Short tandem repeats bind transcription factors to tune eukaryotic gene expression.
Science (New York, N.Y.)
2023; 381 (6664): eadd1250
Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate transcription factor (TF)-DNA affinities and apparent on-rates by about 70-fold by directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density, with impacts well predicted by statistical mechanics. Confirming that STRs also affect TF binding in cells, neural networks trained only on in vivo occupancies predicted effects identical to those observed in vitro. Approximately 90% of TFs preferentially bound STRs that need not resemble known motifs, providing a cis-regulatory mechanism to target TFs to genomic sites.
View details for DOI 10.1126/science.add1250
View details for PubMedID 37733848
A microwell platform for high-throughput longitudinal phenotyping and selective retrieval of organoids.
2023; 14 (9): 764
Organoids are powerful experimental models for studying the ontogeny and progression of various diseases including cancer. Organoids are conventionally cultured in bulk using an extracellular matrix mimic. However, bulk-cultured organoids physically overlap, making it impossible to track the growth of individual organoids over time in high throughput. Moreover, local spatial variations in bulk matrix properties make it difficult to assess whether observed phenotypic heterogeneity between organoids results from intrinsic cell differences or differences in the microenvironment. Here, we developed a microwell-based method that enables high-throughput quantification of image-based parameters for organoids grown from single cells, which can further be retrieved from their microwells for molecular profiling. Coupled with a deep learning image-processing pipeline, we characterized phenotypic traits including growth rates, cellular movement, and apical-basal polarity in two CRISPR-engineered human gastric organoid models, identifying genomic changes associated with increased growth rate and changes in accessibility and expression correlated with apical-basal polarity. A record of this paper's transparent peer review process is included in the supplemental information.
View details for DOI 10.1016/j.cels.2023.08.002
View details for PubMedID 37734323
Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome.
Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40-75% with cargo sizes over 7kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.
View details for DOI 10.1038/s41587-022-01494-w
View details for PubMedID 36217031
Neuronal defects in a human cellular model of 22q11.2 deletion syndrome.
22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.
View details for DOI 10.1038/s41591-020-1043-9
View details for PubMedID 32989314