Seychelle M. Vos

Cyclin-dependent kinases (CDK) dynamically regulate RNA Polymerase (Pol) II during each stage of transcription. Phosphorylation of RNA Pol II and associated transcription factors by CDKs enables stage-specific exchange of factors. CDK9 and a cyclin partner (CYCT1, T2 or K) comprise the Positive Transcription Elongation Factor-b (P-TEFb) complex, which is responsible for releasing RNA Pol II from promoter proximal pausing into processive elongation. P-TEFb activity is stimulated by bromodomain-containing Protein 4 (BRD4); however, the structural basis for BRD4 association with P-TEFb has remained unclear. Using cryogenic-electron microscopy, AlphaFold modeling, and biochemical probing, we show that the P-TEFb interacting domain of BRD4 (BRD4-PID) interacts with P-TEFb via the C-lobe of CDK9 and the second cyclin domain of CYCT1. The BRD4 binding surface on CDK9 is also used by super elongation complex component AFF4. Our analysis indicates that BRD4 may stimulate P-TEFb activity by remodeling the kinase αC helix relative to previous structures. Finally, P-TEFb activity is restricted when bound to the 7SK RNP complex. We show that in addition to BRD4-PID, the AFF4 N-terminus can release P-TEFb from the 7SK RNP complex. Together, these findings define how P-TEFb activity is regulated by its incorporation into distinct complexes. SIGNIFICANCE STATEMENT: The exquisite control of gene expression is required for proper development and cellular response to environmental stress. Positive transcription elongation factor b (P-TEFb) is required for the phosphorylation of RNA Pol II and leads to the coordinated release of RNA Pol II from pause sites near the promoter. P-TEFb is bound to bromo-domain containing protein 4 (BRD4), which stimulates P-TEFb activity; however, it is unknown how BRD4 associates with P-TEFb. Here we report a cryo-EM structure of P-TEFb bound to BRD4, revealing how BRD4 interacts with P-TEFb. Comparison of our structure with previously reported P-TEFb structures shows how P-TEFb is regulated by different transcription factor binding partners.

Chromatin organization underlies essential genome functions, but its nanoscale organization remains challenging to capture and quantify with precision. Atomic force microscopy (AFM) offers direct structural readouts of DNA and chromatin, yet translating these rich images into reproducible biological metrics has been limited by the lack of standardized, scalable analysis tools. Here we present DNAsight, an automated analysis framework that integrates machine learning-based segmentation with modular, base-pair-calibrated quantification of DNA spatial organization, looping, nucleosome spacing, and protein clustering. Applied across diverse chromatin-associated proteins, DNAsight reveals protein-specific organizational signatures, including topology-dependent compaction by integration host factor, condition-dependent changes in loop-like DNA structures in cohesin–CTCF–precocious dissociation of sisters 5A reactions, and promoter-driven multimerization of GAGA factor clusters. The framework further enables direct extraction of nucleosome spacing distributions from raw AFM images, providing a label-free route to investigate chromatin fiber architecture. Together, these advances establish DNAsight as a generalizable and scalable approach for converting AFM measurements into quantitative insights into the physical principles of chromatin organization.

Metazoan RNA polymerase II is prone to pause in the promoter-proximal region of genes. This pausing involves DRB Sensitivity-Inducing Factor (DSIF) and Negative Elongation Factor (NELF). DSIF and NELF, however, are insufficient to fully reconstitute the degree of promoter-proximal pausing observed in cells. Their negative effects are counteracted by transcription initiation factor TFIIF or by physiological nucleotide concentrations, indicating an additional factor is required. Here, we report that Elongation Factor Homolog 1 (ELOF1) is this missing factor. ELOF1 is enriched in the promoter-proximal region of genes, and its rapid degradation reduces pause duration in cells. In reconstituted assays, ELOF1 potently enhances pausing induced by DSIF and NELF at physiological nucleotide concentrations. Cryo-EM structures reveal that DSIF-NELF-ELOF1 sterically clashes with the position of TFIIF on RNA polymerase II. Accordingly, RNA polymerase II-DSIF-NELF-ELOF1, but not RNA polymerase II-DSIF-NELF, counteracts the positive effects of TFIIF. Our results establish ELOF1 as a core component of promoter-proximal paused RNA polymerase II.

Chromatin organization underlies essential genome functions, but its nanoscale organization remains challenging to capture and quantify with precision. Atomic force microscopy (AFM) offers direct structural readouts of DNA and chromatin, yet translating these rich images into reproducible biological metrics has been limited by the lack of standardized, scalable analysis tools. Here we present DNAsight, an automated analysis framework that integrates machine learning (ML)-based segmentation with modular, base-pair-calibrated quantification of DNA spatial organization, looping, nucleosome spacing, and protein clustering. Applied across diverse chromatin-associated proteins, DNAsight reveals protein-specific organizational signatures, including topology-dependent compaction by integration host factor (IHF), condition-dependent changes in loop-like DNA structures in cohesin-CTCF-precocious dissociation of sisters 5A (PDS5A) reactions, and promoter-driven multimerization of GAGA factor (GAF) clusters. The framework further enables direct extraction of nucleosome spacing distributions from raw AFM images, providing a label-free route to investigate chromatin fiber architecture. Together, these advances establish DNAsight as a generalizable and scalable approach for converting AFM measurements into quantitative insights into the physical principles of chromatin organization.

