Reduced Transactivation Activity Research Articles

Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li–Fraumeni syndrome

Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li–Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.

Analysis of IE62 mutations found in Varicella-Zoster virus vaccine strains for transactivation activity.

Live attenuated vaccine strains have been developed for Varicella-Zoster virus (VZV). Compared to clinically isolated strains, the vaccine strains contain several non-synonymous mutations in open reading frames (ORFs) 0, 6, 31, 39, 55, 62, and 64. In particular, ORF62, encoding an immediate-early (IE) 62 protein that acts as a transactivator for viral gene expression, contains six non-synonymous mutations, but whether these mutations affect transactivation activity of IE62 is not understood. In this study, we investigated the role of non-synonymous vaccine-type mutations (M99T, S628G, R958G, V1197A, I1260V, and L1275S) of IE62 in Suduvax, a vaccine strain isolated in Korea, for transactivation activity. In reporter assays, Suduvax IE62 showed 2- to 4-fold lower transactivation activity toward ORF4, ORF28, ORF29, and ORF68 promoters than wild-type IE62. Introduction of individual M99T, S628G, R958G, or V1197A/I1260V/L1275S mutations into wild-type IE62 did not affect transactivation activity. However, the combination of M99T within the N-terminal Sp transcription factor binding region and V1197A/I1260V/L1275S within the C-terminal serine-enriched acidic domain (SEAD) significantly reduced the transactivation activity of IE62. The M99T/V1197A/I1260V/L1275S mutant IE62 did not show considerable alterations in intracellular distribution and Sp3 binding compared to wild-type IE62, suggesting that other alteration(s) may be responsible for the reduced transactivation activity. Collectively, our results suggest that acquisition of mutations in both Met 99 and the SEAD of IE62 is responsible for the reduced transactivation activity found in IE62 of the VZV vaccine strains and contributes to attenuation of the virus.

IRF9-Stat2 Fusion Protein as an Innate Immune Inducer to Activate Mx and Interferon-Stimulated Gene Expression in Zebrafish Larvae.

Virus infection often causes large amounts of mortality during teleost larvae stage. Strong induction of innate immunity to increase survival rates of teleost larvae has been less reported. In this study, we present a zebrafish IRF9-Stat2 fusion protein (zIRF9-S2C) as a strong innate immunity inducer and characterized induction of interferon-stimulated genes (ISGs) in zebrafish larvae. zIRF9-S2C could mimic IFN-stimulated gene factor 3 (ISGF3) complex to constitutively activate transcription of Mx promoter through IFN-stimulatory element (ISRE) sites. Mutation of two ISRE sites on Mx promoter reduced transactivation activities of Mx promoter induced by zIRF9-S2C. An electrophoretic mobility shift assay experiment shows that zIRF9-S2C could directly bind to two ISRE sites of Mx promoter. Induction of transactivation of Mx promoter by zIRF9-S2C shows significantly higher activity than by zebrafish IFN1 (zIFN1), IFNγ (zIFNγ), and Tetraodon IRF9-S2C (TnIRF9-S2C). zIRF9-S2C raises transcription of Mxa, Mxb, Mxc, Ifnφ1, Ifnφ2, and Ifnφ3 in zebrafish liver ((ZFL) cell line) cells and zebrafish larvae. Collectively, we suggest that IRF9-S2C could activate transcription of ISGs with species-specific recognition and could be an innate immunity inducer in teleost larvae.

Association of a low-frequency variant in HNF1A with type 2 diabetes in a Latino population.

