Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA388471 | ENA |
REPOSITORIES: ENA
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Project description:Enhancers are fundamental to gene regulation. Post-translational modifications by the small ubiquitin-like modifiers (SUMO) modify chromatin regulation enzymes, including histone acetylases and deacetylases. However, it remains unclear whether SUMOylation regulates enhancer marks, acetylation at the 27th lysine residue of the histone H3 protein (H3K27Ac). We hypothesize that SUMOylation regulates H3K27Ac. To test this hypothesis, we performed genome-wide ChIP-seq analyses. We discovered that knockdown (KD) of the SUMO activating enzyme catalytic subunit UBA2 reduced H3K27Ac at most enhancers. Bioinformatic analysis revealed that TFAP2C-binding sites are enriched in enhancers whose H3K27Ac was reduced by UBA2 KD. ChIP-seq analysis in combination with molecular biological methods showed that TFAP2C binding to enhancers increased upon UBA2 KD or inhibition of SUMOylation by a small molecule SUMOylation inhibitor. However, this is not due to the SUMOylation of TFAP2C itself. Proteomics analysis of TFAP2C interactome on the chromatin identified histone deacetylation (HDAC) machinery. TFAP2C KD reduced HDAC binding to chromatin and increased H3K27Ac marks at enhancer regions, suggesting that TFAP2C is involved in recruiting HDAC. Taken together, our findings provide important insights into regulation of enhancer marks by SUMOylation.
2024-04-13 | PXD045644 | Pride
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.
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Project description:RNA extracted at 240min. Samples are rRNA depleted. Expression measurement of Homo sapiens genes.
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Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
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Project description:To investigate the impact on global H3K27ac patterns following HDAC1/2 inhibition with romidepsin, we performed using ChIP-seq using formaldehyde crosslinked BT67 romidepsin treated versus vehicle treated cells.
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