Homologous recombination (HR) and Fanconi Anemia (FA) pathway proteins in addition to their DNA repair functions, limit nuclease-mediated processing of stalled replication forks. However, the mechanism by which replication fork degradation results in genome instability is poorly understood. Here, we identify RIF1, a non-homologous end joining (NHEJ) factor, to be enriched at stalled replication forks. Rif1 knockout cells are proficient for recombination, but displayed degradation of reversed forks, which depends on DNA2 nuclease activity. Notably, RIF1-mediated protection of replication forks is independent of its function in NHEJ, but depends on its interaction with Protein Phosphatase 1. RIF1 deficiency delays fork restart and results in exposure of under-replicated DNA, which is the precursor of subsequent genomic instability. Our data implicate RIF1 to be an essential factor for replication fork protection, and uncover the mechanisms by which unprotected DNA replication forks can lead to genome instability in recombination-proficient conditions.
Bloom syndrome is a cancer predisposition disorder caused by mutations in the BLM helicase gene. Cells from persons with Bloom syndrome exhibit striking genomic instability characterized by excessive sister chromatid exchange events (SCEs). We applied single-cell DNA template strand sequencing (Strand-seq) to map the genomic locations of SCEs. Our results show that in the absence of BLM, SCEs in human and murine cells do not occur randomly throughout the genome but are strikingly enriched at coding regions, specifically at sites of guanine quadruplex (G4) motifs in transcribed genes. We propose that BLM protects against genome instability by suppressing recombination at sites of G4 structures, particularly in transcribed regions of the genome.
The eleven zinc finger (ZF) protein CTCF regulates topologically associating domain (TAD) formation and transcription through selective binding to thousands of genomic sites. We replaced endogenous CTCF in mouse embryonic stem cells with GFP-tagged wildtype or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8-11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of TADs, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatintemplated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.Nuclear genomes are folded in three dimensions (3D) in a temporally controlled manner that facilitates essential chromatin-templated processes such as transcription, DNA repair, and replication 1-3 . Chromosomal regions segregate into transcriptionally active euchromatic (A) and inactive heterochromatic (B) compartments 4,5 . Inside compartments, there are submegabase sized regions called topologically associating domains (TADs) [6][7][8] . Genomic regions inside the same TAD show increased interaction frequencies, creating spatially insulated neighborhoods in the genome. Both compartments and TADs play a role in transcriptional control 3 , with TADs attracting significant attention as a major contributor to the specificity of promoter-enhancer interactions 9 . Notably, disrupted TAD organization has been implicated in developmental disorders and cancer 10,11 . CTCF is a highly conserved multifunctional eleven zinc finger (ZF) protein that binds thousands of uniquely oriented sites in the genome, each containing a core (C) motif of ~15 nucleotides 12,13 . Structural analysis of CTCF bound to such a motif revealed that ZFs 3-7 are positioned in the major groove, where each ZF contacts a few bases 14 . By contrast, ZF8 is positioned in the minor groove and does not contribute to binding specificity 14 . Interestingly, a subset of genomic CTCF sites have a small upstream (U) motif, which is separated from the core by a spacer of 5-6 nucleotides 12,13,15 . This extended 'UC' motif is conserved across species and was proposed to represent a high affinity binding site for CTCF 13 . ZFs 9-11 were shown to bind the U motif 15 , and hence ZF8 is thought to act as a linker between ZFs 4-7 and 9-11, not playing an active role in DNA binding.Selective binding to its cognate sites in the genome allows CTCF to control gene expression, for example by modulating promoter-enhancer interactions 16 . CTCF also regulates TAD formation together with the cohesin complex 17-19 in a process termed loop extrusion 20 , where the ring-shaped cohesin complex attaches to chromatin strands either pseudo-topologically or non-topologically 21 to actively extrude these and gene...
Random epigenetic silencing of the X-chromosome in somatic tissues of female mammals equalizes the dosage of X-linked genes between the sexes. Unlike this form of X-inactivation that is essentially irreversible, the imprinted inactivation of the paternal X, which characterizes mouse extra-embryonic tissues, appears highly unstable in the trophoblast giant cells of the placenta. Here, we wished to determine whether such instability is already present in placental progenitor cells prior to differentiation toward lineage-specific cell types. To this end, we analyzed the behavior of a GFP transgene on the paternal X both in vivo and in trophoblast stem (TS) cells derived from the trophectoderm of XX GFP blastocysts. Using single-cell studies, we show that not only the GFP transgene but also a large number of endogenous genes on the paternal X are subject to orchestrated cycles of reactivation/de novo inactivation in placental progenitor cells. This reversal of silencing is associated with local losses of histone H3 lysine 27 trimethylation extending over several adjacent genes and with the topological relocation of the hypomethylated loci outside of the nuclear compartment of the inactive X. The "reactivated" state is maintained through several cell divisions. Our study suggests that this type of "metastable epigenetic" states may underlie the plasticity of TS cells and predispose specific genes to relaxed regulation in specific subtypes of placental cells.
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