Journal: Nature Communications

Article Title: The PTM profiling of CTCF reveals the regulation of 3D chromatin structure by O-GlcNAcylation

doi: 10.1038/s41467-024-47048-3

Figure Lengend Snippet: a Whole-cell lysates were denatured and incubated with RL2-conjugated agarose beads (left) or WGA-conjugated agarose beads (right) to enrich O-GlcNAcylated proteins and bound proteins were analyzed by western blot. b Whole-cell lysates were denatured and immunoprecipitated with antibody for CTCF from WT mESCs. O-GlcNAcylation of proteins was detected by western blot with O-GlcNAc-specific antibody (RL2). c Immunoblot showing the O-GlcNAcylated signal of WT_CTCF and T668A O-GlcNAc-deficient CTCF. d The CTCF was purified by immunoprecipitation with Flag antibody in WT_CTCF mESCs and the O-GlcNAcylated signal of CTCF was decreased when treated with β-N-Acetyl-hexosaminidase (β-hex) in vitro. e Representative quantifications of CTCF O-GlcNAcylation from different tissues of mouse. Bands were quantified with ImageJ and the bands of the RL2 signal were normalized to purified CTCF to control for equal loading. Data were presented as mean ± SD ( n = 3 biologically independent samples). f Representative quantifications of CTCF O-GlcNAcylation from MEF, pre-iPSC, and mESC. Bands were quantified with ImageJ and the bands of the RL2 signal were normalized to purified CTCF to control for equal loading. Data were presented as mean ± SD ( n = 3 biologically independent samples). g Validation of the endogenous interaction between OGT and CTCF by OGT (left) and CTCF (right) antibody-based IP in mESCs. h Predicted protein interaction based on structures of the mouse OGT and CTCF showing that OGT interacted with CTCF in the region of O-GlcNAcylation. OGT was in blue, CTCF was in green and the site of CTCF O-GlcNAcylation was in red. i Quantitative PCR analysis of Ogt expression after the knockdown of Ogt with 3 pairs of siRNA (left) and the signal of CTCF O-GlcNAcylation was decreased (right). This experiment was performed three times, with similar results. Data were presented as mean ± SD ( n = 3 biologically independent samples). p -values by two-tailed t -test, significance levels were indicated by asterisks: ** p < 0.01; *** p < 0.001. All western blot experiments were performed three times, with similar results. Source data are provided as a Source Data file.

Article Snippet: The treatment of β-N-Acetyl-hexosaminidase (β-hex, NEB, #P0721S) was performed according to the manufacturer’s instructions, and 4 μg purified CTCF was used.

Techniques: Incubation, Western Blot, Immunoprecipitation, Purification, In Vitro, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: A novel family of sugar-specific phosphodiesterases that remove zwitterionic modifications of GlcNAc

doi: 10.1016/j.jbc.2023.105437

Figure Lengend Snippet: Metagenomic screening strategy and candidate phosphodiesterase assessment. A , chemical structures of GlcNAc-6-PC and GlcNAc-6-PE. B , a schematic of the coupled assay used to screen for environmental GlcNAc-6-PC phosphodiesterases. A quenched fluorescent GlcNAc analog (4MU-β-GlcNAc-6-PC) is used as substrate. A fluorescent signal ( yellow 4-MU) is generated upon sequential removal of PC by an environmental phosphodiesterase and GlcNAc by β- N -acetylhexosaminidase f provided in the assay. Screening data is provided in Fig. S1 . Glycan structure is represented using the Symbol Nomenclature for Glycans nomenclature. C , SDS-PAGE separation of in vitro produced proteins from eight candidate ORFs identified in the DNA sequence of fosmid “hits” (see Table 1 ). Shown are expressed proteins ( red circles ) and the predicted molecular weight (kDa) of each ( bottom ). D , proteins were assayed for phosphodiesterase activity using 4MU-β-GlcNAc-6-PC ( ± hexosaminidase) and 4MU-PC substrates. Fluorescence was measured at λ ex = 365 nm and λ em = 445 nm after 3 h. 4MU, 4-methylumbelliferyl; DHFR, dihydrofolate receptor; PC, phosphorylcholine; PE, phosphoethanolamine.

Article Snippet: Chemicals, reagents, and enzymes β- N -acetylhexosaminidase f , RNase inhibitor murine, Q5 Hot Start High-Fidelity 2× Master Mix, endoglycoceramidase I (EGCase I), NEB Express I q Competent E. coli, N -Glycosidase F (PNGase F) (glycerol-free, recombinant), α1-2,3,6 mannosidase, and GlycoBuffer 1 were from New England Biolabs.

Techniques: Generated, SDS Page, In Vitro, Produced, Sequencing, Molecular Weight, Activity Assay, Fluorescence