Latino populations have one of the highest prevalences of type 2 diabetes worldwide. To investigate the association between rare protein-coding genetic variants and prevalence of type 2 diabetes in a large Latino population and to explore potential molecular and physiological mechanisms for the observed relationships. Whole-exome sequencing was performed on DNA samples from 3756 Mexican and US Latino individuals (1794 with type 2 diabetes and 1962 without diabetes) recruited from 1993 to 2013. One variant was further tested for allele frequency and association with type 2 diabetes in large multiethnic data sets of 14,276 participants and characterized in experimental assays. Prevalence of type 2 diabetes. Secondary outcomes included age of onset, body mass index, and effect on protein function. A single rare missense variant (c.1522G>A [p.E508K]) was associated with type 2 diabetes prevalence (odds ratio [OR], 5.48; 95% CI, 2.83-10.61; P = 4.4 × 10(-7)) in hepatocyte nuclear factor 1-α (HNF1A), the gene responsible for maturity onset diabetes of the young type 3 (MODY3). This variant was observed in 0.36% of participants without type 2 diabetes and 2.1% of participants with it. In multiethnic replication data sets, the p.E508K variant was seen only in Latino patients (n = 1443 with type 2 diabetes and 1673 without it) and was associated with type 2 diabetes (OR, 4.16; 95% CI, 1.75-9.92; P = .0013). In experimental assays, HNF-1A protein encoding the p.E508K mutant demonstrated reduced transactivation activity of its target promoter compared with a wild-type protein. In our data, carriers and noncarriers of the p.E508K mutation with type 2 diabetes had no significant differences in compared clinical characteristics, including age at onset. The mean (SD) age for carriers was 45.3 years (11.2) vs 47.5 years (11.5) for noncarriers (P = .49) and the mean (SD) BMI for carriers was 28.2 (5.5) vs 29.3 (5.3) for noncarriers (P = .19). Using whole-exome sequencing, we identified a single low-frequency variant in the MODY3-causing gene HNF1A that is associated with type 2 diabetes in Latino populations and may affect protein function. This finding may have implications for screening and therapeutic modification in this population, but additional studies are required.

Mechanistic Deficits Of a Leukemogenic GATA-2 Mutant

Recent studies have demonstrated a role for the master regulator of hematopoiesis GATA-2 in MonoMAC Syndrome, a human immunodeficiency disorder associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Though GATA2 coding region and cis-regulatory element mutations underlie MonoMAC syndrome, many questions remain unanswered regarding how GATA-2 is controlled physiologically and how it is dysregulated in pathological contexts. We dissected how a T354M mutation in the GATA-2 DNA binding zinc finger, which is frequently detected in MonoMAC syndrome and familial MDS/AML, alters GATA-2 activity. The T354M mutation reduced GATA-2 chromatin occupancy, induced GATA-2 hyperphosphorylation, and disrupted GATA-2 subnuclear localization. These molecular phenotypes also characterized an additional familial MDS/AML-associated GATA-2 mutant (Δ355T). T354M hyperphosphorylation and ectopic subnuclear localization were detected in hematopoietic and non-hematopoietic cell lines. We developed a new model system in mouse aortic endothelial (MAE) cells to quantitate GATA-2 activity to regulate endogenous target genes. T354M exhibited significantly reduced activity in this assay (GATA-2: 200-fold activation; T354M: 7.7-fold activation). Mass spectrometric analysis of the phosphorylation states of GATA-2 and T354M revealed that the T354M mutation enhanced phosphorylation at several GATA-2 residues. Analysis of single phosphorylation site mutants indicated that only mutation of S192 (S192A) abolished T354M-induced hyperphosphorylation. The S192A mutation attenuated phosphorylation of sites within wild-type GATA-2 and reduced transactivation activity (50% decrease, p < 0.01). A distinct 60 amino acid (aa) region within the GATA-2 N-terminus was required for T354M hyperphosphorylation and ectopic subnuclear localization. Deletion of this sequence decreased GATA-2 transactivation activity (60 aa deletion: 85% decrease, p < 0.01; 10 aa deletion: 45% decrease, p < 0.05). GATA-1 lacks an analogous subnuclear targeting sequence, and accordingly, a GATA-1(T263M) mutant, which corresponds to the GATA-2(T354M) mutant, localized normally and was not hyperphosphorylated. However, a GATA-1 chimera containing the GATA-2 subnuclear targeting sequence localized to ectopic subnuclear foci in a T263M-dependent manner. The GATA-2 N-terminus endowed GATA-1 with the capacity to induce GATA-2 target genes. By contrast, a GATA-2 chimera containing the GATA-1 N-terminus exhibited normal subnuclear localization. Thus, the leukemogenic T354M mutation utilizes the GATA-2-specific subnuclear targeting sequence to disrupt the normal subnuclear localization pattern, and this disruption is associated with S192-dependent hyperphosphorylation. In addition to its involvement in AML, GATA-2 interfaces with RAS signaling to promote the development of non-small cell lung cancer. We discovered that RAS signaling promotes S192-dependent GATA-2 hyperphosphorylation and ectopic subnuclear localization and propose that GATA-2 is an important component in oncogenic RAS-dependent leukemogenesis, which is being formally tested using innovative mouse models. In summary, dissecting the mechanistic deficits of a leukemogenic GATA-2 mutant revealed unexpected insights into mechanisms underlying physiological GATA-2 function and GATA-2-dependent hematologic pathologies. Disclosures:No relevant conflicts of interest to declare.

