POU domain genes and the immunoglobulin octamer motif in chickens

This research was undertaken to test the hypothesis that multiple POU genes exist and that POU proteins are involved in transcriptional regulation in the chicken. The POU protein Oct-2 has been hypothesized to partially regulate immunoglobulin gene expression through its interaction with the octamer motif found in the promoter of immunoglobulin genes. Transcriptional regulation of immunoglobulin genes provides a model for tissue-specific gene expression in the immune system. In searching for a chicken Oct-2 homologue, two partial chicken POU domain genes were identified. A cDNA clone encoding the POU domain of Brn-3a was isolated and showed near identity to the human Brn-3a sequence (100% at the a.a. level). Chicken Brn-3a was mapped to linkage group E48 in the East Lansing chicken genome mapping reference population. A Skn-1/Epoc-1/Oct-11 genomic clone was identified and mapped to chicken linkage group E49, to an area of synteny with human chromosome 11q23 and mouse chromosome 9. Octamer-binding protein expression patterns in multiple chicken tissues demonstrated that octamer-binding protein complexes existed in ovary, cerebrum, liver, lung, kidney, spleen, thymus, bursa, MSB1 (T cell line), and DT40 (B cell line). Every tissue had at least two octamer-binding proteins, with a total of seven unique chicken protein complexes. A comparison between mouse and chicken octamer-binding proteins identified a unique chicken octamer-binding protein, slightly faster migrating than mouse Oct-2. Chicken Oct-1 was expressed in all tissues except liver. The chicken immunoglobulin lambda light chain promoter contains an octamer motif (-106) and a TATA box (-70). Chicken B and T lymphocyte cell lines were used to determine activity of both the promoter and the octamer motif. The promoter was functional only in the B cell line and required an enhancer sequence. Mutation of the octamer motif abolished the transcriptional activity of the immunoglobulin promoter. These results suggest that the chicken immunoglobulin promoter functions similarly to its mammalian counter parts, in that the chicken immunoglobulin lambda light chain promoter plays a role in tissue-specificity and requires an enhancer and an intact octamer motif

Identification of Melatonin-Regulated Genes in the Ovine Pituitary Pars Tuberalis, a Target Site for Seasonal Hormone Control

The pars tuberalis (PT) of the pituitary gland expresses a high density of melatonin (MEL) receptors and is believed to regulate seasonal physiology by decoding changes in nocturnal melatonin secretion. Circadian clock genes are known to be expressed in the PT in response to the decline (Per1) and onset (Cry1) of MEL secretion, but to date little is known of other molecular changes in this key MEL target site. To identify transcriptional pathways that may be involved in the diurnal and photoperiod-transduction mechanism, we performed a whole genome transcriptome analysis using PT RNA isolated from sheep culled at three time points over the 24-h cycle under either long or short photoperiods. Our results reveal 153 transcripts where expression differs between photoperiods at the light-dark transition and 54 transcripts where expression level was more globally altered by photoperiod (all time points combined). Cry1 induction at night was associated with up-regulation of genes coding for NeuroD1 (neurogenic differentiation factor 1), Pbef / Nampt (nicotinamide phosphoribosyltransferase) , Hif1α (hypoxia-inducible factor-1α), and Kcnq5 (K channel) and down-regulation of Rorβ, a key clock gene regulator. Using in situ hybridization, we confirmed day-night differences in expression for Pbef / Nampt, NeuroD1, and Rorβ in the PT. Treatment of sheep with MEL increased PT expression for Cry1, Pbef / Nampt, NeuroD1, and Hif1α, but not Kcnq5. Our data thus reveal a cluster of Cry1-associated genes that are acutely responsive to MEL and novel transcriptional pathways involved in MEL action in the PT

