TESTING A SUBTYPE-SPECIFIC GP41 AMPLIFICATION METHOD FOR GENOTYPING INDIVIDUALS INFECTED BY HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 IN THE BRAZILIAN POPULATION OF ITAJAÍ, SOUTH BRAZIL

The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itajaí, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.

Using gold nanoparticles in protein crystallography: studies in crystal growth and derivatization

Dissertation for the Master Degree in Structural and Functional Biochemistry

Gd(III) chelates as NMR probes of protein-protein interactions. Case study: rubredoxin and cytochrome c3

Inorganic Chemistry 50(21):10600-7

Analysis of Highly Conserved Regions of the 3'UTR of MECP2 Gene in Patients with Clinical Diagnosis of Rett Syndrome and Other Disorders Associated with Mental Retardation

In this work we explored the role of the 3'UTR of the MECP2 gene in patients with clinical diagnosis of RTT and mental retardation; focusing on regions of the 3'UTR with almost 100% conservation at the nucleotide level among mouse and human. By mutation scanning (DOVAM-S technique) the MECP2 3'UTR of a total of 66 affected females were studied. Five3'UTR variants in the MECP2 were found (c.1461+9G>A, c.1461+98insA, c.2595G>A, c.9961C>G and c.9964delC) in our group of patients. None of the variants found is located in putative protein-binding sites nor predicted to have a pathogenic role. Our data suggest that mutations in this region do not account for a large proportion of the RTT cases without a genetic explanation.

Transcriptional regulation of the mannosyltransferase-encoding gene pigm in inherited glycosylphosphatidylinositol (GPI) deficiency

RESUMO:O glicosilfosfatidilinositol (GPI) é um complexo glicolipídico utlizado por dezenas de proteínas, o qual medeia a sua ancoragem à superfície da célula. Proteínas de superfície celular ancoradas a GPI apresentam várias funções essenciais para a manutenção celular. A deficiência na síntese de GPI é o que caracteriza principalmente a deficiência hereditária em GPI, um grupo de doenças autossómicas raras que resultam de mutações nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispensáveis para a biossíntese do GPI. Uma mutação pontual no motivo rico em GC -270 no promotor de PIGM impede a ligação do factor de transcrição (FT) Sp1 à sua sequência de reconhecimento, impondo a compactação da cromatina, associada à hipoacetilação de histonas, e consequentemente, impedindo a transcrição de PIGM. Desta forma, a adição da primeira manose ao GPI é comprometida, a síntese de GPI diminui assim como as proteínas ligadas a GPI à superficie das células. Pacientes com Deficiência Hereditária em GPI-associada a PIGM apresentam trombose e epilesia, e ausência de hemólise intravascular e anemia, sendo que estas duas últimas características definem a Hemoglobinúria Paroxística Nocturna (HPN), uma doença rara causada por mutações no gene PIGA. Embora a mutação que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de deficiência em GPI varia entre células do mesmo tecido e entre células de tecidos diferentes. Por exemplo nos granulócitos e linfócitos B a deficiência em GPI é muito acentuada mas nos linfócitos T, fibroblastos, plaquetas e eritrócitos é aproximadamente normal, daí a ausência de hemólise intravascular. Os eventos transcricionais que estão na base da expressão diferencial da âncora GPI nas células hematopoiéticas são desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os níveis de PIGM mRNA variam entre células primárias hematopoiéticas normais. Adicionalmente, a configuração dos nucleossomas no promotor de PIGM é mais compacta em células B do que em células eritróides e tal está correlacionado com os níveis de expressão de PIGM, isto é, inferior nas células B. A presença de vários motivos de ligação para o FT específico da linhagem megacariocítica-eritróide GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcrição. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da expressão de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcrição restritamente eritróide, regula a transcrição de PIGM independentemente do motivo -270GC. Em segundo lugar, a investigação do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcrição de PIGM em ambas as células B e eritróide. Curiosamente, ao contrário do que acontece nas células B, em que a transcrição de PIGM requer a ligação do FT geral Sp1 ao motivo -270GC, nas células eritróides Sp1 regula a transcrição de PIGM ao ligar-se a montante e não ao motivo -270GC. Para além disso, demonstrou-se que Sp2 não é um regulador directo da transcrição de PIGM quer nas células B quer nas células eritróides. Estes resultados explicam a ausência de hemólise intravascular nos doentes com IGD associada a PIGM, uma das principais características que define a HPN. Por último, resultados preliminares mostraram que a repressão da transcrição de PIGM devida à mutação patogénica -270C>G está associada com a diminuição da frequência de interacções genómicas em cis entre PIGM e os seus genes “vizinhos”, sugerindo adicionalmente que a regulação de PIGM e desses genes é partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, específico-detecido, por meio de FTs genéricos e específicos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.