Autoimmune regulator is acetylated by transcription coactivator CBP/p300

The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes.

ChemInform Abstract: Non‐Steroidal Dissociated Glucocorticoid Agonists: Indoles as A‐Ring Mimetics and Function‐Regulating Pharmacophores.

AbstractSAR analysis of non‐steroidal glycocorticoid mimetics lead to the discovery of new agonist (I), which shows high progesterone receptor and mineralocorticoid receptor selectivity, potent agonist activity, and reduced transactivation activity in the MMTV (mouse mammary tumor virus) and aromatase assays.

An in vivo proteomic study of the interaction between Salmonella Typhimurium and porcine ileum mucosa

The enteropathogen Salmonella Typhimurium is one of the main causes of porcine and human enterocolitis. We have used a 2-DE, MALDI-TOF/TOF-based approach to characterize in vivo proteome changes in porcine ileum mucosa after pathogen interaction. Ileum samples from non-infected and orally infected animals were collected at 2 days post infection and S. Typhimurium presence was confirmed by immunohistochemistry. Fifty one proteins, involved in immune response (acute phase response, inflammation and immune response regulation), apoptosis and pathogen-mediated cell invasion, were identified as being differentially expressed after pathogen challenge. Overall, anti-inflammatory signals and a possible down-regulation of dendritic cell maturation were observed. According to this, we identified the up-regulation of FK506-binding protein 4 (FKBP4), a negative regulator of the transcription factor IRF4 (interferon regulatory factor 4), implicated in Th2 and Th17 response. Transcriptional analysis using RT-qPCR indicated a general trend toward down-regulation of Th2 and Th17 cytokines genes, which would be in agreement with an IRF4 reduced transactivation activity. On the other hand, proteins that could be involved in maturation of Salmonella-containing vacuole and intracellular pathogen survival were up-regulated. Results derived from this study would be valuable to better characterize a possible pathogen led modulation of host responses in vivo.

Discovery of a Tamoxifen-Related Compound that Suppresses Glial l-Glutamate Transport Activity without Interaction with Estrogen Receptors

We recently found that tamoxifen suppresses l-glutamate transport activity of cultured astrocytes. Here, in an attempt to separate the l-glutamate transporter-inhibitory activity from the estrogen receptor-mediated genomic effects, we synthesized several compounds structurally related to tamoxifen. Among them, we identified two compounds, 1 (YAK01) and 3 (YAK037), which potently inhibited l-glutamate transporter activity. The inhibitory effect of 1 was found to be mediated through estrogen receptors and the mitogen-activated protein kinase (MAPK)/phosphatidylinositol 3-kinase (PI3K) pathway, though 1 showed greatly reduced transactivation activity compared with that of 17β-estradiol. On the other hand, compound 3 exerted its inhibitory effect through an estrogen receptor-independent and MAPK-independent, but PI3K-dependent pathway, and showed no transactivation activity. Compound 3 may represent a new platform for developing novel l-glutamate transporter inhibitors with higher brain transfer rates and reduced adverse effects.

Discovery of selective glucocorticoid receptor modulator MK-5932

A series of partial agonists of the Glucocorticoid Receptor were prepared targeting reduced transactivation activity, while maintaining significant transrepression activity. Incorporation of an ortho-aryl amide produced compounds with the desired in vitro profile. Bioreactors consisting of Suspension cultures of Sf21 cells co expressing a CYP3A4 and NADPH-cytochrome P450 oxireductase were used to prepare the major metabolites of these compounds and revealed that oxidative N-dealkylation provided a pathway for formation of metabolites that were more agonistic than the parent partial agonists. Oxidative N-dealkylation was blocked in a new series of compounds, however oxidation alone was capable of producing full agonist metabolites. Incorporation of an ortho-primary amide and utilization of fluorine to modulate agonism afforded partial agonist MK-5932. Synthesis of the major metabolites of MK-5932 using bioreactor technology revealed that no significant GR-active metabolites were formed. Orally administered MK-5932 displayed anti-inflammatory efficacy in a Rat Oxazolone-induced chronic dermatitis model, while sparing plasma insulin.

