Achieving a quantitative and predictive understanding of 3D genome architecture presents a major challenge and aspiration. However, this milestone will not be achieved without quantitative measurements of the key proteins driving nuclear organization. Here we report the quantification of CTCF and cohesin, two causal regulators of topological associating domains (TADs) in mammalian cells. Within the context of the cohesin/CTCF mediated loop extrusion model and recent imaging studies (Hansen 2017), here we determine the density of extruding cohesins and CTCF boundary permeability. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) confirms the presence of dimers and/or oligomers. Having established cell lines with accurately measured protein abundances, we report a simple method to conveniently count molecules of any Halo-tagged protein in the nucleus. We anticipate that these tools and results will advance a more quantitative understanding of 3D genome organization, and facilitate quantifying proteins involved in diverse biological processes
Abstract Subnuclear compartmentalization has been proposed to play an important role in gene regulation by segregating active and inactive parts of the genome in distinct physical and biochemical environments. During X chromosome inactivation (XCI), the noncoding Xist RNA coats the X chromosome, triggers gene silencing and forms a dense body of heterochromatin from which the transcription machinery appears to be excluded. Phase separation has been proposed to be involved in XCI, and might explain the exclusion of the transcription machinery by preventing its diffusion into the Xist -coated territory. Here, using quantitative fluorescence microscopy and single-particle tracking, we show that RNA polymerase II (RNAPII) freely accesses the Xist territory during the initiation of XCI. Instead, the apparent depletion of RNAPII is due to the loss of its chromatin stably bound fraction. These findings indicate that initial exclusion of RNAPII from the inactive X reflects the absence of actively transcribing RNAPII, rather than a consequence of putative physical compartmentalization of the inactive X heterochromatin domain.
The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP6) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.
Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
Overview This repository contains all the raw and processed trajectory data associated with “paper title”. In this ReadMe file we provide the following information: The cell lines and conditions used in this study A summary of how the data was collected The structure of the chromosome locus tracking data Cell lines and conditions In total, the dataset covers 12 experimental conditions representing the following cell lines and treatment conditions: C36 C65 C27 CTCF-AID (untreated) CTCF-AID (2 hours AID) CTCF-AID (4 hours AID) RAD21-AID (untreated) RAD21-AID (2 hours AID) RAD21-AID (4 hours AID) WAPL-AID (untreated) WAPL-AID (4 hours AID) WAPL-AID (6 hours AID) Data and data processing Trajectories were obtained from 3D timeseries of mouse embryonic stem cell colonies in the conditions listed above using a LSM900 Airyscan 2 Zeiss microscope. For each movie we recorded 365 frames of 49.69 µm x 49.69 µm (584 x 584 pixels, pixel size: 0.085 µm by 0.085 µm), separated by an interval of 20 seconds for a total of just over 2 hours. 3D images were composed of 30 z-stacks separated by 0.25 µm, for a total height of 7.25 µm. Imaging was performed in two colors allowing the tracking of two arrays of fluorophores on Chromosome 18 near the Fbn2 gene. In all conditions, the fluorophore arrays were separated by 515 kb (except the C27 clone where separation was 10 kb). The 3D image time series were processed using ConnectTheDots: https://github.com/ahansenlab/connect_the_dots to obtain paired trajectories of chromosome loci over time. The trajectories have been corrected for chromatic shifts and aberrations. Data are provided in an “unfiltered” format (meaning that individual dot localizations were not quality control filtered) , or a filtered format (the same data set, but having undergone quality control). The filtered (quality controlled) trajectory data was used for all the quantitative analyses in the article “”. File names are formatted follows. Quality controlled data have the structure: {Clone_and_condition_name}.tagged_set.tsv Unfiltered data have the structure: {Clone_and_condition_name}.unfiltered.tagged_set.tsv For example, for RAD21-AID tagged clone, for imaging performed after two hours of protein degradation, the quality-controlled file name is: RAD21_2_hr.tagged_set.tsv. Please note that for all no-treatment conditions, we used “0 hours” as the tag. Thus, the RAD21 (untreated) becomes RAD21_0_hr.tagged_set.tsv. Structure of Data The trajectory data are provided as tab-separated text files consisting of 10 columns. The column headers are: id: a unique dot pair index t: the frame in which the dots were localized x: x-coordinate of the dot in the EGFP channel (units in µm) y: y-coordinate of the dot in the EGFP channel (units in µm) z: z-coordinate of the dot in the EGFP channel (units in µm) x2: x-coordinate of the dot in the mScarlet channel (units in µm) y2: y-coordinate of the dot in the mScarlet channel (units in µm) z2: z-coordinate of the dot in the mScarlet channel (units in µm) dist: 3D distance between the dots across channels (units in µm) movie_index: an identifier used to link the dot pair back to the raw image timeseries.
The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.
