All Publications


  • Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator. eLife Sanborn, A. L., Yeh, B. T., Feigerle, J. T., Hao, C. V., Townshend, R. J., Lieberman-Aiden, E., Dror, R. O., Kornberg, R. D. 2021; 10

    Abstract

    Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

    View details for DOI 10.7554/eLife.68068

    View details for PubMedID 33904398

  • ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesinSTAG1 from WAPL. eLife Wutz, G., Ladurner, R., St Hilaire, B. G., Stocsits, R. R., Nagasaka, K., Pignard, B., Sanborn, A., Tang, W., Varnai, C., Ivanov, M. P., Schoenfelder, S., van der Lelij, P., Huang, X., Durnberger, G., Roitinger, E., Mechtler, K., Davidson, I. F., Fraser, P. J., Lieberman-Aiden, E., Peters, J. 2020; 9

    Abstract

    Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes via extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin's chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesinSTAG1 which binds chromatin for hours, whereas cohesinSTAG2 binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesinSTAG1 complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesinSTAG1 forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesinSTAG2 extrusion activity.

    View details for DOI 10.7554/eLife.52091

    View details for PubMedID 32065581

  • The Energetics and Physiological Impact of Cohesin Extrusion CELL Vian, L., Pekowska, A., Rao, S. P., Kieffer-Kwon, K., Jung, S., Baranello, L., Huang, S., El Khattabi, L., Dose, M., Pruett, N., Sanborn, A. L., Canela, A., Maman, Y., Oksanen, A., Resch, W., Li, X., Lee, B., Kovalchuk, A. L., Tang, Z., Nelson, S., Di Pierro, M., Cheng, R. R., Machol, I., St Hilaire, B., Durand, N. C., Shamim, M. S., Stamenova, E. K., Onuchic, J. N., Ruan, Y., Nussenzweig, A., Levens, D., Aiden, E., Casellas, R. 2018; 173 (5): 1165-+

    Abstract

    Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.

    View details for PubMedID 29706548

  • Cohesin Loss Eliminates All Loop Domains. Cell Rao, S. S., Huang, S. C., Glenn St Hilaire, B. n., Engreitz, J. M., Perez, E. M., Kieffer-Kwon, K. R., Sanborn, A. L., Johnstone, S. E., Bascom, G. D., Bochkov, I. D., Huang, X. n., Shamim, M. S., Shin, J. n., Turner, D. n., Ye, Z. n., Omer, A. D., Robinson, J. T., Schlick, T. n., Bernstein, B. E., Casellas, R. n., Lander, E. S., Aiden, E. L. 2017; 171 (2): 305–20.e24

    Abstract

    The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.

    View details for PubMedID 28985562

    View details for PubMedCentralID PMC5846482

  • Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture. Proceedings of the National Academy of Sciences of the United States of America Darrow, E. M., Huntley, M. H., Dudchenko, O., Stamenova, E. K., Durand, N. C., Sun, Z., Huang, S., Sanborn, A. L., Machol, I., Shamim, M., Seberg, A. P., Lander, E. S., Chadwick, B. P., Aiden, E. L. 2016; 113 (31): E4504-12

    Abstract

    During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the "Barr body." Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called "superdomains," such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called "superloops." DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4 We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.

    View details for DOI 10.1073/pnas.1609643113

    View details for PubMedID 27432957

    View details for PubMedCentralID PMC4978254

  • Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sanborn, A. L., Rao, S. S., Huang, S., Durand, N. C., Huntley, M. H., Jewett, A. I., Bochkov, I. D., Chinnappan, D., Cutkosky, A., Li, J., Geeting, K. P., Gnirke, A., Melnikov, A., McKenna, D., Stamenova, E. K., Lander, E. S., Aiden, E. L. 2015; 112 (47): E6456-E6465

    Abstract

    We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic "tension globule." In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

    View details for DOI 10.1073/pnas.1518552112

    View details for PubMedID 26499245

    View details for PubMedCentralID PMC4664323

  • Structural insights into mu-opioid receptor activation NATURE Huang, W., Manglik, A., Venkatakrishnan, A. J., Laeremans, T., Feinberg, E. N., Sanborn, A. L., Kato, H. E., Livingston, K. E., Thorsen, T. S., Kling, R. C., Granier, S., Gmeiner, P., Husbands, S. M., Traynor, J. R., Weis, W. I., Steyaert, J., Dror, R. O., Kobilka, B. K. 2015; 524 (7565): 315-?

    Abstract

    Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

    View details for DOI 10.1038/nature14886

    View details for Web of Science ID 000359714000028

  • Structural insights into µ-opioid receptor activation. Nature Huang, W., Manglik, A., Venkatakrishnan, A. J., Laeremans, T., Feinberg, E. N., Sanborn, A. L., Kato, H. E., Livingston, K. E., Thorsen, T. S., Kling, R. C., Granier, S., Gmeiner, P., Husbands, S. M., Traynor, J. R., Weis, W. I., Steyaert, J., Dror, R. O., Kobilka, B. K. 2015; 524 (7565): 315-321

    Abstract

    Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

    View details for DOI 10.1038/nature14886

    View details for PubMedID 26245379

  • A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping. Cell Rao, S. S., Huntley, M. H., Durand, N. C., Stamenova, E. K., Bochkov, I. D., Robinson, J. T., Sanborn, A. L., Machol, I., Omer, A. D., Lander, E. S., Aiden, E. L. 2014; 159 (7): 1665-1680

    Abstract

    We use in situ Hi-C to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ∼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs "facing" one another. The inactive X chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats.

    View details for DOI 10.1016/j.cell.2014.11.021

    View details for PubMedID 25497547