Mutation Analysis of NR5A1 Encoding Steroidogenic Factor 1 in 77 Patients with 46, XY Disorders of Sex Development (DSD) Including Hypospadias

BackgroundMutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias.Methodology/Principal FindingsWe evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein.Conclusions/SignificanceMutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations.

Non-steroidal dissociated glucocorticoid agonists: indoles as A-ring mimetics and function-regulating pharmacophores

We report a SAR of non-steroidal glucocorticoid mimetics that utilize indoles as A-ring mimetics. Detailed SAR is discussed with a focus on improving PR and MR selectivity, GR agonism, and in vitro dissociation profile. SAR analysis led to compound (R)-33 which showed high PR and MR selectivity, potent agonist activity, and reduced transactivation activity in the MMTV and aromatase assays. The compound is equipotent to prednisolone in the LPS-TNF model of inflammation. In mouse CIA, at 30 mg/kg compound (R)-33 inhibited disease progression with an efficacy similar to the 3 mg/kg dose of prednisolone.

Abstract 2477: The effect of dissociated glucocorticoids on 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin inflammation in SENCAR mice

Abstract Glucocorticoids are very effective in preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation when applied to skin together with a carcinogen. Their control of cellular functions is mediated by a well-known transcription factor, glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding and protein-protein interaction between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1), which is independent of DNA binding. We hypothesize that only the transrepression activities of the GR are sufficient to suppress skin tumor development. To test our hypothesis, we have synthesized two glucocorticoids (RU24858 and RU24782) that induce only transrepression activities of the GR. These two compounds are able to bind to the GR with high affinity and repress the AP-1 and NF-κB activity while showing reduced transactivation activities in vitro. Different doses (0.001, 0.01 and 0.1 mg) of RU24858, RU24782, desoxymethasone (DES) and fluocinolone acetonide (FA) or acetone were applied to the dorsal skin of female SENCAR mice 20 min. prior to TPA application (2 μg), two times per week for two weeks. Keratinocytes proliferation rates were estimated by measuring epidermis thickness and BrdU incorporation index. Immunohistochemistry and RT-PCR techniques were also used to evaluate expression level of genes involved in inflammation and cell cycle regulation. All studied compounds did not influence keratinocytes proliferation when applied alone. Application of DES, FA and RU24858 reversed TPA induced hyperplasia, in dose-depended manner, while RU24782 treatment had no effect on epidermis thickness or the BrdU incorporation index in TPA treated skins. All tested compounds were able to decrease c-jun mRNA level both in acetone and TPA-treated skins. DES, FA and RU24858 were also able to decrease elevated by TPA mRNA level of IL-6, COX-2 and iNOS. RU24782 did not influence mRNAs levels of these genes. These findings suggest that RU24782 had no effect on TPA-induced inflammation and hyperplasia in SENCAR mice, while RU24858 was able to reduce all tested inflammation markers, having only slight effect on epidermal hyperplasia. Supported by NIH grants CA 0796065-06 and P30 CA 54174-16S1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2477.

