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During embryogenesis the sea urchin early pluteus larva differentiates 40-50 neurons marked by expression of the pan-neural marker synaptotagmin B (SynB) that are distributed along the ciliary band, in the apical plate and pharyngeal endoderm, and 4-6 serotonergic neurons that are confined to the apical plate. Development of all neurons has been shown to depend on the function of Six3. Using a combination of molecular screens and tests of gene function by morpholino-mediated knockdown, we identified SoxC and Brn1/2/4, which function sequentially in the neurogenic regulatory pathway and are also required for the differentiation of all neurons. Misexpression of Brn1/2/4 at low dose caused an increase in the number of serotonin-expressing cells and at higher dose converted most of the embryo to a neurogenic epithelial sphere expressing the Hnf6 ciliary band marker. A third factor, Z167, was shown to work downstream of the Six3 and SoxC core factors and to define a branch specific for the differentiation of serotonergic neurons. These results provide a framework for building a gene regulatory network for neurogenesis in the sea urchin embryo., (© 2016. Published by The Company of Biologists Ltd.)

Long intergenic noncoding RNAs (lincRNAs) play important roles in regulating the biological functions and underlying molecular mechanisms of colorectal cancer (CRC). Here, we investigated the association of linc-POU3F3 and prognosis in CRC. We demonstrated that linc-POU3F3 was overexpressed in CRC tissues and positively correlated with tumor grade and N stage. Inhibition of linc-POU3F3 resulted in inhibition of cell proliferation and G1 cell cycle arrest, which was mediated by cyclin D1, CDK4, p18, Rb, and phosphorylated Rb. Inhibition of linc-POU3F3 induced apoptosis, and suppressed migration and invasion in LOVO and SW480 cell lines. This inhibition also increased the expressions of epithelial markers and decreased the expressions of mesenchymal markers, thus inhibiting the cancer epithelial-mesenchymal transition. The decreased migration and invasion following linc-POU3F3 knockdown were mediated by an increased BMP signal. Furthermore, autophagy was enhanced by linc-POU3F3 knockdown, suggesting the involvement of autophagy in the induced apoptosis. Collectively, linc-POU3F3 might be crucial in pro-proliferation, anti-apoptosis, and metastasis in LOVO and SW480 cells by regulating the cell cycle, intrinsic apoptosis, BMP signaling and autophagy. Thus, linc-POU3F3 is a potential therapeutic target and novel molecular biomarker for CRC.

Maintenance of a stable gut microbial community relies on a delicate balance between immune defense and immune tolerance. We have used Drosophila to study how the microbial gut flora is affected by changes in host genetic factors and immunity. Flies with a constitutively active gut immune system, due to a mutation in the POU transcriptional regulator Pdm1/nubbin (nub) gene, had higher loads of bacteria and a more diverse taxonomic composition than controls. In addition, the microbial composition shifted considerably during the short lifespan of the nub1 mutants. This shift was characterized by a loss of relatively few OTUs (operational taxonomic units) and a remarkable increase in a large number of Acetobacter spp. and Leuconostoc spp. Treating nub1 mutant flies with antibiotics prolonged their lifetime survival by more than 100%. Immune gene expression was also persistently high in the presence of antibiotics, indicating that the early death was not a direct consequence of an overactive immune defense but rather an indirect consequence of the microbial load and composition. Thus, changes in host genotype and an inability to regulate the normal growth and composition of the gut microbiota leads to a shift in the microbial community, dysbiosis and early death., (© 2016 S. Karger AG, Basel.)

Developmental potentials of cells are tightly controlled at multiple levels. The embryonic Drosophila airway tree is roughly subdivided into two types of cells with distinct developmental potentials: a proximally located group of multipotent adult precursor cells (P-fate) and a distally located population of more differentiated cells (D-fate). We show that the GATA-family transcription factor (TF) Grain promotes the P-fate and the POU-homeobox TF Ventral veinless (Vvl/Drifter/U-turned) stimulates the D-fate. Hedgehog and receptor tyrosine kinase (RTK) signaling cooperate with Vvl to drive the D-fate at the expense of the P-fate while negative regulators of either of these signaling pathways ensure P-fate specification. Local concentrations of Decapentaplegic/BMP, Wingless/Wnt, and Hedgehog signals differentially regulate the expression of D-factors and P-factors to transform an equipotent primordial field into a concentric pattern of radially different morphogenetic potentials, which gradually gives rise to the distal-proximal organization of distinct cell types in the mature airway.

