Brand, A. H. | bioRxiv Science

bioRxiv2022

An organoid CRISPRi screen revealed that SOX9 primes human fetal lung tip progenitors to receive WNT and RTK signals

The balance between self-renewal and differentiation in human fetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID we place SOX9 at the centre of a transcriptional network which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new approach for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease. HighlightsO_LIA pooled CRISPRi screen in human fetal lung organoids identified transcription factors controlling progenitor cell self-renewal. C_LIO_LISOX9 promotes tip progenitor cell proliferation and supresses precocious airway differentiation. C_LIO_LITargeted DamID (TaDa) identified SOX9 direct binding targets, revealing that SOX9 lies at the intersection of WNT and RTK signalling. C_LIO_LISOX9 and ETVs co-regulate the human fetal lung progenitor self-renewal programme. C_LI

developmental biology↗

bioRxiv2021

Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID

Genetic studies of autism spectrum disorder (ASD) have revealed a causal role for mutations in chromatin remodeling genes. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeler with one of the highest de novo mutation rates in sporadic ASD. However, the relationship between CHD8 genomic function and autism-relevant biology remains poorly elucidated. CHD8 binding studies have relied on Chromatin Immunoprecipitation followed by sequencing (ChIP-seq), however, these datasets exhibit significant variability. ChIP-seq has technical limitations in the context of weak or indirect protein-DNA interactions or when high-performance antibodies are unavailable. Thus, complementary approaches are needed overall, and, specifically, to establish CHD8 genomic targets and regulatory function. Here we used Targeted DamID in utero to characterize CHD8 binding in developing embryonic mouse cortex. CHD8 Targeted DamID followed by sequencing (CHD8 TaDa-seq) revealed binding at previously identified targets as well as loci sensitive to Chd8 haploinsufficiency. CHD8 TaDa-seq highlighted CHD8 binding distal to a subset of genes specific to neurodevelopment and neuronal function. These studies establish TaDa-seq as a useful alternative for mapping protein-DNA interactions in vivo and provide insights into the relationship between chromatin remodeling by CHD8 and autism-relevant pathophysiology associated with CHD8 mutations.

genomics↗