PROJECT SUMMARY Transcription plays an oversized role in determining proper inflammatory response in immune cells such as macrophages (MF). Transcriptional master regulators of MFs are thought to be the main drivers of chromatin reprogramming during myeloid cell differentiation, polarization and activation. However, recent studies have expanded this “developmental TF-centric” model and suggest that signal-dependent TFs, in particular repressors, likely to have a pioneer role in the shaping the MF epigenome. Such an expanded model also predicts that active transcriptional repression and chromatin bookmarking by signal-dependent master regulators is a key mechanism for the safeguarding the enhancer repertoire and proper deployment and resolution of an inflammatory response. Recently, we identified such a signal-dependent TF, the heme- regulated repressor BACH1 (BTB Domain and CNC Homolog 1), with properties of a master regulator of the MF epigenome. Our preliminary data is contrary to current dogma about this TF and also challenging the current view of inflammatory gene regulation. The central hypothesis of this proposal is that BACH1 acts as a pioneering repressor, signal-dependent master regulator and an epigenomic safeguard that plays a key role during macrophage differentiation and polarization/specification by actively repressing enhancers, genome-wide, controlling inflammatory, metabolic and differentiation-specific genes with effects on the baseline, amplitude and proper kinetics of the inflammatory gene expression. We propose three interrelated and integrated but independent Specific Aims in which we will combine new genetic mouse models with cutting edge molecular biology approaches to establish BACH1 as a critical modulator of MF inflammatory gene expression and subtype specification. Our approaches aim to provide deep mechanistic insights on BACH1 function on active repression, pioneering, transcriptional activation, 3D chromatin structure and inflammatory gene expression and establish BACH1 as a novel signal-dependent master regulator and as part of the core hardwired transcriptional circuit of MFs. Moreover, results from the proposed work are expected to push the field forward by providing a mechanistic framework of how heme, a molecule vital for life and cell metabolism but cytotoxic when in excess, can affect the differentiation and inflammatory potential of MFs.