Journal: Communications Chemistry

Article Title: Human O- GlcNAcase catalytic-stalk dimer anchors flexible histone binding domains

doi: 10.1038/s42004-025-01813-7

Figure Lengend Snippet: a Saturable binding was observed with H3K36 Me3 and H4K 5,8,12,16Ac mononucleosomes but not with K3K27 Me3 , K27 Me3 S28 phos , or unmodified nucleosomes. b Binding parameters were determined for H3K36 Me3 and H4K 5,8,12,16Ac modified nucleosomes using a saturation plot and Scatchard analysis. Nucleosomes with the H4K 5,8,12,16Ac modification ( K d ~ 220 nM; B max = ~ 100 nM; Hill coefficient = 0.8), and the H3K36 Me3 modification binds to OGA-L ( K d ~ 290 nM; B max = ~ 90 nM; Hill coefficient = 1.9). Error bars representing the standard deviation centered on the mean. N = 3 independent experimental replicates. c Using a C-terminal anti-OGA-L, ChIP pulldown of OGA in OGA WT MEFs enriched for H3K36 Me3 but not H3K27 Me3 . d Extracts from OGA KO knockout cells showed no binding to either chromatin mark. N = 4 independent biological replicates.

Article Snippet: The eluted protein, along with input and FT, was subsequently analyzed by immunoblotting using anti-OGA antibody (Santa Cruz, sc-376429) and anti-H3K36 Me3 antibody (Active motif, 61022) or anti-Histone H3K27 Me3 antibody (Active motif, 39055) to detect OGA-L-bound chromatin.

Techniques: Binding Assay, Modification, Standard Deviation, Knock-Out

Journal: Communications Chemistry

Article Title: Human O- GlcNAcase catalytic-stalk dimer anchors flexible histone binding domains

doi: 10.1038/s42004-025-01813-7

Figure Lengend Snippet: Potential binding sites of the pHAT domains to nucleosomes, highlighting proximity and spacing of the H3K36 residues ( a ) as well as the H3K36 and H4K 5,8,12, and 16 residues ( b ) in the nucleosome (PDBID:1kx5: gray). OGA is colored by domain: catalytic domain, dark blue; stalk, yellow; linker, and HAT-like domain: green. The distance between K36 residues (red) is 73.5 Å, and the total distance between pHAT densities is 107 Å (green). The distance between the K36 residue and the H4K 5,8,12,16 is 72.8 Å (pink). c OGA-L pHAT binding to histone modifications such as H3K36 Me and acetylated H4 tails would facilitate recruitment to sites of active transcription and DNA repair. The structural features identified for OGA-L are likely to increase the local concentration of tihe OGA-L and allow flexible movement of the catalytic domain to facilitate O- GlcNAc removal from proteins in proximity. The OGA model is shown with unstructured linkers added from the Alphafold2 colored by domain: catalytic domain, dark blue; stalk, yellow; linker, and pHAT domain, green.

Article Snippet: The eluted protein, along with input and FT, was subsequently analyzed by immunoblotting using anti-OGA antibody (Santa Cruz, sc-376429) and anti-H3K36 Me3 antibody (Active motif, 61022) or anti-Histone H3K27 Me3 antibody (Active motif, 39055) to detect OGA-L-bound chromatin.

Techniques: Binding Assay, Residue, Concentration Assay