Regulation and Function of POU domain transcription factors, Brn-3a and Brn-3b

The generation of distinct cell types during development and their maintainance in adult multicellular organisms is achieved by the selective expression of cell or tissue specific proteins. The expression of the genes encoding these proteins are controlled primarily at the transcriptional level. This regulation is largely achieved by transcription factors which bind to specific DNA elements associated with the gene promoter. Cell-specific transcription has been attributed to families of regulatory proteins which show distinct expression patterns and interactions and which may be activators or repressors that modulate transcription activity. The POU domain family of transcription factors have been shown to be important for the development and function of neuronal cells. Brn-3a, a member of the POU IV subfamily of POU domain transcription factors, was isolated from a brain cDNA library and later shown to be expressed in sensory neurons. Its high homology to the nematode, C.elegans, unc-86 gene which has been shown to be an important factor in differentiation and development of sensory neurons, suggests a conserved role for Brn-3a in sensory neuronal development and function. We have isolated the POU domain of the novel but related Brn-3b protein from the sensory neuron- derived cell line, ND7. The results presented here report on the pattern of expression of these two factors, regulation of expression in ND7 cells and their role in modulation of transcriptional activity. Brn-3a mRNA expression was found predominantly in rat brain and DRG while Brn-3b transcript were detected in these tissues but also in uterus, cervix, ovary and testis. In the ND7 cell line, there was distinct but overlapping expression of these two factors, with Bm-3b being expressed at higher levels in the proliferating ND7 cells while Brn-3a mRNA expression predominated upon differentiatiation of these cells into a sensory neuronal like cell type. The mRNA expression of both Brn-3a and Brn-3b also appeared to be regulated by combinations of growth factors and by a cyclic AMP analogue. Brn-3a may be associated with the neurite outgrowth from these cells while Brn-3b may be characteristic of proliferating cells. Brn-3a and Brn-3b also have opposite and antagonistics effects on gene expression with Brn-3a activating transcription of a heterologous promoter via an octamer-related motif while Brn-3b repressed activity. Conditions which elevated Brn-3a mRNA also increased promoter activity in ND7 cells but not in BHK cells which lack endogenous Brn-3 expression. Brn-3a was also an activator of the cellular ?-internexin gene promoter while Brn-3b repressed its activity. Furthermore, Brn-3b appeared to interact with Brn-3a and modulated its effect on promoter activity, thus suggesting a mechanism of gene regulation by the interaction of these two factors. The amino terminus of the Brn-3a protein appeared to be necessary for efficient activation of the ?-internexin promoter but not of a heterologous promoter containing the octamer-related binding site. The sequence within the ?-internexin promoter that was bound by Brn-3a and Brn-3b to regulate promoter activity was distinct from the classic octamer sites recognized by many other POU proteins. This site was found to be centered at approximately -64 bases from the transcription initiation site. Furthermore, the single stranded 'coding' sequence appeared to bind the Bm-3 proteins with higher affinity than the double stranded oligonucleotide. This therefore represent a novel DNA binding site and binding pattern by these transcription factors. Thus, these transcription factors which have overlapping expression patterns and different effects on transcriptional activity in some cells may interact to modulate gene expression in specific cell types

Identification of genes differentially expressed in rat brain during postnatal development

