Genome organization at the paralogous transcription factors

The emergence of multicellular life over 500 million years ago was accompanied by profound changes in vertebrate genomes, driven in part by at least two rounds of whole-genome duplication. These duplications generated paralogous transcription factors (TFs) that diversified in function, enabling the rise of specialized cell types and contributing to organismal complexity. While most paralogous TFs (>90%) became dispersed across the genome, a small subset remains physically clustered on the same chromosome. The most common arrangement is adjacency of paralogous TFs—a strikingly conserved feature whose functional significance remains poorly understood. Why have certain TF pairs been maintained in close genomic proximity for hundreds of millions of years?

We hypothesize that genomic clustering provides an evolutionary advantage by exploiting 3D chromatin architecture to stabilize TF expression in the face of genetic perturbations—a concept with broad implications that has yet to be rigorously tested.To address this question, we study the ETS1–FLI1 locus, a paralogous TF pair from the ETS family. This required a method capable of resolving chromatin architecture at single-allele resolution—capturing the full spatial configuration of both genes and their regulatory elements simultaneously. Traditional population-based assays such as Hi-C are insufficient, as they fragment chromatin and measure only pairwise contacts. Instead, we implemented Optical Reconstruction of Chromatin Architecture (ORCA), a super-resolution microscopy approach that enables high-resolution chromatin tracing in single alleles.

Using T cells from two genetically engineered mouse strains—one lacking a super-enhancer near Ets1 and another lacking a CTCF boundary element—we generated over 30,000 single-allele chromatin traces, representing the first chromatin tracing study in T cells. Our analyses revealed extensive rewiring of chromatin architecture in mutant T cells, yet expression of both Ets1 and Fli1 was maintained at moderate levels, supporting continued T cell development. These findings suggest that physical clustering of paralogous TFs provides a conserved mechanism to buffer transcriptional output and safeguard essential cellular functions.
Looking ahead, we are extending this paradigm to other ETS paralog pairs (e.g., Ets2–Erg) and to genomic regions harboring STAT paralogs, to test whether clustered paralogs broadly function as evolutionary stabilizers of gene expression programs.