Reduction In Transactivation Research Articles

OR16-04 OTX2 Mutations in Congenital Hypopituitarism Patients

The transcription factor OTX2 is implicated in pituitary, ocular and craniofacial development. Mutations have been described in patients with variable congenital hypopituitarism (CH) ranging from isolated growth hormone deficiency (IGHD) to combined pituitary hormone deficiency (CPHD) with/without an ectopic posterior pituitary (EPP).We aimed (i) to establish the contribution of OTX2 mutations in the etiology of CH in a sub-cohort of patients and to study their functional consequences and (ii) establish a detailed human OTX2 expression profile in a hypothalamo-pituitary (HP) context. We screened 127 patients from national (n=103) and international centers (n=24) on the septo-optic dysplasia (SOD) spectrum with variable eye abnormalities. Eye abnormalities included micro/anophthalmia, retinal dystrophy and/or coloboma in 29 of these patients, with the rest having optic nerve hypoplasia (ONH). An EPP was reported on MRI in 35 patients. The cohort previously tested negative for mutations in HESX1, SOX2, SOX3, PROKR2 and GH1. Transactivation assays involved a dual-luciferase reporter in murine hypothalamic GT1-7 neurons transiently transfected with OTX2 constructs. In situ hybridization was performed to analyze human brain OTX2 expression during embryogenesis. Seven heterozygous OTX2 changes were identified: two chromosomal deletions spanning OTX2 in patients with micro/anophthalmia, GHD and an EPP, with one patient having cerebellar hypoplasia. Three missense substitutions resulting in truncated proteins: (i) the previously reported p.S138* and (ii) p.C170*, in two patients with retinal dystrophy, EPP and IGHD respectively, and (iii) the novel p.E79* in a patient with micro/anophthalmia, EPP and CPHD. A novel insertion-deletion resulting in a truncated protein p.S167* in a patient with microphthalmia, GHD, ONH, EPP and an enlarged abnormal pituitary, and a novel deletion resulting in a frameshift p.Val139Aspfs*39 in a patient with microcephaly, microphthalmia, ONH and an EPP were also identified. The human OTX2 variants caused a significant reduction in transactivation compared to wild type. Our gene expression data identified human OTX2 transcripts in the posterior pituitary, retina, ear, thalamus, choroid plexus, and in the hypothalamus during embryogenesis, but not in RP. To conclude, we identified OTX2 variants in 7 unrelated CH patients with eye abnormalities including 3 with retinal dystrophy and one with a cerebellar abnormality. As OTX2 is involved at multiple levels during HP development, these patients should be monitored for evolving endocrinopathies. Human OTX2 is expressed in the posterior pituitary, the retina and the ear at CS19 and 20 (between 6-7 weeks gestation), and in areas of the hindbrain at CS23, but not in RP at any stage analyzed in this study. The endocrine phenotypes in patients with OTX2 mutations are most likely of hypothalamic origin.

Comparison of E1A CR3-Dependent Transcriptional Activation across Six Different Human Adenovirus Subgroups

The largest E1A isoform of human adenovirus (Ad) includes a C-4 zinc finger domain within conserved region 3 (CR3) that is largely responsible for activating transcription of the early viral genes. CR3 interacts with multiple cellular factors, but its mechanism of action is modeled primarily on the basis of the mechanism for the prototype E1A protein of human Ad type 5. We expanded this model to include a representative member from each of the six human Ad subgroups. All CR3 domains tested were capable of transactivation. However, there were dramatic differences in their levels of transcriptional activation. Despite these functional variations, the interactions of these representative CR3s with known cellular transcriptional regulators revealed only modest differences. Four common cellular targets of all representative CR3s were identified: the proteasome component human Sug1 (hSug1)/S8, the acetyltransferases p300/CREB binding protein (CBP), the mediator component mediator complex subunit 23 (MED23) protein, and TATA binding protein (TBP). The first three factors appear to be critical for CR3 function. RNA interference against human TBP showed no significant reduction in transactivation by any CR3 tested. These results indicate that the cellular factors previously shown to be important for transactivation by Ad5 CR3 are similarly bound by the E1A proteins of other types. This was confirmed experimentally using a transcriptional squelching assay, which demonstrated that the CR3 regions of each Ad type could compete with Ad5 CR3 for limiting factors. Interestingly, a mutant of Ad5 CR3 (V147L) was capable of squelching wild-type Ad5 CR3, despite its failure to bind TBP, MED23, p300/CBP-associated factor (pCAF), or p300/CBP, suggestive of the possibility that an additional as yet unidentified cellular factor is required for transactivation by E1A CR3.

