Lacramioara Bintu

All Publications

• The H3.3K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation. Molecular cell Sinha, J., Nickels, J. F., Thurm, A. R., Ludwig, C. H., Archibald, B. N., Hinks, M. M., Wan, J., Fang, D., Bintu, L. 2024

  Abstract

  Histone H3.3 is frequently mutated in tumors, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the epigenetic landscape, its effects on gene expression dynamics remain unclear. Here, we use a synthetic reporter to measure the effects of H3.3K36M on silencing and epigenetic memory after recruitment of the ZNF10 Kruppel-associated box (KRAB) domain, part of the largest class of human repressors and associated with H3K9me3 deposition. We find that H3.3K36M, which decreases H3K36 methylation and increases histone acetylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a modelfor establishment and maintenance of epigenetic memory, where the H3K36 methylation pathway is necessary to maintain histone deacetylation and convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.

  View details for DOI 10.1016/j.molcel.2024.09.015

  View details for PubMedID 39368466

• 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

• Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model. Acta neuropathologica communications Sinha, S., Huang, M. S., Mikos, G., Bedi, Y., Soto, L., Lensch, S., Ayushman, M., Bintu, L., Bhutani, N., Heilshorn, S. C., Yang, F. 2024; 12 (1): 71

  Abstract

  Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

  View details for DOI 10.1186/s40478-024-01765-4

  View details for PubMedID 38706008

  View details for PubMedCentralID 4161623

• Harmonizing the Generation and Pre-publication Stewardship of FAIR Image data. ArXiv Bialy, N., Alber, F., Andrews, B., Angelo, M., Beliveau, B., Bintu, L., Boettiger, A., Boehm, U., Brown, C. M., Maina, M. B., Chambers, J. J., Cimini, B. A., Eliceiri, K., Errington, R., Faklaris, O., Gaudreault, N., Germain, R. N., Goscinski, W., Grunwald, D., Halter, M., Hanein, D., Hickey, J. W., Lacoste, J., Laude, A., Lundberg, E., Ma, J., Malacrida, L., Moore, J., Nelson, G., Neumann, E. K., Nitschke, R., Onami, S., Pimentel, J. A., Plant, A. L., Radtke, A. J., Sabata, B., Schapiro, D., Schöneberg, J., Spraggins, J. M., Sudar, D., Adrien Maria Vierdag, W. M., Volkmann, N., Wählby, C., Wang, S. S., Yaniv, Z., Strambio-De-Castillia, C. 2024

  Abstract

  Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

  View details for DOI 10.1242/jcs.254151

  View details for PubMedID 38351940

  View details for PubMedCentralID PMC10862930

• Single-molecule chromatin configurations link transcription factor binding to expression in human cells. bioRxiv : the preprint server for biology Doughty, B. R., Hinks, M. M., Schaepe, J. M., Marinov, G. K., Thurm, A. R., Rios-Martinez, C., Parks, B. E., Tan, Y., Marklund, E., Dubocanin, D., Bintu, L., Greenleaf, W. J. 2024

  Abstract

  The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer sequence to TF binding, promoter state, and gene expression levels remain opaque. We applied single-molecule footprinting (SMF) to measure the simultaneous occupancy of TFs, nucleosomes, and components of the transcription machinery on engineered enhancer/promoter constructs with variable numbers of TF binding sites for both a synthetic and an endogenous TF. We find that activation domains enhance a TF's capacity to compete with nucleosomes for binding to DNA in a BAF-dependent manner, TF binding on nucleosome-free DNA is consistent with independent binding between TFs, and average TF occupancy linearly contributes to promoter activation rates. We also decompose TF strength into separable binding and activation terms, which can be tuned and perturbed independently. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the binding microstates observed at the enhancer and subsequent time-dependent gene expression. This work provides a template for quantitative dissection of distinct contributors to gene activation, including the activity of chromatin remodelers, TF activation domains, chromatin acetylation, TF concentration, TF binding affinity, and TF binding site configuration.

