Chromatin structure in transcribed regions poses a barrier for intragenic transcription. In a comprehensive study of the yeast chromatin remodelers and the Mot1p-NC2 regulators of TATA-binding protein (TBP), we detected synthetic genetic interactions indicative of suppression of intragenic transcription. Conditional depletion of Mot1p or NC2 in absence of the ISW1 remodeler, but not in the absence of other chromatin remodelers, activated the cryptic FLO8 promoter. Likewise, conditional depletion of Mot1p or NC2 in deletion backgrounds of the H3K36 methyltransferase Set2p or the Asf1p-Rtt106p histone H3-H4 chaperones, important factors involved in maintaining a repressive chromatin environment, resulted in increased intragenic FLO8 transcripts. Activity of the cryptic FLO8 promoter is associated with reduced H3 levels, increased TBP binding and tri-methylation of H3K4 and is independent of Spt-Ada-Gcn5-acetyltransferase function. These data reveal cooperation of negative regulation of TBP with specific chromatin regulators to inhibit intragenic transcription.