Mental Domain (DD). In this study we characterise an interaction between Spt5 and the transcription factor Pleiohomeotic (Pho) that we uncovered using the yeast 2-hybrid assay. We demonstrate that Spt5 and Pho act together in vivo during adult maturation and PcG repression, and that the majority of sites bound by Pho in the genome colocalize to Spt5 and NELF binding sites.Results Spt5 Interacts with PhoWe performed a yeast 2-hybrid screen using the C-terminal 153 amino acids of Drosophila Spt5 as bait to identify factors that interact with the DD. In frame fragments of Pho were recovered from the screen multiple times and did not retest as false positives. Pho is an ortholog of mammalian Ying Yang 1 (YY1) and like Spt5, is a ubiquitously expressed nuclear protein that has been implicated in both transcriptional activation and repression [12,13]. Full length Spt5 interacts with full length Pho in the yeast 2hybrid assay (Figure 1A) and the DD also interacted weakly but specifically with Pho in GST pull down assays (Figure 1B). The interaction between full length Spt5 and Pho was further validated by expressing tagged proteins in Drosophila S2 cells and performing Title Loaded From File co-immunoprecipitation assays (Figure 1C). We mapped the interaction with Spt5 to the N-terminal 351-amino acids of Pho using co-immunoprecipitation of tagged proteins (Figure 1C). Sequences within this region have previously been shown to interact with Polycomb (Pc), Polyhomeotic (Ph) and the Brahma (BRM) complex [14]. The C-terminal region of Pho (Title Loaded From File remaining 169 amino acids), which does not interact with Spt5, contains the 4 zinc finger motif (C2H2-like) that is highly conserved with human YY1 and has been shown to bind DNA [12]. The DD carrying the W049 (G994D) mutation is able to interact with Pho in the yeast 2-hybrid and GST pull down assays, and full length W049 protein interacts with Pho in the yeast 2hybrid and co-immunoprecipitation assays (Figure 1D and data not shown). Thus a failure of the interaction between Spt5 and Pho is not likely to explain the phenotypes observed in Spt5W049 mutants. The W049 mutation may be affecting the ability of Spt5 to interact with other as yet unidentified factors.for Spt5 alleles to determine if Spt5 interacts with pho during PcG repression in vivo. A two-proportion hypothesis test was applied to determine the significance of any differences observed in these frequencies. There was no significant increase in the frequency of ectopic sex combs observed in phocv/phocv males that are heterozygous for a null allele of Spt5 (Spt5MGE23) [16], indicating that halving the dose of Spt5 does not compromise residual Pho activity (Figure 2 and Table 1). This is perhaps not unexpected as Spt5 is expressed at moderate or moderately high levels during larval development [17] and Spt5MGE23/+ flies appear wild-type. However, Spt5W049/+; phocv/phocv males did show a significant increase in the number of ectopic sex combs, revealing that the W049 variant protein can disrupt the repressive activity of Pho in vivo (Figure 2 and Table 1). We also observed a small but significant increase in the number of ectopic sex combs in phocv males heterozygous for NELF-A[KG] [18] (Figure 2, Table 1). The W049 protein has a significantly reduced repressive activity on transcription in vitro, and in some contexts in vivo [11]. W049 allows RNAP II to continue transcribing through the P-TEFb checkpoint in the presence of a P-TEFb inhibitor [5,6-dichloro-1b-D-ribofuranosyl.Mental Domain (DD). In this study we characterise an interaction between Spt5 and the transcription factor Pleiohomeotic (Pho) that we uncovered using the yeast 2-hybrid assay. We demonstrate that Spt5 and Pho act together in vivo during adult maturation and PcG repression, and that the majority of sites bound by Pho in the genome colocalize to Spt5 and NELF binding sites.Results Spt5 Interacts with PhoWe performed a yeast 2-hybrid screen using the C-terminal 153 amino acids of Drosophila Spt5 as bait to identify factors that interact with the DD. In frame fragments of Pho were recovered from the screen multiple times and did not retest as false positives. Pho is an ortholog of mammalian Ying Yang 1 (YY1) and like Spt5, is a ubiquitously expressed nuclear protein that has been implicated in both transcriptional activation and repression [12,13]. Full length Spt5 interacts with full length Pho in the yeast 2hybrid assay (Figure 1A) and the DD also interacted weakly but specifically with Pho in GST pull down assays (Figure 1B). The interaction between full length Spt5 and Pho was further validated by expressing tagged proteins in Drosophila S2 cells and performing co-immunoprecipitation assays (Figure 1C). We mapped the interaction with Spt5 to the N-terminal 351-amino acids of Pho using co-immunoprecipitation of tagged proteins (Figure 1C). Sequences within this region have previously been shown to interact with Polycomb (Pc), Polyhomeotic (Ph) and the Brahma (BRM) complex [14]. The C-terminal region of Pho (remaining 169 amino acids), which does not interact with Spt5, contains the 4 zinc finger motif (C2H2-like) that is highly conserved with human YY1 and has been shown to bind DNA [12]. The DD carrying the W049 (G994D) mutation is able to interact with Pho in the yeast 2-hybrid and GST pull down assays, and full length W049 protein interacts with Pho in the yeast 2hybrid and co-immunoprecipitation assays (Figure 1D and data not shown). Thus a failure of the interaction between Spt5 and Pho is not likely to explain the phenotypes observed in Spt5W049 mutants. The W049 mutation may be affecting the ability of Spt5 to interact with other as yet unidentified factors.for Spt5 alleles to determine if Spt5 interacts with pho during PcG repression in vivo. A two-proportion hypothesis test was applied to determine the significance of any differences observed in these frequencies. There was no significant increase in the frequency of ectopic sex combs observed in phocv/phocv males that are heterozygous for a null allele of Spt5 (Spt5MGE23) [16], indicating that halving the dose of Spt5 does not compromise residual Pho activity (Figure 2 and Table 1). This is perhaps not unexpected as Spt5 is expressed at moderate or moderately high levels during larval development [17] and Spt5MGE23/+ flies appear wild-type. However, Spt5W049/+; phocv/phocv males did show a significant increase in the number of ectopic sex combs, revealing that the W049 variant protein can disrupt the repressive activity of Pho in vivo (Figure 2 and Table 1). We also observed a small but significant increase in the number of ectopic sex combs in phocv males heterozygous for NELF-A[KG] [18] (Figure 2, Table 1). The W049 protein has a significantly reduced repressive activity on transcription in vitro, and in some contexts in vivo [11]. W049 allows RNAP II to continue transcribing through the P-TEFb checkpoint in the presence of a P-TEFb inhibitor [5,6-dichloro-1b-D-ribofuranosyl.
