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PubMed1991

Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication.

The origin of DNA replication of many human adenoviruses is composed of a highly conserved core origin and an auxiliary region, containing the binding sites for NFI and NFIII/Oct-1. We examined enhancement of DNA replication in vitro by the purified functional DNA-binding domains of NFI (NFI-BD) and NFIII/Oct-1 (the POU domain), using origins in which the positions of the binding sites for these proteins were transposed. Insertion or deletion of two or three base pairs between the core origin and the NFI binding site resulted in a 3-5-fold decrease of stimulation, whereas larger insertions gradually reduced the stimulation further. Mutants in which the NFI binding site was separated approximately one or two helical turns from the core origin by AT-rich sequences could still be stimulated by NFI. In contrast, insertion of two or more base pairs between the NFI and NFIII/Oct-1 binding sites abolished stimulation by NFIII/Oct-1 almost completely. Furthermore, stimulation by this protein was lost when the Ad2 NFIII/Oct-1 binding site was transposed to a position closer to the core origin, destroying the NFI binding site. This shows that the position of the NFIII/Oct-1 binding site is essential for stimulation. Models to explain these position-dependent effects on stimulation are discussed.

Adenoviridae↗
PubMed1989

A pituitary POU domain protein, Pit-1, activates both growth hormone and prolactin promoters transcriptionally.

The anterior pituitary gland provides a model for investigating the molecular basis for the appearance of phenotypically distinct cell types within an organ, a central question in development. The rat prolactin and growth hormone genes are expressed selectively in distinct cell types (lactotrophs and somatotrophs, respectively) of the anterior pituitary gland, reflecting differential mechanisms of gene activation or restriction, as a result of the interactions of multiple factors binding to these genes. We find that when the pituitary-specific 33-kD transcription factor Pit-1, expressed normally in both lactotrophs and somatotrophs, is expressed in either the heterologous HeLa cell line or in bacteria, it binds to and activates transcription from both growth hormone and prolactin promoters in vitro at levels even 10-fold lower than those normally present in pituitary cells. This suggests that a single factor, Pit-1, may be capable of activating the expression of two genes that define different anterior pituitary cell phenotypes. Because a putative lactotroph cell line (235-1) that does not express the growth hormone gene, but only the prolactin gene, appears to contain high levels of functional Pit-1, a mechanism selectively preventing growth hormone gene expression may, in part, account for the lactotroph phenotype.

Animals↗
PubMed1993

Mapping of a major surface-exposed site in herpes simplex virus protein Vmw65 to a region of direct interaction in a transcription complex assembly.

The cellular factor Oct-1 is selectively recruited, together with at least one other cellular protein (CFF), into a multicomponent transcription complex whose assembly is directed by the herpes simplex virus regulatory protein Vmw65 (VP16). The acidic carboxy terminus of Vmw65 is not involved in assembly of the complex but is absolutely required for subsequent transcriptional activation. Elucidation of the mechanism of action of Vmw65 is important for an understanding not only of combinatorial control of gene expression by POU- and homeodomain proteins but also of the interaction(s) between activation domains of regulatory proteins and components of the basal transcriptional apparatus. We used a combination of limited proteolysis with a number of site-specific proteases and immunological detection to demonstrate the presence of two main surface-exposed regions in Vmw65. We mapped these sites to within a few amino acids at positions 365-370 408/409. The site at 408/409 is indicative of a flexible exposed linker region between the acidic carboxy-terminal activation domain (residues 430-480) and an N-terminal domain involved in complex formation with the two cellular factors. The site around residues 365-370 is precisely within a region that results from this and other laboratories have shown to be critical for complex formation. Furthermore, we show that this site is selectively protected from proteolysis after complex assembly. Finally, using a series of overlapping peptide encompassing this region, we show that the eight amino acids, R-E-H-A-Y-S-R-A, from positions 360 through 367 are sufficient to inhibit complex formation by intact Vmw65. We propose that these residues contain sufficient information to selectively bind one of the cellular partners involved in complex assembly and that these residues are located in a physical surface-exposed domain of the protein.

Amino Acid Sequence↗
PubMed2025

TRIM63 Overexpression in FISH-Negative MiTF Family Altered Renal Cell Carcinoma (MiTF RCC).

