POU Domain Transcription Factor Research Articles

The Oct1 transcription factor and epithelial malignancies: Old protein learns new tricks

The metazoan-specific POU domain transcription factor family comprises activities underpinning developmental processes such as embryonic pluripotency and neuronal specification. Some POU family proteins efficiently bind an 8-bp DNA element known as the octamer motif. These proteins are known as Oct transcription factors. Oct1/POU2F1 is the only widely expressed POU factor. Unlike other POU factors it controls no specific developmental or organ system. Oct1 was originally described to operate at target genes associated with proliferation and immune modulation, but more recent results additionally identify targets associated with oxidative and cytotoxic stress resistance, metabolic regulation, stem cell function and other unexpected processes. Oct1 is pro-oncogenic in multiple contexts, and several recent reports provide broad evidence that Oct1 has prognostic and therapeutic value in multiple epithelial tumor settings. This review focuses on established and emerging roles of Oct1 in epithelial tumors, with an emphasis on mechanisms of transcription regulation by Oct1 that may underpin these findings. This article is part of a Special Issue entitled: The Oct Transcription Factor Family, edited by Dr. Dean Tantin.

Critical POU domain residues confer Oct4 uniqueness in somatic cell reprogramming.

The POU domain transcription factor Oct4 plays critical roles in self-renewal and pluripotency of embryonic stem cells (ESCs). Together with Sox2, Klf4 and c-Myc, Oct4 can reprogram any other cell types to pluripotency, in which Oct4 is the only factor that cannot be functionally replaced by other POU family members. To investigate the determinant elements of Oct4 uniqueness, we performed Ala scan on all Ser, Thr, Tyr, Lys and Arg of murine Oct4 by testing their capability in somatic cell reprogramming. We uncovered a series of residues that are important for Oct4 functionality, in which almost all of these key residues are within the POU domains making direct interaction with DNA. The Oct4 N- and C-terminal transactivation domains (TADs) are not unique and could be replaced by the Yes-associated protein (YAP) TAD domain to support reprogramming. More importantly, we uncovered two important residues that confer Oct4 uniqueness in somatic cell reprogramming. Our systematic structure-function analyses bring novel mechanistic insight into the molecular basis of how critical residues function together to confer Oct4 uniqueness among POU family for somatic cell reprogramming.

Induction of Epstein-Barr Virus Oncoprotein LMP1 by Transcription Factors AP-2 and Early B Cell Factor.

Latent membrane protein 1 (LMP1) is a major oncogene essential for primary B cell transformation by Epstein-Barr virus (EBV). Previous studies suggested that some transcription factors, such as PU.1, RBP-Jκ, NF-κB, and STAT, are involved in this expression, but the underlying mechanism is unclear. Here, we identified binding sites for PAX5, AP-2, and EBF in the proximal LMP1 promoter (ED-L1p). We first confirmed the significance of PU.1 and POU domain transcription factor binding for activation of the promoter in latency III. We then focused on the transcription factors AP-2 and early B cell factor (EBF). Interestingly, among the three AP-2-binding sites in the LMP1 promoter, two motifs were also bound by EBF. Overexpression, knockdown, and mutagenesis in the context of the viral genome indicated that AP-2 plays an important role in LMP1 expression in latency II in epithelial cells. In latency III B cells, on the other hand, the B cell-specific transcription factor EBF binds to the ED-L1p and activates LMP1 transcription from the promoter. Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is crucial for B cell transformation and oncogenesis of other EBV-related malignancies, such as nasopharyngeal carcinoma and T/NK lymphoma. Its expression is largely dependent on the cell type or condition, and some transcription factors have been implicated in its regulation. However, these previous reports evaluated the significance of specific factors mostly by reporter assay. In this study, we prepared point-mutated EBV at the binding sites of such transcription factors and confirmed the importance of AP-2, EBF, PU.1, and POU domain factors. Our results will provide insight into the transcriptional regulation of the major oncogene LMP1.

The Multi Regulatory Role of Signal Transducer and Activator of Transcription Factor Brn-3a

In molecular biology and genetics, the transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA. Brn-3a is a POU-domain transcription factor that involve in the neurite outgrowth, induction of synaptic proteins, activation of the neurofilaments genes promoter, neuronal development, protects neuronal cells from apoptosis and plays a vital role in regulating different kinds of cancers. Brn-3a is an antiapoptotic transcription factor and the up-regulation of Brn-3a protects neuronal cells from undergoing apoptosis. Brn-3a specifically triggers the expression of Bcl-2 and Bcl- XL gene in neuronal cells. On the other hand, Brn-3a inhibits the pro-apoptotic factor like Bax. Brn-3a not only expresses in the neuronal cells but also its activity is detected in the cancer cells of non-neuronal nature. Brn-3a also modulates the activity of various others transcription factors, enzymes, receptors, ion channels, antiapoptotic proteins, adhesion molecules and cyclins. In this Review, we describe all the above factors in detail which are regulated by Brn-3a signaling in the neurology and the possible mechanisms underlying the many different functions of the Brn-3a complexes in neurological diseases. It also highlights the potential of Brn-3a as a novel target in heart development and cancer therapy.

