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  • 2021-03
  • br ARv Preferentially Interacts with Transcriptional Corepre


    ARv7 Preferentially Interacts with Transcriptional Corepressors
    Given the codependent binding of ARfl and ARv7 to chromatin, but divergent transcriptional output, we speculated that other factors might contribute to the divergent genomic function of the two receptors. To address this, we first determined the AR isoform-specific chromatin-binding kinetics using fluorescence recovery after photobleaching (FRAP). The experiment was per-formed using wild-type (WT; ARfl or ARv7) or DNA-binding domain mutants of AR (ARfl R585K or ARv7 R585K). Previous studies have shown that due to impaired DNA binding, the
    Figure 2. ARv7, Unlike ARfl, Functions as a Transcriptional Repressor in CRPC Cells
    (A and B) Volcano plots of differentially expressed Acetylcysteine in shARv7 (A) or shARfl (B) cells, compared with shGFP control. Significantly altered genes (fold change >±1.5; adjusted p value <0.05) are highlighted in red (activated) or blue (repressed). Select AR targets and significant outliers are labeled.
    (C) Top: violin plots of log2 fold changes of ARv7-regulated (blue) and ARfl-regulated (red) genes (relative to the shGFP control) in response to DHT stimulation. Only genes with an adjusted p value of <0.05 are shown. Bottom: bar plots of the mean log2 fold changes of the ARv7-regulated (blue) and ARfl-regulated (red) target genes as above. ****p % 0.0001 by ANOVA and Tukey’s honest significant difference test (HSD).
    (D) Comparison of log2 fold changes of significantly dysregulated genes (adjusted p value <0.05) in response to shARv7 or shARfl (as defined in A and B). Colors indicate genes primarily dysregulated by shARfl (red), shARv7 (blue), or both (purple). Select classical AR targets are labeled.
    R585K mutant displays a quicker recovery time after photo-bleaching compared with WT AR, as it does not form long-term interactions with DNA (van Royen et al., 2012). While this was the case for ARfl, ARv7 WT and the R585K mutant FRAP sig-nals were indistinguishable (Figure S4A). This suggests that ARv7, in the absence of ARfl, has few long-term DNA interac-tions, further supported by the finding that ARfl has a speckled nuclear distribution, which was not observed for ARfl-R585K, ARv7, or ARv7-R585K (Figure S4A).
    Next, we examined AR isoform-specific affinities for coregula-tor binding using a MARCoNI peptide-binding assay (Desmet et al., 2014). In this assay, LXXLL or FXXLF motif-encompassing peptides of known coregulators immobilized on an array are exposed to nuclear receptor-containing cell lysates, and binding is detected using specific antibodies. Using a pan-AR antibody we observed that AR bound coregulator peptides similarly in the shGFP and shARv7 cells (Figure S4B). This suggests that these interactions are primarily mediated by the LBD-containing 
    ARfl and are not significantly altered by the loss of ARv7. In com-parison, most interactions were lost in the absence of ARfl, particularly those for classical AR coactivators, such as NCOA1-6 (Figure S4B). However, we also observed increased binding to several corepressor peptides, including those for CNOT1, NCOR1, NCOR2, NRIP1, and PELP1 (Perissi et al., 2010) (Figures 4A and S4B). To test whether this binding was due to the direct interaction of ARv7 with select corepressors, we employed coIP in LNCaP95 cells following ARv7 or ARfl KD. We observed a small but reproducible increase in binding of ARv7 to NCOR1 and NCOR2 upon ARfl depletion (Figures 4B and S4C–S4E), but were unable to validate the ARv7 and NRIP1 interaction (not shown). This supports a model whereby ARv7 preferentially interacts with specific transcriptional core-pressors, such as members of the NCOR family.
    To interrogate the functional link between NCOR- and ARv7-dependent transcription, we investigated the effect of small interfering RNA (siRNA)-mediated corepressor depletion on
    Figure 3. ARfl and ARv7 Binding to Chromatin Is Interdependent
    (A) Euler diagram of the overlap (R1 bp) of ARv7 (blue) and ARfl (red) cistromes, profiled in the absence of hormone (ETOH) or after 4 h of DHT (10 nM) treatment. The numbers of unique and overlapping binding sites are indicated.
    (B) Top: Euler diagram of the overlap of ARv7 (blue), ARfl (red), and AR N-terminal cistromes (ARN; purple) in the absence of hormone. Bottom: signal profiles (500-bp interval around the center of ARv7 peaks) of ARv7, ARfl, and ARN cistromes at different ARv7-binding sites. Left panel: 2,629 ARv7, ARfl, and ARN shared peaks. Middle panel: 199 ARv7 and ARN shared peaks. Right panel: 595 ‘‘ARv7-unique’’ peaks.
    (D) Apparent FRET efficiencies representative of the level of AR isoform interactions, as shown. Values are the mean of 34–48 cells ±SEM.
    (E) Signal profiles of ARfl (left) and ARv7 (right) cistromes centered on AR isoform peaks. Cells were induced for 3 days and treated for 4 h with vehicle (ETOH) or