Mathew J. K. Jones

Chromothripsis: Chromosomes in Crisis

During oncogenesis, cells acquire multiple genetic alterations that confer essential tumor-specific traits, including immortalization, escape from antimitogenic signaling, neovascularization, invasiveness, and metastatic potential. In most instances, these alterations are thought to arise incrementally over years, if not decades. However, recent progress in sequencing cancer genomes has begun to challenge this paradigm, because a radically different phenomenon, termed chromothripsis, has been suggested to cause complex intra- and interchromosomal rearrangements on short timescales. In this Review, we review established pathways crucial for genome integrity and discuss how their dysfunction could precipitate widespread chromosome breakage and rearrangement in the course of malignancy.

Wild-type H- and N-Ras promote mutant K-Ras driven tumorigenesis by modulating the DNA damage response

Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anticancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here, we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways and, consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo.

T cell help controls the speed of the cell cycle in germinal center B cells

B cells have a need for speed High-affinity antibodies provide long-lasting protective immunity against many infections. Generating such antibodies requires help, in the form of T cells, which interact with antibody-producing B cells. As B cells proliferate and mutate their antibody genes, T cells select the cells producing high-affinity antibodies. Gitlin et al. show in mice that B cells that receive T cell help transit through the cell cycle more quickly by increasing the speed at which replication forks progress. Such a rapid cell cycle transition gives high-affinity B cells a selective advantage. Science, this issue p. 643 T cells help select high-affinity antibodies by increasing the speed at which B cells progress through the cell cycle. The germinal center (GC) is a microanatomical compartment wherein high-affinity antibody-producing B cells are selectively expanded. B cells proliferate and mutate their antibody genes in the dark zone (DZ) of the GC and are then selected by T cells in the light zone (LZ) on the basis of affinity. Here, we show that T cell help regulates the speed of cell cycle phase transitions and DNA replication of GC B cells. Genome sequencing and single-molecule analyses revealed that T cell help shortens S phase by regulating replication fork progression, while preserving the relative order of replication origin activation. Thus, high-affinity GC B cells are selected by a mechanism that involves prolonged dwell time in the DZ where selected cells undergo accelerated cell cycles.

APC/CCdh1-dependent proteolysis of USP1 regulates the response to UV-mediated DNA damage

APC/CCdh1-dependent degradation of USP1 allows for PCNA monoubiquitination and subsequent recruitment of trans-lesion synthesis polymerase to UV repair sites.

Dysregulation of DNA polymerase κ recruitment to replication forks results in genomic instability

Translesion synthesis polymerases (TLS Pols) are required to tolerate DNA lesions that would otherwise cause replication arrest and cell death. Aberrant expression of these specialized Pols may be responsible for increased mutagenesis and loss of genome integrity in human cancers. The molecular events that control the usage of TLS Pols in non‐pathological conditions remain largely unknown. Here, we show that aberrant recruitment of TLS Polκ to replication forks results in genomic instability and can be mediated through the loss of the deubiquitinase USP1. Moreover, artificial tethering of Polκ to proliferating cell nuclear antigen (PCNA) circumvents the need for its ubiquitin‐binding domain in the promotion of genomic instability. Finally, we show that the loss of USP1 leads to a dramatic reduction of replication fork speed in a Polκ‐dependent manner. We propose a mechanism whereby reversible ubiquitination of PCNA can prevent spurious TLS Pol recruitment and regulate replication fork speed to ensure the maintenance of genome integrity.

A novel corepressor, BCoR-L1, represses transcription through an interaction with CtBP

Corepressors play a crucial role in negative gene regulation and are defective in several diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and functionally characterize BCoR-L1, a homolog of BCoR. When tethered to a heterologous promoter, BCoR-L1 is capable of strong repression. Like other corepressors, BCoR-L1 associates with histone deacetylase (HDAC) activity. Specifically, BCoR-L1 coprecipitates with the Class II HDACs, HDAC4, HDAC5, and HDAC7, suggesting that they are involved in its role as a transcriptional repressor. BCoR-L1 also interacts with the CtBP corepressor through a CtBP-interacting motif in its amino terminus. Abrogation of the CtBP binding site within BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, BCoR-L1 is located on the E-cadherin promoter, a known CtBP-regulated promoter, and represses the E-cadherin promoter activity in a reporter assay. The inhibition of BCoR-L1 expression by RNA-mediated interference results in derepression of E-cadherin in cells that do not normally express E-cadherin, indicating that BCoR-L1 contributes to the repression of an authentic endogenous CtBP target.

Cohesin recruits the Esco1 acetyltransferase genome wide to repress transcription and promote cohesion in somatic cells.