During neuronal development CNS neurons extend axons over long distances. This high growth potential is lost during postnatal development resulting in very poor axonal outgrowth and regeneration in the adult CNS. This pronounced decline of axon growth potential and regenerative capability might be related to alterations in the expression level of growth-associated genes during postnatal development. The aim of the present study was the identification of candidate molecules that might be associated with axon growth, i.e. which are strongly expressed during axonal outgrowth and are downregulated as neuronal maturation proceeds. As the time periods of developmental axonal outgrowth and decrease in growth potential are well studied in rat cerebellum and entorhinal cortex, these two brain regions were chosen as model systems for analysis of gene expression patterns during axonal extension and after completion of pathway formation. In a first approach the study focused on the identification of transcription factors, because they are known to be involved in the regulation of cellular identity and differentiation and hence might also determine the intrinsic growth state of a neuron. In order to identify transcription factors from rat cerebellum and entorhinal cortex at the time of maximal axonal outgrowth, PCR with degenerate oligonucleotides, specific for the conserved DNA-binding domains of distinct transcription factor classes, was performed with cDNA from cerebellum at E18 and entorhinal cortex at P0, respectively. A limited number of PCR products could be isolated from the above brain regions by the use of primers for the POU and zinc finger family of transcription factors. Because of the small number of candidate molecules and considerable difficulties in constructing cDNA probes for further analysis this approach was not further pursued. A second approach aimed at the comparison of the transcriptional activity of young differentiating CNS neurons, which extend axons, with that of more mature neurons, which have lost growth competence. The method of suppression subtractive hybridisation (SSH) was performed in two distinct CNS tissues, rat cerebellum and entorhinal cortex, at two developmental stages (E18 and P35 for cerebellum and P0 an P10 for entorhinal cortex, respectively) in order to enrich for genes, which are downregulated during postnatal development. Several differentially expressed genes were identified, and the temporal and spatial expression pattern of some of these genes was further examined in rat brain by Northern- and in situ-hybridisation analysis at different developmental stages. One of the identified genes, rMMS2, was not known in the rat before and was characterised in this study for the first time. In addition, CRHSP-24, whose expression pattern had not previously been examined in the developing brain, was identified as a differentially expressed gene. Further analysis showed that rMMS2 and CRHSP-24 were strongly expressed in many brain regions during late embryonic and early postnatal development. Expression of both genes was significantly downregulated during the first postnatal weeks and was only weak or absent in the adult brain. As this regulated distribution correlates well with the time period of establishment of axonal connections in the developing brain, these molecules might play a role in neuronal differentiation processes. However, their function in neuronal development is not yet clear and remains to be elucidated. Because only a fraction of the enriched genes has been analysed by now the pool of subtracted genes might serve as a valuable source for the identification of further candidate genes, which might be associated with neuronal differentiation and axonal outgrowth

Transcriptional regulation of the mouse PAC1 receptor gene

The G-protein coupled receptor PAC₁ has been implicated in playing a role in neural growth, development and stress-protection, cognition, homeostasis, immunomodulation and anti-inflammatory effects. Previously, an investigation of PAC₁ gene (mADCYAP1r1) revealed a minimum promoter region -113bp to +206 relative to the transcriptional start site. Binding of the Zac1 transcription factor has been investigated but no detailed analysis has been carried out of the region downstream or upstream of -2598bp from exon 1 and a full characterisation of the Transcription Factors (TF) that govern expression of the receptor has not been performed. The aim of this study was to characterise the promoter of the mAdcyap1r1 gene and to identify transcription factor binding sites, explore their role in controlling expression. Since the mAdcyap1r1 gene is well conserved across vertebrate species, a cross-species DNA comparison was performed to detect evolutionary conserved regions. Mouse, rat, human and chimpanzee sequences were aligned, and several cross-species conserved regions rich in transcription factor binding sites have been identified and reported. 3 upstream regions AdU1, AdU2 and AdU3; 2 downstream AdD1 and AdD2; and a new minimum promoter region Ad1 -80 and including most of exon 1 to +353. 279 transcription factor binding sites were found in these regions. Luciferase reporter gene assays of these regions was performed in Neuro-2a cell lines, α-T3 neuroendocrine models and Cos-7 cells as a negative control that don't express PAC1. Results indicated a functional new basal promoter region, Ad1, that was expressed in Neuro-2a. Upstream and downstream regions showed Neuro-2a specificity but were 20 fold lower than expression from Ad1. Hydrogen peroxide treatment gave increased expression via the AdU1, Ad1 and AdD2 regions mainly in Neuro-2a cells. Electrophoretic Mobility Shift Assays indicated binding of nuclear extracts to most promoter regions. Promoter Expression data gave functional insight to the bioinformatic analysis, and transcription factor binding sites indicate further roles to be investigated in future studies.The G-protein coupled receptor PAC₁ has been implicated in playing a role in neural growth, development and stress-protection, cognition, homeostasis, immunomodulation and anti-inflammatory effects. Previously, an investigation of PAC₁ gene (mADCYAP1r1) revealed a minimum promoter region -113bp to +206 relative to the transcriptional start site. Binding of the Zac1 transcription factor has been investigated but no detailed analysis has been carried out of the region downstream or upstream of -2598bp from exon 1 and a full characterisation of the Transcription Factors (TF) that govern expression of the receptor has not been performed. The aim of this study was to characterise the promoter of the mAdcyap1r1 gene and to identify transcription factor binding sites, explore their role in controlling expression. Since the mAdcyap1r1 gene is well conserved across vertebrate species, a cross-species DNA comparison was performed to detect evolutionary conserved regions. Mouse, rat, human and chimpanzee sequences were aligned, and several cross-species conserved regions rich in transcription factor binding sites have been identified and reported. 3 upstream regions AdU1, AdU2 and AdU3; 2 downstream AdD1 and AdD2; and a new minimum promoter region Ad1 -80 and including most of exon 1 to +353. 279 transcription factor binding sites were found in these regions. Luciferase reporter gene assays of these regions was performed in Neuro-2a cell lines, α-T3 neuroendocrine models and Cos-7 cells as a negative control that don't express PAC1. Results indicated a functional new basal promoter region, Ad1, that was expressed in Neuro-2a. Upstream and downstream regions showed Neuro-2a specificity but were 20 fold lower than expression from Ad1. Hydrogen peroxide treatment gave increased expression via the AdU1, Ad1 and AdD2 regions mainly in Neuro-2a cells. Electrophoretic Mobility Shift Assays indicated binding of nuclear extracts to most promoter regions. Promoter Expression data gave functional insight to the bioinformatic analysis, and transcription factor binding sites indicate further roles to be investigated in future studies