Insulinoma-associated protein 1 (INSM1) is expressed exclusively in embryonic developing neuroendocrine (NE) tissues. INSM1 gene expression is specific for small-cell lung cancer (SCLC), along with achaete-scute homolog-like 1 (ASCL1) and several NE molecules, such as chromogranin A, synaptophysin, and neural cell adhesion molecule 1. However, the underlying biological role of INSM1 in lung cancer remains largely unknown. We first showed that surgically resected SCLC samples specifically expressed INSM1. Forced expression of the INSM1 gene in adenocarcinoma cell lines (H358 and H1975) induced the expression of ASCL1, brain-2 (BRN2), chromogranin A, synaptophysin, and neural cell adhesion molecule 1; in contrast, knockdown of the INSM1 gene by siRNA in SCLC (H69 and H889) decreased their expression. However, forced/knockdown expression of ASCL1 and BRN2 did not affect INSM1 expression. A chromatin immunoprecipitation study revealed that INSM1 bound to the promoter region of the ASCL1 gene. A xenotransplantation assay using tet-on INSM1 gene-transfected adenocarcinoma cell lines demonstrated that INSM1 induced NE differentiation and growth inhibition. Furthermore, we found that INSM1 was not expressed in non-small-cell lung cancer and some SCLC cell lines expressing Notch1-Hes1. By forced/knockdown expression of Notch1 or Hes1 genes, we revealed that Notch1-Hes1 signaling suppressed INSM1, as well as ASCL1 and BRN2. INSM1, expressed exclusively in SCLC, is a crucial regulator of NE differentiation in SCLCs, and is regulated by the Notch1-Hes1 signaling pathway., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The steroid hormone ecdysone, which controls insect molting and metamorphosis, is synthesized in the prothoracic gland (PG), and several steroidogenic enzymes that are expressed specifically in the PG are involved in ecdysteroidogenesis. In this study, we identified new regulators that are involved in the transcriptional control of the silkworm steroidogenic enzyme genes. In silico analysis predicted several potential cis-regulatory elements (CREs) for the homeodomain transcription factors Antennapedia (Antp) and POU-M2 in the proximal promoters of steroidogenic enzyme genes. Antp and POU-M2 are expressed dynamically in the PG during larval development, and their overexpression in silkworm embryo-derived (BmE) cells induced the expression of steroidogenic enzyme genes. Importantly, luciferase reporter analyses, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Antp and POU-M2 promote the transcription of the silkworm steroidogenic enzyme gene Phantom (Phm) by binding directly to specific motifs within overlapping CREs in the Phm promoter. Mutations of these CREs in the Phm promoter suppressed the transcriptional activities of both Antp and POU-M2 in BmE cells and decreased the activities of mutated Phm promoters in the silkworm PG. In addition, pulldown and co-immunoprecipitation assays demonstrated that Antp can interact with POU-M2. Moreover, RNA interference-mediated down-regulation of either Antp or POU-M2 during silkworm wandering not only decreased the ecdysone titer but also led to the failure of metamorphosis. In summary, our results suggest that Antp and POU-M2 coordinate the transcription of the silkworm Phm gene directly, indicating new roles for homeodomain proteins in regulating insect ecdysteroidogenesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Embryonic stem cell (ESC) identity is orchestrated by co-operativity between the transcription factors (TFs) Sox2 and the class V POU-TF Oct4 at composite Sox/Oct motifs. Neural stem cells (NSCs) lack Oct4 but express Sox2 and class III POU-TFs Oct6, Brn1 and Brn2. This raises the question of how Sox2 interacts with POU-TFs to transcriptionally specify ESCs versus NSCs. Here, we show that Oct4 alone binds the Sox/Oct motif and the octamer-containing palindromic MORE equally well. Sox2 binding selectively increases the affinity of Oct4 for the Sox/Oct motif. In contrast, Oct6 binds preferentially to MORE and is unaffected by Sox2. ChIP-Seq in NSCs shows the MORE to be the most enriched motif for class III POU-TFs, including MORE subtypes, and that the Sox/Oct motif is not enriched. These results suggest that in NSCs, co-operativity between Sox2 and class III POU-TFs may not occur and that POU-TF-driven transcription uses predominantly the MORE cis architecture. Thus, distinct interactions between Sox2 and POU-TF subclasses distinguish pluripotent ESCs from multipotent NSCs, providing molecular insight into how Oct4 alone can convert NSCs to pluripotency., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Degeneration of midbrain dopaminergic (DA) neurons is a key pathological event of Parkinson's disease (PD). Limited adult dopaminergic neurogenesis has led to novel therapeutic strategies such as transplantation of dopaminergic precursors (DPs). However, this strategy is currently restrained by a lack of cell source, the tendency for the DPs to become a glial-restricted state, and the tumor formation after transplantation. Here, we demonstrate the direct conversion of mouse fibroblasts into induced DPs (iDPs) by ectopic expression of Brn2, Sox2 and Foxa2. Besides expression with neural progenitor markers and midbrain genes including Corin, Otx2 and Lmx1a, the iDPs were restricted to dopaminergic neuronal lineage upon differentiation. After transplantation into MPTP-lesioned mice, iDPs differentiated into DA neurons, functionally alleviated the motor deficits, and reduced the loss of striatal DA neuronal axonal termini. Importantly, no iDPs-derived astrocytes and neoplasia were detected in mouse brains after transplantation. We propose that the iDPs from direct reprogramming provides a safe and efficient cell source for PD treatment.

Neuron identity transformations occur upon removal of specific regulatory factors in many different cellular contexts, thereby revealing the fundamental principle of alternative cell identity choices made during nervous system development. One common molecular interpretation of such homeotic cell identity transformations is that a regulatory factor has a dual function in activating genes defining one cellular identity and repressing genes that define an alternative identity. We provide evidence for an alternative, competition-based mechanism. We show that the MEC-3 LIM homeodomain protein can outcompete the execution of a neuropeptidergic differentiation program by direct interaction with the UNC-86/Brn3 POU homeodomain protein. MEC-3 thereby prevents UNC-86 from collaborating with the Zn finger transcription factor PAG-3/Gfi to induce peptidergic neuron identity and directs UNC-86 to induce an alternative differentiation program toward a glutamatergic neuronal identity. Homeotic control of neuronal identity programs has implications for the evolution of neuronal cell types., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Microphthalmia-associated transcription factor (MITF) is a nodal point in melanoma transcriptional network that regulates dozens of genes with critical functions in cell differentiation, proliferation and survival. Highly variable MITF expression levels exist in tumor cell subpopulations conferring marked heterogeneity and plasticity in the tumor tissue. A model has been postulated whereby lower MITF levels favour cell invasion and suppress proliferation, whereas high levels stimulate differentiation and proliferation. Additionally, MITF is considered to be a prosurvival gene and a lineage addiction oncogene in melanoma. Herein, we review how MITF expression may affect the melanoma phenotype with consequences on the survival, invasion and metastasis of melanoma cells, and we discuss the research challenges., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mouse embryonic stem cells (ESCs) treated with all-trans retinoic acid differentiate into a homogenous population of glutamatergic neurons. Although differentiation is initiated through activation of target genes by the retinoic acid receptors, the downstream transcription factors specifying neuronal fate are less well characterised. Here, we show that the transcription factor Brn2 (also known as Pou3f2) is essential for the neuronal differentiation programme. By integrating results from RNA-seq following Brn2 silencing with results from Brn2 ChIP-seq, we identify a set of Brn2 target genes required for the neurogenic programme. Further integration of Brn2 ChIP-seq data from retinoic-acid-treated ESCs and P19 cells with data from ESCs differentiated into neuronal precursors by Fgf2 treatment and that from fibroblasts trans-differentiated into neurons by ectopic Brn2 expression showed that Brn2 occupied a distinct but overlapping set of genomic loci in these differing conditions. However, a set of common binding sites and target genes defined the core of the Brn2-regulated neuronal programme, among which was that encoding the transcription factor Zic1. Small hairpin RNA (shRNA)-mediated silencing of Zic1 prevented ESCs from differentiating into neuronal precursors, thus defining a hierarchical Brn2-Zic1 axis that is essential to specify neural fate in retinoic-acid-treated ESCs., (© 2015. Published by The Company of Biologists Ltd.)

In the nervous system, glial cells need to be specified from a set of progenitor cells. In the developing Drosophila eye, perineurial glia proliferate and differentiate as wrapping glia in response to a neuronal signal conveyed by the FGF receptor pathway. To unravel the underlying transcriptional network we silenced all genes encoding predicted DNA-binding proteins in glial cells using RNAi. Dref and other factors of the TATA box-binding protein-related factor 2 (TRF2) complex were previously predicted to be involved in cellular metabolism and cell growth. Silencing of these genes impaired early glia proliferation and subsequent differentiation. Dref controls proliferation via activation of the Pdm3 transcription factor, whereas glial differentiation is regulated via Dref and the homeodomain protein Cut. Cut expression is controlled independently of Dref by FGF receptor activity. Loss- and gain-of-function studies show that Cut is required for glial differentiation and is sufficient to instruct the formation of membrane protrusions, a hallmark of wrapping glial morphology. Our work discloses a network of transcriptional regulators controlling the progression of a naïve perineurial glia towards the fully differentiated wrapping glia., (© 2015. Published by The Company of Biologists Ltd.)

