ABSTRACT 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. Genomic and computational studies traditionally have focused primarily on predicting and characterizing enhancers. Silencers have been investigated far less and are much less well understood. The overarching goals of this project are to identify and quantify the activities of tissue-specific silencers and their potential bifunctionality as enhancers in alternate cellular contexts, to identify the chromatin signature(s) of silencers, to determine whether silencers exhibit specificity in terms of the promoter and/or enhancer context within which they reduce gene expression, and to elucidate the regulatory roles of silencer-associated repressors, corepressors and DNA sequence motifs. We will also characterize the chromosomal contacts of silencers that are mediated by repressors bound at different classes of silencers. In pursuing these goals, we will develop novel technology for quantitative assays of elements for tissue-specific silencer activity in the context of various enhancer-promoter combinations, and novel 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.