PROJECT SUMMARY
Genome-wide molecular profiling of dynamic DNA and chromatin modifications across diverse cell and disease
states have resulted in comprehensive catalogs of putative non-coding regulatory elements in the human
genome. Perturbation experiments are now critical to learn the causal functions of the DNA & histone
modifications at these genomic elements. However, existing tools to manipulate gene expression and chromatin
state suffer from partial or transient effects, are large and thus difficult to deliver, and exhibit high variability
across loci and cell types. These tools are currently drawn from a tiny fraction of the thousands of natural
chromatin regulatory complexes. A more complete toolbox of compact, efficient domains capable of
manipulating a broad range of chromatin pathways will transform our ability to determine the causal
function of particular chromatin modifications across the human genome and to control gene
expression.
Here, we propose to systematically and comprehensively measure the gene expression effects of
recruiting chromatin regulators and transcription factor protein domains that can interface with human chromatin
- a critical missing dataset. This is made possible by our recent development of the first high-throughput protein
domain recruitment assay in human cells, capable of measuring activity for tens of thousands of effector domains
simultaneously (Tycko et al, bioRxiv 2020). Using this system, we will recruit-and-release effector domains from
the promoter, wait, and then measure the magnitude and permanence of transcriptional silencing or activation
at a reporter locus. We will characterize thousands of domains drawn from human and viral chromatin and gene
regulators. In addition, we will create orthogonal nanobody libraries selected to recruit endogenous chromatin
regulators. We will then measure the function of these domains at a panel of endogenous genomic loci chosen
to represent diverse chromatin states. Finally, we will use genetic screens and epigenomic mapping assays to
determine the molecular networks that underpin the functions of these novel effectors. Therefore, we will create
and share a detailed resource of experimentally-measured, compact, efficient domains that can be fused onto
DNA-binding proteins in order to recruit desired chromatin regulatory complexes to act upon a genomic element.
At the same time, this study will provide detailed functional properties for all human transcriptional and chromatin
regulatory domains, serving as a starting point for future work on understanding the disease implications of
genetic mutations in the coding sequence of these regulators. Further, it will identify novel epigenome and
transcriptional effector domains that can impart permanent epigenetic memory, and identify combinations of
domains to impart a range of chromatin states not currently accessible with available perturbation tools. These
pathway-specific perturbation technologies will be critical to probe the function of the varied chromatin states at
regulatory elements that are currently being discovered and studied across diverse genomic loci and cell types.