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Lamina-associated domains (LADs) are large chromatin regions that are associated with the nuclear lamina (NL) and form a repressive environment for transcription. The molecular players that mediate gene repression in LADs are currently unknown. Here, we performed FACS-based whole-genome genetic screens in human cells using LAD-integrated fluorescent reporters to identify such regulators. Surprisingly, the screen identified very few NL proteins, but revealed roles for dozens of known chromatin regulators. Among these are the negative elongation factor (NELF) complex and interacting factors involved in RNA polymerase pausing, suggesting that regulation of transcription elongation is a mechanism to repress transcription in LADs. Furthermore, the chromatin remodeler complex BAF and the activation complex Mediator can work both as activators and repressors in LADs, depending on the local context and possibly by rewiring heterochromatin. Our data indicate that the fundamental regulators of transcription and chromatin remodeling, rather than interaction with NL proteins, play a major role in transcription regulation within LADs., (© 2024. The Author(s).)

X chromosome inactivation (XCI) generates clonal heterogeneity within XX individuals. Combined with sequence variation between human X chromosomes, XCI gives rise to intra-individual clonal diversity, whereby two sets of clones express mutually exclusive sequence variants present on one or the other X chromosome. Here we ask whether such clones merely co-exist or potentially interact with each other to modulate the contribution of X-linked diversity to organismal development. Focusing on X-linked coding variation in the human STAG2 gene, we show that Stag2 variant clones contribute to most tissues at the expected frequencies but fail to form lymphocytes in Stag2 WT Stag2 variant mouse models. Unexpectedly, the absence of Stag2 variant clones from the lymphoid compartment is due not solely to cell-intrinsic defects but requires continuous competition by Stag2 WT clones. These findings show that interactions between epigenetically diverse clones can operate in an XX individual to shape the contribution of X-linked genetic diversity in a cell-type-specific manner., (© 2024. The Author(s).)

Cohesin holds together the sister chromatids from DNA replication onwards. How cohesion is established has long remained a black box. Through recent studies, a model is emerging in which a replisome-cohesin encounter results in the establishment of cohesive linkages at sites of replication termination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In this issue of Molecular Cell, two papers provide insight into atypical structural maintenance of chromosomes protein complexes (SMCs). Jeppsson et al. 1 link Smc5/6 to supercoiled DNA, and Roisné-Hamelin et al. 2 show how Wadjet SMC bends and cleaves invading DNAs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Genomic instability arising from defective responses to DNA damage 1 or mitotic chromosomal imbalances 2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology 1 ., (© 2024. The Author(s).)

A potential mechanism of DNA loop extrusion by molecular motors is discussed.

Many cellular processes require large-scale rearrangements of chromatin structure. Structural maintenance of chromosomes (SMC) protein complexes are molecular machines that can provide structure to chromatin. These complexes can connect DNA elements in cis, walk along DNA, build and processively enlarge DNA loops and connect DNA molecules in trans to hold together the sister chromatids. These DNA-shaping abilities place SMC complexes at the heart of many DNA-based processes, including chromosome segregation in mitosis, transcription control and DNA replication, repair and recombination. In this Review, we discuss the latest insights into how SMC complexes such as cohesin, condensin and the SMC5-SMC6 complex shape DNA to direct these fundamental chromosomal processes. We also consider how SMC complexes, by building chromatin loops, can counteract the natural tendency of alike chromatin regions to cluster. SMC complexes thus control nuclear organization by participating in a molecular tug of war that determines the architecture of our genome., (© 2023. Springer Nature Limited.)

In the early stages of mitosis, cohesin is released from chromosome arms but not from centromeres. The protection of centromeric cohesin by SGO1 maintains the sister chromatid cohesion that resists the pulling forces of microtubules until all chromosomes are attached in a bipolar manner to the mitotic spindle. Here we present the X-ray crystal structure of a segment of human SGO1 bound to a conserved surface of the cohesin complex. SGO1 binds to a composite interface formed by the SA2 and SCC1 RAD21 subunits of cohesin. SGO1 shares this binding interface with CTCF, indicating that these distinct chromosomal regulators control cohesin through a universal principle. This interaction is essential for the localization of SGO1 to centromeres and protects centromeric cohesin against WAPL-mediated cohesin release. SGO1-cohesin binding is maintained until the formation of microtubule-kinetochore attachments and is required for faithful chromosome segregation and the maintenance of a stable karyotype., (© 2023. The Author(s).)

