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Results for “POU Domain Factors”

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PubMed1995

Functional domains in the oxytocin gene for regulation of expression and biosynthesis of gene products.

In the oxytocin (OT) gene several regions can be discerned that have a function in regulating its expression. Firstly, in the proximal 5' flanking region regulatory elements have been discovered that are targets for transcription factors of the nuclear hormone receptor family. Through these elements the OT gene of rat and man is responsive to estrogens, thyroid hormones and retinoids. Furthermore, these elements can be employed by the nuclear hormone orphan receptor family for repressive or inductive actions. In the distal 5' flanking region the POU class III proteins Brn-1, Brn-2, Brn-4, that are expressed in magnocellular neurons, and Oct-6 are able to bind, but do not display a significant regulatory activity on the OT gene in heterologous expression systems. Secondly, the OT precursor harbours both the biologically active hormone and the protein neurophysin that is able to associate with the hormone. Heterologous expression of wild-type and mutant vasopressin cDNAs in peptidergic cell lines shows that the highly homologous vasopressin-associated neurophysin domain associates with the hormone domain within the prohormone. This intramolecular interaction between two prohormone domains serves an essential intracellular function, i.e. the proper sorting of the prohormone into the regulated secretory pathway.

Animals↗

PubMed2002

Novel function of the transactivation domain of a pituitary-specific transcription factor, Pit-1.

Pit-1 stimulates the expression of growth hormone, prolactin, and thyrotropin beta subunit genes. Consequently, abnormality of the Pit-1 gene results in combined pituitary hormone deficiency (CPHD). In this study, we analyzed the function of Pit-1 with a mutation (proline to leucine at codon 24) in the transactivation domain, P24L, which has a normal POU domain important for binding to DNA, because this mutation had been reported in a patient with CPHD. We found that codon 24 proline in the transactivation domain as well as the POU domain of Pit-1 was crucial to recruit coactivator CREB-binding protein (CBP) in the cultured cells. P24L completely lost the responsiveness to cAMP to stimulate the expression of the Pit-1-targeted genes. Furthermore, CBP and Pit-1, but not P24L, markedly enhanced the expression of the Pit-1-targeted gene to cAMP, and adenovirus E1a that binds to CBP and abrogates its function blocked the induction by cAMP of Pit-1-stimulated gene transcription in the pituitary-derived GH3 cells. These results suggest that CBP and proline at codon 24 in the transactivation domain of Pit-1 are important for the cAMP-induced activation of Pit-1-targeted genes. However, P24L maintained basal transcriptional activity, suggesting that CBP is unlikely to be an essential coactivator for Pit-1.

Animals↗

PubMed2006

Differential utilization of transcription activation subdomains by distinct coactivators regulates Pit-1 basal and Ras responsiveness.

The POU-homeodomain transcription factor Pit-1 governs ontogeny and cell-specific gene expression of pituitary lactotropes, somatotropes, and thyrotropes. The splice isoform, Pit-1beta, inserts a 26-amino acid (AA) repressor at AA48 in the Pit-1 transcription activation domain (TAD). The Pit-1 TAD contains a basal regulatory subregion, R1 (AA1-45), and a basal and Ras-responsive region, R2 (AA46-80). To precisely map these activities, we generated GAL4-Pit-1/Pit-1betaTAD fusions and, in full-length HA-Pit-1, a series of substitution mutants of R2. Analysis in GH4 cells identified an activation domain at AA50-70, followed by an overlapping, dual-function, Ras-responsive-inhibitory domain, located from AA60-80. In contrast, GAL4-Pit-1betaTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the beta-domain, we inserted the beta-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1beta, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, beta-domain insertion at AA61 and 71 resulted in greater repression of Ras responsiveness, defining a critical R2 TAD spanning AA61-71 of Pit-1. Furthermore, Ras activation is augmented by steroid receptor coactivator 1, whereas cAMP response element binding protein-binding protein is not a Ras mediator in this system. In summary, the Pit-1/Pit-1beta TADs are composed of multiple, modular, and transferable subdomains, including a regulatory R1 domain, a basal activation region, a selective inhibitory-Ras-responsive segment, and a beta-specific repressor domain. These data provide novel insights into the mechanisms by which the Pit-1 TAD integrates DNA binding, protein partner interactions, and distinct signaling pathways to fine-tune Pit-1 activity.

