Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only endpoint snapshots of TF binding. Alternatively, TFtagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type-specificity through conditional expression of the TFenzyme fusion. Here we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system, termed Flip-Excision (FLEX) calling cards and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.Proper cellular development and function is a complex process established by elaborate gene expression networks. These networks are fundamentally regulated by transcription factors (TF), which bind to regulatory elements (RE) across the genome and facilitate gene expression through a variety of mechanisms, including recruitment of transcriptional co-factors and modulation of chromatin state 1 . Extensive efforts to profile TF genome occupancy and identify active REs across the genome have highlighted the profound diversity of TF binding, providing important insights into TF-mediated gene regulation 2-5 . Further, a large portion of genetic variation associated with improper cellular function or disease has been shown to exist in TFbound REs 3,6-10 , demonstrating the criticality of proper TF binding in maintaining cellular homeostasis.Several methods exist for profiling TF occupancy across the genome. Antibody-based techniques, such as chromatin immunoprecipitation followed by sequencing (ChIP-seq), and more recently Cleavage Under Targets and Release Using Nuclease (CUT&RUN) 11 or Tagmentation (CUT&Tag) 12 , are widely used and have provided numerous insights into the cellular functions of TFs 2-4,7 . Notably however, these methods require the availability and individual optimization of TF-specific antibodies, limiting the throughput and breadth of genomewide TF profiling. Further, ChIP-seq provides only a snapshot of TF activity at the moment of cell lysis and thus may be inefficient at detecting transient or infrequent TF binding events. Finally, while robust for non-cell typeselective, tissue-level analyses, it is often challenging to interpret ChIP-seq data obtained from complex tissues such as the brain, which is comprised of many different interconnected cell types. Because of this limitation, efforts have recently been made to...
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