Project description:We explored the combinatorial interactions between p53, estrogen receptors and NFkB using the breast adenocarcinoma-derived MCF7 cells. Recently, we have established that p53 can modulate transcription also through non-canonical response elements (REs), consisting of half-sites and ¾ sites. In particular, we identified a half-site p53 response element in the promoter of FLT1/VEGFR-I gene that was required but not sufficient for the p53 responsiveness of the promoter. The transcriptional control required also estrogen receptor (ER) half-sites (EREs) located in close proximity to the p53 RE. Further studies led us to establish that p53-mediated transcription from the FLT1 half site RE is dependent on ER binding at the EREs and strongly influenced by the level of ER proteins, and the specific nature of the p53-inducing stress as well as ER ligand. In another study we found an enrichment of NFkB REs nearby p53 REs in p53 target genes, suggesting an additional mode of functional interactions between these two transcription factors. To identify at a genome scale the transcriptional network that selectively responds to the concomitant activation of p53, ER and/or NFkB, we measured gene expression upon treatment with specific chemotherapeutic agents combined with 17-β estradiol (E2) and/or the inflammatory cytokine TNFα. Firstly we measured using MCF7 cells growing in estrogen-depleted media the transcriptome changes induced by doxorubicin (doxo) and 5-fluorouracil (5FU), two different drugs both able to stabilize and activate the p53 protein. We next obtained the expression profiles in the presence of E2 with or without doxo or 5FU. We also measured transcriptome changes in response to TNFαalone or in combination with doxo and/or E2. All these analyses were conducted using the same batch of MCF7 cells (wild type for p53, ERα, ERβ -weakly-, and NFkB positive) that we had previously used to characterize the cis-mediated transcriptional cooperation between p53 and ER at the FLT1 gene. Keywords: transcription factor, transcriptional networks, p53, ER, NFkB, p53-ER-NFkB crosstalk, doxorubicin, 5-fluorouracil, 17-β estradiol, TNFα, combined genotoxic, estrogenic and inflammatory responses, composite DNA binding site signatures, MCF7. The cooperation at the transcriptional level among p53, ERs and NFkB was analyzed by transcriptome analysis in response to different stimuli (doxo or 5FU, E2, TNFα). To identify mRNA molecules that would be modulated by the coordinated activity of the three TFs, gene expression signals derived from the combinatorial treatment were compared by microarrays analyses to those obtained from each drug when individually administered. Total RNA was isolated from MCF7 cells grown in estrogen-depleted medium and treated so as to stimulate the activity of the three different transcription factors (p53, ERs and NFkB). Cells lysates were collected after 10 hours from the treatments. All experiments were run from triplicate to septuples.
Project description:We explored the combinatorial interactions between p53, estrogen receptors and NFkB using the breast adenocarcinoma-derived MCF7 cells. Recently, we have established that p53 can modulate transcription also through non-canonical response elements (REs), consisting of half-sites and ¾ sites. In particular, we identified a half-site p53 response element in the promoter of FLT1/VEGFR-I gene that was required but not sufficient for the p53 responsiveness of the promoter. The transcriptional control required also estrogen receptor (ER) half-sites (EREs) located in close proximity to the p53 RE. Further studies led us to establish that p53-mediated transcription from the FLT1 half site RE is dependent on ER binding at the EREs and strongly influenced by the level of ER proteins, and the specific nature of the p53-inducing stress as well as ER ligand. In another study we found an enrichment of NFkB REs nearby p53 REs in p53 target genes, suggesting an additional mode of functional interactions between these two transcription factors. To identify at a genome scale the transcriptional network that selectively responds to the concomitant activation of p53, ER and/or NFkB, we measured gene expression upon treatment with specific chemotherapeutic agents combined with 17-β estradiol (E2) and/or the inflammatory cytokine TNFα. Firstly we measured using MCF7 cells growing in estrogen-depleted media the transcriptome changes induced by doxorubicin (doxo) and 5-fluorouracil (5FU), two different drugs both able to stabilize and activate the p53 protein. We next obtained the expression profiles in the presence of E2 with or without doxo or 5FU. We also measured transcriptome changes in response to TNFα alone or in combination with doxo and/or E2. All these analyses were conducted using the same batch of MCF7 cells (wild type for p53, ERα, ERβ -weakly-, and NFkB positive) that we had previously used to characterize the cis-mediated transcriptional cooperation between p53 and ER at the FLT1 gene. Keywords: transcription factor, transcriptional networks, p53, ER, NFkB, p53-ER-NFkB crosstalk, doxorubicin, 5-fluorouracil, 17-β estradiol, TNFα, combined genotoxic, estrogenic and inflammatory responses, composite DNA binding site signatures, MCF7.