The basic helix-loop-helix (bHLH) transcription factor Olig2 is crucial for mammalian central nervous system development. Human ortholog OLIG2 is located in the Down syndrome critical region in trisomy 21. To investigate the effect of Olig2 misexpression on brain development, we generated a developmentally regulated Olig2-overexpressing transgenic line with a Cre/loxP system. The transgenic mice with Olig2 misexpression in cortical neural stem/progenitor cells exhibited microcephaly, cortical dyslamination, hippocampus malformation, and profound motor deficits. Ectopic misexpression of Olig2 impaired cortical progenitor proliferation and caused precocious cell cycle exit. Massive neuronal cell death was detected in the developing cortex of Olig2-misexpressing mice. In addition, Olig2 misexpression led to a significant downregulation of neuronal specification factors including Ngn1, Ngn2 and Pax6, and a defect in cortical neurogenesis. Chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis indicates that Olig2 directly targets the promoter and/or enhancer regions of Nfatc4, Dscr1/Rcan1 and Dyrk1a, the critical neurogenic genes that contribute to Down syndrome phenotypes, and inhibits their expression. Together, our study suggests that Olig2 misexpression in neural stem cells elicits neurogenesis defects and neuronal cell death, which may contribute to developmental disorders including Down syndrome, where OLIG2 is triplicated on chromosomal 21., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Odor perception requires that each olfactory sensory neuron (OSN) class continuously express a single odorant receptor (OR) regardless of changes in the environment. However, little is known about the control of the robust, class-specific OR expression involved. Here, we investigate the cis-regulatory mechanisms and components that generate robust and OSN class-specific OR expression in Drosophila. Our results demonstrate that the spatial restriction of expression to a single OSN class is directed by clusters of transcription-factor DNA binding motifs. Our dissection of motif clusters of differing complexity demonstrates that structural components such as motif overlap and motif order integrate transcription factor combinations and chromatin status to form a spatially restricted pattern. We further demonstrate that changes in metabolism or temperature perturb the function of complex clusters. We show that the cooperative regulation between motifs around and within the cluster generates robust, class-specific OR expression.

Growing number of long intergenic noncoding RNAs (lincRNAs) have recently been identified in mammals as new modulators in cancer origination and progression involved in a broad range of biological processes. Long intergenic noncoding RNA POU3F3 (linc-POU3F3) has been characterized as a highly conserved functional transcription regulator in esophageal squamous cell carcinoma. The contributions of this lincRNA to glioblastoma remain unknown. In this present study, we investigated the expression pattern and functional role of linc-POU3F3 in glioma by using real-time PCR and gain-/loss-of-function studies. The results revealed that linc-POU3F3 levels were extraordinarily associated with the tumor WHO grade. In related biochemical assays, overexpression of linc-POU3F3 promotes cell viability and proliferation in glioma cells, whereas knockdown of linc-POU3F3 showed the opposite effect. As expected, we also found that linc-POU3F3 expression was negatively correlated with the mRNA level of POU3F3 (the evolutionarily conserved neighbor gene of linc-POU3F3). Our results indicate that linc-POU3F3 might affect glioma development via altering expression level of POU3F3, and lead us to believe that linc-POU3F3 may also have a crucial regulatory role in glioma progression., (Copyright © 2014. Published by Elsevier B.V.)

Coordinated intracellular trafficking is critically important for proper timing of major cellular events during embryogenesis. Nuclear import mediated by the karyopherin α/β (importin α/β) heterodimer is perhaps the best characterised nuclear trafficking system in eukaryotic cells. Seven karyopherin α subtypes have been identified in the domestic pig, and although each karyopherin α subtype transports proteins bearing classical nuclear localisation signals (NLSs), individual karyopherin α subtypes have been shown to preferentially transport specific cargoes. The aim of the present study was to determine the mechanism by which BRN2, a transcription factor previously reported to be transported by the karyopherin α/β heterodimer, gains access to the nucleus in porcine oocytes and embryos. Using a combination of in vivo and in vitro assays, we tested the hypothesis that discrete karyopherin α subtypes transport BRN2 into the nuclei of porcine oocytes and cleavage stage embryos. Our results show that ectopically expressed BRN2 adopts a nuclear localisation in all nuclei through the 4-cell stage of development, whereas only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. This pattern is unique to BRN2 because another ectopically expressed NLS-containing protein is able to adopt a nuclear localisation in all blastomeres of 8-cell stage embryos.

Our previous studies indicated that transcription factor Brn-4 is upregulated in the surgically denervated hippocampus in vivo, promoting neuronal differentiation of hippocampal neural stem cells (NSCs) in vitro. The molecules mediating Brn-4 upregulation in the denervated hippocampus remain unknown. In this study we examined the levels of insulin-like growth factor-1 (IGF-1) in hippocampus following denervation. Surgical denervation led to a significant increase in IGF-1 expression in vivo. We also report that IGF-1 treatment on NSCs in vitro led to a marked acceleration of Brn-4 expression and cell differentiation down neuronal pathways. The promotion effects were blocked by PI3K-specific inhibitor (LY294002), but not MAPK inhibitor (PD98059); levels of phospho-Akt were increased by IGF-1 treatment. In addition, inhibition of IGF-1 receptor (AG1024) and mTOR (rapamycin) both attenuated the increased expression of Brn-4 induced by IGF-1. Together, the results demonstrated that upregulation of IGF-1 induced by hippocampal denervation injury leads to activation of the PI3K/Akt signaling pathway, which in turn gives rise to upregulation of the Brn-4 and subsequent stem cell differentiation down neuronal pathways.