  View details for DOI 10.1101/2024.02.02.578660

  View details for PubMedID 38352517

• The H3.3 K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation. bioRxiv : the preprint server for biology Sinha, J., Nickels, J. F., Thurm, A. R., Ludwig, C. H., Archibald, B. N., Hinks, M. M., Wan, J., Fang, D., Bintu, L. 2023

  Abstract

  Histone H3.3 is frequently mutated in cancers, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the cellular epigenetic landscape, it remains unclear how it affects the dynamics of gene expression. Here, we use a synthetic reporter to measure the effect of H3.3K36M on silencing and epigenetic memory after recruitment of KRAB: a member of the largest class of human repressors, commonly used in synthetic biology, and associated with H3K9me3. We find that H3.3K36M, which decreases H3K36 methylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a new model for establishment and maintenance of epigenetic memory, where H3K36 methylation is necessary to convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.

  View details for DOI 10.1101/2023.10.13.562147

  View details for PubMedID 37873347

  View details for PubMedCentralID PMC10592807

• Single-cell chromatin state transitions during epigenetic memory formation. bioRxiv : the preprint server for biology Fujimori, T., Rios-Martinez, C., Thurm, A. R., Hinks, M. M., Doughty, B. R., Sinha, J., Le, D., Hafner, A., Greenleaf, W. J., Boettiger, A. N., Bintu, L. 2023

  Abstract

  Repressive chromatin modifications are thought to compact chromatin to silence transcription. However, it is unclear how chromatin structure changes during silencing and epigenetic memory formation. We measured gene expression and chromatin structure in single cells after recruitment and release of repressors at a reporter gene. Chromatin structure is heterogeneous, with open and compact conformations present in both active and silent states. Recruitment of repressors associated with epigenetic memory produces chromatin compaction across 10-20 kilobases, while reversible silencing does not cause compaction at this scale. Chromatin compaction is inherited, but changes molecularly over time from histone methylation (H3K9me3) to DNA methylation. The level of compaction at the end of silencing quantitatively predicts epigenetic memory weeks later. Similarly, chromatin compaction at the Nanog locus predicts the degree of stem-cell fate commitment. These findings suggest that the chromatin state across tens of kilobases, beyond the gene itself, is important for epigenetic memory formation.

  View details for DOI 10.1101/2023.10.03.560616

  View details for PubMedID 37873344

  View details for PubMedCentralID PMC10592931

• High-throughput functional characterization of combinations of transcriptional activators and repressors. Cell systems Mukund, A. X., Tycko, J., Allen, S. J., Robinson, S. A., Andrews, C., Sinha, J., Ludwig, C. H., Spees, K., Bassik, M. C., Bintu, L. 2023

  Abstract

  Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, whereas combining strong activators often results in weaker activation. In contrast, repressors combine linearly and produce full gene silencing, and repressor domains often overpower activation domains. We use this information to build a synthetic transcription factor whose function can be tuned between repression and activation independent of recruitment to target genes by using a small-molecule drug. Altogether, we outline the basic principles of how effector domains combine to regulate gene expression and demonstrate their value in building precise and flexible synthetic biology tools. A record of this paper's transparent peer review process is included in the supplemental information.

  View details for DOI 10.1016/j.cels.2023.07.001

  View details for PubMedID 37543039

• Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project. Molecular cell Dekker, J., Alber, F., Aufmkolk, S., Beliveau, B. J., Bruneau, B. G., Belmont, A. S., Bintu, L., Boettiger, A., Calandrelli, R., Disteche, C. M., Gilbert, D. M., Gregor, T., Hansen, A. S., Huang, B., Huangfu, D., Kalhor, R., Leslie, C. S., Li, W., Li, Y., Ma, J., Noble, W. S., Park, P. J., Phillips-Cremins, J. E., Pollard, K. S., Rafelski, S. M., Ren, B., Ruan, Y., Shav-Tal, Y., Shen, Y., Shendure, J., Shu, X., Strambio-De-Castillia, C., Vertii, A., Zhang, H., Zhong, S. 2023

  Abstract

  The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

  View details for DOI 10.1016/j.molcel.2023.06.018

  View details for PubMedID 37419111

• High-throughput discovery and characterization of viral transcriptional effectors in human cells. Cell systems Ludwig, C. H., Thurm, A. R., Morgens, D. W., Yang, K. J., Tycko, J., Bassik, M. C., Glaunsinger, B. A., Bintu, L. 2023; 14 (6): 482

  Abstract

  Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression. Given their multifunctional nature and high sequence divergence, it is unclear which viral proteins can affect transcription and which specific sequences contribute to this function. Using a high-throughput assay, we measured the transcriptional regulatory potential of over 60,000 protein tiles across 1,500 proteins from 11 coronaviruses and all nine human herpesviruses. We discovered hundreds of transcriptional effector domains, including a conserved repression domain in all coronavirus Spike homologs, dual activation-repression domains in viral interferon regulatory factors (VIRFs), and an activation domain in six herpesvirus homologs of the single-stranded DNA-binding protein that we show is important for viral replication and late gene expression in Kaposi's sarcoma-associated herpesvirus (KSHV). For the effector domains we identified, we investigated their mechanisms via high-throughput sequence and chemical perturbations, pinpointing sequence motifs essential for function. This work massively expands viral protein annotations, serving as a springboard for studying their biological and health implications and providing new candidates for compact gene regulation tools.

