to date. The class I deacetylases HDAC1 and HDAC2 are components of multisubunit complexes, one of which could associate with the nuclear hormone receptor corepressor, N-CoR. N-CoR also interacts with class II deacetylases HDAC4, HDAC5, and HDAC7. In comparison with HDAC1 and HDAC2, HDAC3 remains relatively uncharacterized, and very few proteins have been shown to interact with HDAC3. Using an affinity purification approach, we isolated an enzymatically active HDAC3 complex that contained members of the nuclear receptor corepressor family. Deletion analysis of N-CoR revealed that HDAC3 binds multiple N-CoR regions in vitro and that all of these regions are required for maximal binding in vivo. The N-CoR domains that interact with HDAC3 are distinct from those that bind other HDACs. Transient overexpression of HDAC3 and microinjection of Abs against HDAC3 showed that a component of transcriptional repression mediated by N-CoR depends on HDAC3. Interestingly, data suggest that interaction with a region of N-CoR augments the deacetylase activity of HDAC3. These results provide a possible molecular mechanism for HDAC3 regulation and argue that N-CoR is a platform in which distinct domains can interact with most of the known HDACs.

A 12(S)-hydroxyeicosatetraenoic acid receptor interacts with steroid receptor coactivator-1.

Lewis lung carcinoma cells contain specific high-affinity binding sites for the eicosanoid 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE]. These binding sites have a cytosolic/nuclear localization and contain the heat shock proteins hsp70 and hsp90 as components of a high molecular weight cytosolic binding complex. The ligand binding subunit of this complex is a protein with an apparent molecular mass of approximately 50 kDa as judged by gel permeation chromatography. In this report, we present data showing that the 50-kDa 12(S)-HETE binding protein interacts as a homodimer with steroid receptor coactivator-1 (SRC-1) in the presence of 12(S)-HETE. Two putative interaction domains were mapped. One of these (amino acids 701-781) was within the nuclear receptor interaction domain in SRC-1 required for binding of various steroid and thyroid hormone receptors. It contains the most C-terminal of the three copies of LXXLL motif present in the nuclear receptor interaction domain. The second interaction domain was present in the N-terminal part of SRC-1 (amino acids 1-221). This region has two LXXLL motifs, one does not bind and the other binds only weakly to steroid and thyroid hormone receptors. Glutathione S-transferase (GST) pulldown experiments and far Western analyses demonstrated that the N-terminal region of SRC-1 (amino acids 1-212) alone does not bind the 50-kDa 12(S)-HETE binding protein, whereas GST/DeltaSRC-1(1-1138) ligand-dependently pulled down a protein of approximately 50 kDa in size. Our results suggest that the 50-kDa 12(S)-HETE binding protein is a receptor that may signal through interaction with a nuclear receptor coactivator protein.