Biology subjects

Groth, A.

Publications and source records attributed to Groth, A..

13 recordsLinked to original sources

bioRxiv2026

CREPAS: a reproducible nascent chromatin sequencing analysis pipeline for epigenome replication studies

Chromatin-based genomics data are essential for understanding genome regulation and the mechanisms underlying epigenetic memory. Recent methods such as ChOR-seq and SCAR-seq assess histone modifications and chromatin-associated proteins during and after replication, capturing chromatin states that contribute to memory across cell divisions. Current tools for chromatin data analysis lack scalability and reproducibility across computing infrastructures, offer limited parameters, and are applicable only to a few sequencing techniques, ignoring the information from nascent chromatin assays. To address these challenges, we developed CREPAS, a Nextflow pipeline for analyzing nascent and parental chromatin sequencing data, including ChIP-seq, ChOR-seq, SCAR-seq, OK-seq, ATAC-seq, CUT&RUN, and CUT&Tag, and derivative protocols. CREPAS provides an end-to-end solution, from quality control to advanced analyses, including downsampling, peak calling, annotation, and visualization. By harnessing quantitative assays such as qChIP-seq and qChOR-seq, the normalization methods in CREPAS allow to compare the restoration kinetics of individual marks or proteins across replication timepoints. Moreover, the pipeline includes calculations such as fork directionality and partitioning using OK-seq and SCAR-seq data, linking replication dynamics to epigenetic inheritance. CREPAS is a valuable resource that enhances the efficiency and reproducibility of nascent chromatin sequencing data analyses, enabling the study of chromatin replication and propagation of epigenetic states. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/732899v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@11634b8org.highwire.dtl.DTLVardef@df2e07org.highwire.dtl.DTLVardef@46d117org.highwire.dtl.DTLVardef@14950a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

FACT depletion results in a temporal cascade of chromatin disruption preceding transcriptional collapse in stem cells

The histone chaperone FACT is essential for chromatin organization and stem cell identity, yet the sequence of events through which FACT maintains chromatin integrity remains unclear. We generate a high-resolution temporal map of events leading to chromatin destabilization and cell identity loss in murine embryonic stem cells following dTAG-mediated depletion of FACT. Loss of FACT initiates a rapid cascade, with nucleosome phasing disruption and reduced H3K4me3 beginning within 10 minutes in a transcription-dependent manner. This architectural destabilization enables pluripotency transcription factors to ectopically occupy gene bodies. FACT loss results in RNA Polymerase II accumulation within 30 minutes and altered H3K36me3 deposition preceding a global transcriptional collapse at 2 hours of FACT depletion. FACT restoration fails to rescue these defects, demonstrating they are largely irreversible. Our findings define a temporal hierarchy in which FACT loss drives progressive deterioration of chromatin architecture, leading to transcriptional collapse in stem cells.

genomics↗

bioRxiv2026

Unified function of FACT in mammalian chromatin replication and transcription, dissolving and restoring nucleosomes to counteract genome aggregation

Nucleosomes with their associated modifications underlie genome organisation and regulation. Replication and transcription require nucleosome disassembly to access the DNA template. How this is orchestrated without jeopardizing chromatin function remains unknown. Here, we reveal a general, global requirement of the histone chaperone FACT in mammalian replication, transcription and chromatin maintenance. Upon acute FACT depletion, replisome and RNA polymerase progression is halted genome-wide and chromatin structure in their wake collapses with reduced nucleosome occupancy, irregular spacing and intermediate assemblies. Chromatin states deteriorate as modified histones are lost due to a lack of histone recycling. Chromatin fiber disorder further manifests in the 3D genome, triggering active genes to coalesce in aberrant microcompartments. This establishes a unifying mechanistic basis for mammalian chromatin replication and transcription, with FACT mediating nucleosome disruption and re-assembly and thereby guarding against spurious chromatin aggregation. Nucleosome organization can therefore dynamically regulate genome architecture.

molecular biology↗

bioRxiv2024

The impact of transplant location on the gut microbiome and resistome in patients undergoing hematopoietic stem cell transplantation at home versus in the hospital

