Biology subjects

Vos, S. M.

Publications and source records attributed to Vos, S. M..

12 recordsLinked to original sources

bioRxiv2026

Structural basis for P-TEFb association with BRD4

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 C 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.

biochemistry↗

bioRxiv2026

A sequence-encoded promoter proximal super pause stabilizes an offline RNA polymerase II state

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.

biochemistry↗

bioRxiv2026

Structural basis for CTCF-mediated chromatin organization

Eukaryotic DNA is organized across multiple scales to support genome compaction, appropriate gene expression, and DNA recombination. A central player in these processes 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 identified CTCF interaction interfaces in cells results in a marked decrease in chromatin looping and functionally 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.

molecular biology↗

bioRxiv2025

Ultrafast CTCF dynamics control cohesin barrier function

Genomes are organized into chromatin loops through cohesin-mediated extrusion, with CTCF acting as a polar boundary element. As cohesin approaches CTCF at kilobase-per-second speeds, it must rapidly choose whether to stall or bypass. How CTCF encodes this probabilistic decision within a brief encounter window has remained unclear. Here we show that CTCF governs this probabilistic outcome by rapidly sampling a dynamic ensemble of conformations generated by spontaneous rearrangements of its DNA-binding zinc fingers. This ensemble is tuned by DNA sequence, CpG methylation, nearby nucleosomes, and the cohesin regulator PDS5A before cohesin engagement. Upon cohesin binding, PDS5A enhances loop-anchor mechanical stability, reinforcing orientation-dependent boundaries. These findings establish conformational ensemble tuning, rather than static occupancy, as a regulatory principle linking base pair-scale motions to megabase-scale genome organization. One sentence summaryChromatin boundary function is governed not by CTCF occupancy alone, but by a tunable ensemble of DNA-bound conformations that probabilistically gates cohesin capture.

biophysics↗

bioRxiv2024

RNA-mediated MYC multimerization suppresses innate immune signaling

In response to perturbed transcription elongation, the MYC oncoprotein multimerizes and undergoes a phase transition; the underlying mechanisms and their function are unknown. Here, we show that MYC re-localizes from its canonical location on DNA to RNA in response to the accumulation of intronic RNA. MYC binds RNA directly, which enhances its multimerization. MYC multimers concentrate the nuclear exosome, a 3-5 RNA exonuclease, and its targeting complexes around double-stranded RNA and R-loops, and promote exosome recruitment to R-loops. RNA binding of MYC suppresses activation of the innate immune kinase TBK1. Upon MYC depletion, intron-derived dsRNAs, including RNA derived from repetitive elements and small nucleolar RNAs, accumulate on TLR3, a pattern recognition receptor that activates TBK1. In MYC-depleted cells, TLR3-bound snoRNAs carry aberrant 3-ends, indicating defective exosomal processing. Our data show that the phase transition of MYC is a RNA-driven stress response that suppresses the accumulation of immunogenic RNAs.

molecular biology↗

bioRxiv2024

The structural basis for RNA slicing by human Argonaute2

Argonaute (AGO) proteins associate with guide RNAs to form complexes that slice transcripts that pair to the guide. This slicing drives post-transcriptional gene-silencing pathways that are essential for many eukaryotes and the basis for new clinical therapies. Despite this importance, structural information on eukaryotic AGOs in a fully paired, slicing-competent conformation--hypothesized to be intrinsically unstable--has been lacking. Here we present the cryogenic-electron microscopy structure of a human AGO-guide complex bound to a fully paired target, revealing structural rearrangements that enable this conformation. Critically, the N domain of AGO rotates to allow the RNA full access to the central channel and forms contacts that license rapid slicing. Moreover, a conserved loop in the PIWI domain secures the RNA near the active site to enhance slicing rate and specificity. These results explain how AGO accommodates targets possessing the pairing specificity typically observed in biological and clinical slicing substrates.

molecular biology↗

bioRxiv2024

Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

The eukaryotic microrchidia (MORC) protein family are DNA gyrase, Hsp90, histidine kinase, MutL (GHKL)-type ATPases involved in gene expression regulation and chromatin compaction. The molecular mechanisms underlying these activities are incompletely understood. Here we studied the full-length human MORC2 protein biochemically. We identified a DNA binding site in the C-terminus of the protein, and we observe that this region is heavily phosphorylated in cells. Phosphorylation of MORC2 reduces its affinity for DNA and appears to exclude the protein from the nucleus. We observe that DNA binding by MORC2 reduces its ATPase activity and that MORC2 can topologically entrap multiple DNA substrates between its N-terminal GHKL and C-terminal coiled coil 3 dimerization domains. Finally, we observe that the MORC2 C-terminal DNA binding region is required for gene silencing in cells. Together, our data provide a model to understand how MORC2 engages with DNA substrates to mediate gene silencing.

biochemistry↗

bioRxiv2023

Direct RNA-binding by MYCN mediates feedback from RNA processing to transcription control