Several Acidic Amino Acids in the N-domain of Insulin-like Growth Factor-binding Protein-5 Are Important for Its Transactivation Activity

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions. IGFBP-5 is also found in the nuclei of cultured cells and has transactivation activity. Here we report the nuclear localization of endogenous IGFBP-5 in mouse embryonic skeletal cells. Chromatin immunoprecipitation experiments indicated that IGFBP-5 interacts with the nuclear histone-DNA complex. Using a series of deletion mutants, the transactivation domain of IGFBP-5 was mapped to its N-terminal region. Intriguingly, the transactivation activity of IGFBP-5 is masked by negative regulatory elements located in the L- and C-domains. Among the other IGFBPs, the N-domains of IGFBP-2 and -3 also had strong transactivation activity, whereas those of IGFBP-1 and -6 had no activity. The IGFBP-4 N-domain had modest activity. Sequence analysis revealed several amino acids in the IGFBP-5 N-domain that are not present in IGFBP-1. The activities of mutants in which these residues were changed to the corresponding IGFBP-1 sequence were determined. Mutations that changed acidic residues to neutral residues (e.g. E8A, D11S, E12A, E30S/P31A, E43L, and E52A) or a polar to a basic residue (e.g. Q56R) significantly reduced transactivation activity. The E8A/D11S/E12A triple mutant and E52A/Q56R double mutants showed further reduced activity. The combinatory mutants had essentially no transactivation activity. Taken together, our results indicate that there are several conserved residues in the IGFBP-5 N-terminal region that are critical for transactivation and that IGFBP-2 and -3 also have strong transactivation activity in their N-domains.

Insulin promoter factor-1 mutations and diabetes in Trinidad: identification of a novel diabetes-associated mutation (E224K) in an Indo-Trinidadian family.

This study investigated the prevalence of insulin promoter factor-1(IPF-1) mutations in familial early-onset diabetes mellitus in Trinidad. We screened 264 unrelated subjects with type 2 diabetes diagnosed before 40 yr of age and a family history of diabetes for mutations in the minimal promoter and coding region of the IPF-1 gene (IPF1). This study population included 169 patients of East Indian descent (Indo-Trinidadians), 66 of African descent (Afro-Trinidadians), and 29 of mixed ancestry. We identified five IPF1 variants, including one new missense mutation E224K, the previously described diabetes-associated duplication P242 P243dupP, two silent mutations in the codons for Leu54 (c.162G>A) and Ala256 (c.768C>A), and a substitution in the 5'-untranslated region (c.-18C>T). The E224K mutation was found in two unrelated diabetic Indo-Trinidadians and 0 of 60 controls. It was present on the same haplotype in both patients suggesting a founder effect. The E224K mutation cosegregated with early-onset diabetes or impaired glucose tolerance in a large family, suggestive of the type 4 form of maturity-onset diabetes of the young rather than type 2 diabetes. Functional studies of E224K showed reduced transactivation activity. IPF1 mutations leading to synthesis of a mutant protein may contribute to the development of familial early-onset diabetes/maturity-onset diabetes of the young in Indo-Trinidadians.

Role of conserved hydrophobic amino acids in androgen receptor AF-1 function.

The intracellular androgen receptor (AR) is a ligand-activated transcription factor. Upon binding the steroids testosterone or dihydrotestosterone, the activated receptor translocates to the nucleus, binds to specific DNA response elements and interacts with the transcription machinery in order to regulate gene transcription. In the present study, we have described a highly conserved region (amino acids 224-258) within the AR AF-1 domain and have investigated the role of conserved bulky hydrophobic residues in gene regulation. Mutating pairs of residues (I229A/L236A; V240A/V242A; L251A/L254A) reduced transactivation activity by 25-40%. Mutating residues M244, L246 and V248 to alanines had a more dramatic affect on receptor activity, disrupting activity by at least 60%. The latter mutations also disrupted binding to the RNA polymerase-associated protein 74 subunit of the general transcription factor TFIIF. The protein conformation and stability of the mutant polypeptide in vitro was not significantly different from the wild type. None of the mutations tested disrupted binding of the AF-1 domain with the coactivator protein steroid receptor coactivator-1a. Thus we have concluded that conserved hydrophobic residues are important for receptor-dependent gene transcription and that M244, L246 and V248 are part of the binding interface for TFIIF.

Phosphorylation of class II transactivator regulates its interaction ability and transactivation function.

