Biology subjects

Ruthenburg, A. J.

Publications and source records attributed to Ruthenburg, A. J..

13 recordsLinked to original sources

bioRxiv2026

Large RNA polymerase II condensates are promoter-centric assemblies associated with early stages of transcription at all expressed genes

Transcriptional condensates concentrate the machinery required for RNA polymerase II mediated transcription. These structures range from numerous small, short-lived species, to a handful of larger, stable assemblages. Large condensates have been implicated in driving potent transcription of several super-enhancer regulated genes, yet the underlying mechanisms and the range of their client genes remain unclear. Here, we developed a biochemical approach which combines density gradient centrifugation and affinity purification to partially purify large transcriptional condensates from nuclei, allowing systematic characterization of their nucleic acid components. We find that transcriptional condensate isolates engage thousands of gene promoters and harbor the nascent transcriptome, but do not stably co-purify with distal enhancers. Binding patterns of RNA polymerase II within condensates suggest these structures could facilitate promoter escape and promoter-proximal pause release. Together, our work supports a promoter-centric condensate organization and paves the way towards understanding the functional link between condensate architecture and nascent transcription.

molecular biology↗

bioRxiv2026

Promiscuous RNA binding by WDR5 remodels the KMT2A (MLL1) histone methyltransferase complex to an inactive state

Chromatin-modifying complexes are critical in gene regulation, yet their proposed interactions with RNA remain poorly understood. Here, we show that WDR5, an essential subunit of the MLL1 (KMT2A) histone methyltransferase complex, binds RNA with high affinity but without sequence specificity. Using a stringent approach, we demonstrate that WDR5 directly engages a diverse pool of RNAs in cells, predominantly as a function of RNA abundance rather than specific motifs. Equilibrium binding assays further show that RNA length, rather than sequence, dictates WDR5 affinity. We map multiple RNA-binding regions on WDR5, some of which overlap with surfaces used in MLL1 complex subunit interfaces. Strikingly, we find that RNA binding disrupts the MLL1 complex by competitively displacing WDR5 from these critical protein-protein interactions, leading to a marked inhibition of MLL1 catalytic activity. This regulatory mechanism provides a layer of control over histone methylation, potentially integrating transcriptional activity with chromatin state.

molecular biology↗

bioRxiv2026

Bound for the nucleus: defining the molecular principles of cargo selection by importin 9

Active nuclear transport of proteins enables essential nuclear processes, such as genome packaging, transcription, splicing, ribosome biogenesis, and DNA repair. To facilitate proper sorting between cytoplasmic and nuclear compartments, the importin class of chaperone proteins transports hundreds of proteins through hydrophobic nuclear pores. While some importins recognize cargos via classical nuclear localization signals (cNLSs), there is not a comprehensive understanding of binding specificity determinants for those that appear to recognize other cargo features. Here we subject one such importin, IPO9, which is known to import H2A-H2B, TFIIB, actin, and the proteasome into the nucleus without cNLS binding, to a detailed analysis of its full set of cargos and the structural elements of IPO9 that confer their recognition. Through cytoplasmic immunoprecipitation followed by mass spectrometry (IP-MS), we stringently and reproducibly identify 79 bona fide IPO9-bound cargos, including 20 previously validated cargos. With this comprehensive cargo list, we find that IPO9 does not appear to use cNLSs, nor any other linear peptide motif, to identify and bind cargos. Unbiased oxidative footprinting of extracted IPO9*cargo complexes reveals that both the inner cavity of IPO9 and the unstructured loops protruding from the main body of the importin are protected by bound cargo, indicative of cargo competition for limiting IPO9 capacity. Guided by these data and evolutionary and phosphoproteomics insights, we employ a systematic IP-MS approach with loop perturbations to define how each element contributes to selective binding of subsets of the whole cargo cohort. These data suggest the H8 and H18-19 loops both specifically mediate IPO9 cargo-recognition by favorable enthalpic contacts. Additionally, these loops, as well as H7, appear to preclude binding of a secondary set of potential cargo to IPO9 which may normally be repelled by them or outcompeted by cognate cargo binding for which the loops provide attractive contacts. We define the continuum of cargo-release factor RanGTP sensitivity for our full cargo set, noting orders of magnitude range of sensitivity that argues that additional release factors may be necessary for efficient unloading in the nucleus. These experiments provide structure-function insight into importin binding specificity dictated by structural elements that recruit and/or restrict protein-protein interactions. Our approach of targeted mutations in cellular contexts coupled to quantitative proteomics affords a thorough biochemical dissection of the discrimination principles undergirding this unique molecular recognition problem of numerous-yet-specific binding events between importins and their many distinct cargos.

