Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet: The FPC is essential for the maintenance of heterochromatic gene silencing (A) Illustration of the FPC (Mrc1, Swi1/Tof1, Swi3/Csm3) at the replisome, showing the CMG helicase (MCM2-6, Cdc45), Mcl1/Ctf4, DNA primase (Polα), and polymerase epsilon (Polε). (B) Heterochromatic mating-type region between the IR-L and IR-R boundaries depicting the silent mating-type cassettes mat2-P and mat3-M , Kint2::YFP and (EcoRV)::mCherry fluorescent reporter genes, RNAi-dependent nucleation center cenH , Atf1 transcription factor binding sites, and REII and REIII silencing elements. (C) Establishment of mCherry silencing following clr4 + reintroduction in FPC mutants. (D) Cells with derepressed reporters in clonal cultures of FPC mutants ( n = 6). Data are represented as mean ± SD. (E) Micrographs of FPC mutants expressing mCherry . Scale bar: 10 μm. (F) Histograms of mCherry fluorescence (mean, black; replicates n = 6, red). (G) Interpretation of loss-of-silencing events in (H). The mCherry locus asymmetrically loses heterochromatic silencing in S phase, and the protein production starts in G2 (top cell). The expressed ( mCherry ON) and repressed ( mCherry OFF) chromatids segregate to sister cells. In subsequent cell divisions, mCherry protein is produced in the ON lineage (blue arrows) but is diluted in the OFF lineage (yellow arrows). (H) Loss of silencing in the mrc1 Δ mutant followed by time-lapse microscopy. White arrowheads point to cells experiencing a loss-of-silencing event. Blue arrowheads point to ON cell lineages and yellow arrows point to OFF lineages. Yellow arrows are omitted at 10 h for clarity. Scale bar: 10 μm.

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques: Binding Assay, Expressing, Fluorescence, Produced, Mutagenesis, Time-lapse Microscopy

Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet: Coordinated function of Mrc1 and Mcm2-HBD in heterochromatin maintenance (A) Aligned S. cerevisiae and S. pombe Mrc1 proteins showing contacts and cross-links to replisome components in S. cerevisiae , and highly conserved residues in Mrc1 HBS (bottom). Boxed residues were mutated in this study. Magenta residues are identified in the cryo-EM structure. (B) Cells expressing mCherry ( n = 6). Data are represented as mean ± SD. (C) Expression of ade6 + reporter in mrc1 mutants. Red color on YE medium indicates repression. (D) Mrc1 contacts with replisome components shown in magenta on PDB 8B9A with the expected positions of the Mcm2 HBD and Mrc1 HBS and DSE indicated. Cross-linked residues (from Baretić et al. [2020] ) are indicated by magenta circles and labeled by their position in S. cerevisiae Mrc1. The conserved F848 in KAF is in green. (E) Conserved tyrosine residues in the Mcm2 HBD mutated to alanine in mcm2-2A . (F) Cells expressing mCherry ( n = 18 [left] [ANOVA, F = 73.9, p = 2.2 × 10 − 16 ]; n = 6 [right]). Data are represented as mean ± SD. (G) H3K9me2 in subtelomeric region Tel1R in mrc1 Δ HBS and mcm2-2A mutants. Centromere 1 is shown for comparison. (H) Major heterochromatic regions of S. pombe . (I) Heatmap depicting H3K9me2 at heterochromatic regions. (J and K) Tethering Pob3 to Mrc1ΔHBS through a GFP-GBP interaction (J) restores silencing of mCherry reporter (K). ( n = 6) [ANOVA, F = 45.9, p = 7.1 × 10 − 7 ]. Data are represented as mean ± SD. See also Figures S2 and .