Animals↗

PubMed2000

Silencing mediator for retinoid and thyroid hormone receptors interacts with octamer transcription factor-1 and acts as a transcriptional repressor.

Octamer transcription factor-1 (Oct-1) is a member of the POU (Pit-1, Oct-1, unc-86) family of transcription factors and is involved in the transcriptional regulation of a variety of gene expressions related to cell cycle regulation, development, and hormonal signals. It has been shown that Oct-1 acts not only as a transcriptional activator but also as a transcriptional repressor for certain genes. The mechanism of the repressive function of Oct-1 has not been well understood. Here we demonstrate by using the glutathione S-transferase pull-down assays and coimmunoprecipitation assays that the POU domain of Oct-1 directly interacts with a silencing mediator for retinoid and thyroid hormone receptors (SMRT). The interaction surfaces are located in the C-terminal region of SMRT, which are different from previously described silencing domains I and II or receptor interacting domains I and II. In transient transfection assays in COS1 cells, overexpression of SMRT attenuated the augmentation of Oct-1 transcriptional activity by OBF-1/OCA-B, activator for Oct-1. In pull-down assays, increasing amounts of SMRT could compete the binding of OCA-B to Oct-1 POU domain. The activity of Oct-1 could be determined by a regulated balance between SMRT and OCA-B. Furthermore, cotransfected unliganded thyroid hormone receptor enhanced the transactivation by Oct-1, and addition of 3,3',5-tri-iodo-l-thyronine obliterated the stimulatory effects. Consequently, in the presence of cotransfected thyroid hormone receptor, the octamer response element acts as an element negatively regulated by 3,3',5-tri-iodo-l-thyronine. The results suggest that the transcriptional activity of Oct-1 can be modulated by interaction through its POU domain by a silencing mediator SMRT resulting in the cross-talk between Oct-1 and nuclear receptors.

Animals↗

PubMed2002

Domains of Pit-1 required for transcriptional synergy with GATA-2 on the TSH beta gene.

Previous studies showed that Pit-1 functionally cooperates with GATA-2 to stimulate transcription of the TSH beta gene. Pit-1 and GATA-2 are uniquely coexpressed in pituitary thyrotropes and activate transcription by binding to a composite promoter element. To define the domains of Pit-1 important for functional cooperativity with GATA-2, we cotransfected a set of Pit-1 deletions with an mTSH beta-luciferase reporter. Plasmids were titrated to express equivalent amounts of protein. A mutant containing a deletion of the hinge region between the POU and homeodomains retained the ability to fully synergize with GATA-2. In contrast, mutants containing deletions of amino acids 2-80 or 72-125 demonstrated 56 or 34% of the synergy found with the full-length protein, suggesting that these regions contributed to cooperativity. Mutants with deletions of the POU-specific or homeodomain further reduced the effect signifying the requirement for DNA binding. GST interaction studies demonstrated that only the homeodomain of Pit-1 interacted with GATA-2. Finally, several mutations between the Pit-1 and GATA-2 sites on the TSH beta promoter reduced binding for each factor and greatly reduced ternary complex formation. Thus multiple domains of Pit-1 are required for full synergy with GATA-2 and sequences between the two binding sites contribute to co-occupancy with both factors on the proximal TSH beta promoter.

Animals↗

PubMed2005

An Oct-1 binding site mediates activation of the gata2 promoter by BMP signaling.

