Biology subjects

Tjian, R.

Publications and source records attributed to Tjian, R..

31 records · Page 2Linked to original sources

bioRxiv2020

EcDNA hubs drive cooperative intermolecular oncogene expression

Extrachromosomal DNAs (ecDNAs) are prevalent in human cancers and mediate high oncogene expression through elevated copy number and altered gene regulation1. Gene expression typically involves distal enhancer DNA elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs, comprised of ~10-100 ecDNAs clustered in the nucleus of interphase cells, drive intermolecular enhancer input for amplified oncogene expression. Single-molecule sequencing, single-cell multiome, and 3D enhancer connectome reveal subspecies of MYC-PVT1 ecDNAs lacking enhancers that access intermolecular and ectopic enhancer-promoter interactions in ecDNA hubs. ecDNA hubs persist without transcription and are tethered by BET protein BRD4. BET inhibitor JQ1 disperses ecDNA hubs, preferentially inhibits ecDNA oncogene transcription, and kills ecDNA+ cancer cells. Two amplified oncogenes MYC and FGFR2 intermix in ecDNA hubs, engage in intermolecular enhancer-promoter interactions, and transcription is uniformly sensitive to JQ1. Thus, ecDNA hubs are nuclear bodies of many ecDNAs tethered by proteins and platforms for cooperative transcription, leveraging the power of oncogene diversification and combinatorial DNA interactions. We suggest ecDNA hubs, rather than individual ecDNAs, as units of oncogene function, cooperative evolution, and new targets for cancer therapy.

cancer biology↗

bioRxiv2019

Super-resolution Imaging Reveals 3D Structure and Organizing Mechanism of Accessible Chromatin

Access to cis-regulatory elements packaged in chromatin is essential for directing gene expression and cell viability. Here, we report a super-resolution imaging strategy, 3D ATAC-PALM, that enables direct visualization of the entire accessible genome. We found that active chromosomal segments are organized into spatially-segregated accessible chromatin domains (ACDs). Rapid depletion of CTCF or Cohesin (RAD21 subunit) induced enhanced ACD clustering, reduced physical separation between intrachromosomal ACDs, and differentially regulated ACD compaction. Experimental perturbations and polymer modeling suggest that dynamic protein-protein and protein-DNA interactions within ACDs couple with loop extrusion to organize ACD topology. Dysorganization of ACDs upon CTCF or Cohesin loss alters transcription factor binding and target search dynamics in living cells. These results uncover fundamental mechanisms underpinning the formation of 3D genome architecture and its pivotal function in transcriptional regulation.

molecular biology↗

bioRxiv2019

Resolving the 3D landscape of transcription-linked mammalian chromatin folding

Chromatin folding below the scale of topologically associating domains (TADs) remains largely unexplored in mammals. Here, we used a high-resolution 3C-based method, Micro-C, to probe links between 3D-genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregate TAD regions into \"microTADs\" with distinct regulatory features. Enhancer-promoter and promoter-promoter interactions extending from the edge of these domains predominantly link co-regulated loci, often independently of CTCF/Cohesin. Acute inhibition of transcription disrupts the gene-related folding features without altering higher-order chromatin structures. Intriguingly, we detect \"two-start\" zig-zag 30-nanometer chromatin fibers. Our work uncovers the finer-scale genome organization that establishes novel functional links between chromatin folding and gene regulation.\n\nONE SENTENCE SUMMARYTranscriptional regulatory elements shape 3D genome architecture of microTADs.

genomics↗

bioRxiv2019

MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions

Loss of function mutations in Methyl-CpG-binding Protein 2 (MeCP2) cause the severe neurological disorder Rett Syndrome. MeCP2 is a highly abundant nuclear protein particularly enriched in neurons. Although biochemical and genomic analyses of MeCP2-DNA interaction and genomic distribution demonstrate that MeCP2 binding on chromatin is dependent on DNA modification state, the dynamic behavior of individual MeCP2 proteins in live neurons has not been explored. Here we use live-cell single-molecule imaging to assess the detailed kinetic features of MeCP2 in distinct sub-nuclear regions at high spatial and temporal resolution. Surprisingly, we found that, in granule cell nuclei, MeCP2 has unique diffusion and chromatin binding kinetics that are distinct from highly mobile sequence-specific transcription factors (TF) and immobile histone proteins. Approximately, half of MeCP2 is bound to DNA in a transiently stable mode that is similar to TF binding to their cognate sites. The binding of meCP2 to DNA requires its methyl-binding domain (MBD) and is sensitive to the levels of both DNA methylation and hydroxymethylation. However, when not stably bound, MeCP2 moves slowly in the nucleus most closely resembling histone H1.0. The rate of MeCP2 diffusion in compact, granule cell nuclei is determined by weak, transient DNA interactions mediated primarily by the MBD and three AT-hook domains located in the C-terminal portion of the protein. Both the fraction of stably bound MeCP2 and its rate of diffusion depend on the level of chromatin compaction and neuronal cell type. Our data reveal new features of MeCP2 that dictate its dynamic behavior in neuronal nuclei and suggest that the limited nuclear diffusion of MeCP2 in live neurons may contribute to its local impact on chromatin structure and gene expression.