Phenanthroimidazoles as fluorimetric and colourimetric chemosensors in aqueous solution

2,4,5-Triaryl-imidazoles are versatile compounds with application in medicine, due to their biological activity, and materials sciences, for their interesting optical properties. These properties can be tuned by careful selection of substituents at positions 2, 4 and 5: replacement of the aryl group by an heterocyclic group results in larger π-conjugated systems with improved optical properties for application in nonlinear optics, OLEDs, DNA intercalators, and chemosensors. Moreover, it is expected that introducing more conjugation and rigidity into the resulting system will further improve its properties. The development of chromo/fluorescent probes that are capable of detecting ions with high sensitivity and selectivity in aqueous media is currently a topic of strong interest and the design of heteroditopic receptors that contain two or more different binding sites for the simultaneous complexation of cationic and anionic guests is a emerging field of supramolecular chemistry. In this communication, we report the synthesis of new phenanthroimidazoles substituted at position 2 with arylthienyl or arylfuryl moieties possessing substituents of different electronic character, in order to tune the chromo/fluoro response in the presence of relevant anions and metal cations. Their photophysical properties and chemosensory ability were studied in acetonitrile and mixtures of acetonitrile and water, and selective detection of cyanide was achieved in aqueous mixtures for some of the derivatives.

Removal of tetracycline from contaminated water by Moringa oleifera seed preparations

The aim of this study was to evaluate tetracycline antibiotic (TA) removal from contaminated water by Moringa oleifera seed preparations. The composition of synthetic water approximate river natural contaminated water and TA simulated its presence as an emerging pollutant. Interactions between TA and protein preparations (extract; fraction and lectin) were also evaluated. TA was determined by solid phase extraction followed by high performance liquid chromatography - mass spectrometry. Moringa extract and flour removed TA from water. Extract removed TA in all concentrations and better removal (40%) was obtained with 40 mg L1; seed flour (particles < 5mm), 1.25 g L1 and 2.50 g L1 removed 28 and 29% of tetracycline, respectively; particles > 5 mm (0.50 g L1) removed 55% of antibiotic. Interactions between TA and seed preparations were assayed by haemagglutinating activity (HA). Specific HA (SHA) of extract (pH 7) was abolished with tetracycline (5 mg L1); fraction (75%) and lectin HA (97%) were inhibited with TA. Extract SHA decreased by 75% at pH 8. Zeta potential (ZP) of extract 700 mg L1 and tetracycline 50 mg L1 , pH range 5 to 8, showed different results. Extract ZP was more negative (10.73 mV to 16.00 mV) than tetracycline ZP (0.27 mV to 20.15 mV); ZP difference was greater in pH 8. The focus of this study was achieved since moringa preparations removed TA from water and compounds interacting with tetracycline involved at least lectin binding sites. This is a natural process, which do not promote environmental damage.

Reconstruction of the regulatory network for Bacillus subtilis and reconciliation with gene expression data

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.00275

Investigation into the potential for establishing a self-sustainable wetland ecosystem over pyritic mine tailings