Partial Dedifferentiation of Murine Radial Glia-Type Neural Stem Cells by Brn2 and c-Myc Yields Early Neuroepithelial Progenitors

Direct cell conversion developed into an important paradigm for generating cells with enhanced differentiation capability. We combined a transcription-factor-based cell fate conversion strategy with the use of pharmacological compounds to derive early neuroepithelial progenitor cells from developmentally more restricted radial glia-type neural stem cells. By combining the small molecules CHIR99021, Tranylcypromine, SB431542 and valproic acid with viral transduction of the transcription factor c-Myc and the POU domain transcription factor Brn2, we dedifferentiated radial glia-type neural stem cells into an early neuroepithelial progenitor cell state within 6days. Reverse transcription PCR analyses showed a rapid down-regulation of the radial glia markers Olig2 and Vimentin during conversion, whereas the neuroepithelial markers Dach1 and Sox1 were fastly up-regulated. Furthermore, a switch from N-Cadherin to E-Cadherin indicates a mesenchymal-to-epithelial transition. The differentiation of cells converted by Brn2/c-Myc yielded smooth muscle actin- and Peripherin-positive cells in addition to the neuronal marker TUJ1 and cells that are positive for the glial marker GFAP. This differentiation potential suggests that the applied reprogramming strategy induced an early neuroepithelial cell population, which might resemble cells of the neural border or even more primitive neuroepithelial cells.

Relationship of Oct-4 to malignant stage: a meta-analysis based on 502 positive/high Oct-4 cases and 522 negative/low case-free controls.

Octamer 4 (Oct-4), an important member of the POU domain transcription factor family, has been suggested to function as a master switch during differentiation of human somatic cells and more recently has come to be linked with neoplastic properties. The aim of this study was to evaluate the relationship between Oct-4 and cancer stage using a meta-analysis approach. Relevant articles published as of May 2015 were retrieved from the following databases: PubMed, ISI Web of Knowledge, Embase, and Chinese National Knowledge Infrastructure (CNKI). The strengths of relationship for outcomes of interest were estimated based on odds ratios (ORs) and 95% confidence intervals (CIs). A total of 11 articles on Oct-4 and cancer staging that collectively included 502 positive/high Oct-4 cases and 522 negative/low case-free controls were chosen. Positive/high Oct-4 was significantly associated with cancer stage in several kinds of cancer. Specifically, positive/high Oct-4 was associated with cancer stage III/IV (fixed effects: OR = 1.53, 95% CI = 1.12-2.10), primary tumor (T3-4) (random effects: OR = 1.93, 95% CI = 0.99-3.77), and cancer grade of differentiation (intermediate-poor) (random effects: OR = 3.45, 95% CI = 1.5-7.61). These findings suggest that positive/high Oct-4 is more strongly linked to stage III/IV cancer and cancer grade of differentiation, and is correlated with malignant characteristics that lead to poor prognosis in different types of cancer, especially in Asian. Given variability related to ethnicity and differences in cancer types, additional studies are warranted to establish the generalizability of our findings.

Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. Adeno-associated virus-mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b-injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP- injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. Adeno-associated virus-mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma.

Regulation of the orphan nuclear receptor Nr2f2 by the DFNA15 deafness gene Pou4f3.

Hair cells are the mechanotransducing cells of the inner ear that are essential for hearing and balance. POU4F3 – a POU-domain transcription factor selectively expressed by these cells – has been shown to be essential for hair cell differentiation and survival in mice and its mutation in humans underlies late-onset progressive hearing loss (DFNA15). The downstream targets of POU4F3 are required for hair cell differentiation and survival. We aimed to identify such targets in order to elucidate the molecular pathways involved in hair cell production and maintenance. The orphan thyroid nuclear receptor Nr2f2 was identified as a POU4F3 target using a subtractive hybridization strategy and EMSA analysis showed that POU4F3 binds to two sites in the Nr2f2 5′ flanking region. These sites were shown to be required for POU4F3 activation as their mutation leads to a reduction in the response of an Nr2f2 5′ flanking region reporter construct to POU4F3. Immunocytochemistry was carried out in the developing and adult inner ear in order to investigate the relevance of this interaction in hearing. NR2F2 expression in the postnatal mouse organ of Corti was shown to be detectable in all sensory epithelia examined and characterised. These data demonstrate that Nr2f2 is a direct target of POU4F3 in vitro and that this regulatory relationship may be relevant to hair cell development and survival.