Screening of some anti-progestin endocrine disruptors using a recombinant yeast based in vitro bioassay

The present study was aimed to develop a sensitive, fast and user friendly progesterone receptor transactivation assay using recombinant yeast cells, Saccharomyces cerevisiae, modified to express human progesterone receptor (PR) and progesterone response element (PRE) driving the expression of green fluorescent protein. Stimulation of cells with increasing concentrations of progesterone resulted in significant elevation in fluorescence activity, with the minimum effective dose of progesterone being 0.1nM. RU486, significantly inhibited progesterone induced transactivation and non-progesterogenic steroids failed to transactivate PR till 10μM concentrations. About 7 different chemicals (mostly pesticides or their metabolites) like DDT and its metabolites, nonylphenol, endosulfan were screened in this assay system for their role in transactivation and they were all found to be anti-progestative and IC50 values within the range of 3–20μM. Further, the assay was used to analyze the endocrine disrupting activity of extracted water samples from leather industries known for their high content of various chemicals and it was found to be rich in anti-progestative compounds. It resulted in about 30% reduction in transactivation. In conclusion, we demonstrated that this yeast based bioassay provides a rapid and robust assay for high throughput screening of (anti)progestative compounds from various sources.

Mutations in the Amino-Terminal Domain of the Human Androgen Receptor may be Associated with Partial Androgen Insensitivity and Impaired Transactivation in vitro

The majority of genetic variations in the androgen receptor (AR) gene are point mutations leading to impairment of the DNA- or hormone-binding domains. The N-terminus encoded by the first exon of the AR-gene usually harbors disruptive mutations associated with complete androgen insensitivity syndrome (CAIS) while missense mutations related with partial androgen insensitivity syndrome (PAIS) are seemingly rare. We present a 46,XY male with scrotal hypospadias in whom we detected a S432 F point mutation within the N-terminus. Transient transfections of an AR expression plasmid carrying the S432 F mutation using Chinese Hamster Ovary (CHO) cells revealed a significant partial reduction in transactivation of the co-transfected androgen responsive (ARE) (2)TATA luciferase reporter gene thus confirming PAIS. In two further 46, XY patients with slight to moderate virilization defects, we detected an S411 N mutation, and a 9 base pair deletion leading to the loss of amino acids 409 to 411 (L-A-S), respectively. These mutations did not compromise AR-function under the chosen experimental settings. The S432 F-patient supports particular significance of the AR-N-terminus for mild forms of AIS while the functional role of the two further mutations remains unclear. The N-terminus is a species-specific AR-domain possibly also involved in contributing to target tissue selectivity of AR-actions via mediating co-regulator interactions. Therefore, mild molecular defects of the AR-N-terminus may not necessarily inhibit general transactivation properties using currently established reporter gene models.

Novel Mutations within the POU1F1 Gene Associated with Variable Combined Pituitary Hormone Deficiency

Mutations within the gene encoding the pituitary-specific transcription factor POU1F1 are associated with combined pituitary hormone deficiency (CPHD). Most of the affected individuals manifest GH, prolactin, and TSH deficiency. We have now screened 129 individuals with CPHD and isolated GH deficiency for mutations within POU1F1. Causative mutations were identified in 10 of 129 individuals (7.8%). Of these, five patients harbored the dominant negative R271W mutation, which is a well-recognized mutational hot spot. We have also identified a second frequently occurring mutation, E230K, which appears to be common in Maltese patients. Additionally, we describe two novel mutations within POU1F1, an insertion of a single base pair (ins778A) and a missense mutation (R172Q). Functional studies have revealed that POU1F1 (E230K) is associated with a reduction in transactivation, although DNA-binding affinity is similar to the wild-type protein. On the other hand, POU1F1 (R172Q) is associated with a reduction in DNA binding and transactivation, whereas POU1F1 (ins778A) is associated with loss of DNA binding and a reduction in transactivation. Our data suggest that the phenotype associated with POU1F1 mutations may be more variable, with the occasional preservation of TSH secretion. Additionally, our data revealed POU1F1 mutations in three patients who were diagnosed as having ACTH deficiency but who, on further evaluation, were found to have normal cortisol secretion. Hence, elucidation of the genotype led to further evaluation of the phenotype, with the cessation of cortisol replacement that had been commenced unnecessarily. These data reflect the importance of mutational analysis in patients with CPHD.

