ABSTRACT Retaining pre-existing patterns of histone modifications during early development and multicellular differentiation is essential for the maintenance of cellular identity. Histone H3 lysine 9 methylation (H3K9me) is associated with the formation of transcriptionally silent, specialized chromatin domains also referred to as heterochromatin. Heterochromatin establishment is essential for normal centromere and telomere function, silencing of transposons and repetitive DNA elements and preserving lineage-specific patterns of gene expression. The loss of H3K9 methylation is associated with genome instability and aneuploidy which are widely recognized as the most common abnormalities associated with cancer. Heterochromatin establishment is a dynamic process which involves weak and transient interactions between histone modifiers and their cognate nucleosome substrates. Counterintuitively, these weak protein-protein interactions produce epigenetic states that remain heritable across generational timescales. These dynamic properties associated with the epigenome exposes significant conceptual and methodological gaps that this proposal will principally address: 1) How do dynamic epigenetic complexes consisting of histone modification readers, writers and erasers assemble in vitro and in living cells? 2) How do histone modifiers traverse a complex chromatin landscape to locate their sites of action? 3) How is epigenetic memory stored and transmitted across subsequent generations? The current belief is that histone modifications function as inert scaffolds which passively promote the localization of histone modifiers to distinct sites in the genome. Based on our recent studies, we propose an inversion of this paradigm. Our results reveal that H3K9 methylation has an active role in catalyzing the cooperative assembly of a heterochromatin regulatory complex. Our studies underscore how the assembly of epigenetic complexes in an H3K9 methylation dependent manner restricts protein-protein interactions to specific chromatin contexts. In this proposal, we will use in vitro biochemistry to reconstitute heterochromatin regulatory complexes, single molecule imaging to define the order and timing of assembly of these complexes in vitro and within a native chromatin context and a high-throughput microfluidic platform to define how epigenetic memory is propagated within individual lineages. The real-time visualization of heterochromatin assembly at high spatial and temporal resolution will illuminate how transient molecular interactions can synergize to establish stable and heritable patterns of gene expression.