To understand how the cortex is built, we need to be able to study how cellular identity evolves in time, ideally at the single cell level. Importantly, chromatin accessibility and 3D genome organization carry unique information that is not provided by single-cell RNAseq and epigenome changes may precede gene expression. Recent breakthroughs in methodology have allowed chromatin structure to be interrogated even at the single-cell level. Therefore, we are in an ideal and timely position to address the spatio-temporal dynamics of gene regulation and 3D nuclear organization in the cortex.

We are developing a highly innovative genomics approach to simultaneously interrogate gene expression and chromatin topology at single-cell level. In addition, we use a combination of single-cell lineage tracing using CRISPR, scATAC-seq and spatial transcriptomics to understand how lineage potential is encoded spatially and temporally in neural stem cells.

We have previously discovered that regulating 3D chromatin architecture and enhancer-promoter interactions plays an important role in the control of gene expression and cell fate in the cortex. Furthermore, several key transcription factors and potentially some long non-coding RNAs are associated at the molecular level with dynamic chromatin loops and may function mechanistically by remodeling genome topology.

However, a key unresolved question in the field is if TF binding and/or lncRNAs can physically affect nuclear 3D architecture or simply exploit it in order to spread and bind on chromatin. To disentangle cause and consequence, we are using transgenic mouse lines and CRISPR-Cas9 genome engineering to determine if TF binding is sufficient to induce an ectopic chromatin looping and rewire 3D genome architecture in vivo.

Cortical evolution in mammals is considered to be a key advance that enabled higher cognitive function such as language. Structural variations including indels, inversions and duplications account for 3-4 times more sequence divergence between the chimpanzee and the human genomes than single-base-pair mutations. Yet, almost all of the comparative evolution studies trying to understand what makes the human brain unique focus on SNPs in coding genes or putative enhancer regions based on proximity to important neural genes. Recent advances in chromatin biology and our own work suggest that changes in 3D architecture can strongly affect gene expression of regions in close physical proximity and not necessarily on the linear 1D genome.

Therefore, we are systematically examining how 3D chromatin organization has changed during primate evolution focusing on the cortex. We use cerebral organoids from mouse, macaque, chimp and human iPSC and compare them with in vivo models of corticogenesis such as the ferret and the human fetal cortex. We will also examine the functional importance of the most promising structural variations using organoids and in mice using the CRISPR-Cas9 system.