The gata2 gene encodes a transcription factor implicated in regulating early patterning of ectoderm and mesoderm, and later in numerous cell-specific gene expression programs. Activation of the gata2 gene during embryogenesis is dependent on the bone morphogenetic protein (BMP) signaling pathway, but the mechanism for how signaling controls gene activity has not been defined. We developed an assay in Xenopus embryos to analyze regulatory sequences of the zebrafish gata2 promoter that are necessary to mediate the response to BMP signaling during embryogenesis. We show that activation is Smad dependent, since it is blocked by expression of the inhibitory Smad6. Deletion analysis identified an octamer binding site that is necessary for BMP-mediated induction, and that interacts with the POU homeodomain protein Oct-1. However, this element is not sufficient to transfer a BMP response to a heterologous promoter, requiring an additional more proximal cooperating element. Based on recent studies with other BMP-dependent promoters (Drosophila vestigial and Xenopus Xvent-2), our studies of the gata2 gene suggest that POU-domain proteins comprise a common component of the BMP signaling pathway, cooperating with Smad proteins and other transcriptional activators.

Animals↗

PubMed1996

A soluble transcription factor, Oct-1, is also found in the insoluble nuclear matrix and possesses silencing activity in its alanine-rich domain.

Expression of the human thyrotropin beta (hTSHbeta) gene is restricted to thyrotrophs, at least in part, by silencing. Using transient-transfection assays, we have localized a silencer element to a region between -128 and -480 bp upstream of the transcription initiation site. The silencing activity was overcome in a thyrotroph-specific manner by an unknown enhancer located in the sequences at -approximately 10000 to -1200 bp. The ubiquitous POU homeodomain protein Oct-1 recognized the A/T-rich silencer element at multiple sites in gel mobility shift assays and in vitro footprinting analyses. The silencing activity of Oct-1 was localized in its C-terminal alanine-rich domain, suggesting that Oct-1 plays a role in silencing of the hTSHbeta promoter. Further, a significant fraction of Oct-1 was shown to be associated with the nuclear matrix, and the hTSHbeta silencer region was tethered to a nuclear matrix of human cells in vivo, suggesting a possible role of the Oct-1-hTSHbeta silencer region interaction in chromatin organization.

Animals↗

PubMed1995

The neuronal nicotinic acetylcholine receptor alpha 2 subunit gene promoter is activated by the Brn-3b POU family transcription factor and not by Brn-3a or Brn-3c.

The regulatory region of the neuronal nicotinic acetylcholine receptor alpha 2 subunit gene, which contains six copies of the octamer-related sequence CCCCATGCAAT, is activated by the Brn-3b POU family transcription factor but not by the closely related factors Brn-3a and Brn-3c. This effect is in contrast to the previously documented inhibitory effect of Brn-3b on octamer-containing promoters that are activated by Brn-3a and Brn-3c. Activation of the alpha 2 gene by Brn-3b requires that both the POU domain and other N-terminal sequences are derived from Brn-3b and is dependent on the intactness of the alpha 2 gene regulatory region, being lost in truncated derivatives containing one, two, or four copies of the octamer-related sequence. Surprisingly, however, these truncated derivatives are activated by Brn-3c. These effects are discussed in terms of both the influence of the target sequence and its context in the promoter on activation by the various forms of Brn-3 as well as of the processes that restrict expression of the alpha 2 subunit gene to a few cells in the nervous system.

Animals↗

PubMed1994

The POU domain protein Tst-1 and papovaviral large tumor antigen function synergistically to stimulate glia-specific gene expression of JC virus.

Synergism between transcriptional activators is a powerful way of potentiating their function. Here we show that the glial POU domain protein Tst-1 (also known as Oct-6 and SCIP) and large tumor antigen (T antigen) synergistically increased transcription from both the early and the late promoters of papovavirus JC in glial cells. Synergism between both proteins did not require T-antigen-mediated DNA replication or direct binding of T antigen to the promoter. The ability of T antigen to functionally cooperate with Tst-1 was contained within its N-terminal region, shown by the fact that small tumor antigen (t antigen) could substitute for T antigen in transfection experiments. In addition to this functional synergism, a direct interaction between Tst-1 and T antigen was observed in vitro. Using deletion mutants of Tst-1 and T antigen, the POU domain of Tst-1 and the N-terminal region of T antigen were found to participate in this interaction. Because of the low levels of Tst-1 present in oligodendrocytes, synergism between Tst-1 and T antigen could be an important factor in establishing the lytic infection of oligodendrocytes by JC virus during the course of the fatal demyelinating disease progressive multifocal leukoencephalopathy.

