SUMMARY Transcription activation relies on the cooperative activity of multiple transcription factors (TFs) that bind cis- regulatory elements (CREs) near their target genes. Although techniques like chromatin immunoprecipitation coupled to sequencing helped uncover the genome-wide location of binding sites for many TFs in different spe- cies and tissues, it still remains unclear how TFs cooperate to compete with histones, bind DNA, regulate CRE activity, and drive transcription activation. In this proposal, we will address this knowledge gap by exploiting the circadian regulation of gene expression by the TF CLOCK:BMAL1. Recent reports from our lab and the litera- ture indicate that the circadian regulation of transcription by CLOCK:BMAL1 relies on the interaction between CLOCK:BMAL1 and other TFs rather than on core clock genes only. CLOCK:BMAL1 rhythmic DNA binding promotes the rhythmic removal of nucleosomes at its CREs, strongly suggesting that CLOCK:BMAL1 gener- ates a permissive chromatin landscape that facilitates the rhythmic recruitment of other TFs at its CREs. This cooperation between CLOCK:BMAL1 and other TFs may explain why less than a third of CLOCK:BMAL1 tar- gets are rhythmically expressed under normal condition, and how their rhythmic transcription can be repro- grammed when animals are exposed to a new environment. This proposal builds upon these findings and the proposed experiments will determine if CLOCK:BMAL1 promotes the formation of partially unwrapped nucleo- somes (Aim 1), the modalities of CLOCK:BMAL1 cooperation with other TFs by using a method of single-mole- cule footprinting (Aim 2), and if high-fat diet alters the cooperativity between CLOCK:BMAL1 and other TFs to reprogram CRE transcriptional activity (Aim 3). Results from these experiments are expected to uncover novel and important mechanisms that control the rhythmic expression of thousands of genes, and to provide a new conceptual framework for how biological functions are coordinated between tissues. They are also expected to provide insights into the mechanisms that underlie the role of TF cooperativity in the control of CRE activity that can be widely relevant to fields beyond the circadian field.