A Cluster of Evolutionarily Recent KRAB Zinc Finger Proteins Protects Cancer Cells from Replicative Stress-Induced Inflammation.

Heterochromatin loss and genetic instability enhance cancer progression by favoring clonal diversity, yet uncontrolled replicative stress leads to mitotic catastrophe and inflammatory responses that promote immune rejection. KRAB domain-containing zinc finger proteins (KZFP) contribute to heterochromatin maintenance at transposable elements (TE). Here, we identified an association of upregulation of a cluster of primate-specific KZFPs with poor prognosis, increased copy-number alterations, and changes in the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL). Depleting two of these KZFPs targeting evolutionarily recent TEs, ZNF587 and ZNF417, impaired the proliferation of cells derived from DLBCL and several other tumor types. ZNF587 and ZNF417 depletion led to heterochromatin redistribution, replicative stress, and cGAS-STING-mediated induction of an interferon/inflammatory response, which enhanced susceptibility to macrophage-mediated phagocytosis and increased surface expression of HLA-I, together with presentation of a neoimmunopeptidome. Thus, cancer cells can exploit KZFPs to dampen TE-originating surveillance mechanisms, which likely facilitates clonal expansion, diversification, and immune evasion. SIGNIFICANCE: Upregulation of a cluster of primate-specific KRAB zinc finger proteins in cancer cells prevents replicative stress and inflammation by regulating heterochromatin maintenance, which could facilitate the development of improved biomarkers and treatments.

Genetic features and genomic targets of human KRAB-zinc finger proteins.

Krüppel-associated box (KRAB) domain-containing zinc finger proteins (KZFPs) are one of the largest groups of transcription factors encoded by tetrapods, with 378 members in human alone. KZFP genes are often grouped in clusters reflecting amplification by gene and segment duplication since the gene family first emerged more than 400 million years ago. Previous work has revealed that many KZFPs recognize transposable element (TE)-embedded sequences as genomic targets, and that KZFPs facilitate the co-option of the regulatory potential of TEs for the benefit of the host. Here, we present a comprehensive survey of the genetic features and genomic targets of human KZFPs, notably completing past analyses by adding data on close to a hundred family members. General principles emerge from our study of the TE-KZFP regulatory system, which point to multipronged evolutionary mechanisms underlaid by highly complex and combinatorial modes of action with strong influences on human speciation.

Nuclear Lipid Droplet Birth during Replicative Stress.

The nuclear membrane defines the boundaries that confine, protect and shape the genome. As such, its blebbing, ruptures and deformations are known to compromise the integrity of genetic material. Yet, drastic transitions of the nuclear membrane such as its invagination towards the nucleoplasm or its capacity to emit nuclear lipid droplets (nLD) have not been evaluated with respect to their impact on genome dynamics. To begin assessing this, in this work we used Saccharomyces cerevisiae as a model to ask whether a selection of genotoxins can trigger the formation of nLD. We report that nLD formation is not a general feature of all genotoxins, but of those engendering replication stress. Exacerbation of endogenous replication stress by genetic tools also elicited nLD formation. When exploring the lipid features of the nuclear membrane at the base of this emission, we revealed a link with the unsaturation profile of its phospholipids and, for the first time, of its sterol content. We propose that stressed replication forks may stimulate nLD birth by anchoring to the inner nuclear membrane, provided that the lipid context is adequate. Further, we point to a transcriptional feed-back process that counteracts the membrane's proneness to emit nLD. With nLD representing platforms onto which genome-modifying reactions can occur, our findings highlight them as important players in the response to replication stress.

A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress.

Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.

A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization.

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.

Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication.

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.

MRX Increases Chromatin Accessibility at Stalled Replication Forks to Promote Nascent DNA Resection and Cohesin Loading.

The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.

qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing.

DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks.

Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

Dbf4 recruitment by forkhead transcription factors defines an upstream rate-limiting step in determining origin firing timing.

Initiation of eukaryotic chromosome replication follows a spatiotemporal program. The current model suggests that replication origins compete for a limited pool of initiation factors. However, it remains to be answered how these limiting factors are preferentially recruited to early origins. Here, we report that Dbf4 is enriched at early origins through its interaction with forkhead transcription factors Fkh1 and Fkh2. This interaction is mediated by the Dbf4 C terminus and was successfully reconstituted in vitro. An interaction-defective mutant, dbf4ΔC, phenocopies fkh alleles in terms of origin firing. Remarkably, genome-wide replication profiles reveal that the direct fusion of the DNA-binding domain (DBD) of Fkh1 to Dbf4 restores the Fkh-dependent origin firing but interferes specifically with the pericentromeric origin activation. Furthermore, Dbf4 interacts directly with Sld3 and promotes the recruitment of downstream limiting factors. These data suggest that Fkh1 targets Dbf4 to a subset of noncentromeric origins to promote early replication in a manner that is reminiscent of the recruitment of Dbf4 to pericentromeric origins by Ctf19.