Biology subjects

Cattoglio, C.

Publications and source records attributed to Cattoglio, C..

12 recordsLinked to original sources

bioRxiv2025

Concomitant acetylation and loading of H2A.Z by NuA4/TIP60 regulate target gene transcription

The human NuA4/TIP60 complex is a multi-subunit, dual enzymatic epigenetic factor and gene regulator. It bears histone acetyltransferase (HAT) activity towards the canonical histones H2A and H4 and the histone variant H2A.Z, a function that has been linked to gene activation. It also acts as a chromatin remodeling enzyme through ATP-dependent exchange of nucleosomal H2A-H2B dimers with H2A.Z-H2B, leading to incorporation of H2A.Z into chromatin at gene regulatory elements. NuA4/TIP60 is unique in merging two enzymatic activities targeting H2A.Z. Both NuA4/TIP60-dependent H2A.Z acetylation and remodeling have been linked to several physiological functions and pathologies, but studies have only focused on either one or the other enzymatic activity, and insights on functional coordination between them are lacking. Here, we leverage our EP400 rapid depletion system to explore and untangle the intricate links between H2A.Z acetylation by Tip60 (the HAT subunit) and loading on chromatin by EP400 (the remodeling subunit) through functional genomic and biochemical analyses. Our data support a mechanism in which H2A.Z is first pre-acetylated to allow for H2A.Zac-H2B dimer association with the complex before incorporation into chromatin, particularly at gene promoters to positively regulate transcription. As both H2A.Z-targeted enzymatic functions of NuA4/TIP60 have been linked to disease, albeit separately, our findings hold important implications for therapeutic intervention, where combinatorial targeting is a promising avenue.

molecular biology↗

bioRxiv2025

Distinct Sox9 single-molecule dynamics characterize adult differentiation and fetal-like reprogrammed states in intestinal organoids

Transcription factors (TFs) mediate gene expression changes during differentiation and development. However, how TF biophysical properties and abundance dynamically regulate specific cell state transitions remains poorly understood. Using automated live-cell single-molecule tracking (SMT) in intestinal organoid models, we revealed an expression level-independent decrease in the fraction of immobile Sox9 molecules during differentiation from [~]48% to [~]38%, largely dependent on DNA binding. Strikingly, long-term Sox9 overexpression caused organoids to transition from budding to spheroid morphology accompanied by increased proliferation and a loss in gene expression signatures for intestinal identity and function. In this fetal-like reprogrammed state, a larger fraction of partially self- interacting Sox9 molecules ([~]61%) binds to DNA. Our results suggest context-dependent Sox9 single-molecule dynamics during adult intestinal differentiation and fetal-like reversion in consequence to long-term Sox9 overexpression. Our work underpins the power of our automated live-cell SMT framework to generate testable hypotheses towards unraveling molecular mechanisms underlying tissue-level phenotypes. Highlights- Heterogenous diffusion behavior of Sox9 across intestinal differentiation states - Concentration-independent decrease in chromatin-bound Sox9 upon differentiation - Sox9 overexpression leads to fetal-like reversion and more immobile Sox9 molecules - Automated SMT in 2D monolayers reveals TF dynamics underlying organoid phenotypes

developmental biology↗

bioRxiv2024

Mesoscale chromatin confinement facilitates target search of pioneer transcription factors in live cells

Pioneer transcription factors (PTFs) possess the unique capability to access closed chromatin regions and initiate cell fate changes, yet the underlying mechanisms remain elusive. Here, we characterized the single-molecule dynamics of PTFs targeting chromatin in living cells, revealing a notable "confined target search" mechanism. PTFs like FOXA1, FOXA2, SOX2, OCT4 and KLF4 sampled chromatin more frequently than non-pioneer factor MYC, alternating between fast free diffusion in the nucleus and slower confined diffusion within mesoscale zones. Super-resolved microscopy showed closed chromatin organized as mesoscale nucleosome-dense domains, confining FOXA2 diffusion locally and enriching its binding. We pinpointed specific histone-interacting disordered regions, distinct from DNA-binding domain, crucial for confined target search kinetics and pioneer activity within closed chromatin. Fusion to other factors enhanced pioneer activity. Kinetic simulations suggested transient confinement could increase target association rate by shortening search time and binding repeatedly. Our findings illuminate how PTFs recognize and exploit closed chromatin organization to access targets, revealing a pivotal aspect of gene regulation.

molecular biology↗

bioRxiv2021

Dynamics of CTCF and cohesin mediated chromatin looping revealed by live-cell imaging

Animal genomes are folded into loops and topologically associating domains (TADs) by CTCF and cohesin, but whether these loops are stable or dynamic is unknown. Here, we directly visualize chromatin looping at the Fbn2 TAD in mouse embryonic stem cells using super-resolution live-cell imaging and quantify looping dynamics by Bayesian inference. Our results are consistent with cohesin-mediated loop extrusion in cells, and with CTCF both stopping and stabilizing cohesin. Surprisingly, the Fbn2 loop is both rare and dynamic, with a looped fraction of ~3-6.5% and a median loop lifetime of ~10-30 minutes. Instead of a stable loop, our results establish a highly dynamic view of TADs and loops where the Fbn2 TAD exists predominantly in a partially extruded conformation. This dynamic and quantitative view of TADs may facilitate a mechanistic understanding of their functions.

biophysics↗

bioRxiv2021

Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors

Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1 and HIF-2 subunit isoforms recognize the same DNA motif in vitro - the hypoxia response element (HRE) - but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF- subunits and their dimerization partner HIF-1{beta} exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2 specific inhibitor, we found that although the DBD and dimerization domains are important, a major determinant of chromatin binding and diffusion behavior is the AD-containing intrinsically disordered region (IDR). Using Cut&Run and RNA-seq as orthogonal genomic approaches we also confirmed IDR-dependent binding and activation of a specific subset of HIF-target genes. These findings reveal a previously unappreciated role of IDRs in regulating the TF search and binding process that contribute to functional target site selectivity on chromatin.

molecular biology↗

bioRxiv2021

Enhancer-promoter interactions and transcription are maintained upon acute loss of CTCF, cohesin, WAPL, and YY1

It remains unclear why acute depletion of CTCF and cohesin only marginally affects expression of most genes despite substantially perturbing 3D genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling to find that enhancer-promoter (E-P) interactions are largely insensitive to acute (3-hour) depletion of CTCF, cohesin, and WAPL. YY1 has been proposed to be a structural regulator of E-P loops, but acute YY1 depletion also had minimal effects on E-P loops, transcription, and 3D genome folding. Strikingly, live-cell single-molecule imaging revealed that cohesin depletion reduced transcription factor binding to chromatin. Thus, although neither CTCF, cohesin, WAPL, nor YY1 are required for the short-term maintenance of most E-P interactions and gene expression, we propose that cohesin may serve as a "transcription factor binding platform" that facilitates transcription factor binding to chromatin.

molecular biology↗

bioRxiv2021

Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes

Early blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here, we identified three major 2 cell (2C) specific endogenous retroviruses (ERVs) as the molecular hallmark of the bi-potential plasticity. Using the LTRs of all three 2C-ERVs, we identified Klf5 as their major upstream regulator. Klf5 is essential for bi-potential cell fate: a single Klf5-overexpressing ESC generated terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces both ICM and TE specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. Altogether, the 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.

developmental 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↗

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

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↗

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↗