ObjectivesHome-based hematopoietic stem cell transplantation (HCT) is a novel approach that has the potential to improve outcomes, however, the impact of transplant location on the gut microbiome remains uncharacterized. We hypothesized that patients randomized to undergo home HCT would have higher gut taxonomic diversity and lower antimicrobial resistance (AMR) gene abundance compared to those undergoing standard hospital HCT. MethodsWe identified 28 patients enrolled in Phase II randomized trials of home (n=16) v. hospital (n=12) HCT at Duke and performed shotgun metagenomic sequencing of stools to compare taxonomic and AMR gene composition between groups. We performed a secondary analysis of patients from each group transplanted at an outpatient infusion clinic with those who underwent standard inpatient HCT ("outpatient" v. "inpatient"). ResultsNo significant differences in duration of hospitalization were found in those randomized to home v. hospital HCT. Taxonomic and AMR gene - and {beta}-diversity were comparable. In contrast, secondary analyses demonstrated that patients from both home and hospital groups transplanted at an outpatient infusion clinic spent significantly less time in the hospital and demonstrated higher taxonomic -diversity and differential {beta}-diversity compared to standard inpatient HCT, although AMR gene -diversity did not differ, and comparisons were confounded by both differences in transplant type and use of antibiotics. ConclusionsRandomization by transplant location did not impact the gut microbiota to the same extent as the duration of hospitalization, although secondary analyses were heavily confounded. Even when taxonomic differences were observed, AMR genes were similar between groups.

microbiology↗

bioRxiv2024

Targeted disruption of linkage-specific ubiquitylation reveals a key role of K29-linked ubiquitylation in epigenome integrity

Linkage-specific ubiquitin chains dictate the functional outcome of numerous critical ubiquitin-dependent signaling processes. However, the functions and targets of several poly-ubiquitin topologies remain poorly defined due to a paucity of tools for their specific detection and manipulation. To remedy this knowledge gap, we applied a cell-based ubiquitin replacement strategy enabling targeted conditional abrogation of each of the seven lysine-based ubiquitin chain types in human cells to profile system-wide impacts of disabling formation of individual chain types. Focusing on K29-linked ubiquitylation, we reveal a strong association of this linkage type with chromatin-associated proteins and show that the H3K9me3 methyltransferase SUV39H1 is a prominent cellular target of this modification. We demonstrate that K29-linked ubiquitylation is essential for proteasomal degradation of SUV39H1 despite its extensive modification by K48-linked ubiquitylation, and that K29-linked ubiquitylation of SUV39H1 is catalyzed and reversed by TRIP12 and TRABID, respectively. Preventing K29-linked ubiquitylation-mediated control of SUV39H1 stability deregulates the H3K9me3 landscape, but not other histone marks. Collectively, our ubiquitin replacement cell line panel and datasets provide valuable resources for illuminating cellular functions of linkage-specific ubiquitin chains and establish a key role of K29-linked ubiquitylation in epigenome integrity.

cell biology↗

bioRxiv2024

CODANIN-1 sequesters ASF1 by using a histone H3 mimic helix to regulate histone supply

ASF1 is a major histone chaperone that regulates the supply of histone H3-H4 and facilitates nucleosome assembly to maintain chromatin structure during DNA replication and transcription. CODANIN-1 negatively regulates the function of ASF1. However, the molecular mechanism by which CODANIN-1 inhibits the ASF1-mediated histone supply remains elusive. Here, we present the electron microscopy (cryo-EM) structure of a human CODANIN-1_ASF1A complex at 3.75 [A] resolution. The structure reveals that CODANIN-1 forms a dimer where each monomer holds two ASF1 molecules, utilizing two B-domains and two histone H3 mimic helices (HMHs). The interaction of CODANIN-1 with ASF1 via the HMH and B domains inhibits the formation of an ASF1/H3-H4 complex and sequesters ASF1 in the cytoplasm. Our study provides a structural and molecular basis for the function of CODANIN-1 as a unique negative regulator that highjacks ASF1 interaction sites with histones and downstream chaperones to inhibit nucleosome assembly.

biophysics↗

bioRxiv2024

Acute multi-level response to defective de novo chromatin assembly in S-phase

Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live-microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyper-accessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. As a result, histone variants usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered. HighlightsO_LIThe histone chaperone CAF-1 sustains DNA replication speed in single cells. C_LIO_LICAF-1 loss alters histone repertoire and delays chromatin maturation. C_LIO_LIH3K9me3 and H3K27me3 regions respond differently to acute CAF-1 depletion. C_LIO_LIImpaired S-phase chromatin assembly triggers an immediate response and a G0 arrest. C_LI

cell biology↗

bioRxiv2023

Repair of DNA double-strand breaks leaves heritable impairment to genome function

Upon DNA breakage, a genomic locus undergoes alterations in 3-D chromatin architecture to facilitate signaling and repair. While cells possess mechanisms to repair damaged DNA, it is unknown whether the surrounding chromatin is restored to its naive state. We show that a single DNA double-strand break (DSB) within a topologically-associated domain (TAD) harboring conformation-sensitive genes causes lasting chromatin alterations, which persist after completion of DNA repair and feature structural changes, chromatin compaction and loss of local RNA species. Unexpectedly, these newly-acquired features of post-repair chromatin are transmitted to daughter cells and manifest as heritable impairments of gene expression. These findings uncover a hitherto concealed dimension of DNA breakage, which we term post-repair chromatin fatigue, and which confers heritable impairment of gene function beyond DNA repair.