Significance The cohesin complex holds sister chromatids together from their duplication in S phase to their separation in anaphase. Cohesin is also involved in other aspects of chromosome structure and function, including the regulation of gene expression. To connect sister chromatids, cohesin must be modified by conserved acetyltransferases, known as Esco1 and Esco2 in humans. We investigated how Esco1 and Esco2 interact with chromosomes. Surprisingly, cohesin recruits Esco1 to numerous sites throughout the genome, while Esco2 is rarely seen at these sites. Esco1’s colocalization with cohesin requires two short regions that have been conserved throughout its evolution. Deleting these regions prevents Esco1 from establishing cohesion, and from silencing genes near its binding sites, a newly identified function of this factor. The cohesin complex links DNA molecules and plays key roles in the organization, expression, repair, and segregation of eukaryotic genomes. In vertebrates the Esco1 and Esco2 acetyltransferases both modify cohesin’s Smc3 subunit to establish sister chromatid cohesion during S phase, but differ in their N-terminal domains and expression during development and across the cell cycle. Here we show that Esco1 and Esco2 also differ dramatically in their interaction with chromatin, as Esco1 is recruited by cohesin to over 11,000 sites, whereas Esco2 is infrequently enriched at REST/NRSF target genes. Esco1’s colocalization with cohesin occurs throughout the cell cycle and depends on two short motifs (the A-box and B-box) present in and unique to all Esco1 orthologs. Deleting either motif led to the derepression of Esco1-proximal genes and functional uncoupling of cohesion from Smc3 acetylation. In contrast, other mutations that preserved Esco1’s recruitment separated its roles in cohesion establishment and gene silencing. We conclude that Esco1 uses cohesin as both a substrate and a scaffold for coordinating multiple chromatin-based transactions in somatic cells.

Insights into phosphorylation-dependent mechanisms regulating USP1 protein stability during the cell cycle

Tight regulation of the cell cycle and DNA repair machinery is essential for maintaining genome stability. The APC/CCdh1 ubiquitin ligase complex is a key regulator of protein stability during the G1 phase of the cell cycle. APC/CCdh1 regulates and promotes the degradation of proteins involved in both cell cycle regulation and DNA repair. In a recent study, we identified a novel APC/CCdh1 substrate, the ubiquitin protease USP1. USP1 is a critical regulator of both the Fanconi anemia (FA) and translesion synthesis (TLS) DNA repair pathways. Here, we provide additional mechanistic insights into the regulation of USP1 during the cell cycle. Specifically, we demonstrate that USP1 is phosphorylated in mitosis by cyclin-dependent kinases (Cdks), and that this phosphorylation event may prevent premature degradation of USP1 during normal cell cycle progression. Finally, we provide a unifying hypothesis integrating the role of G1-specific proteolysis of USP1 with the regulation of the transcriptional repressors, Inhibitor of DNA-binding (ID) proteins.

The Fanconi anemia pathway in replication stress and DNA crosslink repair.

Interstand crosslinks (ICLs) are DNA lesions where the bases of opposing DNA strands are covalently linked, inhibiting critical cellular processes such as transcription and replication. Chemical agents that generate ICLs cause chromosomal abnormalities including breaks, deletions and rearrangements, making them highly genotoxic compounds. This toxicity has proven useful for chemotherapeutic treatment against a wide variety of cancer types. The majority of our understanding of ICL repair in humans has been uncovered through analysis of the rare genetic disorder Fanconi anemia, in which patients are extremely sensitive to crosslinking agents. Here, we discuss recent insights into ICL repair gained using new repair assays and highlight the role of the Fanconi anemia repair pathway during replication stress.

Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair

Fanconi anemia (FA) is a rare familial genome instability syndrome caused by mutations in FA genes that results in defective DNA crosslink repair. Activation of the FA pathway requires the FA core ubiquitin ligase complex-dependent monoubiquitination of 2 interacting FA proteins, FANCI and FANCD2. Although loss of either FANCI or FANCD2 is known to prevent monoubiquitination of its respective partner, it is unclear whether FANCI has any additional domains that may be important in promoting DNA repair, independent of its monoubiquitination. Here, we focus on an FA-I patient-derived FANCI mutant protein, R1299X (deletion of 30 residues from its C-terminus), to characterize important structural region(s) in FANCI that is required to activate the FA pathway. We show that, within this short 30 amino acid stretch contains 2 separable functional signatures, a nuclear localization signal and a putative EDGE motif, that is critical for the ability of FANCI to properly monoubiquitinate FANCD2 and promote DNA crosslink resistance. Our study enable us to conclude that, although proper nuclear localization of FANCI is crucial for robust FANCD2 monoubiquitination, the putative FANCI EDGE motif is important for DNA crosslink repair.