Claudia Cattoglio

How intrinsically disordered regions (IDRs) shape chromatin binding and nuclear organization of transcription factors (TFs) remains unclear. We used proximity-assisted photoactivation (PAPA), a single-molecule protein-protein interaction sensor, to investigate how IDRs might influence TF interactions with each other and with chromatin in live cells. We found that the Sp1 DNA binding domain (DBD) interacted poorly with chromatin and did not colocalize with Sp1. Weak interaction of the isolated IDR with full-length Sp1 was enhanced by fusion to various unrelated DBDs. Live imaging of Drosophila polytene chromosomes confirmed that an IDR could confer sharp locus specificity on an otherwise nonspecific DBD. These findings suggest that TF specificity emerges on chromatin when ensembles of diverse, unstructured interactions are scaffolded by transient DNA contacts.

Summary: 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

The human nucleosome acetyltransferase of histone H4 (NuA4)/Tat-interactive protein, 60 kilodalton (TIP60) coactivator complex, a fusion of the yeast switch/sucrose nonfermentable related 1 (SWR1) and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4, H2A, and H2A.Z to regulate gene expression and maintain genome stability. Our cryo–electron microscopy studies show that, within the NuA4/TIP60 complex, the E1A binding protein P400 (EP400) subunit serves as a scaffold holding the different functional modules in specific positions, creating a distinct arrangement of the actin-related protein (ARP) module. EP400 interacts with the transformation/transcription domain-associated protein (TRRAP) subunit by using a footprint that overlaps with that of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.

Loss-of-function mutations inMECP2cause Rett syndrome (RTT), a severe neurological disorder that mainly affects girls. Mutations inMECP2do occur in males occasionally and typically cause severe encephalopathy and premature lethality. Recently, we identified a missense mutation (c.353G>A, p.Gly118Glu [G118E]), which has never been seen before inMECP2, in a young boy who suffered from progressive motor dysfunction and developmental delay. To determine whether this variant caused the clinical symptoms and study its functional consequences, we established two disease models, including human neurons from patient-derived iPSCs and a knock-in mouse line. G118E mutation partially reduces MeCP2 abundance and its DNA binding, and G118E mice manifest RTT-like symptoms seen in the patient, affirming the pathogenicity of this mutation. Using live-cell and single-molecule imaging, we found that G118E mutation alters MeCP2's chromatin interaction properties in live neurons independently of its effect on protein levels. Here we report the generation and characterization of RTT models of a male hypomorphic variant and reveal new insight into the mechanism by which this pathological mutation affects MeCP2's chromatin dynamics. Our ability to quantify protein dynamics in disease models lays the foundation for harnessing high-resolution single-molecule imaging as the next frontier for developing innovative therapies for RTT and other diseases.

The vast majority of biologic-based therapeutics operate within serum, on the cell surface, or within endocytic vesicles, in large part because proteins and nucleic acids fail to efficiently cross cell or endosomal membranes. The impact of biologic-based therapeutics would expand exponentially if proteins and nucleic acids could reliably evade endosomal degradation, escape endosomal vesicles, and remain functional. Using the cell-permeant mini-protein ZF5.3, here we report the efficient nuclear delivery of functional Methyl-CpG-binding-protein 2 (MeCP2), a transcriptional regulator whose mutation causes Rett syndrome (RTT). We report that ZF-tMeCP2, a conjugate of ZF5.3 and MeCP2(Delta aa13-71, 313-484), binds DNA in a methylation-dependent manner in vitro, and reaches the nucleus of model cell lines intact to achieve an average concentration of 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT corepressor complex, selectively represses transcription from methylated promoters, and colocalizes with heterochromatin in mouse primary cortical neurons. We also report that efficient nuclear delivery of ZF-tMeCP2 relies on an endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-tMeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini -protein ZF5.3. Such a strategy could broaden the impact of multiple families of biologic-based therapeutics.

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 co-factors 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-1α and HIF-2α 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-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, another main 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.

Animal genomes are folded into loops and topologically associating domains (TADs) by CTCF and loop-extruding cohesins, but the live dynamics of loop formation and stability remain unknown. Here, we directly visualized chromatin looping at the Fbn2 TAD in mouse embryonic stem cells using super-resolution live-cell imaging and quantified looping dynamics by Bayesian inference. Unexpectedly, the Fbn2 loop was both rare and dynamic, with a looped fraction of approximately 3 to 6.5% and a median loop lifetime of approximately 10 to 30 minutes. Our results establish that the Fbn2 TAD is highly dynamic, and about 92% of the time, cohesin-extruded loops exist within the TAD without bridging both CTCF boundaries. This suggests that single CTCF boundaries, rather than the fully CTCF-CTCF looped state, may be the primary regulators of functional interactions.

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.

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-1α and HIF-2α 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-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α 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.