cell biology↗

bioRxiv2022

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 cooperates with the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitution, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Pol{varepsilon}) for PCNA binding, but not with the lagging strand DNA polymerase Delta (Pol{delta}). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand both processes are coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

biochemistry↗

bioRxiv2022

DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone network

A multitude of histone chaperones are required to protect histones after their biosynthesis until DNA deposition. They cooperate through the formation of co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using explorative interactomics approaches, we characterize the organization of the histone H3-H4 chaperones network and define the interplay between histone chaperone systems. We identify and validate several novel histone dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX acts separately from the rest of the network, recruiting heterochromatin factors and promoting lysine 9 tri-methylation of new histone H3.3 prior to deposition onto DNA. With its functionality, DAXX provides a molecular mechanism for de novo heterochromatin assembly. Collectively, our findings provide a new framework for understanding how cells orchestrate histone supply and comply with chromatin dynamics throughout the cell cycle.

molecular biology↗

bioRxiv2021

NASP maintains histone H3-H4 homeostasis through two distinct H3 binding modes

Histone chaperones regulate all aspects of histone metabolism. NASP is a major histone chaperone for H3-H4 dimers critical for preventing histone degradation. Here, we identify two distinct histone binding modes of NASP and reveal how they cooperate to ensure histone H3-H4 supply. We determine the structures of a sNASP dimer, a complex of sNASP with an H3 3 peptide, and the sNASP-H3-H4-ASF1b co-chaperone complex. This captures distinct functionalities of NASP and identifies two distinct binding modes involving the H3 3 helix and the H3 N region, respectively. Functional studies demonstrate the H3 N-interaction represents the major binding mode of NASP in cells and shielding of the H3 N region by NASP is essential in maintaining the H3-H4 histone soluble pool. In conclusion, our studies uncover the molecular basis of NASP as a major H3-H4 chaperone in guarding histone homeostasis.

biochemistry↗

bioRxiv2020

BARD1 links histone H2A Lysine-15 ubiquitination to initiation of BRCA1-dependent homologous recombination

Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP1, mediators of the homologous recombination (HR) and non-homologous end joining (NHEJ) DSB repair pathways, respectively. While NHEJ relies on 53BP1 binding to ubiquitinated Lysine 15 on H2A-type histones (H2AK15ub), an RNF168-dependent modification, the mechanism linking RNF168 to BRCA1 recruitment during HR has remained unclear. Here, we identify a tandem BRCT domain ubiquitin-dependent recruitment motif (BUDR) in BARD1 - BRCA1s obligate partner protein - that binds H2AK15ub directly, thereby recruiting BRCA1 to DSBs. BARD1 BUDR mutations compromise HR, and render cells hypersensitive to PARP inhibition and cisplatin treatment. We find that BARD1-nucleosome interactions require BUDR binding to H2AK15ub and ankyrin repeat domain-mediated binding of the histone H4 tail, specifically when unmethylated on Lysine-20 (H4K20me0), a state limited to post replicative chromatin. Finally, we demonstrate that by integrating DNA damagedependent H2AK15ub and DNA replication-dependent H4K20me0 signals at sites of DNA damage, BARD1 coordinates BRCA1-dependent HR with 53BP1 pathway antagonization, establishing a simple paradigm for the governance of DSB repair pathway choice.

molecular biology↗

bioRxiv2019

Domain model explains propagation dynamics and stability of K27 and K36 methylation landscapes

Chromatin states must be maintained during cell proliferation to uphold cellular identity and genome integrity. Inheritance of histone modifications is central in this process. However, the histone modification landscape is challenged by incorporation of new unmodified histones during each cell cycle and the principles governing heritability remain unclear. Here, we take a quantitative computational modeling approach to describes propagation of K27 and K36 methylation states. We measure combinatorial K27 and K36 methylation patterns by quantitative mass spectrometry on subsequent generations of histones. Using model comparison, we reject active global demethylation and invoke the existence of domains defined by distinct methylation endpoints. We find that K27me3 on pre-existing histones stimulates the rate of de novo K27me3 establishment, supporting a read-write mechanism in timely chromatin restoration. Finally, we provide a detailed, quantitative picture of the mutual antagonism between K27 and K37 methylation, and propose that it stabilizes epigenetic states across cell division.

systems biology↗