Promoter proximal pausing by RNA polymerase II is critical for regulating gene expression in multicellular eukaryotes. How nucleic acid sequence and protein factors contribute to pausing remains incompletely understood. We developed Gene-specific Analysis of Transcriptional Output (GATO)-seq, which for the first time enables massively parallel, temporally resolved, reconstituted transcription in an assay that uses direct RNA sequencing to map 3′ends of nascent transcripts from a library of human genes. GATO-seq identified a “super pause” sequence that potently induces RNA polymerase II pausing and is not relieved by rescue factor Transcription Factor (TF) IIS. Cryogenic-electron microscopy (cryo-EM) structures of RNA polymerase II on the super pause sequence reveal a previously unobserved, reversible single-nucleotide backtracked state (“sidetracked”), stabilized by a threonine-lined pocket that limits further backtracking. We introduce a powerful in vitro technique that can be employed to study transcription regulation and through its use show that nucleic acid sequence encodes pausing propensity and traps sequence specific offline states, linking sequence to pausing control.

Eukaryotic DNA is organized across multiple scales to support genome compaction, appropriate gene expression, and DNA recombination. A central player in these roles is the CCCTC binding factor (CTCF), which defines specific chromatin loop structures and insulates enhancer elements from promoters. Chromatin is organized in a distinct pattern around CTCF-bound sites, however, the role of this patterning remains unclear. Here, we report cryo-electron microscopy structures of reconstituted CTCF-nucleosome complexes, revealing that CTCF dimerization promotes the oligomerization of nucleosomes into defined higher-order assemblies involving specific histone-histone and CTCF-CTCF interactions. Notably, CTCF does not oligomerize efficiently on non-chromatinized DNA substrates. Disruption of CTCF-CTCF interaction interfaces in cells results in a marked decrease in chromatin looping and impairs cellular differentiation. These results indicate that chromatin structure at CTCF sites plays an important role in supporting higher-order interactions between distal regions of the genome and that these interactions are important for supporting cell-type-specific gene expression.

CCCTC-binding transcription factor (CTCF) and the cohesin complex shape the genome into loops and topologically associating domains (TADs), yet the mechanisms linking CTCF dynamic behavior to its function as a cohesin barrier remain unclear. Using integrated experimental and computational approaches, we demonstrate that individual CTCF-DNA complexes are intrinsically mobile over distances and timescales compatible with cohesin capture during loop extrusion, driven by the dynamic rearrangements of zinc finger (ZF) domains. Genome-wide single-molecule accessibility and sequencing analyses further reveal that these dynamics occur in cells. This intrinsic flexibility is modulated by DNA sequence, methylation, and nucleosome positioning, and enables a previously uncharacterized single-stranded DNA binding state. Through biochemical dissection of cohesin components, we find that a negative regulator of loop extrusion, PDS5, remodels ZF dynamics and imparts exceptional mechanical stability to CTCF on DNA. Our findings reveal that the conformational dynamics of a transcription factor constitute a fundamental regulatory property that bridges base pair-scale molecular motions to megabase-scale genome organization, offering a kinetic explanation for how CTCF establishes and regulates TAD boundaries.

Ultraviolet (UV) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA- and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA–protein, DNA–protein, and nucleotide–protein complexes in vitro and in vivo. We introduce a specialized nucleotide–protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold, and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid–protein complexes and adds spatial information to the described RNA-binding properties of enzymes, for which crosslinking sites are often observed close to their cofactor-binding domains. In vivo UV and chemical XL-MS data from E. coli cells analysed by NuXL establish a comprehensive nucleic acid–protein crosslink inventory with crosslink sites at amino acid level for >1500 proteins. Our new workflow combined with the dedicated NuXL search engine identified RNA crosslinks that cover most RNA-binding proteins, with DNA and RNA crosslinks detected in transcriptional repressors and activators.

During early transcription, RNA polymerase II (RNAPII) undergoes a series of structural transitions controlled by cyclin-dependent kinases. How protein ubiquitylation and proteasomal degradation control the function of RNAPII is less well understood. Here we show that the deubiquitinating enzyme USP11 forms a complex with TCEAL1, a member of the TFIIS (TCEA)-like protein family. TCEAL1 shares sequence homology with the RNAPII interaction domain of the elongation factor TFIIS (which controls the fate of backtracked RNAPII) and competes with TFIIS for binding to core promoters. USP11 protects TCEAL1 from proteasomal degradation, and TCEAL1 recruits USP11 to RNAPII. Both USP11 and TCEAL1 promote transcription elongation and maintain expression of RPB8, an essential subunit of all three nuclear RNA polymerases. In neuroblastoma, USP11- and TCEAL1-dependent genes define a gene expression program that is characteristic for mesenchymal tumors, which are described as able to escape from many treatments, suggesting that the USP11/TCEAL1 complex promotes transcription elongation to support a critical oncogenic gene expression program.