Extended Data Fig. 2: Histone H3 tyrosine 99 sulfation is catalyzed by SULT1B1.

(a) to (d), H3Y99sulf is not regulated by the TPSTs. TPST1 and TPST2 in HepG2 and LM3 cells were depleted by expressing shRNAs against corresponding mRNAs. The knock-down efficiency was tested by performing real-time PCR (a) and (c), n = 4 biologically independent samples, two-sided t-test were conducted to calculate the P-value, the data is presented as the means±s.d. The level of H3Y99sulf in the cell lines with or without depletion of TPST1 and TPST2 was determined by performing immunoblotting assays with the indicated antibodies (b) and (d). The immunoblotting results represent three independent experiments. (e), Scheme of phenol sulfotransferase activities. (f), Validation of knock-down efficiency of phenol sulfotransferase family members. Real-time PCR assays were performed to confirm the knock-down of SULT1A1, SULT1B1, SULT1C1, SULT1E1, SULT2B1, and SULT4A1 in the culture HepG2 and LM3 cells, n = 4 biologically independent samples, two-sided t-test were conducted to calculate the P-value, the data is presented as the means±s.d. (g) to (i), SULT1B1 catalyzes the sulfation of histone H3Y99 in vitro. Purified histone H3 was incubated with PAPS and purified wide-type SULT1B1, inactive SULT1B1-H109A mutant³⁴, and SULT1A1, respectively. A H3Y99sulf peptide was identified in the SULT1B1-mediated assay by performing HPLC-MS/MS analysis (g). Immunoblotting analyses were performed with the indicated antibodies (f) and (i). Representative images of triplicate experiments are shown. (j), The structure of SULT1B1 and the simulated active SULT1B1. (k), Superimposition of the structures of SULT1B1 in close and open states. (l), SULT1B1 cannot sulfate the H3Y99 in the assembled nucleosomes in vitro. Purified SULT1B1 and PAPS were incubated with in vitro assembled nucleosome. Immunoblotting analyses were performed with the indicated antibodies. Representative images of triplicate experiments are shown.

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