Antigens, Polyomavirus Transforming↗

PubMed2001

A point mutation in the LIM domain of Lhx3 reduces activation of the glycoprotein hormone alpha-subunit promoter.

Lhx3, a member of the LIM homeodomain family of transcription factors, is required for development of the pituitary in mice. A recent report has described a point mutation in the human LHX3 gene that is associated with a combined pituitary hormone disorder. The mutation is predicted to lead to the replacement of a tyrosine residue with a cysteine in the second LIM domain of LHX3. We have characterized the effects of this point mutation (Y114C) when analyzed in the context of the mouse Lhx3 coding sequence. Mobility shift assays demonstrated that the Lhx3 Y114C mutant is capable of binding DNA, although a decrease in the formation of a specific complex was observed. Transfection assays using an expression vector for either full-length Lhx3 or a GAL4-Lhx3 LIM domain fusion provided evidence that the Lhx3 Y114C mutant has a decreased ability to stimulate transcription. In particular, a GAL4-Lhx3 Y114C LIM mutant was unable to support Ras responsiveness of a modified glycoprotein hormone alpha-subunit reporter gene. Protein interaction studies suggest that the Y114C mutation may modestly reduce binding to the POU transcription factor, Pit-1. Interestingly, the Y114C mutation essentially abrogated binding to the putative co-activator/adapter, selective LIM-binding protein. The findings provide insights into the mechanisms mediating transcriptional activation by Lhx3 and suggest that the observed phenotype of the human mutation probably involves reduced transcriptional activity of the mutant LHX3.

Binding Sites↗

PubMed2002

Developmental expression of a class IV POU gene in the gastropod Haliotis asinina supports a conserved role in sensory cell development in bilaterians.

POU-IV genes regulate neuronal development in a number of deuterostomes (chordates) and ecdysozoans (arthropods and nematodes). Currently their function and expression in the third bilaterian clade, the Lophotrochozoa, comprising molluscs, annelids and their affiliates, is unclear. Herein we characterise the developmental expression of HasPOU-IV in the gastropod mollusc, Haliotis asinina. The POU-IV gene is transiently expressed in 11 distinct larval territories during the first 3 days of development. HasPOU-IV is first expressed in sets of ventral epidermal cells in the newly hatched trochophore larvae. As larval morphogenesis proceeds, we observe HasPOU-IV transcripts in cells that putatively form a range of sensory systems including chemo- and mechanosensory cells in the foot, cephalic tentacles, the ctenidia, the geosensory statocyst and the eyes. By comparing HasPOU-IV expression with POU-IV genes in other bilaterians we infer that this class of POU-domain genes had an ancestral role in regulating sensory cell development.

Animals↗

PubMed2006

Identification and functional analysis of the novel S179R POU1F1 mutation associated with combined pituitary hormone deficiency.