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques: Cryo-EM Sample Prep, Expressing, Labeling, Comparison

Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet: Proficient checkpoint in mrc1-DSE and mrc1-KAF mutants and similarities of mrc1ΔHBS and mcm2-2A mutants, related to Figure 3 (A) Ten-fold serial dilutions of cell suspensions were spotted on HU-containing medium to estimate checkpoint proficiency. (B) Ten-fold serial dilutions of cell suspensions were spotted on rich YES medium and incubated at the indicated temperatures to check for bypass of the hsk1-89 mutation at the restrictive temperature of 30°C. (C) Asymmetric loss of heterochromatic gene silencing of (EcoRV)::mCherry reporter in mcm2-2A mutant visualized by time-lapse microscopy. Scale bar: 10 μm. (D) Similar temperature-dependency of heterochromatic silencing in mrc1 Δ HBS and mcm2-2A mutants. In both (C) and (D), microcolonies were grown from single cells under a fluorescence microscope.

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques: Incubation, Mutagenesis, Time-lapse Microscopy, Fluorescence, Microscopy

Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet: Heterochromatin loss away from nucleation sites in Mrc1 and Mcm2 mutants, related to Figure 3 (A) H3K9me2 occupancy at Chromosome 1 (top) and Chromosome 2 (bottom) in histone-recycling mutants. siRNAs are depicted as red blocks across Tel1L/Tel2L (left), centromeres (middle), or Tel1R/Tel2R (right). (B) H3K9me2 occupancy at the mating-type region indicating siRNAs originating from cenH depicted in red and the two Atf1-binding sites in magenta. (C) Ten-fold serial dilutions of cell suspensions were spotted onto the indicated media. Growth on the toxigenic substrate FOA reflects ura4 + repression. (D) H3K9me2 occupancy at the h and ΔK mating-type regions. (E) Quantification of H3K9me2 occupancy at indicated regions within the mating-type region normalized to cnt3 [unpaired Student’s t test used]. (F) Schematic diagram of Pob3 with functional domains annotated and deletion mutations indicated. (G) Proportion of cells expressing mCherry in cells co-expressing Pob3-GBP (full-length and mutants) and Mrc1ΔHBS-GFP (mean ± SD, n = 6) [ANOVA, F = 41.6, P = 8.7 × 10 −9 ].

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques: Binding Assay, Functional Assay, Expressing

Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet: Mrc1 cooperates with Mcm2 in recycling of parental histones to the lagging strand (A) Workflow of xSCAR-seq in fission yeast. (B) Partition of H3K36me3 (top) and H4K20me0 (bottom) at a genomic region. Replication origin centers are depicted as black lines with their respective firing efficiency score. (C) Average partitioning score across replication initiation centers (with score >20) for parental (H3K36me3, top) and newly synthesized (H4K20me0, bottom) histones. (D) Heatmap representing the partitioning score across all replication origin centers (score >20) for H3K36me3 (left) and H4K20me0 (right). Each row represents the partition score of xSCAR-seq sequence reads at one origin. Average of two independent replicates. See also Figure S4 .

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques: Synthesized, Sequencing

Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet: Mrc1 chaperones H3-H4 tetramers in a manner compatible with Mcm2 co-chaperoning (A) AF , prediction of a complex comprising full-length S. pombe Mrc1 (pink) and Mcm2 (light blue) bound to a histone H3-H4 tetramer (gray). Histone tails and unstructured Mrc1 residues predicted with low confidence (residues 1–381, 475–710, 800–804, and 856–1,020) are not depicted for clarity. Closeups: (1) interaction of the Mrc1 KAF motif with a hydrophobic groove in Mcm2 and (2) interaction of the N-terminal alpha helix of the Mrc1 HBS domain with charged aa in histone H3 αN and α2 helices and histone H4 C terminus. (B) AF predictions of Mrc1 histone binding via the HBD, including beginning of the HBS domain (dark pink) and upstream region (710–800). Interaction between Mrc1 (pink) and the histones (gray) remains unchanged in the presence of Mcm2 (light blue). Closeups highlight residues subjected to mutational analysis. (C) Mcm2(34–165) and Mrc1(674–879) polypeptides used for pull-downs. (D) Pull-downs of full-length Mrc1-FLAG and H3-H4 with HA-Mcm2 HBD (34–165). Pulled-down proteins were detected by western blot (WB) or Coomassie staining (CBB). MW in KDa. (E) Pull-downs of H3-H4 dimers or tetramers with His 6 -Mrc1(674–879). (F) Pull-downs of H3-H4 tetramers with His 6 -Mrc1(674–879) WT and indicated mutants. See also Figure S5 and .