Cohesin structures the genome through the formation of chromatin loops and by holding together the sister chromatids. The acetylation of cohesin's SMC3 subunit is a dynamic process that involves the acetyltransferase ESCO1 and deacetylase HDAC8. Here we show that this cohesin acetylation cycle controls the three-dimensional genome in human cells. ESCO1 restricts the length of chromatin loops, and of architectural stripes emanating from CTCF sites. HDAC8 conversely promotes the extension of such loops and stripes. This role in controlling loop length turns out to be distinct from the canonical role of cohesin acetylation that protects against WAPL-mediated DNA release. We reveal that acetylation controls the interaction of cohesin with PDS5A to restrict chromatin loop length. Our data support a model in which this PDS5A-bound state acts as a brake that enables the pausing and restart of loop enlargement. The cohesin acetylation cycle hereby provides punctuation in the process of genome folding., (© 2022. The Author(s).)

SMC protein complexes are molecular machines that provide structure to chromosomes. These complexes bridge DNA elements and by doing so build DNA loops in cis and hold together the sister chromatids in trans. We discuss how drastic conformational changes allow SMC complexes to build such intricate DNA structures. The tight regulation of these complexes controls fundamental chromosomal processes such as transcription, recombination, repair, and mitosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The genome consists of regions of transcriptionally active euchromatin and more silent heterochromatin. We reveal that the formation of heterochromatin domains requires cohesin turnover on DNA. Stabilization of cohesin on DNA through depletion of its release factor WAPL leads to a near-complete loss of heterochromatin domains. We observe the opposite phenotype in cells deficient for subunits of the Mediator-CDK module, with an almost binary partition of the genome into dense H3K9me3 domains, and regions devoid of H3K9me3 spanning the rest of the genome. We suggest that the Mediator-CDK module might contribute to gene expression by limiting the formation of dense heterochromatin domains. WAPL deficiency prevents the formation of heterochromatin domains, and allows for gene expression even in the absence of the Mediator-CDK subunit MED12. We propose that cohesin and Mediator affect heterochromatin in different ways to enable the correct distribution of epigenetic marks, and thus to ensure proper gene expression., (© 2022. The Author(s).)

Cohesin and CTCF are key to the 3D folding of interphase chromosomes. Cohesin forms chromatin loops via loop extrusion, a process that involves the formation and enlargement of DNA loops. The architectural protein CTCF controls this process by acting as an anchor for chromatin looping. How CTCF controls cohesin has long been a mystery. Recent work shows that CTCF dictates chromatin looping via a direct interaction of its N-terminus with cohesin. CTCF's ability to regulate chromatin looping turns out to also be partially dependent on several RNA-binding domains. In this review, we discuss recent insights and consider how cohesin and CTCF together may orchestrate the folding of the genome into chromosomal loops., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Conformation capture-approaches like Hi-C can elucidate chromosome structure at a genome-wide scale. Hi-C datasets are large and require specialised software. Here, we present GENOVA: a user-friendly software package to analyse and visualise chromosome conformation capture (3C) data. GENOVA is an R-package that includes the most common Hi-C analyses, such as compartment and insulation score analysis. It can create annotated heatmaps to visualise the contact frequency at a specific locus and aggregate Hi-C signal over user-specified genomic regions such as ChIP-seq data. Finally, our package supports output from the major mapping-pipelines. We demonstrate the capabilities of GENOVA by analysing Hi-C data from HAP1 cell lines in which the cohesin-subunits SA1 and SA2 were knocked out. We find that ΔSA1 cells gain intra-TAD interactions and increase compartmentalisation. ΔSA2 cells have longer loops and a less compartmentalised genome. These results suggest that cohesin SA1 forms longer loops, while cohesin SA2 plays a role in forming and maintaining intra-TAD interactions. Our data supports the model that the genome is provided structure in 3D by the counter-balancing of loop formation on one hand, and compartmentalization on the other hand. By differentially controlling loops, cohesin SA1 and cohesin SA2 therefore also affect nuclear compartmentalization. We show that GENOVA is an easy to use R-package, that allows researchers to explore Hi-C data in great detail., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Cohesin catalyses the folding of the genome into loops that are anchored by CTCF 1 . The molecular mechanism of how cohesin and CTCF structure the 3D genome has remained unclear. Here we show that a segment within the CTCF N terminus interacts with the SA2-SCC1 subunits of human cohesin. We report a crystal structure of SA2-SCC1 in complex with CTCF at a resolution of 2.7 Å, which reveals the molecular basis of the interaction. We demonstrate that this interaction is specifically required for CTCF-anchored loops and contributes to the positioning of cohesin at CTCF binding sites. A similar motif is present in a number of established and newly identified cohesin ligands, including the cohesin release factor WAPL 2,3 . Our data suggest that CTCF enables the formation of chromatin loops by protecting cohesin against loop release. These results provide fundamental insights into the molecular mechanism that enables the dynamic regulation of chromatin folding by cohesin and CTCF.

Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

From the dynamic interphase genome to compacted mitotic chromosomes, DNA is organized by the conserved SMC complexes cohesin and condensin. The picture is emerging that these complexes structure the genome through a shared basic principle that involves the formation and processive enlargement of chromatin loops. This appears to be an asymmetric process, in which the complex anchors at the base of a loop and then enlarges the loop in a one-sided manner. We discuss the latest insights into how ATPase-driven conformational changes within these complexes may enlarge loops, and consider how asymmetric DNA reeling can bring together genomic elements in a symmetric manner., (Copyright © 2019. Published by Elsevier Ltd.)

Chromatin folding contributes to the regulation of genomic processes such as gene activity. Existing conformation capture methods characterize genome topology through analysis of pairwise chromatin contacts in populations of cells but cannot discern whether individual interactions occur simultaneously or competitively. Here we present multi-contact 4C (MC-4C), which applies Nanopore sequencing to study multi-way DNA conformations of individual alleles. MC-4C distinguishes cooperative from random and competing interactions and identifies previously missed structures in subpopulations of cells. We show that individual elements of the β-globin superenhancer can aggregate into an enhancer hub that can simultaneously accommodate two genes. Neighboring chromatin domain loops can form rosette-like structures through collision of their CTCF-bound anchors, as seen most prominently in cells lacking the cohesin-unloading factor WAPL. Here, massive collision of CTCF-anchored chromatin loops is believed to reflect 'cohesin traffic jams'. Single-allele topology studies thus help us understand the mechanisms underlying genome folding and functioning.

