Peter Suzuki

All Publications

• Mutations to transcription factor MAX allosterically increase DNA selectivity by altering folding and binding pathways. Nature communications Hastings, R., Aditham, A. K., DelRosso, N., Suzuki, P. H., Fordyce, P. M. 2025; 16 (1): 636

  Abstract

  Understanding how proteins discriminate between preferred and non-preferred ligands ('selectivity') is essential for predicting biological function and a central goal of protein engineering efforts, yet the biophysical mechanisms underpinning selectivity remain poorly understood. Towards this end, we study how variants of the promiscuous transcription factor (TF) MAX (H. sapiens) alter DNA specificity and selectivity, yielding >1700Kds and >500 rate constants in complex with multiple DNA sequences. Twenty-two of the 240 assayed MAX point mutations enhance selectivity, yet none of these mutations occur at residues that contact nucleotides in published structures. By applying thermodynamic and kinetic models to these results and previous observations for the highly similar yet far more selective TF Pho4 (S. cerevisiae), we find that these mutations enhance selectivity by altering partitioning between or affinity within conformations with different intrinsic selectivity, providing a mechanistic basis for allosteric modulation of ligand selectivity. These results highlight the importance of conformational heterogeneity in determining sequence selectivity and can guide future efforts to engineer selective proteins.

  View details for DOI 10.1038/s41467-024-55672-2

  View details for PubMedID 39805837

• Development of compact transcriptional effectors using high-throughput measurements in diverse contexts. Nature biotechnology Tycko, J., Van, M. V., DelRosso, N., Ye, H., Yao, D., Valbuena, R., Vaughan-Jackson, A., Xu, X., Ludwig, C., Spees, K., Liu, K., Gu, M., Khare, V., Mukund, A. X., Suzuki, P. H., Arana, S., Zhang, C., Du, P. P., Ornstein, T. S., Hess, G. T., Kamber, R. A., Qi, L. S., Khalil, A. S., Bintu, L., Bassik, M. C. 2024

  Abstract

  Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors. We find that many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. To enable efficient perturbations, we select context-robust domains, including ZNF705 KRAB, that improve CRISPRi tools to silence promoters and enhancers. We engineer a compact human activator called NFZ, by combining NCOA3, FOXO3 and ZNF473 domains, which enables efficient CRISPRa with better viral delivery and inducible control of chimeric antigen receptor T cells.

  View details for DOI 10.1038/s41587-024-02442-6

  View details for PubMedID 39487265

  View details for PubMedCentralID 4494013

• High-throughput affinity measurements of direct interactions between activation domains and co-activators. bioRxiv : the preprint server for biology DelRosso, N., Suzuki, P. H., Griffith, D., Lotthammer, J. M., Novak, B., Kocalar, S., Sheth, M. U., Holehouse, A. S., Bintu, L., Fordyce, P. 2024

  Abstract

  Sequence-specific activation by transcription factors is essential for gene regulation1,2. Key to this are activation domains, which often fall within disordered regions of transcription factors3,4 and recruit co-activators to initiate transcription5. These interactions are difficult to characterize via most experimental techniques because they are typically weak and transient6,7. Consequently, we know very little about whether these interactions are promiscuous or specific, the mechanisms of binding, and how these interactions tune the strength of gene activation. To address these questions, we developed a microfluidic platform for expression and purification of hundreds of activation domains in parallel followed by direct measurement of co-activator binding affinities (STAMMPPING, for Simultaneous Trapping of Affinity Measurements via a Microfluidic Protein-Protein INteraction Generator). By applying STAMMPPING to quantify direct interactions between eight co-activators and 204 human activation domains (>1,500 K ds), we provide the first quantitative map of these interactions and reveal 334 novel binding pairs. We find that the metazoan-specific co-activator P300 directly binds >100 activation domains, potentially explaining its widespread recruitment across the genome to influence transcriptional activation. Despite sharing similar molecular properties (e.g. enrichment of negative and hydrophobic residues), activation domains utilize distinct biophysical properties to recruit certain co-activator domains. Co-activator domain affinity and occupancy are well-predicted by analytical models that account for multivalency, and in vitro affinities quantitatively predict activation in cells with an ultrasensitive response. Not only do our results demonstrate the ability to measure affinities between even weak protein-protein interactions in high throughput, but they also provide a necessary resource of over 1,500 activation domain/co-activator affinities which lays the foundation for understanding the molecular basis of transcriptional activation.