TFE3 and TFEB break-apart fluorescent in situ hybridization (FISH) assays are the "gold standard" for diagnostic confirmation of microphthalmia-associated transcription factor (MiTF) family-altered renal cell carcinoma (MiTF RCC), which includes TFE3-rearranged RCC and TFEB-altered RCC. However, FISH assays, for multiple reasons, may lead to equivocal or false-negative results, especially in cryptic fusions resulting from intrachromosomal inversions involving 5' partner genes, such as non-POU domain-containing octamer-binding protein (NONO); GRIPI-associated protein 1 (GRIPAP1); RNA-binding motif protein, X chromosome (RBMX); and RNA-binding motif protein 10 (RBM10). When FISH results are negative in cases with strong morphological suspicion of the listed tumor entities, pathologists may recommend targeted RT-PCR or panel-based RNA fusion sequencing for diagnostic confirmation. Our recent RNA in situ hybridization (RNA ISH)-based study demonstrated RNA expression of the tripartite motif containing 63 (TRIM63) to be highly enriched in TFE3-rearranged RCC and TFEB-altered RCC, including 2 FISH false-negative RCC cases harboring RBM10::TFE3 fusion. Based on these observations, we hypothesized that TRIM63 positivity could aid in diagnosing cases that are negative by conventional FISH assay but remain morphologically suspicious, representing an unmet clinical need in this area. We collected 20 RCC cases with morphological suspicion (with equivocal/indeterminate immunohistochemistry panel) of MiTF RCC, which were TRIM63 positive, negative/equivocal for TFE3/TFEB gene rearrangement by FISH, and underwent next-generation sequencing (NGS). On NGS correlation, 14 of 20 (70%) FISH-negative TRIM63-positive tumors harbored an MiTF gene rearrangement. In the remaining 6 cases, we were unable to fully ascertain the MiTF rearrangement status due to the inherent limitation of the NGS panel utilized. The cases with MiTF gene rearrangement include TFE3 rearrangement in 60% (12/20) and TFEB low-level copy gains (with an additional missense mutation in 1 case) in 10% (2/20) of samples. RBM10:TFE3 fusion was seen in 67% (8/12) of TFE3-rearranged RCC in this cohort. TRIM63 RNA ISH assay could aid in identifying cases that harbor TFE3 or TFEB rearrangement associated with false-negative or equivocal TFE3/TFEB FISH results, especially those involving gene fusions with a paracentric Xp11 inversion. Overall, employment of TRIM63 RNA ISH coupled with TFE3/TFEB FISH assays and follow-up genomic interrogation enhanced diagnostic accuracy for patients with MiTF RCC.

Carcinoma, Renal Cell↗
PubMed1999

Subnuclear partitioning and functional regulation of the Pit-1 transcription factor.

Subnuclear compartmentation is postulated to play an important role in many aspects of nuclear metabolism. To directly test an application of this model to transcription factor function, we examined the subnuclear partitioning behavior of Pit-1, a tissue-specific, POU-class transactivator. Biochemical and in situ assays indicate the nuclear pool of Pit-1 is normally divided between two compartments: the majority being differentially soluble in detergent, and a significant insoluble fraction (approximately 20%) bound to the nuclear matrix. Examination of Pit-1 deletion mutants and chimeric fusions reveal the highly conserved 66 amino acid POU-specific domain contains a necessary and sufficient nuclear matrix targeting signal. The nuclear partitioning behavior of several natural or engineered point mutations of Pit-1 was also examined. Surprisingly, the inactive point mutants were completely matrix-bound, irrespective of their ability to bind Pit-1 specific DNA. These results suggest that dynamic partitioning of Pit-1 is a component of its normal transactivator function that takes place upon the insoluble nuclear substructure where transcription occurs.

Animals↗
PubMed2002

The embryonic stem cell transcription factors Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression.

The POU homeodomain protein Oct-4 and the Forkhead Box protein FoxD3 (previously Genesis) are transcriptional regulators expressed in embryonic stem cells. Down-regulation of Oct-4 during gastrulation is essential for proper endoderm development. After gastrulation, FoxD3 is generally down-regulated during early endoderm formation, although it specifically remains expressed in the embryonic neural crest. In these studies, we have found that Oct-4 and FoxD3 can bind to identical regulatory DNA sequences. In addition, Oct-4 physically interacted with the FoxD3 DNA-binding domain. Cotransfection of Oct-4 and FoxD3 expression vectors activated the osteopontin enhancer, which is expressed in totipotent embryonic stem cells. FoxA1 and FoxA2 (previously HNF-3alpha and HNF-3beta) are Forkhead Box transcription factors that participate in liver and lung formation from foregut endoderm. Although FoxD3 activated the FoxA1 and FoxA2 promoters, Oct-4 inhibited FoxD3 activation of the FoxA1 and FoxA2 endodermal promoters. These data indicate that Oct-4 functions as a corepressor of FoxD3 to provide embryonic lineage-specific transcriptional regulatory activity to maintain appropriate developmental timing.

