Biology subjects

Mattiroli, F.

Publications and source records attributed to Mattiroli, F..

4 recordsLinked to original sources

bioRxiv2025

The eukaryotic replisome intrinsically generates asymmetric daughter chromatin fibers

DNA replication is molecularly asymmetric, due to distinct mechanisms for lagging and leading strand DNA synthesis. Whether chromatin assembly on newly replicated strands is also asymmetric remains unknown, as visualizing this short-lived state in cells is impossible. To circumvent this limitation, we combine in vitro reconstitution of the Saccharomyces cerevisiae DNA and chromatin replication machineries with replication-aware single-molecule chromatin footprinting, to study how chromatin is re-assembled on replicated DNA. Leveraging the non-destructive, single-molecule, and strand-specific nature of our data, we discover an intrinsic asymmetry in nucleosome positioning patterns and organization between lagging- and leading-strand chromatin created by the yeast replisome. This asymmetry is only partially restored upon addition of chromatin assembly factors involved in de novo histone deposition and the ATP-dependent chromatin remodeler Isw1a, implying that other regulatory factors must resolve this asymmetry in cells. In sum, our data reveal the complexity of chromatin re-establishment following DNA replication, and suggest an asymmetric chromatin assembly intermediate on each daughter chromatid. These pathways have implications for essential chromatin-templated processes such as DNA repair, transcription, and gene silencing at replication forks.

biochemistry↗

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↗

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↗

bioRxiv2021

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B(1-5). Here, we show that the acidic domain of DNA repair factor APLF (APLFAD) can assemble the histone octamer in a single step, and deposit it on DNA to form nucleosomes. The crystal structure of the APLFAD-histone octamer complex shows that APLFAD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLFAD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted. one sentence summaryHistone chaperone APLF assembles histones H2A-H2B/H3-H4 into histone octamers to deposit them onto DNA and form nucleosomes.

biochemistry↗