We aimed to identify cis-regulatory elements that control gene expression in progenitors of the cerebral cortex. A list of 975 putative enhancers were retrieved from a ChIP-Seq experiment performed in NS5 mouse stem cells with antibodies to Sox2, Brn2/Pou3f2, or Brn1/Pou3f3. Through a selection pipeline including gene ontology and expression pattern, we reduced the number of candidate enhancer sequences to 20. Ex vivo electroporation of green fluorescent pProtein (GFP) reporter constructs in the telencephalon of mouse embryos showed that 35% of the 20 selected candidate sequences displayed enhancer activity in the developing cortex at E13.5. In silico transcription factor binding site (TFBS) searches and mutagenesis experiments showed that enhancer activity is related to the presence of Sox/Pou TFBS pairs in the sequence. Comparative genomic analyses showed that enhancer activity is not related to the evolutionary conservation of the sequence. Finally, the combination of in utero electroporation of GFP reporter constructs with immunostaining for Tbr2 (basal progenitor marker) and phospho-histoneH3 (mitotic activity marker) demonstrated that each enhancer is specifically active in precise subpopulations of progenitors in the cortical germinal zone, highlighting the heterogeneity of these progenitors in terms of cis-regulation., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The Apobec/AID family of cytosine deaminases can deaminate cytosine and thereby contribute to adaptive and innate immunity, DNA demethylation, and the modification of cellular mRNAs. Unique among this family is Apobec2, whose enzymatic activity has been questioned and whose function remains poorly explored. We recently reported that zebrafish Apobec2a and Apobec2b (Apobec2a,2b) regulate retina regeneration; however, their mechanism of action remained unknown. Here we show that although Apobec2a,2b lack cytosine deaminase activity, they require a conserved zinc-binding domain to stimulate retina regeneration. Interestingly, we found that human APOBEC2 is able to functionally substitute for Apobec2a,2b during retina regeneration. By identifying Apobec2-interacting proteins, including ubiquitin-conjugating enzyme 9 (Ubc9); topoisomerase I-binding, arginine/serine-rich, E3 ubiquitin protein ligase (Toporsa); and POU class 6 homeobox 2 (Pou6f2), we uncovered that sumoylation regulates Apobec2 subcellular localization and that nuclear Apobec2 controls Pou6f2 binding to DNA. Importantly, mutations in the zinc-binding domain of Apobec2 diminished its ability to stimulate Pou6f2 binding to DNA, and knockdown of Ubc9 or Pou6f2 suppressed retina regeneration., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The temporal bone encases conductive and sensorineural elements of the ear. Mutations of POU3F4 are associated with unique temporal bone abnormalities and X-linked mixed deafness (DFNX2/DFN3). However, the target genes and developmental processes controlled by POU3F4 transcription factor activity have remained largely uncharacterized. Ephrin-B2 (Efnb2) is a signaling molecule with well-documented effects on cell adhesion, proliferation, and migration. Our analyses of targeted mouse mutants revealed that Efnb2 loss-of-function phenocopies temporal bone abnormalities of Pou3f4 hemizygous null neonates: qualitatively identical malformations of the stapes, styloid process, internal auditory canal, and cochlear capsule were present in both mutants. Using failed/insufficient separation of the stapes and styloid process as a quantitative trait, we found that single gene Efnb2 loss-of-function and compound Pou3f4/Efnb2 loss-of-function caused a more severe phenotype than single gene Pou3f4 loss-of-function. Pou3f4 and Efnb2 gene expression domains overlapped at the site of impending stapes-styloid process separation and at subcapsular mesenchyme surrounding the cochlea; at both these sites, Efnb2 expression was attenuated in Pou3f4 hemizygous null mutants relative to control. Results of immunoprecipitation experiments using chromatin isolated from nascent middle ear mesenchyme supported the hypothesis of a physical association between Pou3f4 and specific non-coding sequence of Efnb2. We propose that Efnb2 is a target of Pou3f4 transcription factor activity and an effector of mesenchymal patterning during temporal bone development.

One-third of lung malignancies demonstrate a proneural/neuroendocrine phenotype or type of differentiation. However, it has not been clearly elucidated how proneural/neuroendocrine differentiation is controlled in lung cancers. We recently demonstrated that the POU3F2 gene plays a significant role in proneural/neuroendocrine differentiation of lung cancers. Because class III POU genes (POU3F1, POU3F2, POU3F3, and POU3F4) and class IV POU genes (POU4F1, POU4F2, and POU4F3) share similar properties in neural development, we analyzed the association between class III/IV POU genes and a proneural/neuroendocrine phenotype in lung cancers using seven small cell lung cancer (SCLC) cell lines and twelve non-SCLC (NSCLC) cell lines. Class III/IV POU gene expression was generally restricted to SCLC cells. However, the forced expression of class III/IV POU genes in the NSCLC cell lines induced the expression of neuroendocrine-specific markers (neural call adhesion molecule 1, synaptophysin, and chromogranin A) and proneural transcription factors (achaete-scute homolog-like 1, NeuroD1, and thyroid transcription factor 1) in various degrees. Furthermore, each class III/IV POU gene induced other class III/IV POU genes, suggesting the mutual induction of class III/IV POU genes. These findings suggest that the expression of class III/IV POU genes is important for the proneural/neuroendocrine differentiation of lung cancer cells., (© 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

POU domain proteins are a family of critical regulators of development and differentiation due to their transcriptional activity in the nucleus. Skn‑1a, a member of the POU domain protein family, appears to be expressed predominantly in epidermal keratinocytes and is thought to play a critical role in keratinocyte differentiation and proliferation. In this study, we examined the mechanisms involved in the nuclear localization of Skn‑1a. We transiently expressed enhanced green fluorescent protein (EGFP) reporter constructs encoding EGFP fusions with Skn‑1a deletion and mutation proteins in normal human epidermal keratinocytes (NHEKs). The experiments clearly demonstrated that Skn‑1a contained a functional nuclear localization signal (NLS) domain, and that the smallest domain necessary for Skn‑1a nuclear transport was the GRKRKKR sequence located within amino acids 279‑285. Previous studies have shown that the phosphorylation of specific amino acids neighboring the NLS may regulate nuclear transport and that the amino acid residues threonine (Thr) and serine (Ser) have the potential to undergo phosphorylation. We examined whether the amino acids Thr286 and Ser287, which reside adjacent to the NLS at the carboxy‑terminal side, play a role in Skn‑1a nuclear localization. For this purpose, we generated three EGFP‑Skn‑1a mutation constructs, in which Thr286, Ser287, or both Thr286 and Ser287 residues were replaced with alanine, respectively. The results showed that the Thr286 and Ser287 residues were involved in the regulation of nuclear localization as well as epidermal differentiation. These results suggested that the epidermal differentiation signaling pathway, involving kinase and phosphatase activation, may regulate the NLS activity of Skn‑1a in keratinocytes. Collectively, these data contribute to understanding the mechanisms of nuclear translocation of POU domain proteins and epidermal differentiation.