Research was conducted to investigate the potential for ecologically engineering a sustainable wetland ecosystem over pyritic mine tailings to prevent the generation of acid mine drainage. Ecological engineering is technology with the primary goal being the creation of self-sustainable ecological systems. Work involved the design and construction of a pilot-scale wetland system comprising three wetland cells, each covering 100 m2. Approximately forty tonnes of pyritic mine tailings were deposited on the base of the first cell above a synthetic liner, covered with peat, flooded and planted with emergent wetland macrophytes Typha latifolia, Phragmites australis, and Juncus effusus. The second cell was constructed as a conventional free water surface wetland, planted identically, and used as a reference wetland/experimental control. Wetland monitoring to determine long-term sustainability focused on indicators of ecosystem health including ecological, hydrological, physico-chemical, geochemical, and biotic metrics. An integrated assessment was conducted that involved field ecology in addition to ecological risk assessment. The objective of the field ecology study was to use vegetative parameters as ecological indicators for documenting wetlands success or degradation. The goal of the risk assessment was to determine if heavy-metal contamination of the wetland sediments occurred through metal mobilisation from the underlying tailings, and to evaluate if subsequent water column chemistry and biotic metal concentrations were significantly correlated with adverse wetland ecosystem impacts. Data were used to assess heavy metal bioavailability within the system as a function of metal speciation in the wetland sediments. Results indicate hydrology is the most important variable in the design and establishment of the tailings wetland and suggest a wetland cover is an ecologically viable alternative for pyritic tailings which are feasible to flood. Ecological data indicate that in terms of species richness and diversity, the tailings-wetland was exhibiting the ecological characteristics of natural wetlands within two years. Ata indicate that pH and conductivity in the tailings-wetland were not adversely impacted by the acid-generating potential or sulphate concentration of the tailings substrate and its porewater. Similarly, no enhanced seasonal impacts from sulphate or metals in the water column, nor adverse impacts on the final water quality of the outflows, were detected. Mean total metal concentrations in the sediments of the tailings-wetland indicate no significant adverse mobilisation of metals into the peat substrate from the tailings. Correlation analyses indicate a general increase in sediment metal concentration in this wetland with increasing water depth and pH, and a corresponding decrease in the metal concentrations of the water column. Sediment extractions also showed enrichment of Cd, Fe, Pb and Zn in the oxidisable fraction (including sulphides and organic matter) of the tailings-wetland sediments. These data suggest that adsorption and coprecipitation of metals is occurring from the water column of the tailings wetland with organic material at increasing depths under reducing conditions. The long-term control of metal bioavailability in the tailings wetland will likely be related to the presence and continual build-up of organic carbon binding sites in the developing wetland above the tailings. Metal speciation including free-metal ion concentration and the impact of physico-chemical parameters particularly pH and organic matter, were investigated to assess ecotoxicological risk. Results indicate that potentially bioavailable metals (the sum of the exchangeable and reducible fractions) within the tailings wetland are similar to values cited for natural wetlands. Estimated free-metal ion concentrations calculated from geochemical regression models indicate lower free-metal ion concentrations of Cd in the tailings wetland than natural wetlands and slightly higher free-metal ion concentrations of Pb and Zn. Increased concentrations of metals in roots, rhizomes and stems of emergent macrophytes did not occur in the tailings wetland. Even though a substantial number of Typha latifolia plants were found rooting directly into tailings, elevated metals were not found in these plant tissues. Phragmites also did not exhibit elevated metal concentrations in any plant tissues. Typha and Phragmites populations appear to be exhibiting metal-tolerant behaviour. The chemistry of the water column and sediments in Silvermines wetland were also investigated and were much more indicative of a wetland system impacted by heavy metal contamination than the tailings-wetland. Mean Dc, Fe, Mn, Pb and Zn concentrations in the water column and sediments of Silvermines wetlands were substantially higher than in the pilot wetlands and closely approximate concentrations in these matrices contaminated with metals from mining. In addition, mean sulphate concentration in Silvermines wetland was substantially higher and is closer to sulphate concentrations in waters associated with mining. Potentially bioavailable metals were substantially elevated in Silvermines wetland in comparison to the pilot wetlands and higher than those calculated for natural rive sediments. However, Fe oxy-hydroxide concentrations in Silvermines sediments are also much higher than in the pilot wetlands and this significantly impacts the concentration of free-metal ions in the sediment porewater. The free-metal ion concentrations for Pb and Zn indicate that Silvermines wetland is retaining metals and acting as a treatment wetland for drainage emanating from Silvermines tailings dam.

CLOCK-controlled polyphonic regulation of circadian rhythms through canonical and noncanonical E-boxes.

In mammalian circadian clockwork, the CLOCK-BMAL1 complex binds to DNA enhancers of target genes and drives circadian oscillation of transcription. Here we identified 7,978 CLOCK-binding sites in mouse liver by chromatin immunoprecipitation-sequencing (ChIP-Seq), and a newly developed bioinformatics method, motif centrality analysis of ChIP-Seq (MOCCS), revealed a genome-wide distribution of previously unappreciated noncanonical E-boxes targeted by CLOCK. In vitro promoter assays showed that CACGNG, CACGTT, and CATG(T/C)G are functional CLOCK-binding motifs. Furthermore, we extensively revealed rhythmically expressed genes by poly(A)-tailed RNA-Seq and identified 1,629 CLOCK target genes within 11,926 genes expressed in the liver. Our analysis also revealed rhythmically expressed genes that have no apparent CLOCK-binding site, indicating the importance of indirect transcriptional and posttranscriptional regulations. Indirect transcriptional regulation is represented by rhythmic expression of CLOCK-regulated transcription factors, such as Krüppel-like factors (KLFs). Indirect posttranscriptional regulation involves rhythmic microRNAs that were identified by small-RNA-Seq. Collectively, CLOCK-dependent direct transactivation through multiple E-boxes and indirect regulations polyphonically orchestrate dynamic circadian outputs.

Targeting of secretory IgA to Peyer's patch dendritic and T cells after transport by intestinal M cells.