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques: Binding Assay, Western Blot, Staining

Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet: AlphaFold predicts an Mrc1-Mcm2 co-chaperone complex consistent with the Mcm2 HBD crystal structure and the Mcm2 cryo-EM structure at the replisome, related to Figure 5 (A and B) AF , predictions of complexes comprising full-length Mrc1 and Mcm2 bound to a histone H3-H4 tetramer with Mrc1 (A) and Mcm2 (B) colored according to AF pLDDT (predicted local distance difference test) score to indicate prediction confidence. (C) AF predictions of a complex comprising full-length Mrc1 bound to an H3-H4 tetramer with Mrc1 colored according to AF pLDDT score. (D) The predicted interaction between Mcm2 (light blue) and Mrc1 (pink) mediated by Mrc1 helices (residues 829–845 and 406–437) is in agreement with Mcm2 (yellow)/Mrc1 (dark blue) positions previously experimentally determined by cryo-EM in the context of the replisome (PDB 8B9A ). (E) The predicted interaction of the N-terminal domain of Mcm2 (light blue) with a histone H3-H4 tetramer is in agreement with an available crystal structure (yellow, PDB 5BNV ). (F) Amino acid sequence of the Mrc1(674–879) fragment used in in vitro pull downs with relevant domains and residues highlighted.

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques: Cryo-EM Sample Prep, Sequencing, In Vitro

Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet: PAE plots for AF best scoring models, related to Figure 5 (A) Mrc1 with histone dimer, (B) Mrc1 with histone tetramer, and (C) Mrc1 and Mcm2 with histone tetramer. The predicted interaction interfaces of Mrc1 and Mcm2 with histones are highlighted in pink and light blue, respectively. Predicted aligned error (PAE) plots , were visualized by ChimeraX 1.7 . Tables in (D) show DockQ and QS-score validation scores for Mcm2 and Mrc1 ( Figure 6 C), and Mcm2 and H3/H4 histone ( Figure S5 C) interaction interface in Mrc1-Mcm2-H3/H4 tetramer AF model compared to selected experimental protein structures (PDB: 8B9A and 5BNV, respectively—see STAR Methods).

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques:

Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet: Histone recycling function of Mrc1 is conserved in mammalian cells (A) (top) Mouse CLASPIN with annotated domains (BP1 and BP2: basic patch 1 and 2, CKBD: Chk1 binding domain, AP: acidic patch) and alignment with Polα and Mcm2 HBDs. (Bottom) overview of mutation in CLASPINΔYY. (B) WB analysis of WT and CLASPIN mutant mESCs. (C) Cell-cycle distribution based on mean EdU intensity and total DAPI intensity of WT and CLASPIN mutant mESCs. (D) High-content microscopy of mean EdU intensity in WT and CLASPIN mutant mESCs. (E) Average SCAR-seq profile of H3K27me3 and H4K20me0 in WT and CLASPIN mutant mESCs. (C–E) n = 2 biological replicates. (F) Model: Mrc1 acts as a central coordinator of histone-based inheritance though its ability to bind and transfer H3-H4 tetramers to both leading and lagging strands, with the latter involving joint histone binding with Mcm2 to facilitate transfer to Polα and the lagging strand.

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques: Binding Assay, Mutagenesis, Microscopy

Journal: Cell

Article Title: The fork protection complex promotes parental histone recycling and epigenetic memory

doi: 10.1016/j.cell.2024.07.017

Figure Lengend Snippet:

Article Snippet: Proteins were further purified on a HiLoad 16/600 Superdex 200 Column (Mrc1) and HiLoad 16/60 Superdex 75 column (Mcm2) (Cytiva).

Techniques: Virus, Recombinant, Western Blot, Software, Affinity Column, Membrane