The epigenetic regulation of gene expression is determined through a combinatorial function of multiple epigenetic modalities such as transcription factors, chromatin remodellers, histone modifications, DNA methylation and other factors. We have recently developed methods to uni-modally profile histone marks with single-cell resolution (Bartosovic et al., Nat. Biotech., 2021) and multimodally profile three epigenetic modalities with single-cell resolution at the same time (nano-CUT&Tag; Bartosovic et al., Nat. Biotech., in press). In this project, we aim to further develop these technologies by expanding the number of co-profiled modalities. Ultimately, we aim to develop oligo-modal chromatin profiling technologies for simultaneous profiling of 5-10 histone marks, transcription factors or DNA methylation. For this purpose, we will perform optimizations of CUT&Tag protocol by introducing novel Tn5 fusion proteins and optimizing the conditions of the assay to be better suited for TF profiling. We will then apply the newly developed methods to profile chromatin features of standard encode cell lines such as K562 or GM12878 in bulk and benchmark the obtained data against encode datasets. Finally we will apply the newly developed methods to study epigenetic regulatory logic of brain development.