Simian virus 40 small t antigen mediates conformation-dependent transfer of protein phosphatase 2A onto the androgen receptor.

The tumor antigens simian virus 40 small t antigen (ST) and polyomavirus small and medium T antigens mediate cell transformation in part by binding to the structural A subunit of protein phosphatase 2A (PP2A). The replacement of B subunits by tumor antigens inhibits PP2A activity and prolongs phosphorylation-dependent signaling. Here we show that ST mediates PP2A A/C heterodimer transfer onto the ligand-activated androgen receptor (AR). Transfer by ST is strictly dependent on the agonist-activated conformation of AR, occurs within minutes of the addition of androgen to cells, and can occur in either the cytoplasm or the nucleus. The binding of ST changes the conformation of the A subunit, and ST rapidly dissociates from the complex upon PP2A A/C heterodimer binding to AR. PP2A is transferred onto the carboxyl-terminal half of AR, and the phosphatase activity is directed to five phosphoserines in the amino-terminal activation function region 1, with a corresponding reduction in transactivation. Thus, ST functions as a transfer factor to specify PP2A targeting in the cell and modulates the transcriptional activity of AR.

Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription

BackgroundThe interferon (IFN)-induced, dsRNA-dependent serine/threonine protein kinase, PKR, plays a key regulatory role in the IFN-mediated anti-viral response by blocking translation in the infected cell by phosphorylating the alpha subunit of elongation factor 2 (eIF2). The human immunodeficiency virus type 1 (HIV-1) evades the anti-viral IFN response through the binding of one of its major transcriptional regulatory proteins, Tat, to PKR. HIV-1 Tat acts as a substrate homologue for the enzyme, competing with eIF2α, and inhibiting the translational block. It has been shown that during the interaction with PKR, Tat becomes phosphorylated at three residues: serine 62, threonine 64 and serine 68. We have investigated the effect of this phosphorylation on the function of Tat in viral transcription. HIV-1 Tat activates transcription elongation by first binding to TAR RNA, a stem-loop structure found at the 5' end of all viral transcripts. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR.ResultsWe have investigated the effect of phosphorylation on Tat-mediated transactivation. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR. In vitro phosphorylation experiments with a series of bacterial expression constructs carrying the wild-type tat gene or mutants of the gene with alanine substitutions at one, two, or all three of the serine/threonine PKR phosphorylation sites, showed that these were subject to different levels of phosphorylation by PKR and displayed distinct kinetic behaviour. These results also suggested a cooperative role for the phosphorylation of S68 in conjunction with S62 and T64. We examined the effect of phosphorylation on Tat-mediated transactivation of the HIV-1 LTR in vivo with a series of analogous mammalian expression constructs. Co-transfection experiments showed a gradual reduction in transactivation as the number of mutated phosphorylation sites increased, and a 4-fold decrease in LTR transactivation with the Tat triple mutant that could not be phosphorylated by PKR. Furthermore, the transfection data also suggested that the presence of S68 is necessary for optimal Tat-mediated transactivation.ConclusionThese results support the hypothesis that phosphorylation of Tat may be important for its function in HIV-1 LTR transactivation.

FUNCTIONAL CHARACTERIZATION OF FOUR NATURALLY OCCURRING VARIANTS OF HUMAN PREGNANE X RECEPTOR (PXR): ONE VARIANT CAUSES DRAMATIC LOSS OF BOTH DNA BINDING ACTIVITY AND THE TRANSACTIVATION OF THE CYP3A4 PROMOTER/ENHANCER REGION

