No abstract

Modular Cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of Parallel Adaptor Capture (PAC) proteomics, and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCFFBXL17 in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins.

No abstract

Loss of function of the tumor suppressor protein BRCA1 is responsible for a high percentage of familial and also sporadic breast cancers. Early work identified a stimulatory transcriptional coactivator function for the BRCA1 protein, and more recently, BRCA1 has been implicated in transcriptional repression, although few examples of repressed genes have been characterized. We recently used an in vitro transcription assay to identify a biochemical mechanism that explained the BRCA1 stimulatory activity. In this study, we identified an ubiquitin-dependent mechanism by which BRCA1 inhibits transcription. BRCA1 ubiquitinates the transcriptional preinitiation complex, preventing stable association of TFIIE and TFIIH, and thus blocks the initiation of mRNA synthesis. What is striking about this mechanism of regulation by BRCA1 is that the ubiquitination of the preinitiation complex is not targeting proteins for degradation by the proteasome, nor are ubiquitin receptors modifying the activity, but rather the ubiquitin moiety itself interferes with the assembly of basal transcription factors at the promoter. Using RNAi to knockdown expression of the endogenous BRCA1 protein, we assessed the level of repression dependent on BRCA1 in the cell, and we found that BRCA1 is at least as significant a transcriptional repressor as it is an activator. These results define a biochemical mechanism by which the BRCA1 enzymatic activity regulates a key cellular process. RNA polymerase II ͉ TFIIE ͉ transcription BRCA1 is the breast and ovarian cancer specific tumor suppressor (1). Loss of BRCA1 can occur either by mutation of both alleles of the gene in the tumor cell (Ϸ4% of all breast cancer cases) or by epigenetic down-regulation of the gene by methylation of its promoter (Ϸ14% of sporadic breast cancer cases and up to 30% of ovarian cancer cases) (2, 3).How BRCA1 protein exerts its tumor suppressor function remains unresolved, but it has been found to regulate a number of processes including transcription, repair of DNA damage, cell cycle checkpoints, and centrosome dynamics (4-6). The biochemical mechanism(s) by which BRCA1 regulates these diverse processes is unknown. The BRCA1 protein has the enzymatic activity of an E3 ubiquitin ligase when bound as a heterodimer to BARD1 (7,8), and it is likely that the ubiquitin ligase activity is critical for BRCA1/BARD1 regulation of transcription and other processes. In this paper, the BRCA1/BARD1 heterodimer will be simply referred to as ''BRCA1.'' Previously, we found that BRCA1 strongly stimulated transcription by stabilizing the preinitiation complex (PIC) on the core promoter (9). This activity was observed in either the presence or absence of BARD1 and was independent of ubiquitination function. Our results suggested that BRCA1 enhanced the stability of the PIC on promoter elements relative to bulk DNA (9). Because BRCA1 can ubiquitinate phosphorylated RNA polymerase II (RNAPII) both in vitro and in vivo (10, 11), we wondered whether the E3 ubiquitin ligase activity of BRCA1 might a...