Project description:The polycomb group (PcG) proteins function in gene silencing through histone modifications. They form chromatin-associated multiprotein complexes, termed polycomb repressive complex (PRC) 1 and PRC2. These two complexes work in a coordinated manner in the maintenance of cellular memories through transcriptional repression of target genes. EZH2 is a catalytic component of PRC2 and trimethylates histone H3 at lysine 27 to transcriptionally repress the target genes. PcG proteins have been characterized as general regulators of stem cells, but recent works also unveiled their critical roles in cancer. Wild type and Ezh2 null MEP from fetal liver and adult bone marrow
Project description:The polycomb group (PcG) proteins function in gene silencing through histone modifications. They form chromatin-associated multiprotein complexes, termed polycomb repressive complex (PRC) 1 and PRC2. These two complexes work in a coordinated manner in the maintenance of cellular memories through transcriptional repression of target genes. EZH2 is a catalytic component of PRC2 and trimethylates histone H3 at lysine 27 to transcriptionally repress the target genes. PcG proteins have been characterized as general regulators of stem cells, but recent works also unveiled their critical roles in cancer.
Project description:The polycomb group (PcG) proteins function in gene silencing through histone modifications. They form chromatin-associated multiprotein complexes, termed polycomb repressive complex (PRC) 1 and PRC2. These two complexes work in a coordinated manner in the maintenance of cellular memories through transcriptional repression of target genes. EZH2 is a catalytic component of PRC2 and trimethylates histone H3 at lysine 27 to transcriptionally repress the target genes. PcG proteins have been characterized as general regulators of stem cells, but recent works also unveiled their critical roles in cancer.
Project description:The polycomb group (PcG) proteins function in gene silencing through histone modifications. They form chromatin-associated multiprotein complexes, termed polycomb repressive complex (PRC) 1 and PRC2. These two complexes work in a coordinated manner in the maintenance of cellular memories through transcriptional repression of target genes. EZH2 is a catalytic component of PRC2 and trimethylates histone H3 at lysine 27 to transcriptionally repress the target genes. PcG proteins have been characterized as general regulators of stem cells, but recent works also unveiled their critical roles in cancer. Purified leukemic cells from BM of recipient mice transplanted with wild-type and Ezh2-/- leukemic cells transformed with MLL-AF9 leukemic fusion gene were subjected to RNA extraction and hybridization on Affymetrix microarrays.
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:By combining extensive biochemical fractionation with quantitative mass spectrometry, we directly examined the composition of soluble multiprotein complexes among diverse animal models. The project has been jointly supervised by Andrew Emili and Edward M. Marcotte. Project website: http://metazoa.med.utoronto.ca
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.