  View details for DOI 10.1016/j.cels.2023.05.008

  View details for PubMedID 37348463

• CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping. Genome biology Marinov, G. K., Kim, S. H., Bagdatli, S. T., Higashino, S. I., Trevino, A. E., Tycko, J., Wu, T., Bintu, L., Bassik, M. C., He, C., Kundaje, A., Greenleaf, W. J. 2023; 24 (1): 85

  Abstract

  Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.

  View details for DOI 10.1186/s13059-023-02930-z

  View details for PubMedID 37085898

  View details for PubMedCentralID PMC10120127

• 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

• Single-Molecule Mapping of Chromatin Accessibility Using NOMe-seq/dSMF. Methods in molecular biology (Clifton, N.J.) Hinks, M., Marinov, G. K., Kundaje, A., Bintu, L., Greenleaf, W. J. 2023; 2611: 101-119

  Abstract

  The bulk of gene expression regulation in most organisms is accomplished through the action of transcription factors (TFs) on cis-regulatory elements (CREs). In eukaryotes, these CREs are generally characterized by nucleosomal depletion and thus higher physical accessibility of DNA. Many methods exploit this property to map regions of high average accessibility, and thus putative active CREs, in bulk. However, these techniques do not provide information about coordinated patterns of accessibility along the same DNA molecule, nor do they map the absolute levels of occupancy/accessibility. SMF (Single-Molecule Footprinting) fills these gaps by leveraging recombinant DNA cytosine methyltransferases (MTase) to mark accessible locations on individual DNA molecules. In this chapter, we discuss current methods and important considerations for performing SMF experiments.

  View details for DOI 10.1007/978-1-0716-2899-7_8

  View details for PubMedID 36807067

• The sound of silence: Transgene silencing in mammalian cell engineering. Cell systems Cabera, A., Edelstein, H. I., Glykofrydis, F., Love, K. S., Palacios, S., Tycko, J., Zhang, M., Lensch, S., Shields, C. E., Livingston, M., Weiss, R., Zhao, H., Haynes, K. A., Morsut, L., Chen, Y. Y., Khalil, A. S., Wong, W. W., Collins, J. J., Rosser, S. J., Polizzi, K., Elowitz, M. B., Fussenegger, M., Hilton, I. B., Leonard, J. N., Bintu, L., Galloway, K. E., Deans, T. L. 2022; 13 (12): 950-973

  Abstract

  To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.

  View details for DOI 10.1016/j.cels.2022.11.005

  View details for PubMedID 36549273

• Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Nature biotechnology Durrant, M. G., Fanton, A., Tycko, J., Hinks, M., Chandrasekaran, S. S., Perry, N. T., Schaepe, J., Du, P. P., Lotfy, P., Bassik, M. C., Bintu, L., Bhatt, A. S., Hsu, P. D. 2022

  Abstract

  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

• Dynamic spreading of chromatin-mediated gene silencing and reactivation between neighboring genes in single cells. eLife Lensch, S., Herschl, M. H., Ludwig, C. H., Sinha, J., Hinks, M. M., Mukund, A., Fujimori, T., Bintu, L. 2022; 11

  Abstract

  In mammalian cells genes that are in close proximity can be transcriptionally coupled: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, and when designing synthetic gene regulation circuits. Here, we systematically dissect this process in single cells by recruiting and releasing repressive chromatin regulators at dual-gene synthetic reporters, and measuring how fast gene silencing and reactivation spread as a function of intergenic distance and configuration of insulator elements. We find that silencing by KRAB, associated with histone methylation, spreads between two genes within hours, with a time delay that increases with distance. This fast KRAB-mediated spreading is not blocked by the classical cHS4 insulators. Silencing by histone deacetylase HDAC4 of the upstream gene can also facilitate background silencing of the downstream gene by PRC2, but with a days-long delay that does not change with distance. This slower silencing can sometimes be stopped by insulators. Gene reactivation of neighboring genes is also coupled, with strong promoters and insulators determining the order of reactivation. Our data can be described by a model of multi-gene regulation that builds upon previous knowledge of heterochromatin spreading, where both gene silencing and gene reactivation can act at a distance, allowing for coordinated dynamics via chromatin regulator recruitment.

