Abby R. Thurm

All Publications

• Single-molecule states link transcription factor binding to gene expression. Nature 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 drives gene expression in mammalian cells1. However, the molecular details that link enhancer sequence to TF binding, promoter state and transcription levels remain unclear. Here we applied single-molecule footprinting2,3 to measure the simultaneous occupancy of TFs, nucleosomes and other regulatory proteins on engineered enhancer-promoter constructs with variable numbers of TF binding sites for both a synthetic TF and an endogenous TF involved in the type I interferon response. Although TF binding events on nucleosome-free DNA are independent, activation domains recruit cofactors that destabilize nucleosomes, driving observed TF binding cooperativity. Average TF occupancy linearly determines promoter activity, and we decompose TF strength into separable binding and activation terms. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the enhancer binding microstates and gene expression dynamics. This work provides a template for the quantitative dissection of distinct contributors to gene expression, including TF activation domains, concentration, binding affinity, binding site configuration and recruitment of chromatin regulators.

  View details for DOI 10.1038/s41586-024-08219-w

  View details for PubMedID 39567683

  View details for PubMedCentralID 3514679

• High-throughput development and characterization of new functional nanobodies for gene regulation and epigenetic control in human cells. bioRxiv : the preprint server for biology Wan, J., Thurm, A. R., Allen, S. J., Ludwig, C. H., Patel, A. N., Bintu, L. 2024

  Abstract

  Controlling gene expression and chromatin state via the recruitment of transcriptional effector proteins to specific genetic loci has advanced the potential of mammalian synthetic biology, but is still hindered by the challenge of delivering large chromatin regulators. Here, we develop a new method for generating small nanobodies against human chromatin regulators that can repress or activate gene expression. We start with a large and diverse nanobody library and perform enrichment against chromatin regulatory complexes using yeast display, followed by high-throughput pooled selection for transcriptional control when recruited to a reporter in human cells. This workflow allows us to efficiently select tens of functional nanobodies that can act as transcriptional repressors or activators in human cells.

  View details for DOI 10.1101/2024.11.01.621523

  View details for PubMedID 39554150

  View details for PubMedCentralID PMC11566033

• 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 discovery of regulatory effector domains in human RNA-binding proteins. bioRxiv : the preprint server for biology Thurm, A. R., Finkel, Y., Andrews, C., Cai, X. S., Benko, C., Bintu, L. 2024

  Abstract

  RNA regulation plays an integral role in tuning gene expression and is controlled by thousands of RNA-binding proteins (RBPs). We develop and use a high-throughput recruitment assay (HT-RNA-Recruit) to identify regulatory domains within human RBPs by recruiting over 30,000 protein tiles from 367 RBPs to a reporter mRNA. We discover over 100 unique RNA-regulatory effectors in 86 distinct RBPs, presenting evidence that RBPs contain functionally separable domains that dictate their post-transcriptional control of gene expression, and identify some with unique activity at 5' or 3'UTRs. We identify some domains that downregulate gene expression both when recruited to DNA and RNA, and dissect their mechanisms of regulation. Finally, we build a synthetic RNA regulator that can stably maintain gene expression at desired levels that are predictable by a mathematical model. This work serves as a resource for human RNA-regulatory effectors and expands the synthetic repertoire of RNA-based genetic control tools.Highlights: HT-RNA-Recruit identifies hundreds of RNA-regulatory effectors in human proteins.Recruitment to 5' and 3' UTRs identifies regulatory domains unique to each position.Some protein domains have both transcriptional and post-transcriptional regulatory activity.We develop a synthetic RNA regulator and a mathematical model to describe its behavior.

  View details for DOI 10.1101/2024.07.19.604317

  View details for PubMedID 39071298

• 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 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

• Myeloid lncRNA LOUP mediates opposing regulatory effects of RUNX1 and RUNX1-ETO in t(8;21) AML (vol 138 pg 1331, 2021) BLOOD Trinh, B. Q., Ummarino, S., Zhang, Y., Ebralidze, A. K., Bassal, M. A., Nguyen, T. M., Heller, G., Coffey, R., Tenen, D. E., van der Kouwe, E., Fabiani, E., Gurnari, C., Wu, C., Angarica, V., Yang, H., Chen, S., Zhang, H., Thurm, A. R., Marchi, F., Levantini, E., Staber, P. B., Zhang, P., Voso, M., Pandol, P., Kobayashi, S. S., Chai, L., Di Ruscio, A., Tenen, D. G. 2023; 141 (20): 2542

  View details for DOI 10.1182/blood.2023020299

  View details for Web of Science ID 001001931500001

• The Nonmonotonic Dose Dependence of Protein Expression in Cells Transfected with Self-Amplifying RNA. Journal of virology Tanimoto, C. R., Thurm, A. R., Brandt, D. S., Knobler, C. M., Gelbart, W. M. 2022; 96 (7): e0185821

