Project description:We have performed ChIP-sequencing analysis on human FOXN2 and RFX1 target sequences in human embryonic kidney HEK293T cells stably expressing Streptavidin-S-FLAG (SFB) triple-tagged proteins. The NGS sequencing were performed on Illumina MiSeq desktop sequencer.
Project description:PhosphoTMT experiment on HEK293T cells transiently transfected with either SFB-TLK1, SFB-TLK2, or siRNAs targeting TLK1, TLK2, or TLK1 and TLK2. Data represents 4 biological replicates.
Project description:Histone deacetylation is involved in epigenetically mediated tumor suppressor genes silencing. The screen of genes modulated by histone acetylation in gastric cancer maycontribute to the identification of potential therapeutic targets. In this experiment we aimed to identify differentially expressed genes by comparing Trichostatin A (TSA)-treated and non-treated gastric cancer cell lines. TrichostatinA (TSA) belongs to the hydroxamic acid chemical class histone deacetylaseinhibitors, which selectively inhibit the class I and II mammalian histonedeacetylase families. As expected, TSA acetylates promoter regions, or disables corepressors, resulting in increased gene expression. The ACP02 and ACP03 gastric cancer cell lines used in this experiment were previously established by our research group from primary gastric adenocarcinomas classified as diffuse and intestinal types, respectively. Both cell lines present chromosome 8 trisomy, MYC amplification, and TP53 loss of copy. Moreover, ACP03 is able to start a tumorigenesis process in Cebus apellas.
Project description:To identify acetylation-dependent posttranslational modifications (PTMs) of G6PD, site-specifically acetylated and Flag-tagged G6PD was expressed in HEK293T cells by genetically encoding the incorporation of acetylated lysine in response to an in-frame TAG stop codon. K403-acetylated G6PD (sample) and K414-acetylated G6PD (control) were co-expressed with WT Fyn kinase and a catalytically inactive mutant of Fyn (FynDN). G6PD was immunoprecipitated using anti-Flag beads before MS analysis.
Project description:Pannexin 1 (Panx1) channels can be activated by alpha 1 adrenoceptor-induced pathways for the sympathetic regulation of blood pressure; however, the molecular mechanisms for this form of Panx1 channel activation remain elusive. To identify potential acetyl-lysine residue(s) of Panx1 channels that might be involved in this receptor-mediated Panx1 channel activation, we performed mass-spectrometry on Strep-tagged Panx1 proteins precipitated from the whole cell lysate of HEK293T cells after stable isotope labeling by amino acids in cell culture (SILAC). Those HEK293T cells were transiently transfected with alpha1D adrenoceptors and human Panx1-Strep, with or without phenylephrine stimulation. Equal amounts of light-labeled (control) and heavy-labeled (phenylephrine stimulated) cell lysates were pooled, and Panx1 proteins were precipitated by Strep-Tactin beads, followed by LC-MS-MS analysis. From three biological replicates, we identified a consistent, albeit modest, reduction of acetylation level at K140, following phenylephrine stimulation. Whereas the acetylation level of other lysine residues (K321, K374, K381, K409) were variable among three replicates or were unaffected by phenylephrine treatment. These results implicate Panx1 K140 as a potential regulatory site for receptor-mediated channel activation via an acetylation-deacetylation mechanism.
Project description:Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason cells regulate these levels has remained unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pHi). As pHi decreases, histones are globally deacetylated by histone deacetylases (HDACs) and the released acetate anions are co-exported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pHi. Conversely, global histone acetylation increases at more alkaline pHi, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation at promoters and intergenic regions. Thus acetylation of chromatin functions as a rheostat to regulate pHi with important implications for therapeutic use of HDAC inhibitors. To investigate the redistribution of H4K16ac throughout the genome upon treatment at pH 6.5
Project description:The incubation of a 10,000 member PNA-encoded peptide library with cells over-expressing the alpha(v)beta(3) and alpha(v)beta(5) integrins (D54) or CCR6 (HEK293T-CCR6) followed by microarray analysis allowed detailed information on the interaction between peptide-ligands and cell surface receptors to be extracted. This allowed the identification of new cell specific ligands for alpha(v)beta(3) and alpha(v)beta(5) integrins and CCR6 and offers an approach to ligand discovery that allows the comparative, competitive and simultaneous analysis of different cell types for the identification of differences in surface-receptor ligands and/or receptor expression between cell types.
Project description:METTL3 constitutes the catalytic subunit of the major methyltransferase complex mediating the formation of N6-methyladenosine (m6A) in mRNA. Although the resulting m6A is known to regulate numerous cellular processes, the protein interaction network of this complex remains largely unknown. Here, we employed ascorbate peroxidase-based proximity labeling followed by LC-MS/MS analysis to examine systematically the interaction proteins of METTL3 and METTL14 in HEK293T cells. Aside from many proteins involved in mRNA splicing, a subset of histone regulatory proteins are enriched in the proximity proteome of METTL3, including histone acetyltransferase 1 (HAT1). In addition, genetic ablation of METTL3 led to reduced chromatin occupancy of HAT1 isoform-a, which is expressed exclusively in the nucleus, and diminished H3K9Ac in HEK293T cells, especially in retrotransposons. Depletion of HAT1 led to diminished H3K9Ac, which could be restored by wild-type HAT but not the one fused with the nuclear export sequence (NES) of PKI to remove it from the nucleus. Our in vitro acetylation assay further confirmed the acetylation activity of HAT1 isoform-a on nucleosome histone H3K9. The reduced retrotransposons H3K9Ac in METTL3-/- cells could be restored by ectopic expression of wild-type METTL3, but not its catalytically inactive D395A mutant. Together, we assessed the protein interactome of METTL3, uncovered a new function of HAT1 in acetylating H3K9 in chromatin, and revealed a crosstalk between METTL3-mediated formation of m6A and HAT1-catalyzed formation of H3K9Ac in chromatin.
Project description:METTL3 constitutes the catalytic subunit of the major methyltransferase complex mediating the formation of N6-methyladenosine (m6A) in mRNA. Although the resulting m6A is known to regulate numerous cellular processes, the protein interaction network of this complex remains largely unknown. Here, we employed ascorbate peroxidase-based proximity labeling followed by LC-MS/MS analysis to examine systematically the interaction proteins of METTL3 and METTL14 in HEK293T cells. Aside from many proteins involved in mRNA splicing, a subset of histone regulatory proteins are enriched in the proximity proteome of METTL3, including histone acetyltransferase 1 (HAT1). In addition, genetic ablation of METTL3 led to reduced chromatin occupancy of HAT1 isoform-a, which is expressed exclusively in the nucleus, and diminished H3K9Ac in HEK293T cells, especially in retrotransposons. Depletion of HAT1 led to diminished H3K9Ac, which could be restored by wild-type HAT but not the one fused with the nuclear export sequence (NES) of PKI to remove it from the nucleus. Our in vitro acetylation assay further confirmed the acetylation activity of HAT1 isoform-a on nucleosome histone H3K9. The reduced retrotransposons H3K9Ac in METTL3-/- cells could be restored by ectopic expression of wild-type METTL3, but not its catalytically inactive D395A mutant. Together, we assessed the protein interactome of METTL3, uncovered a new function of HAT1 in acetylating H3K9 in chromatin, and revealed a crosstalk between METTL3-mediated formation of m6A and HAT1-catalyzed formation of H3K9Ac in chromatin.