The MYCN oncoprotein broadly binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3-5 exoribonuclease complex, suggesting a function in RNA metabolism. Here we show that MYCN forms stable high molecular weight complexes with the exosome and multiple RNA-binding proteins. In cells, MYCN binds to thousands of intronic RNAs; recombinant MYCN directly binds RNA via a short, highly conserved sequence termed MYCBoxI. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. At promoters, MYCN is then replaced by the MNT(MXD6) repressor protein, which inhibits MYCN-dependent transcription. MYCN promotes the degradation of its bound introns via the nuclear exosome targeting (NEXT) complex. Our data demonstrate that MYCN is an RNA-binding protein that regulates nascent transcript turnover and show that competition between its RNA- and DNA-bound states links the dynamics of the MYCN/MAX/MXD network to mRNA processing.

cancer biology↗

bioRxiv2023

Structure of the complete S. cerevisiae Rpd3S-nucleosome complex

Acetylation of histones is a key post-translational modification that guides gene expression regulation. In yeast, the class I histone deacetylase containing Rpd3S complex plays a critical role in the suppression of spurious transcription by removing histone acetylation from actively transcribed genes. The Saccharomyces cerevisiae Rpd3S complex has five subunits (Rpd3, Sin3, Rco1, Eaf3, and Ume1) but its subunit stoichiometry and how the complex engages nucleosomes to achieve substrate specificity remains elusive. Here we report the cryo-EM structure of the complete Rpd3S complex bound to a nucleosome. Sin3 and two copies of subunits Rco1 and Eaf3 encircle the deacetylase subunit Rpd3 and coordinate the binding of Ume1. The Rpd3S complex binds both trimethylated H3 tails at position lysine 36 and makes multiple additional contacts with the nucleo-somal DNA, the H2A-H2B acidic patch, and histone H3. Direct regulation via the Sin3 subunit coordinates binding of the acetylated histone substrate to achieve substrate specificity.

molecular biology↗

bioRxiv2022

Optimized path planning surpasses human efficiency in cryo-EM imaging

Cryo-electron microscopy (cryo-EM) represents a powerful technology for determining atomic models of biological macromolecules(Kuhlbrandt, 2014). Despite this promise, human-guided cryo-EM data collection practices limit the impact of cryo-EM because of a path planning problem: cryo-EM datasets typically represent 2-5% of the total sample area. Here, we address this fundamental problem by formalizing cryo-EM data collection as a path planning optimization from low signal data. Within this framework, we incorporate reinforcement learning (RL) and deep regression to design an algorithm that uses distributed surveying of cryo-EM samples at low magnification to learn optimal cryo-EM data collection policies. Our algorithm - cryoRL - solves the problem of path planning on cryo-EM grids, allowing the algorithm to maximize data quality in a limited time without human intervention. A head-to-head comparison of cryoRL versus human subjects shows that cryoRL performs in the top 10% of test subjects, surpassing the majority of users in collecting high-quality images from the same sample. CryoRL establishes a general framework that will enable human-free cryo-EM data collection to increase the impact of cryo-EM across life sciences research.

biophysics↗

bioRxiv2020

Structure of a transcribing RNA polymerase II-U1 snRNP complex

To initiate co-transcriptional splicing, RNA polymerase II (Pol II) recruits U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent pre-mRNA. Here we report the cryo-EM structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the 5 splice site in pre-mRNA near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5 splice site, leading to formation of an intron loop. Loop formation may facilitate scanning of the nascent pre-mRNA for the 3 splice site and enable prespliceosome assembly and functional pairing of distant intron ends. Our results provide a starting point for a mechanistic analysis of co-transcriptional splicing and the biogenesis of mRNA isoforms during alternative splicing.

molecular biology↗

bioRxiv2018

Structure of transcribing RNA polymerase II-nucleosome complex

Transcription of eukaryotic protein-coding genes requires passage of RNA polymerase II (Pol II) through chromatin. Pol II passage is impaired by nucleosomes and requires elongation factors that help Pol II to efficiently overcome the nucleosomal barrier1-4. How the Pol II machinery transcribes through a nucleosome remains unclear because structural studies have been limited to Pol II elongation complexes formed on DNA templates lacking nucleosomes5. Here we report the cryo-electron microscopy (cryo-EM) structure of transcribing Pol II from the yeast Saccharomyces cerevisiae engaged with a downstream nucleosome core particle (NCP) at an overall resolution of 4.4 [A] with resolutions ranging from 4-6 [A] in Pol II and 6-8 [A] in the NCP. Pol II and the NCP adopt a defined orientation that could not be predicted from modelling. Pol II contacts DNA of the incoming NCP on both sides of the nucleosomal dyad with its domains clamp head and lobe. Comparison of the Pol II-NCP structure to known structures of Pol II complexes reveals that the elongation factors TFIIS, DSIF, NELF, PAF1 complex, and SPT6 can be accommodated on the Pol II surface in the presence of the oriented nucleosome. Further structural comparisons show that the chromatin remodelling enzyme Chd1, which is also required for efficient Pol II passage6,7, could bind the oriented nucleosome with its motor domain. The DNA-binding region of Chd1 must however be released from DNA when Pol II approaches the nucleosome, and based on published data8,9 this is predicted to stimulate Chd1 activity and to facilitate Pol II passage. Our results provide a starting point for a mechanistic analysis of chromatin transcription.

biochemistry↗