Beyond Gene Dosage: Understanding Down Syndrome via 4D Genome Organization

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ABSTRACT Chromosomal aneuploidy underlies a variety of human diseases. The most prominent paradigm among these is Down syndrome (DS) that is caused by an extra copy of homo sapiens chromosome 21 (HSA21). As the most common genetic disease of human cognitive impairment, DS affects about 1 in 750 live-born infants in the US, and it pre-disposes patients to muscle hypotonia, dysmorphic features, congenital heart defects and early onset Alzheimer's disease. Despite many progress, our conceptual understanding of the pathological basis of such chromosomal abnormality is so far largely limited to the “gene dosage hypothesis”, which however cannot explain broad gene deregulation that takes place throughout the genome in specific cell types. It has been unexplored that whether 3D genome mal-folding may play yet unrealized roles in DS and other aneuploidies. Here, we assembled a strong team to test an overall hypothesis that the presence of trisomy 21 deregulates 3D genome as an entirety and changes gene expression in DS cells, particularly via forming aberrant inter-chromosomal interactions (ICIs). We have two specific aims. In Aim-1, in multiple pairs of isogenic iPSC cells and their derived neuron/glia cells that contain disomic versus trisomy HSA21, we will conduct assays to systematically characterize their 3D genome (in situ Hi-C and PLAC-Seq), transcriptome and 1D epigenome (PRO-Seq, ATAC-Seq, and histone modification ChIP-Seq). Integrative analyses will dissect the aberrant chromatin interactomes, particularly these interchromosomal interactions altered in trisomy nucleus, and correlate those with gene deregulation in specific developmental stages or cell types (neurons, astrocytes or microglia). We will use leading-edge new techniques based on long reads sequencing to further characterize aberrant inter-chromosomal interactions, and will validate them using DNA and/or RNA FISH. In Aim-2, we focus on functionally dissecting the roles of aberrant inter-chromosomal interactions in gene deregulation. This will be first investigated by chemical and epigenetic perturbation in both cultured primary neural progenitor cells and in brain cortical organoids. We will then use novel optogenetic tools to model disease-relevant formation of ICIs to deduce their potential causal roles in gene deregulation. The expected results from this proposal are significant not only to our understanding of the 4D genome, but also to human brain developmental disorders. The knowledge generated here will shed light on many forms of aneuploidy, providing a new conceptual framework beyond “gene dosage effects” to understand gene deregulation, and inspire strategies to ameliorate these diseases via restoring 3D genome architecture.