In addition to being instrumental to the protection of mucosal epithelia, secretory IgA (SIgA) adheres to and is transported by intestinal Peyer's patch (PP) M cells. The possible functional reason for this transport is unknown. We have thus examined in mice the outcome of SIgA delivered from the intestinal lumen to the cells present in the underlying organized mucosa-associated lymphoreticular tissue. We show selective association of SIgA with dendritic cells and CD4(+) T and B lymphocytes recovered from PP in vitro. In vivo, exogenously delivered SIgA is able to enter into multiple PP lining the intestine. In PP, SIgA associates with and is internalized by dendritic cells in the subepithelial dome region, whereas the interaction with CD4(+) T cells is limited to surface binding. Interaction between cells and SIgA is mediated by the IgA moiety and occurs for polymeric and monomeric molecular forms. Thus, although immune exclusion represents the main function of SIgA, transport of the Ab by M cells might promote Ag sampling under neutralizing conditions essential to the homeostasis of mucosal surfaces.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

Directed integration of new insulated lentiviral vectors to heterochromatin towards safer gene transfer to stem cells

The need for better gene transfer systems towards improved risk=benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT). We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing genetic integration to heterochromatin. We have designed SIN-insulated retrovectors with two candidate GIEs and could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) (see Duros et al, abstract ibid). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested with chimeric HIV-1 derived integrases which comprise C-ter chromodomains targeting heterochromatin through either histone H3 (ML6chimera) or methylatedCpGislands (ML10). With DCaro4 only and both chimeras, a homogeneous expression is evidenced in over 20% of the cells which is sustained over time. With control lentivectors, less than 2% of cells express GFP as compared to background using a control double-mutant in both catalytic and ledgf binding-sites; in addition, a two-times increase of expression can be induced with histone deacetylase inhibitors. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity, according to X-SCIDs patients data.Work performed with the support of EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933

Combinatorial Synthesis and Biological Evaluation of Inhibitors of D-Ala-D-Ala-Ligase

Report for the scientific sojourn carried out at the Max Planck Institut of Molecular Phisiology, Germany, from 2006 to 2008.The work carried out during this postdoctoral stage was focused on two different projects. Firstly, identification of D-Ala D-Ala Inhibitors and the development of new synthethic approaches to obtain lipidated peptides and proteins and the use of these lipidated proteins in biological and biophysical studies. In the first project, new D-Ala D-Ala inhibitors were identified by using structural alignments of the ATP binding sites of the bacterial ligase DDl and protein and lipid kinases in complex with ATP analogs. We tested a series of commercially available kinase inhibitors and found LFM-A13 and Tyrphostine derivatives to inhibit DDl enzyme activity. Based on the initial screening results we synthesized a series of malononitrilamide and salicylamide derivatives and were able to confirm the validity of these scaffolds as inhibitors of DDl. From this investigation we gained a better understanding of the structural requirements and limitations necessary for the preparation of ATP competitive DDl inhibitors. The compounds in this study may serve as starting points for the development of bi-substrate inhibitors that incorporate both, an ATP competitive and a substrate competitive moiety. Bisubstrate inhibitors that block the ATP and D-Ala binding sites should exhibit enhanced selectivity and potency profiles by preferentially inhibiting DDl over kinases. In the second project, an optimized synthesis for tha alkylation of cysteins using the thiol ene reaction was establisehd. This new protocol allowed us to obtain large amounts of hexadecylated cysteine that was required for the synthesis of differently lipidated peptides. Afterwards the synthesis of various N-ras peptides bearing different lipid anchors was performed and the peptides were ligated to a truncated N-ras protein. The influence of this differently lipidated N-ras proteins on the partioning and association of N-Ras in model membrane subdomains was studied using Atomic Force Microscopy.

Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus.

The beta thyroid hormone receptor (TRbeta), but not TRalpha1, plays a specific role in mediating T(3)-dependent repression of hypothalamic TRH transcription. To investigate the structural basis of isoform specificity, we compared the transcriptional regulation and DNA binding obtained with chimeric and N-terminally deleted TRs. Using in vivo transfection assays to follow hypothalamic TRH transcription in the mouse brain, we found that TRbeta1 and chimeras with the TRbeta1 N terminus did not affect either transcriptional activation or repression from the rat TRH promoter, whereas N-terminally deleted TRbeta1 impaired T(3)-dependent repression. TRalpha1 or chimeras with the TRalpha1 N terminus reduced T(3)-independent transcriptional activation and blocked T(3)-dependent repression of transcription. Full deletion of the TRalpha1 N terminus restored ligand-independent activation of transcription. No TR isoform specificity was seen after transcription from a positive thyroid hormone response element. Gel mobility assays showed that all TRs tested bound specifically to the main negative thyroid hormone response element in the TRH promoter (site 4). Addition of neither steroid receptor coactivator 1 nor nuclear extracts from the hypothalamic paraventricular nuclei revealed any TR isoform specificity in binding to site 4. Thus N-terminal sequences specify TR T(3)-dependent repression of TRH transcription but not DNA recognition, emphasizing as yet unknown neuron-specific contributions to protein-promoter interactions in vivo.