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Summary The recent availability of somatic haploid cell lines has provided a unique tool for genetic studies in mammals. However, the percentage of haploid cells rapidly decreases in these cell lines, which we recently showed is due to their overgrowth by diploid cells present in the cultures. Based on this property, we have now performed a phenotypic chemical screen in human haploid HAP1 cells aiming to identify compounds that facilitate the maintenance of haploid cells. Our top hit was 10-Deacetyl-baccatin-III (DAB), a chemical precursor in the synthesis of Taxol, which selects for haploid cells in HAP1 and mouse haploid embryonic stem cultures. Interestingly, DAB also enriches for diploid cells in mixed cultures of diploid and tetraploid cells, including in the colon cancer cell line DLD-1, revealing a general strategy for selecting cells with lower ploidy in mixed populations of mammalian cells., Graphical Abstract, Highlights • Mammalian haploid cell cultures become progressively enriched in diploid cells • DAB, a precursor of Taxol, facilitates the maintenance of haploidy • DAB selects for cells with lower ploidy in mixed cultures of mammalian cells • Statins accelerate the gradual loss of haploid cells in culture, Cultures of haploid animal cell lines become progressively enriched in diploid cells. By conducting a chemical screen in HAP1 cells, Olbrich et al. identify compounds that facilitate the maintenance of haploid cells and a general strategy to select for cells with lower ploidy in mixed cultures of mammalian cells.

MEK inhibition in combination with a glycogen synthase kinase-3β (GSK3β) inhibitor, referred as the 2i condition, favors pluripotency in embryonic stem cells (ESCs). However, the mechanisms by which the 2i condition limits ESC differentiation and whether RAS proteins are involved in this phenomenon remain poorly understood. Here we show that RAS nullyzygosity reduces the growth of mouse ESCs (mESCs) and prohibits their differentiation. Upon RAS deficiency or MEK inhibition, ERF (E twenty-six 2 [Ets2]-repressive factor), a transcriptional repressor from the ETS domain family, translocates to the nucleus, where it binds to the enhancers of pluripotency factors and key RAS targets. Remarkably, deletion of Erf rescues the proliferative defects of RAS-devoid mESCs and restores their capacity to differentiate. Furthermore, we show that Erf loss enables the development of RAS nullyzygous teratomas. In summary, this work reveals an essential role for RAS proteins in pluripotency and identifies ERF as a key mediator of the response to RAS/MEK/ERK inhibition in mESCs. We thank Cian Lynch, Jorge Monsech, and Diego Megias for their help with microarray, ChIP-seq, and high-throughput microscopy analyses. We also thank Dr. Manuel Serrano and Dr. André Nussenzweig for their input on the manuscript, and Dr. Diego Sanz for his support throughout the project. C.M.-R. was funded by a PhD fellowship from La Caixa Foundation, T.O. was funded by a PhD fellowship from the Boehringer Ingelheim Fonds, and S.R. was funded by a Ramon y Cajal contract (RYC-2011-09242). Research was funded by Fundación Botín and Banco Santander through its Santander Universities Global Division; grants from the Spanish Ministry of Economy and Competitiveness (SAF2011-23753 and SAF2014- 57791-REDC; these projects were cofinanced with European Fonds Européen de Développement Économique et Régional [FEDER] funds), Fundació La Marato de TV3, Howard Hughes Medical Institute, and the European Research Council (ERC- 617840) to O.F.-C.; and grants from the Spanish Ministryof Economy and Competitiveness (SAF2013-49147-P and SAF2016-80874-P; these projects were cofinanced with European FEDER funds) to S.R. Author contributions: C.M.-R. and S.R. conducted most of the experiments; T.O. helped with the characterization of RASlox/lox mESCs and with ERF localization studies; E.L. helped with ChIP-seq experiments; M.D., S.O., and M.B. contributed to the work on RAS-deficient cells; M.V.-S. provided technical help; O.D. helped with genomics experiments and bioinformatics analysis; and S.R. and O.F.-C. coordinated the study and wrote the manuscript. Sí

The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include near-haploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell-sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells.

Oncogene-induced senescence is a permanent cell cycle arrest characterized by extensive chromatin reorganization. Here, we investigated the specific targeting and dynamics of histone H3 variants in human primary senescent cells. We show that newly synthesized epitope-tagged H3.3 is incorporated in senescent cells but does not accumulate in senescence-associated heterochromatin foci (SAHF). Instead, we observe that new H3.3 colocalizes with its specific histone chaperones within the promyelocytic leukemia nuclear bodies (PML-NBs) and is targeted to PML-NBs in a DAXX-dependent manner both in proliferating and senescent cells. We further show that overexpression of DAXX enhances targeting of H3.3 in large PML-NBs devoid of transcriptional activity and promotes the accumulation of HP1, independently of H3K9me3. Loss of H3.3 from pericentromeric heterochromatin upon DAXX or PML depletion suggests that the targeting of H3.3 to PML-NBs is implicated in pericentromeric heterochromatin organization. Together, our results underline the importance of the replication-independent chromatin assembly pathway for histone replacement in non-dividing senescent cells and establish PML-NBs as important regulatory sites for the incorporation of new H3.3 into chromatin.