Biology subjects

Farnung, L.

Publications and source records attributed to Farnung, L..

16 recordsLinked to original sources

bioRxiv2026

ssDNA hand-off from RPA to Polθ drives microhomology-mediated end-joining

DNA polymerase theta-mediated end joining (TMEJ) is the primary mechanism of double strand break repair in mitosis and becomes essential in BRCA-deficient cells. RPA, the ssDNA-binding protein that coats resected DNA ends, has long been thought to inhibit this pathway by blocking the initial microhomology search in TMEJ. Here we find instead that RPA plays an essential activating role in TMEJ. Structural, biochemical, and genetic analyses support a model in which Pol{theta} binds directly to RPA at resected DNA ends and then competes with RPA for ssDNA. This releases ssDNA into the Pol{theta} helicase domain. We propose that threading two DNA ends through the Pol{theta} dimer in this manner initiates the search for microhomology. Together, our findings establish a detailed mechanism of how proteins can harness RPA to access ssDNA for critical genome maintenance transactions.

biochemistry↗

bioRxiv2026

A Modular Framework for Automated Segmentation and Analysis of AFM Imaging of Chromatin Organization

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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/708946v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1225883org.highwire.dtl.DTLVardef@1e1041dorg.highwire.dtl.DTLVardef@1d53d18org.highwire.dtl.DTLVardef@9e0a2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

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↗

bioRxiv2025

Catalytic pocket of Clr4 (Suv39h) methyltransfer-ase serves as a substrate receptor for Cullin 4-dependent histone H3 ubiquitination

Histone H3 lysine 9 (H3K9) methylation must be regulated to prevent inappropriate heterochromatin formation. Regulation of the conserved fission yeast H3K9 methyltransferase Clr4 (Suv39h) involves an automethylation-induced conformational switch and interaction of its catalytic SET domain with mono-ubiquitinated histone H3 lysine 14 (H3K14ub), a modification catalyzed by the Cul4 subunit of the CLRC complex. Using reconstituted CLRC, we show that Clr4 catalytic pocket serves as a substrate receptor for Cul4-dependent H3K14 ubiquitination. H3K14ub activates Clr4 to catalyze cis methylation of H3K9 on the same histone tail, while Clr4 auto-methylation enables H3K14ub-bound Clr4 to methylate H3K9 on an unmodified H3 tail in trans. Crosslinking and structural modeling reveal interactions between Clr4 chromo and SET domains, and between the chromo-domain and H3K14ub, suggesting that the chromodomain reads H3K9me3 and H3K14ub to allosterically regulate Clr4 activity. H3K14 ubiquitination therefore regulates Clr4 by promoting its recruitment and by positioning H3K9 in the active site.

biochemistry↗

bioRxiv2025

Structure and function of IWS1 in transcription elongation

Transcription elongation by RNA polymerase II is a tightly regulated process that requires coordinated interactions between transcription elongation factors. IWS1 (Interacts with SPT6) has been implicated as a core elongation factor, but its molecular role remains unclear. We show that the intrinsically disordered C-terminal region of IWS1 contains short linear motifs (SLiMs) that multivalently engage the elongation machinery. Using cryo-electron microscopy, we map SLiMs in IWS1 that interact with Pol II subunits RPB1, RPB2, and RPB5, as well as elongation factors DSIF, SPT6, and ELOF1. Functional assays demonstrate that distinct IWS1 SLiMs specify IWS1 recruitment and IWS1-dependent transcription stimulation. IWS1 recruitment to the transcription elongation complex depends on association via the RPB1 jaw and binding of downstream DNA. Transcription elongation stimulation requires interactions with the RPB2 lobe and ELOF1. We identify other transcription elongation factors including ELOA and RECQL5 that bind the RPB1 jaw and demonstrate that IWS1 protects the activated transcription elongation complex from RECQL5 inhibition. We also reveal the binding of the histone reader and IWS1 interactor LEDGF to a transcribed downstream nucleosome. Our findings establish IWS1 as a modular scaffold that helps organize the transcription elongation complex, illustrating how disordered regions regulate transcription elongation.

molecular biology↗

bioRxiv2025

Structural basis of human replisome progression into a nucleosome

Epigenetic inheritance requires the transfer of parental histones to newly synthesized DNA during eukaryotic chromosome replication, yet the structural mechanisms underlying replisome engagement with nucleosomes remain unclear. Here we establish an in vitro chromatin replication system and report four cryo-EM structures of the human replisome in complex with a parental nucleosome. The structures capture distinct states of nucleosomal DNA unwrapping and nucleosome integrity during nucleosome disassembly by the encroaching replisome.