Metabolism of administered drugs is determined by expression and activity of many drug-metabolizing enzymes, such as the cytochrome P450 (P450s) family members. Pregnane X receptor (PXR) is a master transcriptional regulator of many drug/xenobiotic-metabolizing enzymes, including P450s and drug transporters. In this study, we describe the functional analysis of four naturally occurring human PXR (hPXR) variants (R98C, R148Q, R381W, and I403V) that we have recently identified. By a reporter gene assay using the CYP3A4 promoter/enhancer reporter in COS-7 or HepG2 cells, it was found that the R98C variant failed to transactivate the CYP3A4 reporter. The R381W and I403V variants also showed varying degrees of reduction in transactivation, depending on the dose of PXR activators, rifampicin, clotrimazole, and paclitaxel. The transcriptional activities of the R148Q variant were not significantly different from that of the wild-type hPXR. The electrophoretic mobility shift assay revealed that only the R98C variant lacked DNA binding. Furthermore, the cellular localization of the hPXR proteins was analyzed. All four variants as well as the wild-type hPXR localized exclusively to the nucleus, regardless of the presence or absence of rifampicin. These data suggest that the R98C, R381W, and I403V hPXR variants, especially R98C, may influence the expression of drug-metabolizing enzymes and transporters, which are transactivated by PXR.

Modular organization of the E2F1 activation domain and its interaction with general transcription factors TBP and TFIIH.

The transcriptional activator E2F1 regulates the expression of genes at the G1/S boundary. We have characterized interactions of the E2F1 activation domain with two general transcription factors, the TATA-box binding protein (TBP) and TFIIH. Two distinct binding sites on E2F1 were identified for TBP (amino acids 386-417 and 415-437) each of which supported activation in mammalian cells when expressed as a fusion to a heterologous DNA-binding domain. Neither of these minimal activation domains independently bound TFIIH; rather, the TFIIH binding site of E2F1 overlaps both domains. Loss of TFIIH-binding by E2F1 resulted in a 60-65% reduction in transactivation, suggesting that the E2F1/TFIIH interaction is important, but not essential, for transactivation. The retinoblastoma protein (Rb) binds directly to E2F1 and represses E2F1-mediated transactivation. We have demonstrated that recombinant Rb can compete with TBP and the p62 subunit of TFIIH for binding to immobilized E2F1. A tumorigenic form of Rb deficient in repressing E2F1-mediated transactivation is likewise deficient in displacing TBP from E2F1. We propose that competition between Rb and both TBP and TFIIH for binding to E2F1 is a mechanism by which Rb inhibits transactivation by E2F1.

Estrogen receptor residues required for stereospecific ligand recognition and activation.

The mouse estrogen receptor (mER) has been shown to exhibit stereospecific binding of certain stilbene estrogen agonists. The region of the mER involved in the stereochemical recognition of ligands was further defined using a stilbene isomer, Indenestrol B (IB). The IB compound has a chiral carbon bearing an ethyl substituent, and the wild type uterine mER has been shown to bind the enantiomers, IB-S and IB-R, with similar affinity. The wild type mER expressed in yeast exhibited a very minor preference for IB-S in transactivation (1.5-fold lower half-maximal dose than IB-R). The IB enantiomers could then be used to determine whether stereochemically distinct compounds with similar transcriptional activity utilize different amino acids in AF-2 for transactivation. Mutant mERs with glycine substitutions at Met521, His528, Met532, and Val537 were expressed in yeast and measured for IB-S- and IB-R-induced transactivation and ligand binding. The M532G mER showed a 124-fold and 50-fold reduction in transactivation induced by IB-S and IB-R, respectively, without a corresponding change in their ligand-binding affinities. Therefore, Met532 is required for transactivation induced by both IB enantiomers but does not discriminate based on stereospecificity. In contrast, the H528G mER displayed a gross change in stereospecific ligand recognition as illustrated by a 110-fold reduction in transactivation by IB-S and only a 8.8-fold decrease in activity by IB-R. As a result, H528G mER displayed a switch in ligand preference such that IB-R was now 8-fold more active than IB-S in transactivation. Therefore, His528 appears largely involved in transactivation specifically induced by IB-S but has a minimal influence in IB-S ligand binding. The remaining mutant mERs displayed wild type ligand binding and transactivation properties for the IB enantiomers illustrating no stereospecific recognition. These results imply that individual IB enantiomers bind to the mER with similar affinity but utilize at least one different amino acid within the AF-2 domain for signal transduction. The binding of stereochemically distinct ligands may alter the tertiary structure of the mER and cause repositioning of the AF-2 region that mediates transcription of specific genes and/or affect the binding of receptor-associated proteins, such as coactivators, which could influence transcription.

