Factors involved in chromatin modification The transcription based screening method using an endogenous E m3 promoter sequence was particu larly useful for identifying chromatin components. We identified several chromatin Nutlin-3a Mdm2 inhibitor factors previously shown to affect Notch dependent transcription. A component of the SAGA histone acetyltransferase complex, Nipped A, was identified. Nipped A, the Drosophila homologue of yeast Tra1 and mammalian TRAP proteins, is a key fac tor of the SAGA complex. It has been shown previously that reduced Nipped A dosage enhances the wing notching phenotype of both mastermind and Notch mutants. The RNAi treated cell culture data demonstrates that Nipped A promotes transcription at the E m3 promoter both in the presence and absence of activated Notch.

This shows that the result of Nipped A function is independent of whether active Nicd is localized on the target promoter. We also identified several homologues of components of the Rpd3 histone deacetylase co repressor complex, including Sin3a, Sds3, a putative ortholog of SAP130, and Rpd3 itself. When these factors were targeted by RNAi, there was an increase in Notch induced reporter transcription, consis tent with the role of the Rpd3 complex and histone deacetylation as a transcriptional inhibitor. Conver sely, knocking down Sin3a had the opposite effect on the uninduced baseline activity of the E m3 promo ter. Thus, unlike the histone acetylation complex, the activity of the deacetylation com plex on the E m3 promoter is dependent on the presence of activated Notch.

The screen identified several components of the chro matin remodeling complex Brahma, Brm Associated Protein 55, Brm Associated Protein 170, polybromo, and moira. A previous Dro sophila phenotype based screen has found a genetic interaction between the Notch ligand Delta and another component of the Brahma complex, brahma. Loss of function brm alleles were found to enhance Delta mutant phenotypes in eye and bristle development. The various Brahma components identified in this study show a complex array of effects on the transcrip tion of the E m3 promoter, some consistent with previously described loss of function brm alleles while others opposing. RNAi directed against Bap55 and poly bromo demonstrated Anacetrapib a specific reduction in Notch induced transcription that is consis tent with the previously observed role of brm in Notch signaling during Drosophila development. Unex pected are the Brahma subunits identified that modulate transcription from the uninduced E m3 promoter, Bap170 and mor. The screen reveals that both of these components specifically mediate transcription from the uninduced E m3 promoter, while Bap170 activates and mor represses.