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

Neural stem cells (NSCs) are pluripotent cells capable of differentiation into dopaminergic (DA) neurons, which are the major cell types damaged in Parkinson's disease (PD). Therefore, NSCs are considered the most promising cell source for cell replacement therapy of PD. However, the poor differentiation and maturation of DA neurons and decreased cell survival after transplantation are a challenge. We have previously demonstrated that Brn4, a member of the POU domain family of transcription factors, induced the differentiation of NSCs into neurons and promoted their maturation. In this study, we directly transduced tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, into NSCs to induce DA neuronal differentiation. However, these DA neurons were morphologically immature and seldom expressed dopamine transporter (DAT), a late marker of mature DA neurons. In contrast, TH co-transfected with Brn4 generated increased number of mature DA neurons. Furthermore, Brn4 significantly induced the expression of glial cell line-derived neurotrophic factor (GDNF) with its receptors GFRα-1 and Ret, which may contribute to the maturation and survival of differentiated DA neurons. Our findings may be of future importance for the use of NSCs in cell replacement therapy of PD., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background & Aims: Thousands of long intergenic non-protein coding RNAs (lincRNAs) have been identified in mammals via genome-wide sequencing studies. Many are functional, but are expressed aberrantly by cancer cells. We investigated whether levels of lincRNAs are altered during the development of esophageal squamous cell carcinoma (ESCC)., Methods: We used quantitative real-time polymerase chain reaction to measure levels of 26 highly conserved lincRNAs in ESCC and surrounding nontumor tissues. A total of 182 ESCC and paired adjacent nontumor tissue samples were collected from patients undergoing tylectomy at The First Affiliate Hospital of Soochow University from 2001 through 2009; another 178 ESCC tissue pairs were collected from Guangzhou Medical University from 2002 through 2009. LincRNAs were expressed from lentiviral vectors or knocked down with small hairpin RNAs in Eca-109 and TE-1 cells., Results: Levels of a lincRNA encoded by a gene located next to POU3F3 (linc-POU3F3) were significantly higher in ESCC than neighboring nontumor tissues. In RNA immunoprecipitation assays, linc-POU3F3 was associated with the EZH2 messenger RNA (mRNA). Overexpression of linc-POU3F3 in cell lines increased their proliferation and ability to form colonies, and reduced the expression of POU3F3 mRNA, whereas knockdown of linc-POU3F3 increased the levels of POU3F3 mRNA. CpG islands in POU3F3 were densely hypermethylated in cell lines that overexpressed linc-POU3F3; methylation at these sites was reduced by knockdown of linc-POU3F3. Pharmacologic inhibition of EZH2 increased the levels of POU3F3 mRNA and significantly reduced binding of DNA methyltransferase (DNMT)1, DNMT3A, and DNMT3B to POU3F3. ESCC cells with knockdown of linc-POU3F3 formed xenograft tumors more slowly in mice than control ESCC cells., Conclusions: Levels of linc-POU3F3 are increased in ESCC samples from patients compared with nontumor tissues. This noncoding RNA contributes to the development of ESCC by interacting with EZH2 to promote methylation of POU3F3, which encodes a transcription factor., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling., (©AlphaMed Press.)

Various mutations have occurred during evolution among orthologs, genes in different species that diverged from a common ancestral gene by speciation. Here, we report the remarkable deterioration of a characteristic mammalian maternal behavior, pup retrieval, in nonmammalized mice, in which the transcription factor Pou3f2 was replaced with the Xenopus ortholog lacking all of the homopolymeric amino acid repeats of mammalian POU3F2. Most of the pups born to the nonmammalized mice died within days after birth, depending on the dam genotype alone. Quantitative immunohistochemical analysis revealed decreases in the rate-limiting enzymes of dopamine and serotonin synthesis in various brain structures. Similar results were obtained in knock-in mice in which all of the homopolymeric amino acid repeats of mammalian POU3F2 were removed. Pup retrieval behavior in mammals is thus strongly related to monoamine neurotransmitter levels via the acquisition of homopolymeric amino acid repeats during mammalian evolution., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Homeobox transcription factors are known to play crucial roles in the anterior lobe of the pituitary gland. During molecular cloning with the Yeast One-Hybrid System using a 5'-upstream region of the porcine Fshβ as a bait sequence, we have cloned a cDNA encoding a partial sequence of the retina-derived POU domain factor 1 (RPF1) from the porcine pituitary cDNA library and confirmed its specific binding to the bait sequence. In situ hybridization was performed to examine localization of Rpf1 and showed that this gene is expressed in the stem/progenitor cells of the rat pituitary primordium as well as the diencephalon and retina. In addition, real-time PCR demonstrated that Rpf1 transcripts are abundant in early embryonic periods but that this is followed by a decrease during pituitary development, indicating that this factor plays a role in differentiating cells of the pituitary. The transcriptional activity of RPF1 for genes of Prop1, Prrx1 and Prrx2, which were characterized as genes participating in the pituitary stem/progenitor cells by our group, was then examined with full-length cDNA obtained from the rat pituitary. RPF1 showed regulatory activity for Prop1 and Prrx2, but not for Prrx1. These results indicate the involvement of this retina-derived factor in pituitary development.

Transcription factors that drive neuron type-specific terminal differentiation programs in the developing nervous system are often expressed in several distinct neuronal cell types, but to what extent they have similar or distinct activities in individual neuronal cell types is generally not well explored. We investigate this problem using, as a starting point, the C. elegans LIM homeodomain transcription factor ttx-3, which acts as a terminal selector to drive the terminal differentiation program of the cholinergic AIY interneuron class. Using a panel of different terminal differentiation markers, including neurotransmitter synthesizing enzymes, neurotransmitter receptors and neuropeptides, we show that ttx-3 also controls the terminal differentiation program of two additional, distinct neuron types, namely the cholinergic AIA interneurons and the serotonergic NSM neurons. We show that the type of differentiation program that is controlled by ttx-3 in different neuron types is specified by a distinct set of collaborating transcription factors. One of the collaborating transcription factors is the POU homeobox gene unc-86, which collaborates with ttx-3 to determine the identity of the serotonergic NSM neurons. unc-86 in turn operates independently of ttx-3 in the anterior ganglion where it collaborates with the ARID-type transcription factor cfi-1 to determine the cholinergic identity of the IL2 sensory and URA motor neurons. In conclusion, transcription factors operate as terminal selectors in distinct combinations in different neuron types, defining neuron type-specific identity features.

