Biomedical subjects

Iannis Talianidis

Publications and source records attributed to Iannis Talianidis.

16 recordsLinked to original sources

PubMed2006

Plasticity and expanding complexity of the hepatic transcription factor network during liver development.

Cross-regulatory cascades between hepatic transcription factors have been implicated in the determination of the hepatic phenotype. Analysis of recruitments to regulatory regions and the temporal and spatial expression pattern of the main hepatic regulators during liver development revealed a gradual increase in complexity of autoregulatory and cross-regulatory circuits. Within these circuits we identified a core group of six transcription factors, which regulate the expression of each other and the expression of other downstream hepatic regulators. Changes in the promoter occupancy patterns during development included new recruitments, release, and exchange of specific factors. We also identified promoter and developmental stage-specific dual regulatory functions of certain factors as an important feature of the network. Inactivation of HNF-4alpha in embryonic, but not in adult, liver resulted in the diminished expression of most hepatic factors, demonstrating that the stability of the network correlates with its complexity. The results illustrate the remarkable flexibility of a self-sustaining transcription factor network, built up by complex dominant and redundant regulatory motifs in developing hepatocytes.

Animals↗

PubMed2006

Mitogen-activated protein kinase-mediated disruption of enhancer-promoter communication inhibits hepatocyte nuclear factor 4alpha expression.

Hepatocyte nuclear factor 4 (HNF-4) is a key member of the transcription factor network regulating hepatocyte differentiation and function. Activation of the HNF-4 gene involves physical interaction between a distant enhancer and the proximal promoter region, bound by distinct sets of transcription factors. Here we report that, upon mitogen-activated protein (MAP) kinase activation, HNF-4 expression is downregulated in human hepatoma cells. This effect is mediated by the loss of CEBPalpha expression. During MAP kinase signaling, the recruitment of HNF-3beta and HNF-1alpha to the HNF-4 enhancer and RNA polymerase II to the proximal HNF-4 promoter was compromised. CBP, Brg1, and TFIIB were also dissociated from the HNF-4 regulatory regions, and the enhancer-promoter complex was disrupted. Interestingly, the extent of nucleosome acetylation did not decrease at either regulatory region, and HNF-6 and HNF-1alpha, as well as components of the TFIID, remained associated with the proximal promoter during the repressed state. The results point to an absolute requirement of enhancer-promoter communication for maintaining the active state of the HNF-4 gene and provide evidence for a molecular bookmarking mechanism, which may contribute to the prevention of permanent silencing of the locus during the repressed state.

Acetylation↗

PubMed2005

Regulation of hepatic metabolic pathways by the orphan nuclear receptor SHP.

SHP (small heterodimer partner) is an important component of the feedback regulatory cascade, which controls the conversion of cholesterol to bile acids. In order to identify the bona fide molecular targets of SHP, we performed global gene expression profiling combined with chromatin immunoprecipitation assays in transgenic mice constitutively expressing SHP in the liver. We demonstrate that SHP affects genes involved in diverse biological pathways, and in particular, several key genes involved in consecutive steps of cholesterol degradation, bile acid conjugation, transport and lipogenic pathways. Sustained expression of SHP leads to the depletion of hepatic bile acid pool and a concomitant accumulation of triglycerides in the liver. The mechanism responsible for this phenotype includes SHP-mediated direct repression of downstream target genes and the bile acid sensor FXRalpha, and an indirect activation of PPARgamma and SREBP-1c genes. We present evidence for the role of altered chromatin configurations in defining distinct gene-specific mechanisms by which SHP mediates differential transcriptional repression. The multiplicity of genes under its control suggests that SHP is a pleiotropic regulator of diverse metabolic pathways.

Animals↗

PubMed2005

SUMO-dependent compartmentalization in promyelocytic leukemia protein nuclear bodies prevents the access of LRH-1 to chromatin.