CONTEXT: The pituitary-specific transcription factor 1 plays a key role in the development and differentiation of three pituitary cell types: somatotrophs, lactotrophs, and thyrotrophs. Several mutations of the human gene (called POU1F1) have been shown to be responsible for a phenotype of combined pituitary hormone deficiency involving GH, prolactin (PRL), and TSH. OBJECTIVE: We have identified a novel homozygous C to G mutation in exon 4 of the POU1F1 gene (S179R) in a patient with this rare phenotype. We analyzed the functional consequences of this S179R mutation associated with a single-amino acid change in the POU-specific domain. METHODS: Consequences of this mutation on transcriptional activities by transfection studies in alphaT3 cells, DNA binding ability by EMSA, structural properties, and nuclear accumulation of POU1F1 were investigated. RESULTS: The transactivation capacity of this mutant was markedly decreased on the GH1, PRL, TSHbeta, and POU1F1 genes. Interestingly, this mutation abolished the functional interaction of POU1F1 on the PRL promoter with the coactivator cAMP response element-binding protein-binding protein but not with the transcription factor LIM homeodomain transcription factor 3. The S179R mutant displayed normal nuclear accumulation but a markedly decreased binding to a DNA response element in keeping with crystallographic data, suggesting that the S179R mutation might interfere with DNA binding. CONCLUSIONS: Together with previous data, our study indicates that both DNA binding and interaction with cofactors like cAMP response element-binding protein-binding protein are critical for POU1F1 function and that functional and structural properties of abnormal POU1F1 proteins are variously influenced by the type of mutations.

Adult↗

PubMed2001

Expression of the Oct-1 transcription factor and characterization of its interactions with the Bob1 coactivator.

The Oct-1 transcription factor regulates a variety of tissue-specific and general housekeeping genes by recruiting specialized coactivators of transcription. It acts synergistically with the B-cell-specific coactivator Bob1 (OCA-B, OBF-1) to stimulate transcription of immunoglobulin genes. To analyze Oct-1's interactions with Bob1 and other regulatory proteins, we have overexpressed and purified different functional domains of the recombinant proteins. A version of Oct-1 that encompasses the amino-terminal activation region and the POU DNA-binding domain was extensively characterized (OctDeltaC1; comprising residues 1-445). Using an in vitro transcription assay, we demonstrate that this fragment is sufficient and necessary to stimulate transcription from an immunoglobulin promoter with Bob1. It also coactivates from the herpes simplex virus ICPO promoter element in the presence of VP16. Using a range of spectroscopic and biophysical techniques, we demonstrate that the activation domains of Oct-1 and Bob1 have little globular structure and that they do not physically interact. Thus, their functional synergy is likely to arise by the co-recruitment of common factors as part of a larger regulatory assembly. We propose a hypothesis to explain why the activation domains of these and other transcription factors of metazoans have little if any intrinsic structure.

Amino Acid Sequence↗

PubMed1990

The octamer binding factor Oct6: cDNA cloning and expression in early embryonic cells.

We have cloned a cDNA encoding a novel octamer binding factor Oct6 that is expressed in undifferentiated ES cells. Expression of the Oct6 gene is downregulated upon differentiation of these cells by aggregate formation. Furthermore the gene is transiently up regulated during retinoic acid induced differentiation of P19 EC cells, reaching maximum levels of expression one day after RA addition. Sequence analysis of the cDNA encoding the Oct6 protein indicated that the Oct6 gene is a member of the POU-HOMEO domain gene family. The gene expresses a 3 kb mRNA encoding a 449 amino acid protein with an apparent molecular weight of 45 kD. The sequence of the Oct6 POU domain is identical to that of the rat SCIP (Tst-1) gene. The Oct6 expression pattern suggests a role for this DNA binding protein in neurogenesis as well as early embryogenesis.

Amino Acid Sequence↗

PubMed1999

Herpes simplex virus transactivator VP16 discriminates between HCF-1 and a novel family member, HCF-2.