  View details for DOI 10.7554/eLife.75115

  View details for PubMedID 35678392

• Temporal signaling, population control, and information processing through chromatin-mediated gene regulation. Journal of theoretical biology Mukund, A., Bintu, L. 1800: 110977

  Abstract

  Chromatin regulation is a key pathway cells use to regulate gene expression in response to temporal stimuli, and is becoming widely used as a platform for synthetic biology applications. redHere, we build a mathematical framework for analyzing the response of genetic circuits containing chromatin regulators to temporal signals in mammalian cell populations. Chromatin regulators can silence genes in an all-or-none fashion at the single-cell level, with individual cells stochastically transitioning between active, reversibly silent, and irreversibly silent gene states at constant rates over time. redWe integrate this mode of regulation with classical gene regulatory motifs, such as autoregulatory and incoherent feedforward loops, to determine the types of responses achievable with duration-dependent signaling. We demonstrate that repressive regulators without long-term epigenetic memory can filter out high frequency noise, and as part of an autoregulatory loop can precisely tune the fraction of cells in a population that expresses a gene of interest. redAdditionally, we find that repressive regulators with epigenetic memory can sum up and encode the total duration of their recruitment in the fraction of cells irreversibly silenced and, when included in a feed forward loop, enable perfect adaptation. redLast, we use an information theoretic approach to show that all-or-none stochastic silencing can be used by populations to transmit information reliably and with high fidelity even in very simple genetic circuits. redAltogether, we show that chromatin-mediated gene control enables a repertoire of complex cell population responses to temporal signals and can transmit higher information levels than previously measured in gene regulation.

  View details for DOI 10.1016/j.jtbi.2021.110977

  View details for PubMedID 34919934

• Nanobody-mediated control of gene expression and epigenetic memory. Nature communications Van, M. V., Fujimori, T. n., Bintu, L. n. 2021; 12 (1): 537

  Abstract

  Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

  View details for DOI 10.1038/s41467-020-20757-1

  View details for PubMedID 33483487

• 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

• Mapping chromatin modifications at the single cell level. Development (Cambridge, England) Ludwig, C. H., Bintu, L. n. 2019; 146 (12)

  Abstract

  Understanding chromatin regulation holds enormous promise for controlling gene regulation, predicting cellular identity, and developing diagnostics and cellular therapies. However, the dynamic nature of chromatin, together with cell-to-cell heterogeneity in its structure, limits our ability to extract its governing principles. Single cell mapping of chromatin modifications, in conjunction with expression measurements, could help overcome these limitations. Here, we review recent advances in single cell-based measurements of chromatin modifications, including optimization to reduce DNA loss, improved DNA sequencing, barcoding, and antibody engineering. We also highlight several applications of these techniques that have provided insights into cell-type classification, mapping modification co-occurrence and heterogeneity, and monitoring chromatin dynamics.

  View details for DOI 10.1242/dev.170217

  View details for PubMedID 31249006

• Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements. Nature communications Tycko, J. n., Wainberg, M. n., Marinov, G. K., Ursu, O. n., Hess, G. T., Ego, B. K., Aradhana, n. n., Li, A. n., Truong, A. n., Trevino, A. E., Spees, K. n., Yao, D. n., Kaplow, I. M., Greenside, P. G., Morgens, D. W., Phanstiel, D. H., Snyder, M. P., Bintu, L. n., Greenleaf, W. J., Kundaje, A. n., Bassik, M. C. 2019; 10 (1): 4063

  Abstract

  Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.

  View details for DOI 10.1038/s41467-019-11955-7

  View details for PubMedID 31492858

• Advancing towards a global mammalian gene regulation model through single-cell analysis and synthetic biology Current Opinion in Biomedical Engineering Tycko, J., Van, M. V., Elowitz, M. B., Bintu, L. 2017; 4: 174-193

  View details for DOI 10.1016/j.cobme.2017.10.011