Re-activation of molecules active in embryogenesis can play an important role in cancer, since they can provide tumor cells with malignant properties. Several members of the family of POU transcription factors are essential for the development of the nervous system and several are expressed in the neural crest, which is the same location where melanocytic cells develop from. Here, the POU transcription factor family and its role in melanocytic transformation and melanoma are reviewed., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The upper layers (II-IV) are the most prominent distinguishing feature of mammalian neocortex compared with avian or reptilian dorsal cortex, and are vastly expanded in primates. Although the time-dependent embryonic generation of upper-layer cells is genetically instructed within their parental progenitors, mechanisms governing cell-intrinsic fate transitions remain obscure. POU-homeodomain transcription factors Pou3f3 and Pou3f2 (Brn1 and Brn2) are known to label postmitotic upper-layer cells, and are redundantly required for their production. We find that the onset of Pou3f3/2 expression actually occurs in ventricular zone (VZ) progenitors, and that Pou3f3/2 subsequently label neural progeny switching from deep-layer Ctip2(+) identity to Satb2(+) upper-layer fate as they migrate to proper superficial positions. By using an Engrailed dominant-negative repressor, we show that sustained neurogenesis after the deep- to upper-layer transition requires the proneual action of Pou3fs in VZ progenitors. Conversely, single-gene overexpression of any Pou3f in early neural progenitors is sufficient to specify the precocious birth of Satb2(+) daughter neurons that extend axons to the contralateral hemisphere, as well as exhibit robust pia-directed migration that is characteristic of upper-layer cells. Finally, we demonstrate that Pou3fs influence multiple stages of neurogenesis by suppressing Notch effector Hes5, and promoting the expression of proneural transcription factors Tbr2 and Tbr1.

Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types., (Copyright © 2013 Elsevier Inc. All rights reserved.)

As a crucial pluripotency-related factor, the epigenetic regulation of Oct4 has been studied intensively in mammalians. However, its dynamic changes of DNA methylation and histone modification in avians remain poorly understood. In the present study, we first described the alterations of DNA methylation and histone acetylation in the promoter of chicken PouV (cPouV; the homologue of Oct4 in avian) during chick embryonic germ (EG) cell differentiation. The epigenetic modification analysis showed that DNA methylation in the cPouV promoter increased obviously, while histone acetylation decreased dramatically detected by chromatin immunoprecipitation assay in the process of differentiation. Gene expression analysis detection indicated that the levels of DNA methyltransferase 3a (Dnmt 3a), Dnmt 3b, and histone deacetylase 3 (HDAC 3) transcripts were significantly high, whereas the relative abundance of Dnmt 1, histone acetyltransferase (HAT), and cPouV mRNA was significantly decreased during the conversion of EG to embryoid body-like structures (EBs), which was correlated with the increased level of methylation and reduced level of H3 acetylation. Moreover, in vitro methylation assay indicated that the reporter gene was remarkably inhibited by the methylated promoter of cPouV. To further understand the effect of epigenetic modifiers on cPouV expression, we performed an analysis of EB cells treated with trichostatin A (TSA), Aza-2'-deoxycytidine (Aza), or TSA plus Aza (TSA/Aza). We observed that the effect of TSA/Aza is more sensitive to the reactivation of cPouV compared with TSA or Aza, indicating that these epigenetic inhibitors can function synergistically to facilitate the reprogramming process. The present study provided evidences that a critical role for cPouV activation/repression by DNA methylation and/or histone modifications is involved in the pluripotency maintenance and differentiation process of chick EG.

JAK/STAT signaling is localized to the wing hinge, but its function there is not known. Here we show that the Drosophila STAT Stat92E is downstream of Homothorax and is required for hinge development by cell-autonomously regulating hinge-specific factors. Within the hinge, Stat92E activity becomes restricted to gap domain cells that lack Nubbin and Teashirt. While gap domain cells lacking Stat92E have significantly reduced proliferation, increased JAK/STAT signaling there does not expand this domain. Thus, this pathway is necessary but not sufficient for gap domain growth. We show that reduced Wingless (Wg) signaling dominantly inhibits Stat92E activity in the hinge. However, ectopic JAK/STAT signaling does not perturb Wg expression in the hinge. We report negative interactions between Stat92E and the notum factor Araucan, resulting in restriction of JAK/STAT signaling from the notum. In addition, we find that the distal factor Nub represses the ligand unpaired as well as Stat92E activity. These data suggest that distal expansion of JAK/STAT signaling is deleterious to wing blade development. Indeed, mis-expression of Unpaired within the presumptive wing blade causes small, stunted adult wings. We conclude that JAK/STAT signaling is critical for hinge fate specification and growth of the gap domain and that its restriction to the hinge is required for proper wing development., (© 2013 Elsevier Inc. All rights reserved.)

Background: Innate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-κB transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood., Results: We show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-κB/Relish-driven antimicrobial peptide gene expression in flies. In nub1 mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism., Conclusions: This study demonstrates that a large number of genes that are activated by NF-κB/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.

POU3F4 is a POU domain transcription factor that is required for hearing. In the ear, POU3F4 is essential for mesenchymal remodeling of the bony labyrinth and is the causative gene for DFNX2 human nonsyndromic deafness. Ear abnormalities underlie this form of deafness, characterized previously in multiple spontaneous, radiation-induced and transgenic mouse mutants. Here, we report three novel mutations in the POU3F4 gene that result in profound hearing loss in both humans and mice. A p.Gln79* mutation was identified in a child from an Israeli family, revealed by massively parallel sequencing (MPS). This strategy demonstrates the strength of MPS for diagnosis with only one affected individual. A second mutation, p.Ile285Argfs*43, was identified by Sanger sequencing. A p.Cys300* mutation was found in an ENU-induced mutant mouse, schwindel (sdl), by positional cloning. The mutation leads to a predicted truncated protein, similar to the human mutations, providing a relevant mouse model. The p.Ile285Argfs*43 and p.Cys300* mutations lead to a shift of Pou3f4 nuclear localization to the cytoplasm, demonstrated in cellular localization studies and in the inner ears of the mutant mice. The discovery of these mutations facilitates a deeper comprehension of the molecular basis of inner ear defects due to mutations in the POU3F4 transcription factor., (© 2013 WILEY PERIODICALS, INC.)

We recently demonstrated that the expression of the importin α subtype is switched from α2 to α1 during neural differentiation in mouse embryonic stem cells (ESCs) and that this switching has a major impact on cell differentiation. In this study, we report a cell-fate determination mechanism in which importin α2 negatively regulates the nuclear import of certain transcription factors to maintain ESC properties. The nuclear import of Oct6 and Brn2 was inhibited via the formation of a transport-incompetent complex of the cargo bound to a nuclear localization signal binding site in importin α2. Unless this dominant-negative effect was downregulated upon ESC differentiation, inappropriate cell death was induced. We propose that although certain transcription factors are necessary for differentiation in ESCs, these factors are retained in the cytoplasm by importin α2, thereby preventing transcription factor activity in the nucleus until the cells undergo differentiation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

PQBP1 is a nuclear-cytoplasmic shuttling protein that is engaged in RNA metabolism and transcription. In mouse embryonic brain, our previous in situ hybridization study revealed that PQBP1 mRNA was dominantly expressed in the periventricular zone region where neural stem progenitor cells (NSPCs) are located. Because the expression patterns in NSPCs are related to the symptoms of intellectual disability and microcephaly in PQBP1 gene-mutated patients, we investigated the transcriptional regulation of PQBP1 by NSPC-specific transcription factors. We selected 132 genome sequences that matched the consensus sequence for the binding of Sox2 and POU transcription factors upstream and downstream of the mouse PQBP1 gene. We then screened the binding affinity of these sequences to Sox2-Pax6 or Sox2-Brn2 with gel mobility shift assays and found 18 genome sequences that interacted with the NSPC-specific transcription factors. Some of these sequences had cis-regulatory activities in Luciferase assays and in utero electroporation into NSPCs. Furthermore we found decreased levels of expression of PQBP1 protein in NSPCs of heterozygous Sox2-knockout mice in vivo by immunohistochemistry and western blot analysis. Collectively, these results indicated that Sox2 regulated the transcription of PQBP1 in NSPCs.