Herpes simplex virus infection is initiated by VP16, a viral transcription factor that activates the viral immediate-early (IE) genes. VP16 does not recognize the IE gene promoters directly but instead forms a multiprotein complex with Oct-1 and HCF-1, a ubiquitous nuclear protein required for progression through the G1 phase of the cell cycle. The functional significance of recruiting HCF-1 to the VP16-induced complex is not understood. Here we describe the identification of a second HCF-like protein, designated HCF-2. HCF-2 is smaller than HCF-1 but shares three regions of strong amino acid sequence homology, including the beta-propeller domain required for association with VP16. HCF-2 is expressed in many tissues, especially the testis, and shows a more dynamic pattern of subcellular localization than HCF-1. Although HCF-2 associates with VP16 and can support complex assembly with Oct-1 and DNA, it is significantly less efficient than HCF-1. A similar preference is shown by LZIP, a cellular counterpart of VP16. Analysis of chimeric proteins showed that differences between the fifth and sixth kelch repeats of the beta-propeller domains from HCF-1 and HCF-2 dictate this selectivity. These results reveal an unexpected level of specificity in the recruitment of HCF-1 to the VP16-induced complex, paralleling the preferential selection of Oct-1 rather than the closely related POU domain protein Oct-2. Implications for regulation of the viral life cycle are discussed.

Amino Acid Sequence↗

PubMed1992

An alternatively spliced Pit-1 isoform altered in its ability to trans-activate.

Although alternative splicing has been shown to give rise to isoforms of a number of transcription factors, such isoforms have not previously been detected for the POU homeodomain protein Pit-1. Screening of a rat pituitary GH3 cell cDNA expression library yielded a clone, termed pCMVPit-1a, encoding a 35.8 kD protein (Pit-1a) containing a 26 amino acid insert in the Pit-1 trans-activation domain. The position of the insert, plus Southern blot analysis, implied that Pit-1a mRNA arises by alternative splicing of the Pit-1 gene transcript. Pit-1a mRNA was detected in GH3 rat pituitary tumor cells at levels about 1/7 that of Pit-1 mRNA. Pit-1a mRNA-specific sequences were also detected in rat and mouse pituitary, and in mouse thyrotropic tumor TtT cells. DNA mobility shift assays showed that Pit-1a binds specifically to Pit-1 binding sites in the proximal prolactin promoter, but produces DNA-protein complexes of markedly different mobilities than Pit-1. In stably transfected CHO cells which accumulated approximately equal levels of either of the two proteins, Pit-1 trans-activated a prolactin promoter-driven CAT construct, while Pit-1a yielded no detectable transactivation, implying a trans-activation ratio for Pit-1a/Pit-1 of less than 0.05. Thus, the insertion of 26 amino acids of similar composition into the activation domain of Pit-1 has at once affected both the mode of binding of this protein and its ability to function as a trans-activator.

Amino Acid Sequence↗

PubMed1992

Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity.

Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif. Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells, however. We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line. Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells. This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation. Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation. Moreover, this domain conferred enhancing activity when fused to the Oct1 protein, which by itself was unable to stimulate from a remote position. The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position.

Animals↗

PubMed2004

A new single nucleotide polymorphism in the chicken pituitary-specific transcription factor (POU1F1) gene associated with growth rate.

The pituitary-specific transcription factor (POU1F1) is a protein which binds to and transactivates promoters of growth hormone (GH), prolactin (PRL) and thyroid-stimulating hormone chain (TSHB)-encoding genes. Ten chicken populations (n = 662), including six Chinese indigenous breeds, White Leghorn, paternal/maternal lines of brown egg layer and a paternal line of broiler, were used to detect single nucleotide polymorphisms in the pituitary-specific transcription factor gene (PIT1) by means of PCR-SSCP. A nucleotide transversion from adenine (A) to thymidine (T) at position 980 of the open reading frame of the PIT1 cDNA (GenBank accession no. AF029892) was identified. This nucleotide transversion results in an alteration of codon 299 from AAC to ATC, which leads to a change from asparagine (Asn) to isoleucine (Ile) in the POU domain of POU1F1. The distribution of allele and genotype frequencies differed significantly between meat-type chickens (higher frequencies of A and A/A) and layer-type chickens (P < 0.01). Another experimental population with growth records was used to evaluate the relationship between this polymorphism and growth rate. The results revealed a positive relationship between genotype A/A and body weight at 8 weeks of age, indicating that the SNP in PIT1 gene is a potential molecular marker for early growth rate in chicken.

Animals↗