Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism
Fig 4
BAHD1 binds to MIER and HDACs.
A-B. TAP-MS purification of the His6-Protein-C-BAHD1-associated complex. Solubilized chromatin extracts (“Inputs”) from HPT-CT cells (“Control”) or HPT-BAHD1 cells (“BAHD1”) were processed on anti–protein C affinity matrix (E1) and nickel-Sepharose (E2). Eluted fractions were separated by SDS-PAGE and analyzed by Mass Spectrometry. A. Silver staining of E2. B. Immunoblots of inputs E1 and E2, using antibodies against the specified proteins (for G9a, also see S4 Fig). C. Schematic representation of MTA1, RERE, MIER1 and BAHD1. ELM2, SANT, BAH domains, cPRR region and amino-acid numbers are indicated. D. Schematic diagrams of NurD and BAHD1 co-repressor complexes. Different protein paralogs can be part of distinct complexes. E-F. Nuclear extracts from HEK293-FT cells expressing BAHD1-V5 or YFPc-BAHD1, YFPc-BAHD1- ΔcPRR, YFPc-BAHD1- ΔBAH were used in immunoprecipitations (IP) assays with MIER1 or V5 antibodies or IgG control (E) or YFP antibodies. Vertical lines indicate cropping sites in original blots (see S5 Fig). (F). Eluted fractions were separated by SDS-PAGE and analyzed by immunoblot with the indicated antibodies (α-). In (F), the MIER1 blot was stripped and reprobed with YFP antibodies (see S5 Fig for a replicate experiment).