The homeobox transcription factor DUXBL controls exit from totipotency.

In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.

Genome architecture and totipotency: An intertwined relation during early embryonic development.

Chromosomes are not randomly packed and positioned into the nucleus but folded in higher-order chromatin structures with defined functions. However, the genome of a fertilized embryo undergoes a dramatic epigenetic reprogramming characterized by extensive chromatin relaxation and the lack of a defined three-dimensional structure. This reprogramming is followed by a slow genome refolding that gradually strengthens the chromatin architecture during preimplantation development. Interestingly, genome refolding during early development coincides with a progressive loss of developmental potential suggesting a link between chromatin organization and cell plasticity. In agreement, loss of chromatin architecture upon depletion of the insulator transcription factor CTCF in embryonic stem cells led to the upregulation of the transcriptional program found in totipotent cells of the embryo, those with the highest developmental potential. This essay will discuss the impact of genome folding in controlling the expression of transcriptional programs involved in early development and their plastic-associated features.

The ETS transcription factor ERF controls the exit from the naïve pluripotent state in a MAPK-dependent manner.

The naïve epiblast transitions to a pluripotent primed state during embryo implantation. Despite the relevance of the FGF pathway during this period, little is known about the downstream effectors regulating this signaling. Here, we examined the molecular mechanisms coordinating the naïve to primed transition by using inducible ESC to genetically eliminate all RAS proteins. We show that differentiated RASKO ESC remain trapped in an intermediate state of pluripotency with naïve-associated features. Elimination of the transcription factor ERF overcomes the developmental blockage of RAS-deficient cells by naïve enhancer decommissioning. Mechanistically, ERF regulates NANOG expression and ensures naïve pluripotency by strengthening naïve transcription factor binding at ESC enhancers. Moreover, ERF negatively regulates the expression of the methyltransferase DNMT3B, which participates in the extinction of the naïve transcriptional program. Collectively, we demonstrated an essential role for ERF controlling the exit from naïve pluripotency in a MAPK-dependent manner during the progression to primed pluripotency.

CTCF is a barrier for 2C-like reprogramming.

Totipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.

TRF2-mediated telomere protection is dispensable in pluripotent stem cells.

In mammals, telomere protection is mediated by the essential protein TRF2, which binds chromosome ends and ensures genome integrity1,2. TRF2 depletion results in end-to-end chromosome fusions in all cell types that have been tested so far. Here we find that TRF2 is dispensable for the proliferation and survival of mouse embryonic stem (ES) cells. Trf2-/- (also known as Terf2) ES cells do not exhibit telomere fusions and can be expanded indefinitely. In response to the deletion of TRF2, ES cells exhibit a muted DNA damage response that is characterized by the recruitment of γH2AX-but not 53BP1-to telomeres. To define the mechanisms that control this unique DNA damage response in ES cells, we performed a CRISPR-Cas9-knockout screen. We found a strong dependency of TRF2-null ES cells on the telomere-associated protein POT1B and on the chromatin remodelling factor BRD2. Co-depletion of POT1B or BRD2 with TRF2 restores a canonical DNA damage response at telomeres, resulting in frequent telomere fusions. We found that TRF2 depletion in ES cells activates a totipotent-like two-cell-stage transcriptional program that includes high levels of ZSCAN4. We show that the upregulation of ZSCAN4 contributes to telomere protection in the absence of TRF2. Together, our results uncover a unique response to telomere deprotection during early development.

A Chemical Screen Identifies Compounds Capable of Selecting for Haploidy in Mammalian Cells.

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.

ERF deletion rescues RAS deficiency in mouse embryonic stem cells.

MEK inhibition in combination with a glycogen synthase kinase-3ß (GSK3ß) 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.

A p53-dependent response limits the viability of mammalian haploid cells.

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.

Dynamics of histone H3.3 deposition in proliferating and senescent cells reveals a DAXX-dependent targeting to PML-NBs important for pericentromeric heterochromatin organization.

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.

Antiproliferative effects of kisspeptin­10 depend on artificial GPR54 (KISS1R) expression levels.

Kisspeptins are peptides derived from the metastasis suppressor gene KISS1 interacting with GPR54 as their corresponding receptor. The KISS1/GPR54 system is one regulator of cellular motility mechanisms leading to decreased migration and invasion. Its role in cell proliferation processes is not clearly understood. In this study, breast cancer cell lines, T47D, ZR75-1, MDA­MB­231, MDA­MB­435s, MDA­MB­453, HCC 70, HCC 1806, HCC 1937 and MCF­7, were investigated for their endogenous GPR54 expression by immunocytochemistry, RT­PCR and western blot analysis. The effect of kisspeptin­10 on proliferation was measured in MDA­MB­231, MDA­MB­435s, HCC 1806 and MCF­7 cells. Further experiments on proliferation were carried out with cells transfected with GPR54. All of the tested breast cancer cell lines expressed GPR54 in different amounts. No effects on proliferation were detected in the breast cancer cells expressing the receptor endogenously. In transfected neuronal cells overexpressing GPR54, proliferation was significantly inhibited by kisspeptin­10. The results indicate that the antiproliferative action of kisspeptin depends on the nature of GPR54 expression. The effect was detected in an artificial system of cells transfected with GPR54 and not in cells expressing the receptor endogenously. Thus, the antiproliferative action of kisspeptin seems not to be important for pathophysiological processes.

Kisspeptin-10 inhibits bone-directed migration of GPR54-positive breast cancer cells: Evidence for a dose-window effect.

OBJECTIVES: The KiSS-1 gene product is absent or expressed at low level in metastatic breast cancer compared with their nonmetastatic counterparts. A deca-peptide derived from the KiSS-1 gene product, designated kisspeptin-10 (Kp-10), activates a receptor coupled to Gαq subunits (GPR54 or KiSS-1R). In this study we have analyzed whether Kp-10 treatment affects bone-directed migration of GPR54-positive breast cancer cells. METHODS: GPR54 expression was analyzed using immune cytochemistry. Bone-directed breast cancer cell invasion was measured by assessment of the breast cancer cell migration rate through an artificial basement membrane. Chemokine receptor CXCR4 and stromal cell-derived factor-1 (SDF-1) mRNA expression was quantified using semi-quantitative RT-PCR. CXCR4 protein expression and SDF-1 protein secretion were measured using the western blot technique. RESULTS: Breast cancer cell invasion was increased when cocultured with MG63 osteoblast-like cells. Treatment with KP-10 reduced the ability to invade a reconstituted basement membrane and to migrate in response to the cellular stimulus. This effect was significant in a dose-window of 10⁻9 M to 10⁻¹¹ M. Searching for the molecular mechanisms we found that KP-10 treatment significantly reduces expression of the chemokine receptor CXCR4 by the breast cancer cells. In addition, expression and secretion of its ligand SDF-1 by the MG63 cells were significantly reduced. Furthermore, SDF-1-induced CXCR4 signaling was down-regulated. CONCLUSIONS: These data represent the first report that KP-10 inhibits bone-directed migration of GPR54-positive breast cancer cells. In addition, we found evidence for a KP-10 dose-window effect. Furthermore, the SDF-1/CXCR4 system seems to be involved in the anti-migratory action of KP-10.