molecular biology↗

bioRxiv2025

A revised model of nuclear actin import: Importin 9 competes with cofilin, profilin, and RanGTP for actin binding

While predominantly studied in cytoplasmic contexts, actin plays critical roles in the nucleus, regulating chromatin accessibility and remodeling, transcription, and DNA damage repair. Cell- based studies have contributed to a widely accepted model in which the import factor Importin 9 (IPO9) acts in concert with the actin filament-severing protein cofilin to transport actin into the nucleus. The classical nuclear localization signal on cofilin is thought to anchor IPO9 to cofilin- bound actin monomers, driving the formation of an import-competent tripartite actin*cofilin*IPO9 complex. In striking contradiction to this established model of actin import, we demonstrate that IPO9 directly binds to monomeric actin with mid-nanomolar affinity and, rather than promoting IPO9*actin complex formation, cofilin competitively inhibits binding of IPO9 to actin. We further report competitive binding for monomeric actin between IPO9 and the canonical actin monomer- sequestering protein, profilin. As cofilin and profilin are both capable of binding actin monomers at the barbed face, our results are consistent with a model in which IPO9 binds an overlapping portion of this interface. In further support, we demonstrate that IPO9 modestly occludes the barbed face of actin monomers, decreasing the rate of filament formation, and exhibits minimal filamentous actin binding. Finally, we identify unexpected affinity between the nuclear import release factor RanGTP and monomeric actin; however, a tripartite IPO9*actin*RanGTP complex does not form. The competitive interactions observed between IPO9 and cytoplasmic actin- binding proteins suggest dynamically coupled equilibria mediate the nuclear transport of actin monomers.

biochemistry↗

bioRxiv2024

The N-terminus of YY1 regulates DNA and RNA binding affinity for both the zinc-fingers and an unexpected nucleic acid binding domain

Transcription factors (TFs) play central roles in dictating cellular identity and function by regulating gene expression programs. Beyond their well-folded DNA binding domains (DBDs) which recognize cognate DNA elements in the genome, TFs are enriched for intrinsically disordered regions (IDRs), which have a host of proposed functions including facilitating protein-protein interactions, aiding in binding site search, and binding RNA. Defining intrinsic regulatory properties of TFs requires further mechanistic investigation. We chose to investigate the DNA and RNA binding properties of Yin Yang 1 (YY1), a ubiquitously expressed TF directly involved in transcriptional activation, repression and genome architecture. Through systematic in vitro nucleic acid binding experiments we resolve conflicting literature defining the RNA binding interface of YY1, demonstrating that there are two RNA binding domains within YY1: its canonical 4 zinc finger DBD and a previously unannotated nucleic acid binding domain, which we term the REPO-NAB. Furthermore, we discover surprising autoinhibitory properties that the N-terminus of the protein imparts on each of these binding domains. Our results provide a new example of IDR-mediated regulation within TFs and enables future mechanistically precise functional investigations.

molecular biology↗

bioRxiv2023

The Kinase Module of the Mediator regulates cell cycle re-entry and transcriptional response following DNA damage

The Cdk8 kinase module (CKM) is a non-obligate and dissociable subcomplex of Mediator of transcription, a key regulator of RNA polymerase II (RNAPII). Through a genetic screen in yeast, we discovered a surprising role for Mediator CKM in the DNA damage response (DDR) and mitotic re-entry. Remarkably, we find that a single DNA break is sufficient for CKM-dependent global transcriptional attenuation. Upon DDR activation, the kinase activity of CKM antagonizes RNAPII binding to core Mediator, thereby reducing the transcriptionally-engaged RNAPII pool. This transcriptional attenuation is essential for DDR inactivation and limits the spreading of {psi}-H2AX into gene bodies. Furthermore, CKM localizes to DNA breaks to impede RNAPII binding. Importantly, we demonstrate that the role of CKM on DDR and transcriptional attenuation is conserved from yeast to mammals, establishing a multifaceted and essential function for CKM in transcriptional regulation of DNA-damage response.