molecular biology↗

bioRxiv2024

STK19 positions TFIIH for cell-free transcription-coupled DNA repair

In transcription-coupled repair, stalled RNA polymerase II (Pol II) is recognized by CSB and CRL4CSA, which co-operate with UVSSSA and ELOF1 to recruit TFIIH for nucleotide excision repair (TC-NER). To explore the mechanism of TC-NER, we recapitulated this reaction in vitro. When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4CSA, UVSSA, and ELOF1. Repair also depends on STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9 [A] cryo-electron microscopy structure shows that STK19 joins the TC-NER complex by binding CSA and the RPB1 subunit of Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of Pol II for lesion verification. In summary, our analysis of cell-free TC-NER suggests that STK19 couples RNA polymerase II stalling to downstream repair events.

biochemistry↗

bioRxiv2024

In silico screening identifies SHPRH as a novel nucleosome acidic patch interactor

Nucleosomes are the fundamental unit of eukaryotic chromatin. Diverse factors interact with nucleosomes to modulate chromatin architecture and facilitate DNA repair, replication, transcription, and other cellular processes. An important platform for chromatin binding is the H2A-H2B acidic patch. Here, we used AlphaFold-Multimer to screen over 7000 human proteins for nucleosomal acidic patch binding and identify 41 potential acidic patch binders. We determined the cryo-EM structure of one hit, SHPRH, with the nucleosome at 2.8 [A]. The structure confirms the predicted acidic patch interaction, reveals that the SHPRH ATPase engages a different nucleosomal DNA location than other SF2-type ATPases, and clarifies the roles of SHPRHs domains in nucleosome recognition. Our results illustrate the use of in silico screening as a high throughput method to identify specific interaction types and expands the set of potential acidic patch binding factors. All the screening data is freely available athttps://predictomes.org/view/acidicpatch

molecular 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↗

bioRxiv2020

Structural basis of nucleosome transcription mediated by Chd1 and FACT

Transcription of eukaryotic protein-coding genes requires passage of RNA polymerase II (Pol II) through nucleosomes. Efficient Pol II passage through nucleosomes depends on the chromatin remodelling factor Chd11 and the histone chaperone FACT2. How Chd1 and FACT mediate Pol II passage through nucleosomes remains unclear. Here we first show that Chd1 and FACT cooperate with the elongation factors Spt4/5 and TFIIS to facilitate Pol II transcription through a nucleosome in a defined biochemical system. We then determine cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II-Spt4/5-nucleosome complexes with bound Chd1 or FACT at 2.9 [A] and 3.1 [A] resolution, respectively. In the first structure, transcribing Pol II has partially unwrapped nucleosomal DNA and exposed the proximal histone H2A/H2B dimer, which is bound by the acidic N-terminal region of Spt5 (Spt5N). The inhibitory DNA-binding region of Chd1 is released3 and the Chd1 translocase adopts an activated state that is poised to pump DNA towards Pol II. In the second structure, transcribing Pol II has generated a partially unravelled nucleosome that binds FACT in a manner that excludes Chd1 and Spt5N. These results suggest a dynamic model of Pol II passage through a nucleosome. In the model, Pol II enters the nucleosome4, activates Chd1 by releasing its DNA-binding region, and thereby stimulates its own progression. Pol II progression then enables FACT binding, liberates Chd1 and Spt5N, and eventually displaces a complex of FACT with histones that is transferred to upstream DNA.

biochemistry↗

bioRxiv2020

Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients1-4. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-25-7. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls6,8. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3-exonuclease that recognizes mismatches9,10. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

biochemistry↗

bioRxiv2020

Structure of replicating SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here we present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in its replicating form. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the RNA duplex as it exits. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged sliding poles that may enable processive replication of the long coronavirus genome. Our results will allow for a detailed analysis of the inhibitory mechanisms used by antivirals such as remdesivir, which is currently in clinical trials for the treatment of coronavirus disease 2019 (COVID-19).

biophysics↗

bioRxiv2020

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the ruler that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

biochemistry↗

bioRxiv2019

Nucleosome-CHD4 chromatin remodeller structure explains human disease mutations

Chromatin remodelling plays important roles in gene regulation during development, differentiation and in disease. The chromatin remodelling enzyme CHD4 is a component of the NuRD and ChAHP complexes that are involved in gene repression. Here we report the cryo-electron microscopy (cryo-EM) structure of Homo sapiens CHD4 engaged with a nucleosome core particle in the presence of the non-hydrolysable ATP analogue AMP-PNP at an overall resolution of 3.1 [A]. The ATPase motor of CHD4 binds and distorts nucleosomal DNA at super-helical location (SHL) +2, supporting the twist defect model of chromatin remodelling. CHD4 does not induce unwrapping of terminal DNA, in contrast to its homologue Chd1, which functions in gene activation. Our results also rationalize the effect of CHD4 mutations that are associated with cancer or the intellectual disability disorder Sifrim-Hitz-Weiss syndrome.

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↗