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    2022-05-23


    Given that the majority of ARv7-binding sites overlap with the ARfl cistrome (Figure 3A), we compared sites shared by ARfl and ARv7 (n = 2629) and sites occupied by ARfl only (n = 4,737) (Table S3). This revealed that the AR motif was more prevalent at ARfl/ ARv7 sites than at ARfl-only sites (Figure S3D). Although we did 
    not observe any differences in gene expression in response to ARfl KD for targets in the vicinity of ARfl/ARv7 or ARfl-only sites, shARv7-regulated genes associated with the two sites were significantly differentially expressed (Figure S3E). To investigate whether ARv7 and ARfl co-occupy the same genomic loci, we carried out sequential ARfl/ARv7 ChIP-re-ChIP experiments at select target genes. We detected positive signal enrichment (Fig-ure S3F), suggesting a potential functional interaction (i.e., heter-odimerization) of the two receptors. To investigate this further, we next employed acceptor photobleaching fluorescence reso-nance energy transfer (FRET) (Figure 3D). We observed strong FRET signals for ARfl/ARfl and ARfl/ARv7 or ARv7/ARfl interac-tions, but not for ARv7/ARv7 homotypic interaction. Although these results reinforce a model of ARfl and ARv7 heterodimeriza-tion, they do not establish codependent binding on chromatin, as this is not required for the FRET signal. To further investigate Metronidazole binding, we performed ChIP-seq of both AR variants in the shGFP, shARv7, and shARfl LNCaP95 cells. Here, KD of ARv7 significantly reduced ARfl chromatin binding in both the vehicle and DHT condition (Figure 3E). Concordantly, loss of ARfl also reduced ARv7 binding in both treatment conditions (Figure 3E). Similar results were obtained in 22Rv1 cells, where codependent binding of ARfl and ARv7 was observed at ARE-containing sites with high levels of both factors (Figures S3G– S3I). Taken together, these results suggest that ARfl and ARv7 form heterodimers and can modulate their respective DNA-bind-ing affinities.
    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 genes 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).