All Publications

  • 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


    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


    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


    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


    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
  • 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


    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


    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