Our group investigates how chromatin is modified and packaged to prevent aberrant transcription. There are numerous genes in the genome, but a cell uses only a subset of genes to establish and maintain its identity. Much attention has focused on the gene selective activation mechanisms needed to express the correct set of genes. However, repression mechanisms play equally essential roles because misexpression of inappropriate genes can reprogram cell fate, especially in development. Furthermore, in pathological conditions such as cancer, transcription can be indiscriminately initiated from incorrect genes or non-coding loci, driving malignant transformation. Tissue-specific Zinc Finger Proteins (ZFPs), which interact with the chromatin remodeler Mi-2, are critical repressive chromatin regulators for cell fate in diverse cell types. However, ZFP/Mi-2 complexes exhibit distinct patterns of genomic distributions and transcriptional phenotypes compared to classical repressors, and their regulatory mechanisms remain poorly understood. One such ZFP, Kumgang, is a testis-specific Mi-2-interacting protein we discovered in D. melanogaster. In kumgang mutants, hundreds of genes normally restricted to differentiated somatic tissues were mis-expressed from inter- or intragenic cryptic promoters. Building on this foundational discovery, we will uncover the fundamental principles of Mi-2-interacting ZFP mechanisms. Over the next five years, we aim to: (1) dissect the molecular functions of ZFPs by systematically characterizing Kumgang’s domains, including zinc finger arrays, to identify distinct regions critical for genomic targeting, Mi-2 interaction, and transcriptional repression; and (2) test the “compartmentalization hypothesis,” which posits that Kumgang/Mi-2 segregates active and inactive genomic regions to prevent activation machinery from accessing cryptic promoters. We have established in vivo genetic replacement and acute depletion systems, which will allow us to do a mechanistic investigation of Kumgang’s function in its native context. Mi-2 interacting ZFPs have similar functions of safeguarding lineage identity across diverse metazoans, from worms to humans. Our findings will uncover a deeply conserved and distinct layer of gene regulatory principles mediated by higher-order chromatin organization.