Posttranslational modification by SUMO elicits a repressive effect on many transcription factors. In principle, sumoylation may either influence transcription factor activity on promoters, or it may act indirectly by targeting the modified factors to specific cellular compartments. To provide direct experimental evidence for the above, not necessarily mutually exclusive models, we analyzed the role of SUMO modification on the localization and the activity of the orphan nuclear receptor LRH-1. We demonstrate, by using fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) assays, that sumoylated LRH-1 is exclusively localized in promyelocytic leukemia protein (PML) nuclear bodies and that this association is a dynamic process. Release of LRH-1 from nuclear bodies correlated with its desumoylation, pointing to the pivotal role of SUMO conjugation in keeping LRH-1 in these locations. SUMO-dependent shuttling of LRH-1 into PML bodies defines two spatially separated pools of the protein, of which only the soluble, unmodified one is associated with actively transcribed target genes. The results suggest that SUMO-PML nuclear bodies may primarily function as dynamic molecular reservoirs, controlling the availability of certain transcription factors to active chromatin domains.

Cell Line↗

PubMed2004

Histone modifications defining active genes persist after transcriptional and mitotic inactivation.

We examined various histone modifications across the promoter and the coding regions of constitutively active hepatic genes in G0/G1-enriched, mitotically arrested and alpha-amanitin-blocked cells. Gene activation correlated with localized histone hyperacetylation, H3-K4 tri- or dimethylation and H3-K79 dimethylation and localized nucleosome remodeling at the promoter and the 5' portion of the coding regions. Nucleosomes at more downstream locations were monomethylated at H3-K4. CBP, PCAF, Brg-1, SNF2H and FACT were recruited to the coding regions in a gene-specific manner, in a similarly restricted promoter-proximal pattern. Elongator, however, associated with the more downstream regions. While all factors were dissociated from the chromatin after transcriptional inactivation by alpha-amanitin, the histone modifications remained stable. In mitotic cells, histone modifications on parental nucleosomes were preserved and were regenerated in a transcription-dependent manner at the newly deposited nucleosomes, as the cells entered the next G1 phase. The findings suggest that histone modifications may function as molecular memory bookmarks for previously active locations of the genome, thus contributing to the maintenance of active chromatin states through cell division.

Acetyltransferases↗

PubMed2004

Transcriptional regulation of the mouse deoxycytidine kinase: identification and functional analysis of nuclear protein binding sites at the proximal promoter.

Deoxycytidine kinase (EC 2.7.1.74, dCK) catalyzes the phosphorylation of deoxynucleosides and several nucleoside analogues that are important in antiviral and cancer chemotherapy. The enzyme is predominantly expressed in lymphoid tissue by as yet poorly defined mechanisms. In this work, we have studied the mouse dCK regulatory region to understand the molecular details of the tissue specific expression of the enzyme. DNase I footprinting and electrophoretic mobility shift assays using nuclear extracts from mouse lymphocytes (EL-4, T cells; J558, B cells) and non-lymphoid cells (L929, fibroblasts) demonstrated the existence of at least six cis-acting elements (FP-1-FP-6) within the proximal promoter region. Functional analysis revealed that all the elements necessary to promote high level transcription of the mdCK gene are located downstream the transcription start site. 5'-Deletion and site-directed mutagenesis assays demonstrated the importance of four GC-rich regions, which bind Sp-1 and Sp-3 transcription factors. In addition, we identified a site (FP-3) located at the -282 to -310 nucleotide region of the promoter, which binds NF-1, only in B cells. Analysis of point mutations introduced at the different regions revealed functional differences in their role in mdCK transcription in the cell lines used.

5' Flanking Region↗

PubMed2004

Functional role of G9a-induced histone methylation in small heterodimer partner-mediated transcriptional repression.

Site-specific modification of nucleosomal histones plays a central role in the formation of transcriptionally active and inactive chromatin structures. These modifications may serve as specific recognition motifs for chromatin proteins, which act as a signal for the adoption of the appropriate regulatory responses. Here, we show that the orphan nuclear receptor SHP (small heterodimer partner), a coregulator that inhibits the activity of several nuclear receptors, can associate with unmodified and lysine 9-methylated histone-3, but not with the acetylated protein. The naturally occurring SHP mutant (R213C), which exhibits decreased transrepression potential, interacts less avidly with K9-methylated histone 3. We demonstrate that SHP can functionally interact with histone deacetylase-1 and the G9a methyltransferase and that it is localized exclusively in nuclease-sensitive euchromatin. The results point to the involvement of a multistep mechanism in SHP-dependent transcriptional repression, which includes histone deacetylation, followed by H3-K9 methylation and stable association of SHP itself with chromatin.