  View details for DOI 10.1101/2024.08.19.608698

  View details for PubMedID 39229005

  View details for PubMedCentralID PMC11370418

• Short tandem repeats bind transcription factors to tune eukaryotic gene expression. Science (New York, N.Y.) Horton, C. A., Alexandari, A. M., Hayes, M. G., Marklund, E., Schaepe, J. M., Aditham, A. K., Shah, N., Suzuki, P. H., Shrikumar, A., Afek, A., Greenleaf, W. J., Gordân, R., Zeitlinger, J., Kundaje, A., Fordyce, P. M. 2023; 381 (6664): eadd1250

  Abstract

  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

• Large-scale mapping and mutagenesis of human transcriptional effector domains. Nature DelRosso, N., Tycko, J., Suzuki, P., Andrews, C., Mukund, A., Liongson, I., Ludwig, C., Spees, K., Fordyce, P., Bassik, M. C., Bintu, L. 2023

  Abstract

  Human gene expression is regulated by more than 2,000 transcription factors and chromatin regulators1,2. Effector domains within these proteins can activate or repress transcription. However, for many of these regulators we do not know what type of effector domains they contain, their location in the protein, their activation and repression strengths, and the sequences that are necessary for their functions. Here, we systematically measure the effector activity of more than 100,000 protein fragments tiling across most chromatin regulators and transcription factors in human cells (2,047 proteins). By testing the effect they have when recruited at reporter genes, we annotate 374 activation domains and 715 repression domains, roughly 80% of which are new and have not been previously annotated3-5. Rational mutagenesis and deletion scans across all the effector domains reveal aromatic and/or leucine residues interspersed with acidic, proline, serine and/or glutamine residues are necessary for activation domain activity. Furthermore, most repression domain sequences contain sites for small ubiquitin-like modifier (SUMO)ylation, short interaction motifs for recruiting corepressors or are structured binding domains for recruiting other repressive proteins. We discover bifunctional domains that can both activate and repress, some of which dynamically split a cell population into high- and low-expression subpopulations. Our systematic annotation and characterization of effector domains provide a rich resource for understanding the function of human transcription factors and chromatin regulators, engineering compact tools for controlling gene expression and refining predictive models of effector domain function.

  View details for DOI 10.1038/s41586-023-05906-y

  View details for PubMedID 37020022

  View details for PubMedCentralID 4494013

• High throughput measurements of direct activation domain-coactivator interactions DelRosso, N., Suzuki, P., Griffith, D., Holehouse, A. S., Bintu, L., Fordyce, P. CELL PRESS. 2023: 68A

  View details for Web of Science ID 000989629700339

• High throughput measurements of direct activation domain-coactivator interactions. Biophysical journal DelRosso, N., Suzuki, P., Griffith, D., Holehouse, A. S., Bintu, L., Fordyce, P. 2023; 122 (3S1): 68a

  View details for DOI 10.1016/j.bpj.2022.11.576

  View details for PubMedID 36784957

• Versatile phenotype-activated cell sorting SCIENCE ADVANCES Lee, J., Liu, Z., Suzuki, P. H., Ahrens, J. F., Lai, S., Lu, X., Guan, S., St-Pierre, F. 2020; 6 (43)

  Abstract

  Unraveling the genetic and epigenetic determinants of phenotypes is critical for understanding and re-engineering biology and would benefit from improved methods to separate cells based on phenotypes. Here, we report SPOTlight, a versatile high-throughput technique to isolate individual yeast or human cells with unique spatiotemporal profiles from heterogeneous populations. SPOTlight relies on imaging visual phenotypes by microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. To illustrate SPOTlight's ability to screen cells based on temporal properties, we chose to develop a photostable yellow fluorescent protein for extended imaging experiments. We screened 3 million cells expressing mutagenesis libraries and identified a bright new variant, mGold, that is the most photostable yellow fluorescent protein reported to date. We anticipate that the versatility of SPOTlight will facilitate its deployment to decipher the rules of life, understand diseases, and engineer new molecules and cells.

  View details for DOI 10.1126/sciadv.abb7438

  View details for Web of Science ID 000582114600021

  View details for PubMedID 33097540

• Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS. Analytical chemistry Brower, K. K., Khariton, M., Suzuki, P. H., Still, C. 2., Kim, G., Calhoun, S. G., Qi, L. S., Wang, B., Fordyce, P. M. 2020

  Abstract

  In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5-25 mum in diameter) within highly monodisperse double emulsions (35 mum in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.

  View details for DOI 10.1021/acs.analchem.0c02499

  View details for PubMedID 32900183

• High-Throughput Discovery and Characterization of Human Transcriptional Effectors. Cell Tycko, J. n., DelRosso, N. n., Hess, G. T., Aradhana, n. n., Banerjee, A. n., Mukund, A. n., Van, M. V., Ego, B. K., Yao, D. n., Spees, K. n., Suzuki, P. n., Marinov, G. K., Kundaje, A. n., Bassik, M. C., Bintu, L. n. 2020

  Abstract

  Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.

  View details for DOI 10.1016/j.cell.2020.11.024

  View details for PubMedID 33326746