Modeling uniquely human gene regulatory function via targeted humanization of the mouse genome

Emily V Dutrow,Kristina Yim,Martina Krenzer,James P Noonan,Daniel B Burkhardt,Smita Krishnaswamy,Angeliki Louvi,Severin Uebbing,Deena Emera,Acadia A Kocher,Timothy Nottoli

doi:10.1038/s41467-021-27899-w

Abstract

The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Human Accelerated Regions (HARs), which include transcriptional enhancers harboring a significant excess of human-specific sequence changes, are leading candidates for driving gene regulatory modifications in human development. However, insight into whether HARs alter the level, distribution, and timing of endogenous gene expression remains limited. We examined the role of the HAR HACNS1 (HAR2) in human evolution by interrogating its molecular functions in a genetically humanized mouse model. We find that HACNS1 maintains its human-specific enhancer activity in the mouse embryo and modifies expression of Gbx2, which encodes a transcription factor, during limb development. Using single-cell RNA-sequencing, we demonstrate that Gbx2 is upregulated in the limb chondrogenic mesenchyme of HACNS1 homozygous embryos, supporting that HACNS1 alters gene expression in cell types involved in skeletal patterning. Our findings illustrate that humanized mouse models provide mechanistic insight into how HARs modified gene expression in human evolution.

Highlights

The evolution of uniquely human traits likely entailed changes in developmental gene regulation
We found that HACNS1 maintains its human-specific enhancer activity in the mouse embryo and alters the expression of the nearby transcription factor encoding gene Gbx[2] in limb chondrocytes, a cell type required for skeletal morphogenesis
This in vivo approach enabled us to identify spatial and temporal changes in gene expression driven by HACNS1 and to characterize the specific cell types affected by these changes, providing insight into the developmental processes modified due to human-specific alterations in enhancer activity

Summary

Introduction

The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Over the last decade comparative studies have begun to reveal potential genetic drivers underlying novel human biological features These efforts have identified a prominent class of elements in the genome that are highly conserved across many species but show a significant excess of human-specific sequence changes[3,4,5,6,7]. HACNS1 exhibits increased levels of histone H3K27 acetylation (H3K27ac), which is correlated with enhancer activity, in human versus rhesus macaque and mouse embryonic limb[11] Together, these findings suggest that HACNS1 may have contributed to changes in limb development during human evolution. Our findings support that HARs can direct changes in endogenous gene expression during development and illustrate the power of genetically humanized mouse models to provide insight into regulatory pathways and developmental mechanisms modified in human evolution

Methods

Results

Conclusion