  Abstract

  Self-amplifying (sa) RNA molecules-"replicons"-derived from the genomes of positive-sense RNA viruses are receiving increasing attention as gene and vaccine delivery vehicles. This is because mRNA forms of genes of interest can be incorporated into them and strongly amplified, thereby enhancing target protein expression. In this report, we demonstrate a nonmonotonic dependence of protein expression on the mass of transfected replicon, in contrast to the usual, monotonic case of non-saRNA transfections. We lipotransfected a variety of cell lines with increasing masses of enhanced yellow fluorescent protein (eYFP) as a reporter gene in sa form and found that there is a "sweet spot" at which protein expression and cell viability are optimum. To control the varying mass of transfected replicon RNA for a given mass of Lipofectamine, the replicons were mixed with a "carrier" RNA that is neither replicated nor translated; the total mass of transfected RNA was kept constant while increasing the fraction of the replicon from zero to one. Fluorescence microscopy studies showed that the optimum protein expression and cell viability are achieved for replicon fractions as small as 1/10 of the total transfected RNA, and these results were quantified by a systematic series of flow cytometry measurements. IMPORTANCE Positive-sense RNA viruses often have a cytotoxic effect on their host cell because of the strength of their RNA replicase proteins, even though only one copy of their genome begins the viral life cycle in each cell. Noninfectious forms of them-replicons-which include just their RNA replication-related genes, are also strongly self-amplifying and cytotoxic. Accordingly, when replicons fused with nonviral genes of interest are transfected into cells to amplify expression of proteins of interest, one needs to keep the replicon "dose" sufficiently low. We demonstrate how to control the number of RNA replicons getting into transfected cells and that there is a sweet spot for the replicon dose that optimizes protein expression and cell viability. Examples are given for the case of Nodamura viral replicons with fluorescent protein reporter genes in a variety of mammalian cell lines, quantified by flow cytometry and live/dead cell assays.

  View details for DOI 10.1128/jvi.01858-21

  View details for PubMedID 35293773

  View details for PubMedCentralID PMC9006910

• Myeloid lncRNA LOUP Mediates Opposing Regulatory Effects of RUNX1 and RUNX1-ETO in t(8;21) AML. Blood Trinh, B. Q., Ummarino, S. n., Zhang, Y. n., Ebralidze, A. K., Bassal, M. A., Nguyen, T. M., Heller, G. n., Coffey, R. n., Tenen, D. E., van der Kouwe, E. n., Fabiani, E. n., Gurnari, C. n., Wu, C. S., Espinosa Angarica, V. n., Yang, H. n., Chen, S. n., Zhang, H. n., Thurm, A. R., Marchi, F. n., Levantini, E. n., Staber, P. B., Zhang, P. n., Voso, M. T., Pandolfi, P. P., Kobayashi, S. S., Chai, L. n., Di Ruscio, A. n., Tenen, D. G. 2021

  Abstract

  The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA- and DNA-interactions with the broadly expressed transcription factor RUNX1, we identified the long noncoding RNA LOUP. This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia, wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein RUNX1-ETO limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression. These findings highlight the important role of the interplay between cell type-specific RNAs and transcription factors as well as their oncogenic derivatives in modulating lineage-gene activation and raise the possibility that RNA regulators of transcription factors represent alternative targets for therapeutic development.

  View details for DOI 10.1182/blood.2020007920

  View details for PubMedID 33971010

• RNA Homopolymers Form Higher-Curvature Virus-like Particles Than Do Normal-Composition RNAs. Biophysical journal Thurm, A. R., Beren, C., Duran-Meza, A. L., Knobler, C. M., Gelbart, W. M. 2019; 117 (7): 1331-1341

  Abstract

  Unlike double-stranded DNA, single-stranded RNA can be spontaneously packaged into spherical capsids by viral capsid protein (CP) because it is a more compact and flexible polymer. Many systematic investigations of this self-assembly process have been carried out using CP from cowpea chlorotic mottle virus, with a wide range of sequences and lengths of single-stranded RNA. Among these studies are measurements of the relative packaging efficiencies of these RNAs into spherical capsids. In this work, we address a fundamental issue that has received very little attention, namely the question of the preferred curvature of the capsid formed around different RNA molecules. We show in particular that homopolymers of RNA-polyribouridylic acid and polyriboadenylic acid-form exclusively T = 2-sized (∼22-nm diameter) virus-like particles (VLPs) when mixed with cowpea chlorotic mottle virus CP, independent of their length, ranging from 500 to more than 4000 nucleotides. This is in contrast to "normal-composition" RNAs (i.e., molecules with comparable numbers of each of the four nucleotides and hence capable of developing a large amount of secondary structure because of intramolecular complementarity/basepairing); a curvature corresponding to T = 3-size (∼28 nm in diameter) is preferred for the VLPs formed with such RNAs. Our work is consistent with the preferred curvature of VLPs being a consequence of interaction of CP with RNA-in particular, the presence or absence of short RNA duplexes-and suggests that the equilibrium size of the capsid results from a trade-off between this optimum size and the cost of confinement.

  View details for DOI 10.1016/j.bpj.2019.08.012

  View details for PubMedID 31514968

  View details for PubMedCentralID PMC6818174