POU3F2 (POU class 3 homeobox 2)

POU3F2, also known as BRN2, Oct7, and N-Oct3, is a member of the neural cell-specific class III POU domain transcription factors (Ryan and Rosenfeld, 1997). POU3F2-knockout causes the loss of specific neuronal lineages in the endocrine hypothalamus and the subsequent loss of the posterior pituitary gland (Nakai et al., 1995; Schonemann et al., 1995; Alvarez-Bolado et al., 1995). And also, transgenes of POU3F2 and some other few factors converted non-neural cells to neural cells in vitro (Ambasudhan et al., 2011; Lujan et al., 2012; Pang et al., 2011). These results indicate that POU3F2 is an indispensable transcription factor for neural differentiation and generation of normal nervous system, especially hypothalamus. There have been a few reports regarding the functions of POU3F2 in association with tumorigenesis. POU3F2 has been demonstrated to be an oncogene in malignant melanomas derived from the neuroectodermal cell lineage and to accelerate the growth of melanoma cells (Cook and Sturm, 2008). POU3F2 are also highly expressed in small cell lung cancers and closely associated with the cancer specific neural/neuroendocrine phenotype (Schreiber et al., 1992; Ishii et al., 2013; Sakaeda et al., 2013).

Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells.

Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). We demonstrate here that loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1.

Germ cell nuclear factor regulates gametogenesis in developing gonads.

Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.

Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

Precise regulation of gene expression during biological processes, including development, is often achieved by combinatorial action of multiple transcription factors. The mechanisms by which these factors collaborate are largely not known. We have shown previously that Isl1, a Lim-Homeodomain transcription factor, and Pou4f2, a class IV POU domain transcription factor, co-regulate a set of genes required for retinal ganglion cell (RGC) differentiation. Here we further explore how these two factors interact to precisely regulate gene expression during RGC development. By GST pulldown assays, co-immunoprecipitation, and electrophoretic mobility shift assays, we show that Isl1 and Pou4f2 form a complex in vitro and in vivo, and identify the domains within these two proteins that are responsible for this interaction. By luciferase assay, in situ hybridization, and RNA-seq, we further demonstrate that the two factors contribute quantitatively to gene expression in the developing RGCs. Although each factor alone can activate gene expression, both factors are required to achieve optimal expression levels. Finally, we discover that Isl1 and Pou4f2 can interact with other POU and Lim-Homeodomain factors respectively, indicating the interactions between these two classes of transcription factors are prevalent in development and other biological processes.

HIF‐2α/ARNT complex regulates hair development via induction of p21 Waf1/Cip1 and p27 Kip1

The hypoxia-inducible factors HIF-1α or HIF-2α form heterodimeric complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-1α/ARNT and HIF-2α/ARNT complexes activate hypoxia-inducible genes that play critical roles in angiogenesis, anaerobic metabolism, and other processes in response to O2 deprivation. HIF-2α is known to regulate the function and/or differentiation of stem cells by activating the POU domain transcription factor Oct4; however, the precise underlying mechanism is unknown. This study examined the role of HIF-2α/ARNT in hair development using conditional-knockout mice, in which Arnt was specifically deleted in keratinocytes. In wild-type mice, HIF-2α and ARNT were highly expressed in the precortex above the hair matrix, an area containing differentiating stem cells. An analysis of hair size and type in these mice showed that loss of ARNT decreased the production of zigzag hairs, corresponding to reduced expression of HIF-2α and induction of the mammalian cyclin-dependent kinase inhibitors p21(Waf1/Cip1) and p27 (Kip1). The results suggest that the HIF-2α/ARNT complex regulates hair follicle differentiation via induction of p21(Waf1/Cip1) and possibly p27(Kip1), as p27(Kip1) expression was not altered in ARNT knockout mice. The findings provide insight into a possible mechanism underlying hair growth disorders and can be useful for future studies on hair follicle response to insults, such as chemotherapy and ionizing radiation.