Binding Sites↗
PubMed1993

Differential expression of four members of the POU family of proteins in activated and phorbol 12-myristate 13-acetate-treated Jurkat T cells.

The POU family of proteins binds specifically to octamer DNA motifs present in the promoters of several genes and regulates their expression. We identified the presence of four members of the POU family of proteins, Oct-1, Oct-2, Oct-T1, and Oct-T2, in the human T-cell line Jurkat. To obtain insight into the physiological role played by these proteins in T cells, we studied the time course of expression of these genes in resting, activated, and phorbol 12-myristate 13-acetate (PMA)-treated cells. The expression of the gene encoding Oct-1 (now assigned the name OTF1 for octamer-binding transcription factor 1) remained unchanged and the levels of Oct-T2 mRNA decreased with increasing time of incubation to undetectable amounts in all three states of T-cell growth. The levels of Oct-2 mRNA and protein were increased in activated cells, were increased to a lesser extent in the PMA-treated cells, and were undetectable in resting cells. The levels of the Oct-T1 transcripts increased dramatically in PMA-treated cells but not in resting or activated cells. Sequence analysis of the Oct-T1 cDNA showed an open reading frame coding for a POU domain-containing protein of 42.7 kDa. Transient transfection of the gene encoding Oct-T1 decreased the activity of the interleukin 2 gene promoter in activated Jurkat cells. Further, there is evidence for an additional octamer-binding protein, Oct-T3, in Jurkat T cells.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of fish Pit-1 cDNA and its functional binding to promoter of gene expressed in the pituitary.

Pit-1 is a pituitary-specific transcription factor responsible for activating growth hormone (GH) and prolactin genes. Here, we describe the isolation of a rainbow trout cDNA clone that contains the entire Pit-1 coding region. The deduced amino acid sequence contains 358 residues, encoding a 39-kDa protein. Comparison of the protein sequences of the rainbow trout and rat Pit-1 shows that the 160 residue POU domain at the C terminus is highly conserved (86% identical). However, homology is much weaker in the N-terminal region (56% identical), and the rainbow trout Pit-1 contains segments of 29 and 33 amino acids that are not present in rat Pit-1. The protein produced by expression of rainbow trout Pit-1 cDNA in Escherichia coli binds specifically to at least four sites in the rainbow trout GH gene promoter. Moreover, we demonstrate that the promoter region of salmon somatolactin gene, which belongs to the GH prolactin gene family and is also expressed specifically in the pituitary, has at least five rainbow trout Pit-1 binding sites. The consensus sequence of these binding sites closely matches the 9-base pair motif, (T/A)(T/A)TATNCAT, recognized by rat Pit-1. Rainbow trout Pit-1 specifically activates rainbow trout GH promoter fusion gene expression, confirming the ability of Pit-1 to bind in a transcriptionally active conformation in GH gene promoter.

Amino Acid Sequence↗
PubMed1993

Genomic structure of the POU-related hepatic transcription factor HNF-1 alpha.

Hepatocyte Nuclear Factor-1 alpha (HNF-1 alpha) is a vertebrate transcriptional regulatory protein which plays a prominent role in the activation of a large family of hepatocyte-specific genes. It is the prototype of a new subfamily of POU-related proteins important in embryogenesis and cell-type specification. Here we report on the genomic structure and DNA sequence of HNF-1 alpha of the hen. Functional domains are non-uniformly conserved between hen and mammals consistent with the notion of functions in the hen oviduct that were lost in mammals after avian mammalian separation. The unusual homeo-related DNA-binding domain is well preserved between birds and mammals. The exon/intron pattern of the gene changed during evolution. The rat gene consists of 9 exons, the chicken gene of 10. In mammals one intron was eliminated that in hens subdivides the serine-rich transactivation domain. Comparison of the sequences at this junction indicates the involvement of elements of the V(D)J recombination system in the process of intron elimination.

Amino Acid Sequence↗
PubMed1990

Characterization of a cellular factor which interacts functionally with Oct-1 in the assembly of a multicomponent transcription complex.