What drives the formation of chromatin loops has been a long-standing question in chromosome biology. Recent work provides major insight into the basic principles behind loop formation. Structural maintenance of chromosomes (SMC) complexes, that are conserved from bacteria to humans, are key to this process. The SMC family includes condensin and cohesin, which structure chromosomes to enable mitosis and long-range gene regulation. We discuss novel insights into the mechanism of loop formation and the implications for how these complexes ultimately shape chromosomes. A picture is emerging in which these complexes form small loops that they then processively enlarge. It appears that SMC complexes act by family-wide basic principles, with complex-specific levels of control., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin's DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesin's Scc1, Smc1, and Smc3 subunits is created during S and destroyed at anaphase through Scc1 cleavage by separase. Cohesin's association with chromosomes is controlled by opposing activities: loading by Scc2/4 complex and release by a separase-independent releasing activity as well as by cleavage. Coentrapment of sister DNAs at replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We show that mutations capable of bypassing Eco1 in Smc1, Smc3, Scc1, Wapl, Pds5, and Scc3 subunits reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. This process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The X shape of chromosomes is one of the iconic images in biology. Cohesin actually connects the sister chromatids along their entire length, from S phase until mitosis. Then, cohesin's antagonist Wapl allows the separation of chromosome arms by opening a DNA exit gate in cohesin rings. Centromeres are protected against this removal activity, resulting in the X shape of mitotic chromosomes. The destruction of the remaining centromeric cohesin by Separase triggers chromosome segregation. We review the two-phase regulation of cohesin removal and discuss how this affects chromosome alignment and decatenation in mitosis and cohesin reloading in the next cell cycle., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Chromosomal instability is a driving force for heterogeneity within tumours. A recent study shows that boosting sister chromatid cohesion corrects chromosomal instability in pRB-deficient cancer cells. This key finding provides an important lead to make tumours more susceptible to anti-cancer drugs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The classical X shape of mitotic human chromosomes is the consequence of two distinct waves of cohesin removal. First, during prophase and prometaphase, the bulk of cohesin is driven from chromosome arms by the cohesin antagonist WAPL. This arm-specific cohesin removal is referred to as the prophase pathway [1-4]. The subsequent cleavage of the remaining centromeric cohesin by Separase is known to be the trigger for anaphase onset [5-7]. Remarkably the biological purpose of the prophase pathway is unknown. We find that this pathway is essential for two key mitotic processes. First, it is important to focus Aurora B at centromeres to allow efficient correction of erroneous microtubule-kinetochore attachments. In addition, it is required to facilitate the timely decatenation of sister chromatids. As a consequence, WAPL-depleted cells undergo anaphase with segregation errors, including both lagging chromosomes and catenanes, resulting in micronuclei and DNA damage. Stable WAPL depletion arrests cells in a p53-dependent manner but causes p53-deficient cells to become highly aneuploid. Our data show that the WAPL-dependent prophase pathway is essential for proper chromosome segregation and is crucial to maintain genomic integrity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Biallelic mutations of the von Hippel-Lindau (VHL) gene are the most common cause of sporadic and inherited renal cell carcinoma (RCC). Loss of VHL has been reported to affect cell proliferation by deregulating cell cycle-associated proteins. We report that the VHL gene product (pVHL) inhibits E2F1 expression at both mRNA and protein level in zebrafish and human RCC cells, while loss of VHL increases E2F1 expression in patient kidney tumour tissue and RCC cells, resulting in a delay of cell cycle progression. RCCs from von Hippel-Lindau patients with known germline VHL mutations express significantly more E2F1 compared to sporadic RCCs with either clear-cell (cc) or non-cc histology. Analysis of 138 primary RCCs reveals that E2F1 expression is significantly higher in tumours with a diameter ≤7 cm and with a favourable American Joint Committee on Cancer (AJCC) stage. The expression of E2F1 in RCC significantly correlates with p27 expression, suggesting that increased expression of E2F1 in RCC induces tumour cell senescence via p27. Cox regression analysis shows significant prediction of E2F1 expression for disease-free survival and overall survival, implying that E2F1 expression in kidney tumour is a novel prognostic factor for patients with RCC., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Mutant RAS (RAS(V12)) is known to transform most immortal cells but to induce premature senescence in primary cells. RAS(V12)-induced senescence in murine cells depends on the induction of the ARF/p53 and the retinoblastoma (Rb) family tumor suppressor pathways. We and others have shown previously that oncogene-induced senescence in vitro can be used as a tool to identify new cancer-related genes. In addition, we have shown that oncogene-induced senescence corresponds to an in vivo tumor suppressive mechanism. Therefore, we extended our search for novel genes that bypass of RAS(V12)-induced senescence, with the help of a previously designed unbiased functional screen with cDNA expression libraries. In this screen, we expected to find new mediators feeding into the p53 or Rb pathways or novel signaling factors. We report here the identification of leukemia/lymphoma related factor (Lrf) encoding a transcription factor with a BTB/POZ domain and Krüppel-like zinc fingers. This gene was previously identified as a potential oncogene that is overexpressed in human cancer. We find that LRF enhances E2F-dependent transcription and that it synergizes with RAS(V12) in activating E2F. Indeed, LRF-mediated bypass of RAS(V12)-induced senescence is accompanied by the induction of several E2F-target genes, including Cyclin E, Cyclin A and p107. Unexpectedly, LRF exerted this activity independent of several critical senescence inducers, such as p19(ARF), p21(CIP) and p16(INK4A). We show that CYCLIN E is necessary for LRF-mediated bypass, suggesting that it corresponds to a critical mediator of LRF-driven oncogenic transformation. Thus, LRF bypasses RAS(V12)-induced senescence in a CYCLIN E-dependent manner, which conceivably contributes to its role in cancer.