DIFFERENTIAL ABILITIES OF THE CHICKEN PIT1 ISOFORMS TO REGULATE THE CHICKEN GROWTH HORMONE PROMOTER

Pit1, a pituitary-specific transcription factor, regulates differentiation of cells of the PIT1 lineage in the anterior pituitary. PIT1 also regulates the synthesis of peptide hormones from these cell types, including growth hormone (GH). A founding member of the POU-homeodomain family of transcription factors, PIT1 is characterized by a serine-threonine rich N-terminal transactivation domain and a C-terminal POU-domain. Alternative forms of PIT1, differing from each other in the N-terminal domain have been reported in several species, but the functional implication of having multiple isoforms is not known. Several Pit1 isoform mRNAs exist in chickens which have not been characterized. The main aim of this study was to determine which, if any, of the chicken PIT1 isoforms regulated the chicken Gh (cGh) promoter. PIT1β2, a novel isoform of chicken PIT1 was discovered, and known and novel isoforms (PIT1α, PIT1β1, PIT1β2 and PIT1γ) were characterized. A luciferase reporter construct containing 1775bp of the cGh promoter driving expression of firefly luciferase was used to determine the ability of the isoforms to regulate the target gene promoter activity in chicken LMH cells. We showed that three of the isoforms, PIT1α, PIT1β1 and PIT1β2, expressed from recombinant plasmids, regulated the cGh promoter, while PIT1γ did not. All the isoforms localized to the nucleus in both non-pituitary and pituitary cells. Results from gel-shift assays show that PIT1γ did not bind the proximal PIT1-binding site of the cGh promoter as well as the other isoforms, suggesting a possible mechanism behind the inactivity. Our result did not suggest a negative regulatory role for this isoform. In contrast, we found a functional advantage for having multiple isoforms. PIT1β1, the isoform that activated the promoter most strongly, when co-transfected with other activating isoforms, such as PIT1α and PIT1β2, induced significantly higher level of activation than one isoform alone. Whether this increased activation required, or was facilitated by, heterodimerization of two isoforms is not known. Nevertheless, identification of isoforms with specific functions will facilitate identification of their respective interacting partners, which are essential for GH gene expression

c-ABL gene expression and spermatogenesis : investigations into the possible role of octamer transcription factors

