SET1B interacts with the RNA Pol II complex and requires multiple functional domains to coordinate HIF activity. A, Schematic representation of full-length (FL) SET1B and truncation mutants. Key functional domains are highlighted, including the SET domain (associated with methylation activity), RRM, and DPR motif (involved in RNA Pol II binding). B, HEK293T cells were transfected with 4 µg of the indicated expression constructs for 48 hours. Constructs were immunoprecipitated using FLAG-tagged magnetic beads, and their interaction with other members of the SET complex was evaluated by immunoblotting. C, A549 cells stably expressing full-length SET1B or SET1B truncation mutants were transfected with an SET1B siRNA (siSET1B) targeting the 3′-UTR and incubated under 21% or 1% O2 for 24 hours. mRNA expression of HIF target genes (CA9, VEGFA, and ANGPTL4) was assessed using qPCR. Graphs represent the mean ± SD from 3 biological replicates. Statistical analysis was performed using two-way ANOVA. EV; empty vector. D, Coimmunoprecipitation of SET1B and RNA Pol II. SET1B was immunoprecipitated from HeLa cells incubated under 21% or 1% O2 conditions for 6 hours. Samples were analyzed by immunoblotting with the indicated antibodies. Results are representative of 3 biological replicates. Graph depicts densitometric analysis of 3 independent experiments quantifying the interaction between SET1B and RNA Pol II under normoxic (21% O2) and hypoxic (1% O2) conditions. E, ChIP-PCR analysis of SET1B and RNA Pol II in A549 cells exposed to 21% or 1% O2 for 6 hours. Primers targeted the promoter and exon 3 regions of the CA9 and VEGFA genes. Data represent the mean ± SD from 3 biological replicates. Statistical significance was determined using two-way ANOVA. TSS; transcriptional start site. F, HEK293T cells were transfected with 4 µg of the indicated expression constructs for 48 hours. Constructs were immunoprecipitated using FLAG-tagged magnetic beads, and their interaction with other members of the SET complex and RNA Pol II was assessed by immunoblotting. G, ChIP-PCR analysis of FLAG-tagged SET1B and SET1B mutants in A549 cells exposed to 21% or 1% O2 for 6 hours. Primers targeted the promoters of the CA9 and VEGFA genes. Data represent the mean ± SD from 3 biological replicates. Statistical significance was determined using two-way ANOVA.