Acetylation↗

PubMed2004

HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment.

Mutations in the HNF1beta gene, encoding the dimeric POU-homeodomain transcription factor HNF1beta (TCF2 or vHNF1), cause various phenotypes including maturity onset diabetes of the young 5 (MODY5), and abnormalities in kidney, pancreas and genital tract development. To gain insight into the molecular mechanisms underlying these phenotypes and into the structure of HNF1beta, we functionally characterized eight disease-causing mutations predicted to produce protein truncations, amino acids substitutions or frameshift deletions in different domains of the protein. Truncated mutations, retaining the dimerization domain, displayed defective nuclear localization and weak dominant-negative activity when co-expressed with the wild-type protein. A frameshift mutation located within the C-terminal QSP-rich domain partially reduced transcriptional activity, whereas selective deletion of this domain abolished transactivation. All five missense mutations, which concern POU-specific and homeodomain residues, were correctly expressed and localized to the nucleus. Although having different effects on DNA-binding capacity, which ranged from complete loss to a mild reduction, these mutations exhibited a severe reduction in their transactivation capacity. The transcriptional impairment of those mutants, whose DNA-binding activity was weakly or not affected, correlated with the loss of association with one of the histone-acetyltransferases CBP or PCAF. In contrast to wild-type HNF1beta, whose transactivation potential depends on the synergistic action of CBP and PCAF, the activity of these mutants was not increased by the synergistic action of these two coactivators or by treatment with the specific histone-deacetylase inhibitor TSA. Our findings suggest that the complex syndrome associated with HNF1beta-MODY5 mutations arise from either defective DNA-binding or transactivation function through impaired coactivator recruitment.

DNA↗

PubMed2004

Detection of an alternatively spliced form of deoxycytidine kinase mRNA in the 2'-2'-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000.

Gemcitabine (2'-2'-difluorodeoxycytidine (dFdC)) is a deoxycytidine analogue that is effective against solid tumors, including lung cancer and ovarian cancer. dFdC requires the phosphorylation by deoxycytidine kinase (dCK) as a primary step in its activation. Deficiency of dCK is associated with resistance against this compound both in vitro in cancer cell lines and in clinical practice in acute myeloid leukemia and solid tumors. The human ovarian cancer cell line AG6000 is 100,000-fold resistant against dFdC compared to its parent cell line A2780. This cell line proved to be dCK deficient in enzyme activity assays and by Western blot analysis, but by RT-PCR, a normal and a truncated dCK mRNA was found. Sequencing revealed that exon 3 was deleted from the dCK cDNA, resulting in a 74-aa-long open-reading frame due to the generation of a premature stop codon. No gross genomic alteration was observed at the dCK locus, suggesting the involvement of post-transcription mechanisms. Transient transfection experiments indicated that the truncated dCK transcripts are not translated to protein. To study the functional role of the truncated dCK transcripts, both A2780 cells and AG6000 cells were stably transfected with human and rat dCK. The results indicated that over-expression of full-length dCK genes in AG6000 failed to completely reverse the sensitivity to dFdC or other drugs.

Alternative Splicing↗

PubMed2004

Gene-specific modulation of TAF10 function by SET9-mediated methylation.

SET9 is a member of the SET domain-containing histone methyltransferase family that can specifically methylate histone 3 at lysine 4 position. Although nucleosomal histones are poor substrates for SET9, the active enzyme can stimulate activator-induced transcription. Here, we show that SET9 can monomethylate the TBP-associated factor TAF10 at a single lysine residue located at the loop 2 region within the putative histone-fold domain of the protein. Methylated TAF10 has an increased affinity for RNA polymerase II, pointing to a direct role of this modification in preinitiation complex formation. Reporter assays and studies on TAF10 null F9 cells expressing a methylation-deficient TAF10 mutant revealed that SET9-mediated methylation of TAF10 potentiates transcription of some but not all TAF10-dependent genes. This gene specificity correlated with SET9 recruitment. The promoter-specific effects of SET9-methylated TAF10 may have important implications regarding the biological function of SET domain-containing lysine methylases, whose primary targets have been presumed to be histones.