Induction of transcription of the immediate-early (IE) genes of herpes simplex virus involves the assembly of a DNA-binding complex containing the viral protein Vmw65 and the cellular transcription factor Oct-1. We show that Oct-1 is not sufficient for complex formation and that another cellular factor(s) which is absolutely required for complex formation can be separated from Oct-1 under native conditions. We have purified this factor by approximately 100-fold using DNA-cellulose, ion-exchange and size-exclusion chromatographies. The assay used throughout the purification procedure follows the ability of the cellular factor to form a complex when added to purified Oct-1, Vmw65 and an IE specific DNA probe. The complex forming factor (CFF) had a sedimentation coefficient of about 4.4 S (i.e. molecular mass of about 70K, under non-denaturing conditions) and the polypeptide profile of highly purified CFF demonstrated two major species with molecular masses of 80K and 70K. Unequivocal association of either of these two species with CFF activity could not presently be demonstrated due to the sensitivity of CFF to denaturation. CFF, when tested on its own or in the presence of Vmw65, did not bind to the IE-specific consensus motif. We have also used deletion mutants of Oct-1 to show that the POU domain of this protein was sufficient for CFF-dependent complex formation with Vmw65. Deletions of the POU specific region of Oct-1 significantly reduced the complex forming ability, although detectable levels of complex were reconstituted using Vmw65, CFF and just the homeodomain of Oct-1.

Animals↗
PubMed2003

A novel nonsense mutation in the Pit-1 gene: evidence for a gene dosage effect.

The POU transcription factor Pit-1 functions in the development of somatotrophs, lactotrophs, and thyrotrophs of the anterior pituitary gland. It also plays a role in cell-specific gene expression and regulation of the gene products from these cell types, GH, prolactin, and TSH, respectively. In the present report we studied a patient with severe growth failure. Provocative studies revealed undetectable GH, prolactin, and TSH levels, and her pituitary gland was hypoplastic on magnetic resonance imaging. She had a novel homozygous nonsense mutation in the 3' end of the first alpha-helix of the POU-specific domain of the Pit-1 gene. This mutation results in a truncated protein with loss of most of the Pit-1 DNA-binding domains. Interestingly, her parents, who each have one mutant allele, have evidence of mild endocrine dysfunction. Thus, two normal copies of the Pit-1 gene appear necessary for full Pit-1 gene function.

Child↗
PubMed1993

Synergistic interactions between Pit-1 and other elements are required for effective somatotroph rat growth hormone gene expression in transgenic mice.

The role of the pituitary-specific POU-domain protein, Pit-1, in GH gene activation has been established by in vitro analyses and by the observation that mutations affecting the Pit-1 genomic locus result in genetically transmitted dwarfism. To define the quantitative contribution of the two Pit-1 response elements and the potential role of other factors in GH gene activation, we systematically assessed the ability of a series of GH promoter regions to activate transgenes in the mouse anterior pituitary gland. These studies revealed that the two GH Pit-1 binding sites are necessary, but not sufficient, for efficient transcriptional activation. Transgenes containing information including only these cis-active regions are expressed at extremely low levels in the pituitary glands of transgenic mice. The addition of 35 base pairs of 5'-flanking information, contributing other elements including a thyroid hormone/retinoic acid response element, results in much higher levels of transgene expression. Sequences located upstream of this segment contribute a further 5- to 10-fold activation. Thus, while Pit-1 is required for GH gene activation, it alone can only direct minimal expression in transgenic animals. Rather, synergistic interactions between other promoter elements and Pit-1 appear to be required for expression of the transgenes at approximately the 100-fold higher levels that are characteristic of somatotrophs, and are therefore likely to be critical components of somatotroph-specific expression of the GH gene.

Animals↗
PubMed1995

Activation of the alpha-internexin promoter by the Brn-3a transcription factor is dependent on the N-terminal region of the protein.

The Brn-3a, Brn-3b, and Brn-3c proteins are closely related POU (Pit-Oct-Unc) family transcription factors which are expressed predominantly in neuronal cells. We have identified the alpha-internexin gene as the first reported, neuronally expressed, target gene whose promoter activity is modulated by these factors. Both the Brn-3a and Brn-3c factors can activate the alpha-internexin promoter while Brn-3b represses it and can prevent activation by Brn-3a. Using chimeric constructs containing different regions of Brn-3a or Brn-3b, we show that activation of the alpha-internexin promoter requires the N-terminal region of Brn-3a. In contrast the activation by Brn-3a but not Brn-3b of an artificial promoter containing a synthetic Brn-3 binding site can be shown using the same constructs to be dependent on the POU domain of Brn-3a. Moreover, the isolated POU domain of Brn-3a can activate this artificial promoter but not the alpha-internexin promoter. Hence Brn-3a contains two distinct transactivation domains, at the N terminus and within the POU domain, whose effect is dependent upon the target promoter. The relationship of gene transactivation by Brn-3a to its ability to transform primary cells which is also dependent on the N-terminal region of the protein is discussed.