With the advent of high throughput sequencing and high resolution transcriptomic technologies, there exists today an unprecedented opportunity to understand gene regulation at a quantitative level. State of the art models of the relationship between regulatory sequence and gene expression have shown great promise, but also suffer from some major shortcomings. In this paper, we identify and address methodological challenges pertaining to quantitative modeling of gene expression from sequence, and test our models on the anterior-posterior patterning system in the Drosophila embryo. We first develop a framework to process cellular resolution three-dimensional gene expression data from the Drosophila embryo and create data sets on which quantitative models can be trained. Next we propose a new score, called 'weighted pattern generating potential' (w-PGP), to evaluate model predictions, and show its advantages over the two most common scoring schemes in use today. The model building exercise uses w-PGP as the evaluation score and adopts a systematic strategy to increase a model's complexity while guarding against over-fitting. Our model identifies three transcription factors--ZELDA, SLOPPY-PAIRED, and NUBBIN--that have not been previously incorporated in quantitative models of this system, as having significant regulatory influence. Finally, we show how fitting quantitative models on data sets comprising a handful of enhancers, as reported in earlier work, may lead to unreliable models., (Copyright © 2013. Published by Elsevier Inc.)

Fish egg envelopes consist of several glycoproteins, called zona pellucida (ZP) proteins, which are conserved among chordates. Euteleosts synthesize ZP proteins in the liver, while elopomorphs synthesize them in the ovaries. In Cypriniformes, zp genes are expressed in the ovaries. We investigated the zp genes of two Otocephalan orders: Clupeiformes (Pacific herring and Japanese anchovy) and Gonorynchiformes (milkfish), which diverged earlier than Cypriniformes. cDNA cloning of zp gene homologs revealed that Pacific herring and Japanese anchovy possessed both ovary- and liver-expressed zp genes; however, the zp genes of milkfish were only expressed in the ovaries. Molecular phylogenetic analysis showed that ovary- and liver-expressed zpc genes of two the Clupeiformes formed independent clades. Based on this, we hypothesized the evolution of teleostean zp genes, focusing on the organ expressing zp gene. As in other chordates, the original site of expression of zp genes was likely the ovary. In the early stage of teleostean evolution, the ancestral zp genes acquired the ability to express in the liver. Later, one of the two expression sites became dominant. The liver-expressed zp genes are component proteins of the egg envelope in the Euteleostei. In Otocephala, Clupeiformes possess both ovary- and liver-expressed genes that presumably participate in egg envelope formation, whereas the Gonorynchiformes and Cypriniformes have primarily preserved ovary expressed zp genes., (Copyright © 2013 Wiley Periodicals, Inc.)

Purpose: Ras-like without CAAX 2 (RIT2), a member of the Ras superfamily of small guanosine triphosphatases, is involved in regulating neuronal function. RIT2 is a unique member of the Ras family in that RIT2 is preferentially expressed in various neurons, including retinal neurons. The mechanisms that regulate RIT2 expression in neurons were studied., Methods: Reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry, western blotting, bioinformatic prediction, electrophoretic mobility shift assay (EMSA), and cell transfection methods were used., Results: With immunohistochemistry of the mouse retina, RIT2 protein was detected in the ganglion cell layer (GCL), inner plexiform layer, inner nuclear layer, and outer plexiform layer, with the strongest staining in the GCL and the inner plexiform layer. RT-qPCR combined with laser capture microdissection detected Rit2 messenger RNA in the GCL and the inner nuclear layer. Western blot analysis showed a large increase in the RIT2 protein in the retina during maturation from newborn to adult. Transient transfection identified the 1.3 kb upstream region of human RIT2 as capable of driving expression in neuronal cell lines. Based on the known expression pattern and biological activity, we hypothesized that POU4 family factors might modulate RIT2 expression in retinal ganglion cells (RGCs). Bioinformatic analyses predicted six POU4 factor-binding sites within the 1.3 kb human RIT2 promoter region. EMSA analyses showed binding of POU4 proteins to three of the six predicted sites. Cotransfection with expression vectors demonstrated that POU4 proteins can indeed modulate the human RIT2 promoter, and that ISL1, a LIM homeodomain factor, can further modulate the activity of the POU4 factors., Conclusions: These studies confirm the expression of RIT2 in retinal neuronal cells, including RGCs, begin to reveal the mechanisms responsible for neuronal expression of RIT2, and suggest a role for the POU4 family factors in modulating RIT2 expression in RGCs.

In many situations animals are confronted with approaching objects. Depending on whether the approach represents a potential threat or is intended during a goal-oriented approach, the adequate behavioral strategies differ. In all of these cases the visual system experiences an expanding or looming shape. The neuronal machinery mediating looming elicited behavioral responses has been studied most comprehensively in insects but is still far from being fully understood. It is particularly unknown how insects adjust their behavior to objects approaching from different directions. A brain structure that is thought to play an important role in spatial orientation in insects is the central complex (CC). We investigated whether CC neurons process information about approaching objects on a collision course. We recorded intracellularly from CC neurons in the locust Schistocerca gregaria during visual stimulation via lateral LCD screens. Many neurons in the locust CC, including columnar and tangential neurons, were sensitive to looming stimuli. Some of the neurons also responded to small moving targets. Several cell types showed binocular responses to looming objects, and some neurons were excited or inhibited depending on which eye was stimulated. These neurons may, therefore, detect the gross azimuthal direction of approaching objects and may mediate directional components of escape or steering movements.