Amino Acid Sequence↗

PubMed2004

Activation of deoxycytidine kinase in lymphocytes is calcium dependent and involves a conformational change detectable by native immunostaining.

Deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, plays a seminal role in the bioactivation of a wide array of cytotoxic nucleoside analogues. Recently, activation of dCK has been considered as a protective cellular response to a number of DNA-damaging agents in lymphocytes. Regarding the molecular mechanism of the enzyme activation, a post-translational modification by protein phosphorylation has been suggested. Here we provide evidence that both the activation process and the maintenance of the activated state require free cytosolic calcium. BAPTA-AM, a cell-permeable calcium chelator selectively inhibited the activation of dCK in a time- and concentration-dependent manner while extracellular calcium depletion had no effect. On the other hand, elevation of cytoplasmic calcium levels by thapsigargin did not potentiate the enzyme, referring to the permissive function of calcium in the activation process. Denaturing Western blots of extracts from lymphocytes incubated with 2-chlorodeoxyadenosine, aphidicolin and/or BAPTA-AM clearly demonstrated that dCK protein levels were unchanged during these treatments. However, a striking correlation was found between enzyme activity and the intensity of dCK-specific signals in native Western blots. Extracts from CdA-treated cells were much better recognized by the antibody raised against the C-terminal peptide of dCK than the BAPTA-AM-treated samples. These results indicate that the calcium-dependent activation of dCK is accompanied by a conformational change that renders the C-terminal epitope more accessible to the antibody.

Calcium↗

PubMed2003

Activation of deoxycytidine kinase by gamma-irradiation and inactivation by hyperosmotic shock in human lymphocytes.

Deoxycytidine kinase (dCK) is a key enzyme in the intracellular metabolism of deoxynucleosides and their analogues, phosphorylating a wide range of drugs used in the chemotherapy of leukaemia and solid tumours. Previously, we found that activity of dCK can be enhanced by incubating primary cultures of lymphocytes with substrate analogues of the enzyme, as well as with various genotoxic agents. Here we present evidence that exposure of human lymphocytes to 0.5-2 Gy dosage of gamma-radiation as well as incubation of cells with calyculin A, a potent inhibitor of protein phosphatase 1 and 2A, both elevate dCK activity without changing the level of dCK protein. When cells were gamma-irradiated in the presence of calyculin A, a more pronounced activation of dCK was observed. In contrast, both basal and stimulated dCK activities were reduced by hyperosmotic treatment of the cells. DNA repair determined by the Comet assay and by thymidine incorporation was induced by irradiation. Complete repair of gamma-irradiated DNA was detected within 1 hr following the irradiation along with dCK activation, but the rate of repair was not accelerated by calyculin A. These data provide evidence for the activation of dCK upon DNA damage and repair that seems to be mediated by phosphorylation of the enzyme, suggesting the role of dCK in DNA repair processes.

Comet Assay↗

PubMed2003

Synergism between nuclear receptors bound to specific hormone response elements of the hepatic control region-1 and the proximal apolipoprotein C-II promoter mediate apolipoprotein C-II gene regulation by bile acids and retinoids.

We have shown previously that the hepatic control region 1 (HCR-1) enhances the activity of the human apolipoprotein C-II (apoC-II) promoter in HepG2 cells via two hormone response elements (HREs) present in the apoC-II promoter. In the present paper, we report that the HCR-1 selectively mediates the transactivation of the apoC-II promoter by chenodeoxycholic acid (CDCA) and 9- cis -retinoic acid. CDCA, which is a natural ligand of farnesoid X receptor alpha (FXRalpha), increases the steady-state apoC-II mRNA levels in HepG2 cells. This increase in transcription requires the binding of retinoid X receptor alpha (RXRalpha)-FXRalpha heterodimers to a novel inverted repeat with one nucleotide spacing (IR-1) present in the HCR-1. This element also binds hepatocyte nuclear factor 4 and apoA-I regulatory protein-1. Transactivation of the HCR-1/apoC-II promoter cluster by RXRalpha-FXRalpha heterodimers in the presence of CDCA was abolished by mutations either in the IR-1 HRE of the HCR-1 or in the thyroid HRE of the proximal apoC-II promoter, which binds RXRalpha-thyroid hormone receptor beta (T3Rbeta) heterodimers. The same mutations also abolished transactivation of the HCR-1/apoC-II promoter cluster by RXRalpha-T3Rbeta heterodimers in the presence of tri-iodothyronine. The findings establish synergism between nuclear receptors bound to specific HREs of the proximal apoC-II promoter and the HCR-1, and suggest that this synergism mediates the induction of the HCR-1/apoC-II promoter cluster by bile acids and retinoids.