Base Sequence↗
PubMed1989

The Oct-2 protein binds cooperatively to adjacent octamer sites.

Recombinant proteins derived from the cloned human oct-2 gene were used to investigate cooperative binding by Oct-2 to adjacent DNA-binding sites. Oct-2, a B-cell-specific transcription factor, binds tightly to the octamer sequence in immunoglobulin promoters. A second apparently unrelated consensus sequence in heavy chain promoters, the heptamer site, also is recognized by the Oct-2 protein but with 1000-fold lower affinity. Simultaneous occupancy of both the octamer and heptamer sites is favored by cooperative interactions. The heptamer site is probably recognized by the same binding surface in the Oct-2 protein as the octamer site and thus is conserved as a lower-affinity binding site. This permits the immunoglobulin promoter to respond to a much broader range of levels of Oct-2 protein. Substitution of prototype octamer sequences for heptamer sequences yields a probe with two octamer sites spaced by 2 nucleotides, which also binds Oct-2 protein cooperatively. Only the POU domain in the Oct-2 protein is required for this cooperative interaction. Similar protein-protein interactions between bound Oct-2 proteins may promote promoter-enhancer synergism in the heavy chain gene.

Base Sequence↗
PubMed2006

Oct-2 DNA binding transcription factor: functional consequences of phosphorylation and glycosylation.

Phosphorylation and O-GlcNAc modification often induce conformational changes and allow the protein to specifically interact with other proteins. Interplay of phosphorylation and O-GlcNAc modification at the same conserved site may result in the protein undergoing functional switches. We describe that at conserved Ser/Thr residues of human Oct-2, alternative phosphorylation and O-GlcNAc modification (Yin Yang sites) can be predicted by the YinOYang1.2 method. We propose here that alternative phosphorylation and O-GlcNAc modification at Ser191 in the N-terminal region, Ser271 and 274 in the linker region of two POU sub-domains and Thr301 and Ser323 in the POUh subdomain are involved in the differential binding behavior of Oct-2 to the octamer DNA motif. This implies that phosphorylation or O-GlcNAc modification of the same amino acid may result in a different binding capacity of the modified protein. In the C-terminal domain, Ser371, 389 and 394 are additional Yin Yang sites that could be involved in the modulation of Oct-2 binding properties.

Amino Acid Sequence↗
PubMed1994

An alternatively spliced form of Pit-1 represses prolactin gene expression.

The pituitary-specific transcription factor Pit-1 is required for expression of the PRL gene. Transcription of the PRL gene in the anterior pituitary is both activated and repressed in response to neuroendocrine signals. The molecular events that mediate repression are unknown. Transplantation of GH3 pituitary tumor cells from culture to female Wistar-Furth rats resulted in repression of PRL gene expression. When the transplanted cells were returned to culture, PRL gene expression was rapidly activated. We used this model to study potential mechanisms by which PRL gene expression was silenced. In addition to the appropriate size Pit-1 proteins of 33 and 31 kilodaltons, smaller forms of the transcription factor, migrating at approximately 27 and 24 kilodaltons, were found in transplanted cells in which PRL gene expression was repressed. These smaller forms of Pit-1 protein were observed to disappear when transplanted cells were returned to culture, coincident with the activation of PRL gene expression. A transcript approximately 170 base pairs shorter than expected for that encoding full-length Pit-1 was detected in transplanted GH3 cell RNA by polymerase chain reaction. The shorter Pit-1 transcript was abundant only in GH3 cells after in vivo passage and was not readily detected in transplanted cells after as little as 12 h in culture. This shorter transcript was found to result from excision of sequence corresponding to exon IV and encodes a Pit-1 protein lacking 54 amino acids of the POU-specific domain. Gene transfer studies demonstrated this alternative form of Pit-1 inhibited PRL promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1.

A pituitary LIM homeodomain factor, P-Lim, is expressed as Rathke's pouch forms and as specific pituitary cell phenotypes are established, suggesting functional roles throughout pituitary development. While selectively expressed in both anterior and intermediate pituitary in mature mice, P-Lim is also transiently expressed in the developing ventral neural cord and brainstem. P-Lim binds to and activates the promoter of the alpha-glycoprotein subunit gene, a marker of early pituitary development, and synergizes with Pit-1 in transcriptional activation of genes encoding terminal differentiation markers. The LIM domain of P-Lim specifically interacts with the Pit-1 POU domain and is required for synergistic interactions with Pit-1, but not for basal transcriptional activation events.

Amino Acid Sequence↗