molecular biology↗

bioRxiv2018

An RNA-binding region regulates CTCF clustering and chromatin looping

Mammalian genomes are folded into Topologically Associating Domains (TADs), consisting of cell-type specific chromatin loops anchored by CTCF and cohesin. Since CTCF and cohesin are expressed ubiquitously, how cell-type specific CTCF-mediated loops are formed poses a paradox. Here we show RNase-sensitive CTCF self-association in vitro and that an RNA-binding region (RBR) mediates CTCF clustering in vivo. Intriguingly, deleting the RBR abolishes or impairs almost half of all chromatin loops in mouse embryonic stem cells. Disrupted loop formation correlates with abrogated clustering and diminished chromatin binding of the RBR mutant CTCF protein, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least 2 classes: RBR-independent and RBR-dependent loops. We suggest that evidence for distinct classes of RBR-dependent loops may provide a mechanism for establishing cell-specific CTCF loops regulated by RNAs and other RBR partner.

biophysics↗

bioRxiv2018

Guided nuclear exploration increases CTCF target search efficiency

Mammalian genomes are enormous. For a DNA-binding protein, this means that the number of non-specific, off-target sites vastly exceeds the number of specific, cognate sites. How mammalian DNA-binding proteins overcome this challenge to efficiently locate their target sites is not known. Here through live-cell single-molecule tracking, we show that CCCTC-binding factor, CTCF, is repeatedly trapped in small zones in the nucleus in a manner that is largely dependent on its RNA-binding region (RBR). Integrating theory, we devise a new model, Anisotropic Diffusion through transient Trapping in Zones (ADTZ), to explain this. Functionally, transient RBR-mediated trapping increases the efficiency of CTCF target search by [~]2.5 fold. Since the RBR-domain also mediates CTCF clustering, our results suggest a \"guided\" mechanism where CTCF clusters concentrate diffusing CTCF proteins near cognate binding sites, thus increasing the local ON-rate. We suggest that local \"guiding\" may represent a general target search mechanism in mammalian cells.

biophysics↗

bioRxiv2018

Transient DNA Binding Induces RNA Polymerase II Compartmentalization During Herpesviral Infection Distinct From Phase Separation

During lytic infection, Herpes Simplex Virus 1 generates replication compartments (RCs) in host nuclei that efficiently recruit protein factors, including host RNA Polymerase II (Pol II). Pol II and other cellular factors form hubs in uninfected cells that are proposed to phase separate via multivalent protein-protein interactions mediated by their intrinsically disordered regions. Using a battery of live cell microscopic techniques, we show that although RCs superficially exhibit many characteristics of phase separation, the recruitment of Pol II instead derives from nonspecific interactions with the viral DNA. We find that the viral genome remains nucleosome-free, profoundly affecting the way Pol II explores RCs by causing it to repetitively visit nearby binding sites, thereby creating local Pol II accumulations. This mechanism, distinct from phase separation, allows viral DNA to outcompete host DNA for cellular proteins. Our work provides new insights into the strategies used to create local molecular hubs in cells.

bioinformatics↗

bioRxiv2018

Architectural Features of 3D Genome Organization Revealed by Counting CTCF and Cohesin Molecules

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.

biophysics↗

bioRxiv2018

mRNA expression-mediated gene repression in human cells

We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference were used in an integrated manner to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that blocked translation of the ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2. We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.

genetics↗

bioRxiv2018

A stable mode of bookmarking by TBP recruits RNA Polymerase II to mitotic chromosomes

How a cell maintains transcriptional fidelity across mitosis has remained an enduring mystery in biology. One challenge arises during mitosis when chromatin becomes condensed and transcription is shut off. How do the daughter cells re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, remains bound globally to active promoters in ES cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding is highly stable, with an average residence time of minutes. This stable binding is in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery maintains global transcriptional memory during mitosis.

molecular biology↗

bioRxiv2017

Dynamic and Selective Low-Complexity Domain Interactions Revealed by Live-Cell Single-Molecule Imaging

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs) but how they perform transactivation functions remains unclear. Recent studies report that TF-LCDs can undergo hydrogel formation or liquid-liquid phase separation in vitro. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II) and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid reversible and multivalent LCD-LCD interactions occur between TFs and the Pol II machinery, which underpins a central mechanism for transactivation and plays a key role in gene expression and disease.

biophysics↗

bioRxiv2017

Spot-On: robust model-based analysis of single-particle tracking experiments

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce \"Spot-On\", an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.\n\nIMPACT STATEMENTSpot-On is an easy-to-use website that makes a rigorous and bias-corrected modeling framework for analysis of single-molecule tracking experiments available to all.

biophysics↗

bioRxiv2016

CTCF and Cohesin Regulate Chromatin Loop Stability with Distinct Dynamics

Abstract/summaryFolding of mammalian genomes into spatial domains is critical for gene regulation. CTCF and cohesin control domain location by folding domains into loop structures, which are thought to be highly stable. Combining genomic, biochemical and single-molecule imaging approaches, we show that although CTCF and cohesin can physically interact, CTCF binds chromatin much more dynamically than cohesin (~1 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging \"dynamic complex\" rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops constantly break and reform throughout the cell cycle.

biophysics↗