All Publications

  • Rewriting regulatory DNA to dissect and reprogram gene expression. bioRxiv : the preprint server for biology Martyn, G. E., Montgomery, M. T., Jones, H., Guo, K., Doughty, B. R., Linder, J., Chen, Z., Cochran, K., Lawrence, K. A., Munson, G., Pampari, A., Fulco, C. P., Kelley, D. R., Lander, E. S., Kundaje, A., Engreitz, J. M. 2023


    Regulatory DNA sequences within enhancers and promoters bind transcription factors to encode cell type-specific patterns of gene expression. However, the regulatory effects and programmability of such DNA sequences remain difficult to map or predict because we have lacked scalable methods to precisely edit regulatory DNA and quantify the effects in an endogenous genomic context. Here we present an approach to measure the quantitative effects of hundreds of designed DNA sequence variants on gene expression, by combining pooled CRISPR prime editing with RNA fluorescence in situ hybridization and cell sorting (Variant-FlowFISH). We apply this method to mutagenize and rewrite regulatory DNA sequences in an enhancer and the promoter of PPIF in two immune cell lines. Of 672 variant-cell type pairs, we identify 497 that affect PPIF expression. These variants appear to act through a variety of mechanisms including disruption or optimization of existing transcription factor binding sites, as well as creation of de novo sites. Disrupting a single endogenous transcription factor binding site often led to large changes in expression (up to -40% in the enhancer, and -50% in the promoter). The same variant often had different effects across cell types and states, demonstrating a highly tunable regulatory landscape. We use these data to benchmark performance of sequence-based predictive models of gene regulation, and find that certain types of variants are not accurately predicted by existing models. Finally, we computationally design 185 small sequence variants (≤10 bp) and optimize them for specific effects on expression in silico. 84% of these rationally designed edits showed the intended direction of effect, and some had dramatic effects on expression (-100% to +202%). Variant-FlowFISH thus provides a powerful tool to map the effects of variants and transcription factor binding sites on gene expression, test and improve computational models of gene regulation, and reprogram regulatory DNA.

    View details for DOI 10.1101/2023.12.20.572268

    View details for PubMedID 38187584

    View details for PubMedCentralID PMC10769263

  • Domain adaptive neural networks improve cross-species prediction of transcription factor binding. Genome research Cochran, K., Srivastava, D., Shrikumar, A., Balsubramani, A., Hardison, R. C., Kundaje, A., Mahony, S. 1800


    The intrinsic DNA sequence preferences and cell-type specific cooperative partners of transcription factors (TFs) are typically highly conserved. Hence, despite the rapid evolutionary turnover of individual TF binding sites, predictive sequence models of cell-type specific genomic occupancy of a TF in one species should generalize to closely matched cell types in a related species. To assess the viability of cross-species TF binding prediction, we train neural networks to discriminate ChIP-seq peak locations from genomic background and evaluate their performance within and across species. Cross-species predictive performance is consistently worse than within-species performance, which we show is caused in part by species-specific repeats. To account for this domain shift, we use an augmented network architecture to automatically discourage learning of training species-specific sequence features. This domain adaptation approach corrects for prediction errors on species-specific repeats and improves overall cross-species model performance. Our results demonstrate that cross-species TF binding prediction is feasible when models account for domain shifts driven by species-specific repeats.

    View details for DOI 10.1101/gr.275394.121

    View details for PubMedID 35042722