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Chromosomes have an intrinsic tendency to segregate into compartments, forming long‐distance contacts between loci of similar chromatin states. How genome compartmentalization is regulated remains elusive. Here, comparison of mouse ground‐state embryonic stem cells (ESCs) characterized by open and active chromatin, and advanced serum ESCs with a more closed and repressed genome, reveals distinct regulation of their genome organization due to differential dependency on BAZ2A/TIP5, a component of the chromatin remodeling complex NoRC. On ESC chromatin, BAZ2A interacts with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. BAZ2A associates with chromatin sub‐domains within the active A compartment, which intersect through long‐range contacts. We found that ground‐state chromatin selectively requires BAZ2A to limit the invasion of active domains into repressive compartments. BAZ2A depletion increases chromatin accessibility at B compartments. Furthermore, BAZ2A regulates H3K27me3 genome occupancy in a TOP2A‐dependent manner. Finally, ground‐state ESCs require BAZ2A for growth, differentiation, and correct expression of developmental genes. Our results uncover the propensity of open chromatin domains to invade repressive domains, which is counteracted by chromatin remodeling to establish genome partitioning and preserve cell identity., The NoRC chromatin remodeller subunit BAZ2A restricts active chromatin compartments and secures developmental gene expression in ESCs.

Naive pluripotency is established in preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of naive pluripotency. 2i culture has optimized this state, leading to a gene signature and DNA hypomethylation closely comparable to preimplantation epiblast, the developmental ground state. Here we show that Pramel7 (PRAME-like 7), a protein highly expressed in the inner cell mass (ICM) but expressed at low levels in ESCs, targets for proteasomal degradation UHRF1, a key factor for DNA methylation maintenance. Increasing Pramel7 expression in serum-cultured ESCs promotes a preimplantation epiblast-like gene signature, reduces UHRF1 levels and causes global DNA hypomethylation. Pramel7 is required for blastocyst formation and its forced expression locks ESCs in pluripotency. Pramel7/UHRF1 expression is mutually exclusive in ICMs whereas Pramel7-knockout embryos express high levels of UHRF1. Our data reveal an as-yet-unappreciated dynamic nature of DNA methylation through proteasome pathways and offer insights that might help to improve ESC culture to reproduce in vitro the in vivo ground-state pluripotency.

Chromosomes have an intrinsic tendency to segregate into compartments, forming long-distance contacts between loci of similar chromatin states. However, how genome compartmentalization is regulated remains elusive. We analyzed two closely and developmentally related pluripotent cell types: ground-state ESCs that have an open and active chromatin and developmentally advanced ESCs that display a more closed and repressed state. We show that these two ESC types differ in their regulation of genome organization due to their differential dependency on TIP5, a component of the chromatin remodeling complex NoRC. We show that TIP5 interacts on ESC chromatin with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. TIP5 associates with sub-domains within the active A compartment that strongly intersect through long-range contacts in ESCs. We found that only ground-state chromatin requires TIP5 to limit the invasion of active domains into repressive compartments. Depletion of TIP5 increased chromatin accessibility particularly at B compartments and decreased their repressive features. Furthermore, TIP5 acts as a barrier for the repressive H3K27me3 spreading, a process that also requires TOP2A activity. Finally, ground-state ESCs require TIP5 for growth, differentiation capacity, and correct expression of developmental genes. Our results revealed the propensity of open and active chromatin domains to invade repressive domains, an action counteracted by chromatin remodeling and the relief of chromatin torsional stress. This effort in controlling open/active chromatin domains is required to establish active and repressed genome partitioning and preserves cell function and identity.

Introduction The transcription factor Sall4 is a well-known developmental regulator involved in embryonic patterning and stem cell maintenance. More recently Sall4 has been suggested to be an oncofetal gene due to being expressed in many fetal tissues and malignant tumours but only rarely in normal adult tissues. The re-expression of such genes in cancer might reflect crucial processes during development, which are reactivated in neoplasias such as increased migratory and invasive capacities. Sall4 is discussed as novel marker and target in some solid tumours and haematological malignancies, however its role in melanoma is unknown. Material and methods To address a putative role of Sall4 in melanoma, we used the Tyr::Nras Q61K INK4a −/− murine melanoma model, which spontaneously develops metastatic melanoma. Those mice were crossed with Tyr::Cre ERT2 Sall4 lox/lox R26R::GFP mice, allowing us to conditionally ablate Sall4 in melanocytes and to trace Cre activity via GFP expression. We further used human melanoma patient-derived primary cell cultures for in vitro experiments and the same cell lines as xenographs in immunocompromised mice. Results and discussions Mice lacking Sall4 did not form primary tumours compared to the control animals however strikingly had more GFP +melanoma metastases in the lungs. Sall4 lox/wt animals formed tumours like control animals but also showed more lung metastases. Through RNA Sequencing on siSALL4-treated human melanoma cell lines we found that SALL4 decrease correlates with upregulation of melanoma invasiveness genes and functionally increased invasiveness in vitro . We further found by Co-IP that SALL4 binds to members of the histone deacetylase family (HDACs) and by running a ChIP-Seq for H3K27ac, we could indeed verify that knocking down SALL4 lead to increased histone acetylation of enhancer regions of the mentioned invasiveness genes. Interestingly, HDAC inhibitor (HDACi) treatment in vitro resulted in an invasiveness gene expression pattern very similar to SALL4 knock down and HDACi treatment of xenograph tumours also lead to an expression profile hinting towards increased invasiveness in vivo . Conclusion All in all we found that downregulation of Sall4 in melanoma leads to increased acetylation and expression of invasiveness genes in vitro and increased metastasis in vivo, with both phenotypes being closely mimicked by HDAC inhibitor application.

Genotoxic treatments in human cells consistently induce uncoupling of replication forks and their remodeling into four-way junctions by the RAD51 recombinase., Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy.

Nature Cell Biology http://dx.doi.org/10.1038/ncb3554 (2017); published online 12 June 2017; corrected after print 21 June 2017 In the version of this Article originally published, the following affiliation was omitted for Sarah Wyck: Clinic of Reproductive Medicine, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland.

Long non‐coding RNAs (lncRNAs) have been implicated in the regulation of chromatin conformation and epigenetic patterns. lncRNA expression levels are widely taken as an indicator for functional properties. However, the role of RNA processing in modulating distinct features of the same lncRNA is less understood. The establishment of heterochromatin at rRNA genes depends on the processing of IGS‐rRNA into pRNA, a reaction that is impaired in embryonic stem cells (ESCs) and activated only upon differentiation. The production of mature pRNA is essential since it guides the repressor TIP5 to rRNA genes, and IGS‐rRNA abolishes this process. Through screening for IGS‐rRNA‐binding proteins, we here identify the RNA helicase DHX9 as a regulator of pRNA processing. DHX9 binds to rRNA genes only upon ESC differentiation and its activity guides TIP5 to rRNA genes and establishes heterochromatin. Remarkably, ESCs depleted of DHX9 are unable to differentiate and this phenotype is reverted by the addition of pRNA, whereas providing IGS‐rRNA and pRNA mutants deficient for TIP5 binding are not sufficient. Our results reveal insights into lncRNA biogenesis during development and support a model in which the state of rRNA gene chromatin is part of the regulatory network that controls exit from pluripotency and initiation of differentiation pathways.