As bivalent BET inhibitors simultaneously engage BD1 and BD2 bromodomains , we hypothesized that trivalent SIM1 would also display a cis intramolecular engagement of a BET protein. To explore this, we first employed biophysical binding assays with recombinant proteins using size-exclusion chromatography (SEC) with varying tandem BD1-BD2 constructs from BRD4. These included wild-type (WT), capable of cis intramolecular binding, or point mutations in either BD1 or BD2 at a conserved asparagine residue in the ligand-binding pocket (N140F in BRD4 BD1 , or N433F in BRD4 BD2 ), to abrogate cis binding . SIM1 and MT1 both shifted the SEC profile of BRD4 WT BD1-BD2 tandem construct to a higher elution volume, compared to free or MZ-1-bound BRD4, consistent with the formation of a more compact intramolecular 1:1 complex (Fig. ). In contrast, when either N140F or N433F mutant tandems were used, we observed a significant shift to lower elution volumes with both SIM1 and MT1, consistent with the formation of higher-molecular weight 2:1 species in solution (Fig. and Extended Data Fig. ). Having established that SIM1 engages BD1 and BD2 in a cis fashion, we next asked whether SIM1 could form a 1:1:1 complex between the BD1-BD2 tandem domain and VHL. Indeed, a sample containing 1:1:1 equivalents of SIM1, BD1-BD2, and VHL-Elongin B-Elongin C complex (VCB) ran at lower elution volumes compared to either of the two peaks observed from a sample containing the same equivalent ratio of cis-SIM1, BD1-BD2 tandem and VCB, where only the 1:1 cis-SIM1:BD1-BD2 complex and unbound VCB are formed (Fig. ).