Nicole DelRosso

All Publications

• 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

• Using High-Throughput Measurements to Identify Principles of Transcriptional and Epigenetic Regulators. Methods in molecular biology (Clifton, N.J.) DelRosso, N., Bintu, L. 2024; 2842: 79-101

  Abstract

  To achieve exquisite control over the epigenome, we need a better predictive understanding of how transcription factors, chromatin regulators, and their individual domain's function, both as modular parts and as full proteins. Transcriptional effector domains are one class of protein domains that regulate transcription and chromatin. These effector domains either repress or activate gene expression by interacting with chromatin-modifying enzymes, transcriptional cofactors, and/or general transcriptional machinery. Here, we discuss important design considerations for high-throughput investigations of effector domains, recent advances in discovering new domains in human cells and testing how domain function depends on amino acid sequence. For every effector domain, we would like to know the following: What role does the cell type, signaling state, and targeted context have on activation, silencing, and epigenetic memory? Large-scale measurements of transcriptional activities can help systematically answer these questions and identify general rules for how all these parameters affect effector domain activities. Last, we discuss what steps need to be taken to turn a newly discovered effector domain into a robust, precise epigenome editor. With more carefully considered high-throughput investigations, soon we will have better predictive control over the epigenome.

  View details for DOI 10.1007/978-1-0716-4051-7_4

  View details for PubMedID 39012591

  View details for PubMedCentralID 4151296

• 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 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

• High-Throughput Affinity Measurements of Transcription Factor and DNA Mutations Reveal Affinity and Specificity Determinants. Cell systems Aditham, A. K., Markin, C. J., Mokhtari, D. A., DelRosso, N. n., Fordyce, P. M. 2020

  Abstract

  Transcription factors (TFs) bind regulatory DNA to control gene expression, and mutations to either TFs or DNA can alter binding affinities to rewire regulatory networks and drive phenotypic variation. While studies have profiled energetic effects of DNA mutations extensively, we lack similar information for TF variants. Here, we present STAMMP (simultaneous transcription factor affinity measurements via microfluidic protein arrays), a high-throughput microfluidic platform enabling quantitative characterization of hundreds of TF variants simultaneously. Measured affinities for ∼210 mutants of a model yeast TF (Pho4) interacting with 9 oligonucleotides (>1,800 Kds) reveal that many combinations of mutations to poorly conserved TF residues and nucleotides flanking the core binding site alter but preserve physiological binding, providing a mechanism by which combinations of mutations in cis and trans could modulate TF binding to tune occupancies during evolution. Moreover, biochemical double-mutant cycles across the TF-DNA interface reveal molecular mechanisms driving recognition, linking sequence to function. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.

  View details for DOI 10.1016/j.cels.2020.11.012

  View details for PubMedID 33340452