cell biology↗

bioRxiv2023

Specificity Guides Interpretation: On H3K4 Methylation at Enhancers and Broad Promoters

In 2018, we used internally calibrated chromatin immunoprecipitation (ICeChIP) to find that many of the most commonly used antibodies against H3K4 methylforms had significant off-target binding, which compromised the findings of at least eight literature paradigms that used these antibodies for ChIP-seq (Shah et al., 2018). In many cases, we were able to recapitulate the prior findings in K562 cells with the original, low-quality antibody, only to find that the models did not hold up to scrutiny with highly specific reagents and quantitative calibration. In a recent preprint originally prepared as a Letter to the Editor of Molecular Cell, though they agree with our overarching conclusions, Pekowska and colleagues take issue with analyses presented for two relatively minor points of the paper (Pekowska et al., 2023). We are puzzled by the assertion that these two points constitute the "bulk" of our findings, nor is it clear which components of our "analytical design" they find problematic. We feel their critique, however mild, is misguided.

molecular biology↗

bioRxiv2022

Merging short and stranded long reads improves transcript assembly

Long-read RNA sequencing has arisen as a counterpart to short-read sequencing, with the potential to capture full-length isoforms, albeit at the cost of lower depth. Yet this potential is not fully realized due to inherent limitations of current long-read assembly methods and underdeveloped approaches to integrate short-read data. Here, we critically compare the existing methods and develop a new integrative approach to characterize a particularly challenging pool of low-abundance long noncoding RNA (lncRNA) transcripts from short- and long-read sequencing in two distinct cell lines. Our analysis reveals severe limitations in each of the sequencing platforms. For short-read assemblies, coverage declines at transcript termini resulting in ambiguous ends, and uneven low-coverage results in segmentation of a single transcript into multiple transcripts. Conversely, long-read transcript assembly lacks strand-of-origin information and depth, culminating in erroneous assembly and quantitation of transcripts. We also discover a cDNA synthesis artifact in long-read datasets that markedly impacts the identity and quantitation of assembled transcripts. Towards remediating these problems, we develop a computational pipeline to "strand" long-read cDNA libraries that rectifies inaccurate mapping and assembly of long-read transcripts. Leveraging the strengths of each platform and our computational stranding, we present and benchmark a hybrid assembly approach that drastically increases the sensitivity and accuracy of full-length transcript assembly on the correct strand and improves detection of biological features of the transcriptome. When applied to a challenging set of under-annotated and cell-type variable lncRNA, our method resolves the segmentation problem of short-read sequencing and the depth problem of long-read sequencing, resulting in the assembly of coherent transcripts with precise 5 and 3 ends. Our workflow can be applied to existing datasets for superior demarcation of transcript ends and refined isoform structure, which can enable better differential gene expression analyses and molecular manipulations of transcripts.

bioinformatics↗

bioRxiv2021

The cardiac lncRNA Chantico directly regulates Cxcl1 chemokine transcription

Withdrawal StatementThe corresponding author has withdrawn this preprint owing to inability to reproduce some of the data, instances of inappropriate data exclusion, and loss of much of the primary experimental records/data. Specifically, Figures 2B,E,H; 3; 4; 5A,B,D; 6; S2A,C; S4A; S5; and S6A,B and attendant text contain analyses for which the primary record and/or raw data no longer exist; the analyses, where still available, suffer from inappropriate data exclusion and thus should not be construed to be an accurate reflection of the experiments. Attempts by others in the lab to repeat several of the experiments in these indicated panels have failed reproduce the presented effects, despite showing much greater precision. Therefore the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

molecular biology↗

bioRxiv2021

Profiling of Nascent Lariat Intermediates Reveals Key Genetic Determinants of the Timing of Human Co-transcriptional Splicing

