ABSTRACT Heterochromatin, a gene repressive nuclear structure, patterns the genome into active and inactive regions. This patterning is necessary to preserve the structural integrity of the genome and to drive and maintain developmental fates. Heterochromatin is seeded by DNA-sequences, but heterochromatin assembly can continue distal to those seeding sites via a process called “spreading”. In spreading, “writer” enzymes propagate key chemical histone marks along chromatin that attract the gene repressive machinery required for the normal function of heterochromatin. Spreading is responsible for tha majority of heterochromatin assembly. The parent grant set out to investigate three major questions about heterochromatin spreading: 1. What are the biochemical mechanisms underlying it? 2. How can heterochromatin spread over loci of vastly different chemical, structural, and stability regimes? And 3. How is the reaction tuned to expand or contract during development to stabilize cell fate switches? In this supplement application, we will train a Junior Specialist, Nathan Ho to develop methods relevant to the 1st question above, concerning the biochemical mechanisms of the “writer”-mediated spreading process. Specially, Nathan will continue to develop a simple and robust, but powerful, single molecule approach we have devised together with the Mullins lab at UCSF. In this approach, chromatin templates are deposited onto patterns on coverslips. These patterns, which can be of any shape and, crucially, density, are formed by a micropatterning system that removes passivating agents in a UV light-dependent process. Molecules on the coverslip are visualized via Total Internal Reflection microscopy. Using this micropatterning system we want to ask 1. How “writer” enzymes engage with different chromatin substrates and 2. how different forms of the “writer” enzymes behave on chromatin. Finally, we want to 3. reconstitute part of the spreading process on this platform. Importantly, the basics of the approach have been worked out by a prior research technician, setting up Nathan for a productive time in the laboratory. The supplement application is further focused on Nathan’s mentoring, training, and career development. We have designed an integrative research experience plan that is motivated by Nathan’s Individual Development Plan. The plan focusses on the following three main areas: 1. In-lab training and development of data analysis and experimental design skills, 2. Development of science communication skills, and 3. Preparation for successful application to graduate school for the fall of 2025, the central goal of Nathan’s IDP. We believe the scientific, mentoring and career development aims are well balanced to enable substantial new insight into “writer” enzyme activity on chromatin and development of Nathan as an independent, critically thinking young scientist.