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:As the evolution of miRNA genes has been found to be one of the important factors in formation of the modern type of man, we performed a comparative analysis of the evolution of miRNA genes in two archaic hominines, Homo sapiens neanderthalensis and Homo sapiens denisova, and elucidated the expression of their target mRNAs in bain.A comparative analysis of the genomes of primates, including species in the genus Homo, identified a group of miRNA genes having fixed substitutions with important implications for the evolution of Homo sapiens neanderthalensis and Homo sapiens denisova. The mRNAs targeted by miRNAs with mutations specific for Homo sapiens denisova exhibited enhanced expression during postnatal brain development in modern humans. By contrast, the expression of mRNAs targeted by miRNAs bearing variations specific for Homo sapiens neanderthalensis was shown to be enhanced in prenatal brain development.Our results highlight the importance of changes in miRNA gene sequences in the course of Homo sapiens denisova and Homo sapiens neanderthalensis evolution. The genetic alterations of miRNAs regulating the spatiotemporal expression of multiple genes in the prenatal and postnatal brain may contribute to the progressive evolution of brain function, which is consistent with the observations of fine technical and typological properties of tools and decorative items reported from archaeological Denisovan sites. The data also suggest that differential spatial-temporal regulation of gene products promoted by the subspecies-specific mutations in the miRNA genes might have occurred in the brains of Homo sapiens denisova and Homo sapiens neanderthalensis, potentially contributing to the cultural differences between these two archaic hominines.
Project description:PurposeWe investigated the evidence of recent positive selection in the human phototransduction system at single nucleotide polymorphism (SNP) and gene level.MethodsSNP genotyping data from the International HapMap Project for European, Eastern Asian, and African populations was used to discover differences in haplotype length and allele frequency between these populations. Numeric selection metrics were computed for each SNP and aggregated into gene-level metrics to measure evidence of recent positive selection. The level of recent positive selection in phototransduction genes was evaluated and compared to a set of genes shown previously to be under recent selection, and a set of highly conserved genes as positive and negative controls, respectively.ResultsSix of 20 phototransduction genes evaluated had gene-level selection metrics above the 90th percentile: RGS9, GNB1, RHO, PDE6G, GNAT1, and SLC24A1. The selection signal across these genes was found to be of similar magnitude to the positive control genes and much greater than the negative control genes.ConclusionsThere is evidence for selective pressure in the genes involved in retinal phototransduction, and traces of this selective pressure can be demonstrated using SNP-level and gene-level metrics of allelic variation. We hypothesize that the selective pressure on these genes was related to their role in low light vision and retinal adaptation to ambient light changes. Uncovering the underlying genetics of evolutionary adaptations in phototransduction not only allows greater understanding of vision and visual diseases, but also the development of patient-specific diagnostic and intervention strategies.
Project description:Cortical thickness has been investigated since the beginning of the 20th century, but we do not know how similar the cortical thickness profiles among humans are. In this study, the local similarity of cortical thickness profiles was investigated using sliding window methods. Here, we show that approximately 5% of the cortical thickness profiles are similarly expressed among humans while 45% of the cortical thickness profiles show a high level of heterogeneity. Therefore, heterogeneity is the rule, not the exception. Cortical thickness profiles of somatosensory homunculi and the anterior insula are consistent among humans, while the cortical thickness profiles of the motor homunculus are more variable. Cortical thickness profiles of homunculi that code for muscle position and skin stimulation are highly similar among humans despite large differences in sex, education, and age. This finding suggests that the structure of these cortices remains well preserved over a lifetime. Our observations possibly relativize opinions on cortical plasticity.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.