Alitretinoin↗

PubMed2002

Coordination of PIC assembly and chromatin remodeling during differentiation-induced gene activation.

We analyzed the ordered recruitment of factors to the human alpha1 antitrypsin promoter around the initial activation of the gene during enterocyte differentiation. We found that a complete preinitiation complex, including phosphorylated RNA pol II, was assembled at the promoter long before transcriptional activation. The histone acetyltransferases CBP and P/CAF were recruited subsequently, but local histone hyperacetylation was delayed. After transient recruitment of the human Brahma homolog hBrm, remodeling of the neighboring nucleosome coincided with transcription initiation. The results suggest that, at this promoter, chromatin reconfiguration is a defining step of the initiation process, acting after the assembly of the Pol II machinery.

Acetylation↗

PubMed2002

Dynamics of enhancer-promoter communication during differentiation-induced gene activation.

We analyzed the order of recruitment of factors to the HNF-4alpha regulatory regions upon the initial activation of the gene during enterocyte differentiation. An initially independent assembly of regulatory complexes at the proximal promoter and the upstream enhancer regions was followed by the tracking of the entire DNA-protein complex formed on the enhancer along the intervening DNA until it encountered the proximal promoter. This movement correlated with a unidirectional spreading of histone hyperacetylation. Transcription initiation coincided with the formation of a stable enhancer-promoter complex and remodeling of the nucleosome situated at the transcription start site. The results provide experimental evidence for the involvement of a dynamic process culminating in enhancer-promoter communication during long-distance gene activation.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2002

Pretreatment deoxycytidine kinase levels predict in vivo gemcitabine sensitivity.

Deoxycytidine kinase (dCK) is essential for the phosphorylation of gemcitabine (2',2'-difluorodeoxycytidine), a deoxycytidine analogue active against various solid tumors. Cytidine deaminase (CDA) catalyzes the degradation of gemcitabine. We determined whether dCK and/or CDA levels would predict response to gemcitabine. Activities of dCK and CDA were measured in a panel of eight gemcitabine-sensitive and -resistant tumors of a different origin (pancreas, lung, colon, ovary, and head and neck) grown as s.c. tumors in mice. Sensitivity to gemcitabine was expressed as treated versus control (tumor volume treated mice/control mice). Gemcitabine was given on days 0, 3, 6, and 9 (q3dx4) at its maximum tolerated dose. In addition, we measured the mRNA expression and protein levels of dCK in seven human tumor xenografts. dCK activity (mean +/- SE) ranged from 3.3+/-0.3 to 18.4+/-1.2 nmol/h/mg protein. Sensitivity to gemcitabine, expressed as treated versus control, ranged from 0.98 to 0.02, and the activity of CDA varied from 2+/-2 to 411+/-4 nmol/h/mg protein. In contrast to CDA, dCK activity was clearly related to gemcitabine sensitivity (p = -0.93; P < 0.001). This indicates that dCK might be an important prognostic marker for gemcitabine sensitivity. Protein levels were significantly related to both dCK activity (r = 0.96; P < 0.001) and gemcitabine sensitivity (rho = -0.96; P < 0.001). dCK expression as determined by competitive template reverse transcriptase PCR was significantly related with the dCK activity (r = 0.88; P = 0.025) and protein levels (p = 0.80; P = 0.052) but not with gemcitabine sensitivity, suggesting a post-translational regulation of dCK. In conclusion, the clear correlation between dCK levels and gemcitabine sensitivity in various murine tumors and human tumor xenografts may be a prognostic parameter when considering gemcitabine therapy.

Animals↗