As splicing is intimately coupled with transcription, understanding splicing mechanisms requires an understanding of splicing timing, which is currently limited. Here, we developed CoLa-seq (co-transcriptional lariat sequencing), a genomic assay that reports splicing timing relative to transcription through analysis of nascent lariat intermediates. In human cells, we mapped 165,282 branch points and characterized splicing timing for over 70,000 introns. Splicing timing varies dramatically across introns, with regulated introns splicing later than constitutive introns. Machine learning-based modeling revealed genetic elements predictive of splicing timing, notably the polypyrimidine tract, intron length, and regional GC content, which illustrate the significance of the broader genomic context of an intron and the impact of co-transcriptional splicing. The importance of the splicing factor U2AF in early splicing rationalizes surprising observations that most introns can splice independent of exon definition. Together, these findings establish a critical framework for investigating the mechanisms and regulation of co-transcriptional splicing. HighlightsO_LICoLa-seq enables cell-type specific, genome-wide branch point annotation with unprecedented efficiency. C_LIO_LICoLa-seq captures co-transcriptional splicing for tens of thousands of introns and reveals splicing timing varies dramatically across introns. C_LIO_LIModeling uncovers key genetic determinants of splicing timing, most notably regional GC content, intron length, and the polypyrimidine tract, the binding site for U2AF2. C_LIO_LIEarly splicing precedes transcription of a downstream 5 SS and in some cases accessibility of the upstream 3 SS, precluding exon definition. C_LI

molecular biology↗

bioRxiv2021

Re-evaluating the role of nucleosomal bivalency in early development

Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome1; posttranslational modifications of these histone proteins influence the activity of the associated genomic regions to regulate cell identity2-4. Traditionally, trimethylation of histone 3-lysine 4 (H3K4me3) is associated with transcriptional initiation5-10, whereas trimethylation of H3K27 (H3K27me3) is considered transcriptionally repressive11-15. The apparent juxtaposition of these opposing marks, termed "bivalent domains"16-18, was proposed to specifically demarcate of small set transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status19-22. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure H3K4me3/H3K27me3 bivalency genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model16,18: from naive mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread, does not resolve with differentiation, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against interpreting bivalent domains as specific markers of developmentally poised genes.

molecular biology↗

bioRxiv2020

Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

MLL-rearranged leukemia depends on H3K79 methylation. Depletion of this transcriptionally-activating mark by DOT1L deletion or high concentrations of the inhibitor pinometostat downregulates HOXA9 and MEIS1, and consequently reduces leukemia survival. Yet some MLL-rearranged leukemias are inexplicably susceptible to low-dose pinometostat, far below concentrations that downregulate this canonical proliferation pathway. In this context, we define alternative proliferation pathways that more directly derive from H3K79me2 loss. By ICeChIP-seq, H3K79me2 is markedly depleted at pinometostat-downregulated and MLL-fusion targets, with paradoxical increases of H3K4me3 and loss of H3K27me3. Although downregulation of polycomb components accounts for some of the proliferation defect, transcriptional downregulation of FLT3 is the major pathway. Loss-of-FLT3-function recapitulates the cytotoxicity and gene expression consequences of low-dose pinometostat, whereas overexpression of constitutively active STAT5A, a target of FLT3-ITD-signalling, largely rescues these defects. This pathway also depends on MLL1, indicating combinations of DOT1L, MLL1 and FLT3 inhibitors should be explored for treating FLT3-mutant leukemia.

cancer biology↗

bioRxiv2019

WDR76 promotes MLL-rearranged leukemia via selective recognition of 5-hydroxymethylcytosine in DNA

Although rare, the distribution of the 5-hydroxymethylcytosine (hmC) modification in mammalian DNA is tissue- and gene-specific, yet distinct from its transcriptionally-repressive methylcytosine (mC) precursor, suggesting unique signaling potential. To examine this possibility, we fractionated mammalian brain extracts to discover binding partners specific for oxidized states of mC. We demonstrate that one such factor, WDR76, is a highly hmC-specific binding protein that modulates gene expression within chromosomal regions enriched in hmC where it binds. We demonstrate direct transcriptional activation of several target genes in mouse embryonic stem cells as a function of hmC levels and contingent upon WDR76. In human cell lines and mouse models, WDR76 recruitment by hmC is critical for the initiation and maintenance of MLL-rearranged leukemias. Beyond its canonical role as an intermediate in mC remediation, we show that hmC can be an epigenetic mark whose recognition drives leukemogenesis, portending analogous signaling pathways for other rare DNA modifications.

biochemistry↗