Studies have shown that despite overlapping functions observed among the E

Studies have shown that despite overlapping functions observed among the E2F proteins, individual E2Fs also possess unique transcriptional regulatory functions. Specificity of function is dictated in part by the specificity in the proteins recruited by individual E2Fs. E2F3, for example, was previously observed to bind TFE3 to dictate specific binding of E2F3 to the DNA polymerase alpha p68 promoter [11]. E2F1 specifically bound to Jab1 and this interaction was found to be important for E2F1’s role in apoptosis [33]. To determine if BRG1 binds specifically to E2F6, we determined the ability of HA-tagged E2F1, 2, 3, and 4 to interact with BRG1. As shown in Figure 2, only HA-tagged E2F4 and HA-tagged E2F6 were able to associate with BRG1. Although E2F4 did not interact with BRG1 in yeast (Table 1), we assume that the interaction between BRG1 with E2F4 may be weaker and therefore not detected unless ectopically MedChemExpress ��-Sitosterol ��-D-glucoside expressed in cells.whether its interaction with BRG1 is independent of the SWI/ SNF complex, we determined E2F6’s interaction with BAF 155 and BAF180. As shown in Figure 3B and 3C, immunoprecipitation with an antibody to BAF155 and BAF180 showed an association of these BAFs with E2F6. Our results suggest that E2F6 is a component of the polybromo-containing SWI/SNF complex, PBAF [36].E2F6 and BRG1 associates with the E2F1 promoterOur prior work has shown that E2F6 specifically Homotaurine custom synthesis recognizes promoters of E2F target genes that are activated at G1/S of the cell cycle. This interaction during S phase is coincident with a decline in expression of the genes [5]. To test the possibility that BRG1 may interact with E2F6 on these promoters, we performed a chromatin immunoprecipitation assay to assess the presence of Brg1 on the promoters of genes previously found to be activated during G1-S phase of the cell cycle. Cells were synchronized by serum deprivation followed by induction with serum for 24 hours whereby cells are in S phase of the cell cycle. As shown in Figure 4, BRG1 associates with the G1/S promoters concurrently with E2F6 during S phase of the cell cycle. BRG1, however, was not observed on the CYCA2, CDC2 and CYCB1 promoters (G2/M promoters), where E2F6 does not bind.BRG1 functions in a complex with other proteinsPrior work has shown that members of the E2F family, including E2F6, dimerize with DP proteins for efficient DNA binding activity [18,19,24]. To determine if BRG1 is in a complex with E2F6/DP or whether it may be functioning in transcriptional regulation independent of DP, we assessed an association between DP and BRG1. An antibody to DP1 was able to immunoprecipitate both BRG1 and E2F6 (Figure 3A). BRG1 is a component of the SWI/SNF complexes. In the mammalian system, SWI/SNF complexes contain, in addition to BRM or BRG1, as many as 8?0 subunits referred to as BRM- or BRG1- associated factors or BAFs [34,35]. To determine if E2F6 is also associated with any BAFs via its interaction with BRG1, orE2F6 repression of the E2F1 promoter is blocked by a dominant negative BRGTo understand the potential function of the E2F6 and BRG1 complex, we used a luciferase reporter construct that consists of the E2F1 promoter [37]. Consistent with a role for E2F6 as a transcriptional repressor, we observed repression of the E2F1 reporter upon ectopic expression of E2F6 (Figure 5A). FurtherE2F6 and BRG1 in Transcriptional RegulationFigure 3. BRG1 and E2F6 functions in a complex with other proteins. A) DP1, previously shown to interact with E2.Studies have shown that despite overlapping functions observed among the E2F proteins, individual E2Fs also possess unique transcriptional regulatory functions. Specificity of function is dictated in part by the specificity in the proteins recruited by individual E2Fs. E2F3, for example, was previously observed to bind TFE3 to dictate specific binding of E2F3 to the DNA polymerase alpha p68 promoter [11]. E2F1 specifically bound to Jab1 and this interaction was found to be important for E2F1’s role in apoptosis [33]. To determine if BRG1 binds specifically to E2F6, we determined the ability of HA-tagged E2F1, 2, 3, and 4 to interact with BRG1. As shown in Figure 2, only HA-tagged E2F4 and HA-tagged E2F6 were able to associate with BRG1. Although E2F4 did not interact with BRG1 in yeast (Table 1), we assume that the interaction between BRG1 with E2F4 may be weaker and therefore not detected unless ectopically expressed in cells.whether its interaction with BRG1 is independent of the SWI/ SNF complex, we determined E2F6’s interaction with BAF 155 and BAF180. As shown in Figure 3B and 3C, immunoprecipitation with an antibody to BAF155 and BAF180 showed an association of these BAFs with E2F6. Our results suggest that E2F6 is a component of the polybromo-containing SWI/SNF complex, PBAF [36].E2F6 and BRG1 associates with the E2F1 promoterOur prior work has shown that E2F6 specifically recognizes promoters of E2F target genes that are activated at G1/S of the cell cycle. This interaction during S phase is coincident with a decline in expression of the genes [5]. To test the possibility that BRG1 may interact with E2F6 on these promoters, we performed a chromatin immunoprecipitation assay to assess the presence of Brg1 on the promoters of genes previously found to be activated during G1-S phase of the cell cycle. Cells were synchronized by serum deprivation followed by induction with serum for 24 hours whereby cells are in S phase of the cell cycle. As shown in Figure 4, BRG1 associates with the G1/S promoters concurrently with E2F6 during S phase of the cell cycle. BRG1, however, was not observed on the CYCA2, CDC2 and CYCB1 promoters (G2/M promoters), where E2F6 does not bind.BRG1 functions in a complex with other proteinsPrior work has shown that members of the E2F family, including E2F6, dimerize with DP proteins for efficient DNA binding activity [18,19,24]. To determine if BRG1 is in a complex with E2F6/DP or whether it may be functioning in transcriptional regulation independent of DP, we assessed an association between DP and BRG1. An antibody to DP1 was able to immunoprecipitate both BRG1 and E2F6 (Figure 3A). BRG1 is a component of the SWI/SNF complexes. In the mammalian system, SWI/SNF complexes contain, in addition to BRM or BRG1, as many as 8?0 subunits referred to as BRM- or BRG1- associated factors or BAFs [34,35]. To determine if E2F6 is also associated with any BAFs via its interaction with BRG1, orE2F6 repression of the E2F1 promoter is blocked by a dominant negative BRGTo understand the potential function of the E2F6 and BRG1 complex, we used a luciferase reporter construct that consists of the E2F1 promoter [37]. Consistent with a role for E2F6 as a transcriptional repressor, we observed repression of the E2F1 reporter upon ectopic expression of E2F6 (Figure 5A). FurtherE2F6 and BRG1 in Transcriptional RegulationFigure 3. BRG1 and E2F6 functions in a complex with other proteins. A) DP1, previously shown to interact with E2.