In metazoans, gene expression is regulated in a tissue/cell-type-specific manner predominantly via stretches of noncoding sequence referred to as cis regulatory modules (CRMs). CRMs contain 1 or more DNA binding sites for 1 or more sequence-specific, regulatory transcription factors that function to modulate the expression of target gene(s). CRMs that activate gene expression are typically referred to as enhancers, while those that repress gene expression are referred to as silencers. Transcriptional enhancers activate gene expression in a tissue-specific manner in development and also in adult cells in response to cellular or environmental stimuli. Like enhancers, silencers can function in a cell-type-specific manner. Indeed, silencers may contribute a crucial role in the specification of precise gene expression patterns, thus enabling the establishment of sharp expression domains, such as during development. Numerous genomic and computational studies have focused primarily on predicting and characterizing enhancers. In contrast to enhancers, silencers are much less well understood. Few transcriptional silencers have been described, and there have been no large-scale efforts to catalog silencers nor to identify predictive features of silencers, such as chromatin marks. The overarching goals of this project are to identify and quantify the activities of tissue-specific silencers, to identify the chromatin signatures of silencers, and to elucidate the regulatory roles of silencer-associated (co-)repressors and DNA sequence motifs. In pursuing these goals, we will develop novel “quantitative silencer-FACS-Seq” technology for quantitative screening of CRMs for tissue-specific silencer activity, and BioTAP-XL-TALE technology for identification of proteins found in tissue-specific chromatin assembled at specific cis-regulatory elements in cells where they act as silencers versus in cells where they act as enhancers. We will focus on the developing embryonic mesoderm in Drosophila melanogaster as our model system. We anticipate that the features and chromatin signatures of silencers identified in this project will be evolutionarily conserved across metazoans, including human.