Cohesin's Smc1, Smc3, and Scc1 subunits form a tripartite ring that entraps sister DNAs. Scc3, Pds5, and Rad61 (Wapl) are regulatory subunits that control this process. We describe here smc3, scc3, pds5, and rad61 mutations that permit yeast cell proliferation and entrapment of sister DNAs by cohesin rings in the absence of Eco1, an acetyl transferase normally essential for establishing sister chromatid cohesion. The smc3 mutations cluster around and include a highly conserved lysine (K113) close to Smc3's ATP-binding pocket, which, together with K112, is acetylated by Eco1. Lethality caused by mutating both residues to arginine is suppressed by the scc3, pds5, and rad61 mutants. Scc3, Pds5, and Rad61 form a complex and inhibit entrapment of sister DNAs by a process involving the "K112/K113" surface on Smc3's ATPase. According to this model, Eco1 promotes sister DNA entrapment partly by relieving an antiestablishment activity associated with Scc3, Pds5, and Rad61.

Activation of E2F transcription factors is thought to drive the expression of genes essential for the transition of cells from G1 to S phase and for the initiation of DNA replication. However, this textbook view of E2Fs is increasingly under challenge. Here we discuss an alternative model for how E2Fs may work.

Krüppel-like factors are transcriptional regulators that influence several cellular functions, including proliferation. Recent studies have shown that one family member, KLF4, can function both as a tumour suppressor and an oncogene. The ability of KLF4 to affect the levels of expression of the cell-cycle regulator p21 seems to be involved, in that this protein might function as a switch that determines the outcome of KLF4 signalling. Is this role of p21 restricted to KLF4, or does p21 represent a nodal point for signals from multiple other factors with opposing functions in cancer?

KLF4 (GKLF/EZF) encodes a transcription factor that is associated with both tumour suppression and oncogenesis. We describe the identification of KLF4 in a functional genomic screen for genes that bypass RAS(V12)-induced senescence. However, in untransformed cells, KLF4 acts as a potent inhibitor of proliferation. KLF4-induced arrest is bypassed by oncogenic RAS(V12) or by the RAS target cyclin-D1. Remarkably, inactivation of the cyclin-D1 target and the cell-cycle inhibitor p21CIP1 not only neutralizes the cytostatic action of KLF4, but also collaborates with KLF4 in oncogenic transformation. Conversely, KLF4 suppresses the expression of p53 by directly acting on its promoter, thereby allowing for RAS(V12)-mediated transformation and causing resistance to DNA-damage-induced apoptosis. Consistently, KLF4 depletion from breast cancer cells restores p53 levels and causes p53-dependent apoptosis. These results unmask KLF4 as a regulator of p53 that oncogenically transforms cells as a function of p21CIP1 status. Furthermore, they provide a mechanistic explanation for the context-dependent oncogenic or tumour-suppressor functions of KLF4.

The p16(INK4a)/pRB/E2F and p19(ARF)/p53 tumor suppressor pathways are disrupted in most human cancers. Both p19(ARF) and p53 are required for the induction of senescence in primary mouse embryonic fibroblasts (MEFs), but little is known about their downstream targets. Disruption of E2F-mediated transcriptional repression in MEFs caused a general increase in the expression of E2F target genes, including p19ARF. We detected no contribution of E2F-mediated transactivation in this setting, indicating that a predominant role of endogenous E2F in asynchronously growing primary MEFs is to repress its target genes. Moreover, relief of transcriptional repression by E2F rendered MEFs resistant to senescence induced by either p19(ARF), p53, or RAS(V12). Thus, E2F transcriptional repressor complexes are critical downstream targets of antiproliferative p19(ARF)/p53 signaling.