Thyroid transcription factor 1 (TTF1) plays crucial roles in thyroid, lung, and developing brain morphogenesis. Because TTF1-expressing neoplasms are generated from organs and tissues that normally express TTF1, such as the thyroid follicular epithelium and peripheral lung airway epithelium, TTF1 is widely used as a cell lineage-specific and diagnostic marker for thyroid carcinomas and for lung adenocarcinomas with terminal respiratory unit (TRU) differentiation. However, among lung neuroendocrine tumors, small-cell carcinomas (small-cell lung cancers (SCLCs)), most of which are generated from the central airway, also frequently express TTF1 at high levels. To clarify how SCLCs express TTF1, we investigated the molecular mechanisms of its expression using cultivated lung cancer cells and focusing upon neural cell-specific transcription factors. Both SCLC cells and lung adenocarcinoma cells predominantly expressed isoform 2 of TTF1, and TTF1 promoter assays in SCLC cells revealed that the crucial region for activation of the promoter, which is adjacent to the transcription start site of TTF1 isoform 2, has potent FOX-, LHX-, and BRN2-binding sites. Transfection experiments using expression vectors for FOXA1, FOXA2, LHX2, LHX6, and BRN2 showed that BRN2 substantially upregulated TTF1 expression, whereas FOXA1/2 weakly upregulated TTF1 expression. BRN2 and FOXA1/2 binding to the TTF1 promoter was confirmed through chromatin immunoprecipitation experiments, and TTF1 expression in SCLC cells was considerably downregulated after BRN2 knockdown. Furthermore, the TTF1 promoter in SCLC cells was scarcely methylated, and immunohistochemical examinations using a series of primary lung tumors indicated that TTF1 and BRN2 were coexpressed only in SCLC cells. These findings suggest that TTF1 expression in SCLC is a cell lineage-specific phenomenon that involves the developing neural cell-specific homeoprotein BRN2.

The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

BRN2 is a developmental neural cell-specific POU domain transcription factor and is crucial for cell lineage determination. We investigated the importance of BRN2 in the expression of the lineage-specific transcription factors (achaete-scute homolog-like 1 (ASCL1) and NeuroD1 (ND1)) and neural/neuroendocrine marker molecules (neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYP) and chromogranin A (CHGA)) in small cell lung cancer (SCLC) using cultured lung cancer cells. All examined SCLC cell lines expressed BRN2, as well as ASCL1, ND1, NCAM1, SYP and CHGA. The expression levels of ASCL1, ND1, NCAM1, SYP and CHGA considerably decreased when BRN2 was knocked down in SCLC cells, and the addition of a BRN2 transgene into non-SCLC (NSCLC) cells induced the expression of ASCL1, ND1, NCAM1, SYP and CHGA. However, the BRN2 gene was not activated by the forced expression of ASCL1 or ND1 in NSCLC cells. The knockdown of BRN2 caused significant growth retardation with decrease of S to G2 phase population and mitotic cell rates and unaltered Ki-67-labeled or apoptotic cell rates in SCLC cells, indicating increase of G1 phase population. These findings suggest that BRN2 is a higher level regulator than ASCL1 and ND1 and BRN2 might be involved in aggressiveness of SCLC., (© 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Nephron morphogenesis is a complex process that generates blood-filtration units (glomeruli) connected to extremely long and patterned tubular structures. Hepatocyte nuclear factor 1β (HNF1β) is a divergent homeobox transcription factor that is expressed in kidney from the first steps of nephrogenesis. Mutations in HNF1B (OMIM #137920) are frequently found in patients with developmental renal pathologies, the mechanisms of which have not been completely elucidated. Here we show that inactivation of Hnf1b in the murine metanephric mesenchyme leads to a drastic tubular defect characterized by the absence of proximal, distal and Henle's loop segments. Nephrons were eventually characterized by glomeruli, with a dilated urinary space, directly connected to collecting ducts via a primitive and short tubule. In the absence of HNF1β early nephron precursors gave rise to deformed S-shaped bodies characterized by the absence of the typical bulge of epithelial cells at the bend between the mid and lower segments. The lack of this bulge eventually led to the absence of proximal tubules and Henle's loops. The expression of several genes, including Irx1, Osr2 and Pou3f3, was downregulated in the S-shaped bodies. We also observed decreased expression of Dll1 and the consequent defective activation of Notch in the prospective tubular compartment of comma- and S-shaped bodies. Our results reveal a novel hierarchical relationship between HNF1β and key genes involved in renal development. In addition, these studies define a novel structural and functional component of S-shaped bodies at the origin of tubule formation.

Most X-linked nonsyndromic hearing loss is caused by various types of mutations of the POU domain class 3 transcription factor 4 gene (POU3F4). We found five unique missense and frameshift truncation and extension mutations in Korean patients. Two missense mutations (p.Thr211Met and p.Gln229Arg) disturbed transcriptional activity. Two frameshift extension mutations (p.Thr354GlnfsX115 and p.X362ArgextX113) were located outside of POU domain and nuclear localization signal (NLS) at the C-terminus. POU3F4 protein levels were low and could be restored by MG132, a proteasome inhibitor, in vitro. These mutant POU3F4 proteins were exclusively localized to the cytoplasm and did not have transcriptional activity. Frameshift mutation (p.Leu317PhefsX12) in POU3F4 leads to the truncation of the C-terminal 44 amino acids spanning the POU domain and NLS. This frameshift truncation mutant protein was located in both the nucleus and cytoplasm and was present at low protein levels. This mutant was also transcriptionally inactive, even in the presence of MG132. From these results, we conclude that frameshift truncation and extension mutations in the C-terminus of POU3F4 lead to cytoplasmic localization and subsequent proteosomal degradation due to structural aberrations, which cause transcriptional inactivity and thus nonsyndromic hearing loss., (© 2012 Wiley Periodicals, Inc.)

Neural stem cells (NSCs) tranplantation has great potential for the treatment of neurodegenerative disease such as Parkinson's disease (PD). However, the usage of NSCs is limited because the differentiation of NSCs into specific dopaminergic neurons has proven difficult. We have recently demonstrated that transgenic expression of Nurr1 could induce the differentiation of NSCs into tyrosine hydroxylase (TH) immunoreactive dopaminergic neurons, and forced co-expression of Nurr1 with Brn4 caused a dramatic increase in morphological and phenotypical maturity of these neurons. In this study, we investigated the effect of transplanted NSCs in PD model rats. The results showed that overexpression of Nurr1 promoted NSCs to differentiate into dopaminergic neurons in vivo, increased the level of dopamine (DA) neurotransmitter in the striatum, resulting in behavioral improvement of PD rats. Importantly, co-expression of Nurr1 and Brn4 in NSCs significantly increased the maturity and viability of dopaminergic neurons, further raised the DA amount in the striatum and reversed the behavioral deficit of the PD rats. Our findings provide a new potential and strategy for the use of NSCs in cell replacement therapy for PD., (Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.)

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state., Competing Interests: The authors have declared that no competing interests exist.