Decoding Positional Epigenetic Memory - PROJECT SUMMARY / ABSTRACT: DECODING POSITIONAL EPIGENETIC MEMORY Hox gene expression is crucial for patterning the main body axis of all bilaterian species. In a classic epigenetic memory fashion, positional identity is acquired early in development and is inherited through cell lineage via Hox expression. Misexpression of even a single Hox gene can lead to homeotic transformations or diseases like cancer, making stable and accurate Hox positional epigenetic memory essential for proper cell differentiation and disease prevention. Building on a decade of seminal contributions to the field, our research aims to decipher how stable Hox positional identity is generated. In mammals, the Hox genes are organized into four chromosomal clusters, each containing a subset of 13 genes within approximately 100 kb. Polycomb Repressive Complexes (PRCs) decorate and suppress Hox clusters in undifferentiated cells. Transient patterning signals such as retinoic acid partition Hox clusters into active (PRC-removed) and repressed (PRC-high) domains at early differentiation stages, establishing precise and stable boundaries that convey positional identity across cell generations. We made three critical contributions to the accepted Hox positional epigenetic memory model. First, we identified that patterning signals result in the binding of activating transcription factors to Hox cluster domains. Second, we discovered that this binding results in PRC eviction from CTCF-established chromatin boundaries. Lastly, we demonstrated that the Hox cluster is sufficient to establish positional identity. In other words, a 100 kb Hox cluster engages with the three elements required for positional memory: repression (PRC recruitment), activation (signaling transcription factors), and boundary formation (CTCF). The ability of CTCF to engage in boundary formation is an active area of research and the best-understood component. However, there is a knowledge gap in the mechanisms that control activation and repression: Challenge 1: Activation. The current working model cannot explain how Retinoic Acid signaling evicts PRC while initiating a progressive colinear Hox gene transcription. Further, the model lacks definitive proof of the physical distribution of the activating Retinoic Acid Receptor and CDX2 factors along Hox cluster domains. We will challenge the Hox regulatory paradigm to the core by building novel Hox regulatory units with synthetic DNA technology. Challenge 2: Repressors. Although it is among the earliest examples of PRC repression, there is no mechanistic understanding of how Hox clusters recruit PRC in mammalian cells. With our powerful synthetic DNA approach, we will identify the mammalian Hox polycomb recruitment element (PRE). By merging synthetic DNA, new genomic engineering tools, and stem cell differentiation systems, we aim to understand positional memory through Hox chromatin boundary formation and build the smallest genetic unit sufficient to establish and sustain positional epigenetic memory.
