SERBP1 is a component of the Liver Receptor Homolog-1 transcriptional complex|核受体数据集|癌症转录调控数据集

Liver receptor homolog-1 (NR5A2) is an orphan nuclear receptor whose expression is essential for normal liver, intestine, and pancreas function. Nuclear receptors are ligand regulated transcription factors and NR5A2 has been shown to play a role in the transcriptional regulation of pathways involved in cancer. Elucidating the components of the NR5A2 transcriptional complex to better understand endogenous regulation of the receptor as well as its role in cancer remains a high priority. A sub-cellular enrichment strategy coupled with proteomic approaches was employed to identify putative NR5A2 coregulators. Nuclear fractionation protocol was essential for detection of NR5A2 peptides by MS, with most peptides being observed in the insoluble fraction (receptor bound to DNA). Duplicate data-dependent MS/MS runs using an inclusion/exclusion list improved the number of NR5A2 peptide identifications, including isoform-specific and fusion-specific peptides. Moreover, increased NR5A2 sequence coverage and a greater number of protein hits were observed with on-bead trypsin digestion of affinity captured protein. SERBP1 and ILF3 were identified as NR5A2 interacting partners. Both western blot and MS/MS analysis of endogenous receptor confirmed these binding partners are present in the NR5A2 transcriptional complex in established cell lines. Receptor knockdown by siRNA showed an increased in SERBP1 expression while ILF3 expression was unchanged. In contrast, receptor overexpression decreased only SERBP1 mRNA levels. Consistent with these data, in a promoter:reporter assay, binding of NR5A2 to the promoter region of SERBP1 resulted in a decrease in the expression level of the reporter gene, and subsequently, inhibiting transcription. In conclusion, using a sub-cellular enrichment strategy coupled with proteomic approaches, functionally relevant NR5A2 coregulators were identified. Given the receptor’s role in cancer progression, the study here elucidates additional transcriptional machinery involved in NR5A2 signaling and potentially provides new targets for therapeutics development.