The MHC class II transactivator (CIITA) plays a central role in adaptive immune responses by controlling the expression of MHC class II genes. CIITA binds DNA-binding proteins and co-activator proteins to form an enhanceosome complex necessary for MHC class II gene expression. Here we demonstrate that CIITA interactions depend upon the phosphorylation status of CIITA. Hyper-phosphorylated CIITA interacts with co-activator p300, RFX5 and CIITA itself, which in turn results in induction of MHC class II promoter activity. Moreover, the C-terminal region of CIITA containing leucine-rich repeats (LRR) is a regulatory domain for CIITA self-association and LRR binding to CIITA is negatively regulated by phosphorylation. cAMP-dependent protein kinase (PKA) phosphorylates CIITA, and serine residues residing in a region between the proline/serine/threonine-rich domain and the GTP-binding domain are phosphorylated by PKA in vitro. The maximum transactivation potential of CIITA requires PKA phosphorylation as demonstrated by reduced transactivation activities of the mutant bearing substitutions of serine residues at the PKA site.

Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder.

Novel N-Terminal Variant of Human VDR

The importance of N-terminal regions of nuclear hormone receptors in transcriptional regulation is increasingly recognized. As variant VDR gene transcripts indicated possible N-terminally extended receptors, we investigated their natural occurrence, transactivation capacity, and subcellular localization. A novel 54-kDa VDRB1 protein, in addition to the previously recognized 48-kDa VDRA form, was detected in human kidney tissue as well as in osteoblastic (MG63), intestinal (Int-407, DLD-1, and COLO 206F), and kidney epithelial (786) human cell lines by Western blots using isoform-specific and nonselective anti-VDR antibodies. VDRB1 was present at approximately one-third the level of VDRA. Isoform-specific VDRB1 expression constructs produced lower ligand-dependent transactivation than VDRA when transiently transfected with a vitamin D-responsive promoter into cell lines with low endogenous VDR. Intracellular localization patterns of the green fluorescent protein-tagged VDR isoforms differed. VDRB1 appeared as discrete intranuclear foci in the absence of 1,25-dihydroxyvitamin D3, whereas VDRA produced diffuse nuclear fluorescence. After 1,25-dihydroxyvitamin D3 treatment, both VDR isoforms exhibited similar diffuse nuclear signal. In the absence of 1,25-dihydroxyvitamin D3, the VDRB1 foci partially colocalized with SC-35 speckles and a subset of promyelocytic leukemia nuclear bodies. These data provide the first evidence of VDRB1, a novel N-terminally variant human VDR that is expressed at a level comparable to VDRA in human tissue and cell lines. It is characterized by reduced transactivation activity and a ligand-responsive speckled intranuclear localization. The intranuclear compartmentalization and altered functional activity of VDRB1 may mediate a specialized physiological role for this receptor isoform.

Novel N-Terminal Variant of Human VDR

The importance of N-terminal regions of nuclear hormone receptors in transcriptional regulation is increasingly recognized. As variant VDR gene transcripts indicated possible N-terminally extended receptors, we investigated their natural occurrence, transactivation capacity, and subcellular localization. A novel 54-kDa VDRB1 protein, in addition to the previously recognized 48-kDa VDRA form, was detected in human kidney tissue as well as in osteoblastic (MG63), intestinal (Int-407, DLD-1, and COLO 206F), and kidney epithelial (786) human cell lines by Western blots using isoform-specific and nonselective anti-VDR antibodies. VDRB1 was present at approximately one-third the level of VDRA. Isoform-specific VDRB1 expression constructs produced lower ligand-dependent transactivation than VDRA when transiently transfected with a vitamin D-responsive promoter into cell lines with low endogenous VDR. Intracellular localization patterns of the green fluorescent protein-tagged VDR isoforms differed. VDRB1 appeared as discrete intranuclear foci in the absence of 1,25-dihydroxyvitamin D3, whereas VDRA produced diffuse nuclear fluorescence. After 1,25-dihydroxyvitamin D3 treatment, both VDR isoforms exhibited similar diffuse nuclear signal. In the absence of 1,25-dihydroxyvitamin D3, the VDRB1 foci partially colocalized with SC-35 speckles and a subset of promyelocytic leukemia nuclear bodies. These data provide the first evidence of VDRB1, a novel N-terminally variant human VDR that is expressed at a level comparable to VDRA in human tissue and cell lines. It is characterized by reduced transactivation activity and a ligand-responsive speckled intranuclear localization. The intranuclear compartmentalization and altered functional activity of VDRB1 may mediate a specialized physiological role for this receptor isoform.