The work described in this thesis aims at the elucidation of mechanisms that govern cellular differentiation events in male germ cell development (spermatogenesis), especially during the post-meiotic phase (spermiogenesis). and in embryonal carcinoma cells. Chapter II describes the molecular characterization of a variant c-abl mANA (TSabl) that is specifically expressed at high levels during spermiogenesis and was suggested to play an important role in this proces. The TSabl mANA is transcribed from the proximal of the two c-abl promoters and is alternatively processed resulting in a removal of most of the 3'UTA, without an effect on the coding capicity of the mANAs. The high levels of this shortened mANA in post-meiotic male germ cells could be due to two not mutually exclusive mechanisms, i.e. a higher mANA stability or/and continued transcription of the gene during the later phases of spermiogenesis. Chapter Ill describes experiments that tried to address the question whether the TSabl mANA has a longer half life as a consequence of the removal of most of the 3'UTA. In chapter IV a preliminary analysis of the c-abl promoter is presented, using DNAsel footprinting and gel retardation assays. The results of these experiments hinted at the possibility that there exists a testis specific octamer binding factor that could be involved in the haploid specific regulation of gene expression. This stimulated us to undertake the experiments described in chapter V that aimed at the cloning of testis specific cDNAs encoding octamer binding factors. We show that the POU domain gene Oct2 is highly expressed in spermatogenic cells, generating two transcripts through a mechanism of alternative processing and/or promoter usage. This chapter closes with a discussion of testis specific gene expression. The temporally regulated expression of a family of octamer binding factors during 'neuronal 'differentiation of P19 EC cells is described in chapter VI. One factor, Oct6, is expressed in a bi-phasic pattern, suggesting that it might play a role at different stages of development. This factor is further characterized by cloning of the cognate eDNA and was found to be the mouse homologue of the rat Tst-1 POU gene [29]. This gene is highly expressed in rat testis but not in mouse testis (this thesis). Chapter VII describes the functional mapping of the protein domains involved in transcriptional activation and DNA binding. In chapter VIII the genomic organization of the Oct6 gene is described. Furthermore, we present an initial characterization of the Oct6 promoter, to begin to address the important question of how this transcriptional regulator is regulated itself. In the last chapter some aspects of the Oct6 gene are discussed in relation to its possible function in differentiation, drawing on examples from other members of the POU domain gene famil

Structure and expression pattern of Oct4 gene are conserved in vole Microtus rossiaemeridionalis

<p>Abstract</p> <p>Background</p> <p>Oct4 is a POU-domain transcriptional factor which is essential for maintaining pluripotency in several mammalian species. The mouse, human, and bovine <it>Oct4 </it>orthologs display a high conservation of nucleotide sequence and genomic organization.</p> <p>Results</p> <p>Here we report an isolation of a common vole (<it>Microtus rossiaemeridionalis) Oct4 </it>ortholog. Organization and exon-intron structure of vole <it>Oct4 </it>gene are similar to the gene organization in other mammalian species. It consists of five exons and a regulatory region including the minimal promoter, proximal and distal enhancers. Promoter and regulatory regions of the vole <it>Oct4 </it>gene also display a high similarity to the corresponding regions of <it>Oct4 </it>in other mammalian species, and are active during the transient transfection within luciferase reporter constructs into mouse P19 embryonic carcinoma cells and TG-2a embryonic stem cells. The vole <it>Oct4 </it>gene expression is detectable starting from the morula stage and until day 17 of embryonic development.</p> <p>Conclusion</p> <p>Genomic organization of this gene and its intron-exon structure in vole are identical to those in all previously studied species: it comprises five exons and the regulatory region containing several conserved elements. The activity of the <it>Oct4 </it>gene in vole, as well as in mouse, is confined only to pluripotent cells.</p