Do all roads lead to Oct4? The emerging concepts of induced pluripotency

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

A Conserved Oct4/POUV-Dependent Network Links Adhesion and Migration to Progenitor Maintenance

SummaryBackgroundThe class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarily conserved regulator of progenitor cell differentiation during embryonic development.ResultsHere we examine the function of Oct4 homologs in Xenopus embryos and compare this to the role of Oct4 in maintaining mammalian embryo-derived stem cells. Based on a combination of expression profiling of Oct4/POUV-depleted Xenopus embryos and in silico analysis of existing mammalian Oct4 target data sets, we defined a set of evolutionary-conserved Oct4/POUV targets. Most of these targets were regulators of cell adhesion. This is consistent with Oct4/POUV phenotypes observed in the adherens junctions in Xenopus ectoderm, mouse embryonic, and epiblast stem cells. A number of these targets could rescue both Oct4/POUV phenotypes in cellular adhesion and multipotent progenitor cell maintenance, whereas expression of cadherins on their own could only transiently support adhesion and block differentiation in both ESC and Xenopus embryos.ConclusionsCurrently, the list of Oct4 transcriptional targets contains thousands of genes. Using evolutionary conservation, we identified a core set of functionally relevant factors that linked the maintenance of adhesion to Oct4/POUV. We found that the regulation of adhesion by the Oct4/POUV network occurred at both transcriptional and posttranslational levels and was required for pluripotency.

Buffalo alpha S1-casein gene 5′-flanking region and its interspecies comparison

The expression of milk protein genes is tightly regulated in a spatio-temporal manner through the combinatorial interaction of lactogenic hormones and a set of transcription factors mediating developmental and tissue-specific gene expression. The recruitment of a unique set of transcription factors is determined by the cis-regulatory motifs present in the gene promoter region. Here, we report the isolation, sequencing, structural analysis and interspecies comparison of the 5'cis-regulatory region of the buffalo alpha S1 (αS1)-casein gene. The proximal promoter region of the buffalo αS1-casein gene harbored the insertion of a 72-bp fragment of long interspersed nuclear element of the L1_BT retrotransposon family. Among the core and vertebrate-specific promoter elements, the motifs for the binding of Brn POU domain factors (BRNF), Lim homeodomain factors (LHXF), NK6 homeobox transcription factors (NKX6), nuclear factor kappa B/c-rel (NFKB), AT-rich interactive domain factor (ARID), Brn POU domain factor 5 (BRN5), pancreatic and intestinal homeodomain transcription factor (PDX1), Distal-less homeodomain transcription factors (DLXF), T-cell factor/lymphoid enhancer-binding factor-1 (LEFF) and GHF-1 pituitary-specific POU domain transcription factor (PIT1) were over-represented in the αS1-casein gene regulatory region (Z score >4.0). The Multiple EM for Motif elicitation predicted three motifs which consisted of the sequences known to bind mammary gland factor/signal transducer and activator of transcription 5 (MGF/STAT5), estrogen receptor-related alpha (ERα), steroidogenic factor 1 (SF1) and glucocorticoid receptor (GR), indicating their potential role in the mammary gland-specific gene expression. The interspecies comparison of the proximal promoter region revealed conserved sequences for TATA boxes and MGF/STAT5 in all species, whereas activator protein 1 (AP1), pregnancy-specific mammary nuclear factor (PMF), CCAAT/enhancer binding protein (C/EBP), double-stranded and single-stranded DNA-binding protein 1 (DS1 and SS), ying and yang factor 1 (YY1), and GR half-sites were among ruminants. The functional significance of the L1_BT retrotransposon insertion on the buffalo αS1-casein gene expression needs to be experimentally validated.

Effect of triiodothyronine on developmental competence of bovine oocytes

Developmental competence of in vitro–matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro–matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo– and in vitro–produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development.

Cytoplasmic Mislocalization of POU3F4 Due to Novel Mutations Leads to Deafness in Humans and Mice

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.

POU domain transcription factor BRN2 is crucial for expression of ASCL1, ND1 and neuroendocrine marker molecules and cell growth in small cell lung cancer

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.

The POU domain transcription factors Oct-6 and Oct-11 negatively regulate loricrin gene expression in keratinocytes: association with AP-1 and Sp1/Sp3

Loricrin is a major component of the epidermal cornified cell envelope, and is expressed only in terminally differentiated keratinocytes. This cell differentiation-specific expression pattern suggests specific regulatory mechanisms for activation and suppression of loricrin gene transcription in differentiated keratinocytes. Here, we identified a regulatory element in the proximal promoter region of the loricrin gene involved in suppression of its expression in keratinocytes. A database search indicated that this sequence contained a POU transcription factor binding motif. Electrophoretic mobility shift assay revealed that Oct-1, Oct-6, and Oct-11 actually bind to the motif. Constructs with point mutations in the POU-binding motif showed increased reporter activity, indicating that the POU factors negatively regulate loricrin gene transcription. Cotransfection experiments suggested that Oct-6 and Oct-11 suppress loricrin gene transcription in a cooperative manner with AP-1 and Sp1. Furthermore, in vitro experiments indicated that the Oct-6 and Oct-11 can physically associate with both AP-1 factors and Sp1/Sp3. These findings indicate that Oct-6 and Oct-11 contribute to the regulation of loricrin gene transcription via interaction with AP-1 factors and Sp1/Sp3.