Alteration in the Retinoid Specificity of Retinoic Acid Receptor-β by Site-directed Mutagenesis of Arg269 and Lys220

Retinoic acid receptor-beta (RAR-beta) specifically binds retinoic acid (RA) and functions as a RA-inducible transcriptional regulatory factor. Simultaneous mutation of Arg269 and Lys220 of RAR-beta to Ala results in a dramatic reduction in both transactivation and affinity for RA along with creating a RA concentration-dependent dominant negative mutant. In this report, we found that mutation of these two amino acid residues singly and simultaneously to Gln results in mutant RAR-beta s, each displaying a more dramatic reduction in transactivation and affinity for RA than their corresponding Ala mutant, with the R269Q more profoundly affected than K220Q. Furthermore, we examined both the Ala and Gln mutants for their ability to transactivate and bind two other retinoids with different functional end groups (all-trans-retinol and all-trans-retinal). Mutation of Lys220 to either an Ala or a Gln favors transactivation and binding of retinal, while mutation of either Lys220 or Arg269 to Gln favors retinol transactivation and binding. Taken together, these results suggest that Arg269 and Lys220 lie within the ligand binding pocket of RAR-beta and Lys220 lie within the ligand binding pocket of RAR-beta and that these two amino acid residues play an important role in determining retinoid specificity most likely by directly interacting with the carboxylate group of RA.

The HIV-1 regulatory protein Nef has a specific function in viral expression in a murine macrophage cell line.

Expression of reporter genes under the control of the HIV-1 long terminal repeat (LTR) was up-regulated in the murine macrophage cell line RAW264 by cotransfection of a plasmid coding for the viral regulatory protein Nef. To determine if a discrete section of the LTR was exclusively responsive to Nef, a series of promoters was produced by successive 5' deletions from the LTR up to the boundary of the enhancer region. These truncated promoters were as active as the full-length sequence in the RAW264 cells, but elimination of the direct repeats and one of the three Sp1 sites reduced promoter activity to minimal levels. Transcription driven by all constructs was equally susceptible to the trans-activating effect of Nef and could be increased further by the addition of a Tat-expressing plasmid to the cotransfection. Open reading frames of nef from NL4-3, from HXB2, which has a premature stop, and a fully functional hybrid of the two under the control of the SR alpha artificial promoter (SV40 early promoter plus HTLV-I R-U5') were able to transactivate the LTR in RAW264 cells to the same degree as HXB3 nef under the control of the cytomegalovirus (CMV) immediate-early promoter. A frameshift mutation of Nef at the XhoI site at position 8475 did not abrogate trans-activation of the LTR in macrophages. To further define the effective trans-activation region of Nef, internal deletions were made. Changes downstream of the XhoI site at amino acid 35 resulted in little or no reduction in trans-activation, whereas a deletion between the CMV promoter of the expression plasmid and the XhoI site largely abolished activity. Nef trans-activation of the LTR may be restricted to macrophages. Parallel cotransfection experiments in COS-1 simian fibroblast-like cells showed repression of reporter expression by Nef. Results suggested that the section of nef responsible for transactivation of the LTR in macrophages differed slightly from that sufficient for trans-repression in fibroblasts. Translation of the protein from the first translation start site (Met-1) rather than from the second in-frame ATG (Met-20) appears to be necessary for the trans-activating effect of Nef in RAW264 cells. Mutation of the initial ATG to ATA led to loss of trans-activating activity. Expression of Nef also has a cytostatic/cytotoxic effect on RAW264 cells indicated by a reduced rate of establishment of stably transfected clones. The cytostatic effect of Nef was not relieved by internal deletions in the coding sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Translation of the mRNA of the maize transcriptional activator Opaque-2 is inhibited by upstream open reading frames present in the leader sequence.

The protein encoded by the Opaque-2 (O2) gene is a transcription factor, translated from an mRNA that possesses an unusually long 5' leader sequence containing three upstream open reading frames (uORFs). The efficiency of translation of O2 mRNA has been tested in vivo by a transient assay in which the level of activation of the b32 promoter, a natural target of O2 protein, is measured. We show that uORF-less O2 alleles possess a higher transactivation value than the wild-type allele and that the reduction in transactivation due to the uORFs is a cis-dominant effect. The data presented indicate that both uORF1 and uORF2 are involved in the reducing effect and suggest that both are likely to be translated.