Thymine DNA glycosylase regulates cell-cycle-driven p53 transcriptional control in pluripotent cells.

Cell cycle progression is linked to transcriptome dynamics and variations in the response of pluripotent cells to differentiation cues, mostly through unknown determinants. Here, we characterized the cell-cycle-associated transcriptome and proteome of mouse embryonic stem cells (mESCs) in naive ground state. We found that the thymine DNA glycosylase (TDG) is a cell-cycle-regulated co-factor of the tumor suppressor p53. Furthermore, TDG and p53 co-bind ESC-specific cis-regulatory elements and thereby control transcription of p53-dependent genes during self-renewal. We determined that the dynamic expression of TDG is required to promote the cell-cycle-associated transcriptional heterogeneity. Moreover, we demonstrated that transient depletion of TDG influences cell fate decisions during the early differentiation of mESCs. Our findings reveal an unanticipated role of TDG in promoting molecular heterogeneity during the cell cycle and highlight the central role of protein dynamics for the temporal control of cell fate during development.

RETRACTED: Vulnerability of glioblastoma cells to catastrophic vacuolization and death induced by a small molecule.

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer with marginal life expectancy. Based on the assumption that GBM cells gain functions not necessarily involved in the cancerous process, patient-derived glioblastoma cells (GCs) were screened to identify cellular processes amenable for development of targeted treatments. The quinine-derivative NSC13316 reliably and selectively compromised viability. Synthetic chemical expansion reveals delicate structure-activity relationship and analogs with increased potency, termed Vacquinols. Vacquinols stimulate death by membrane ruffling, cell rounding, massive macropinocytic vacuole accumulation, ATP depletion, and cytoplasmic membrane rupture of GCs. The MAP kinase MKK4, identified by a shRNA screen, represents a critical signaling node. Vacquinol-1 displays excellent in vivo pharmacokinetics and brain exposure, attenuates disease progression, and prolongs survival in a GBM animal model. These results identify a vulnerability to massive vacuolization that can be targeted by small molecules and point to the possible exploitation of this process in the design of anticancer therapies.

EPOP Functionally Links Elongin and Polycomb in Pluripotent Stem Cells.

The cellular plasticity of pluripotent stem cells is thought to be sustained by genomic regions that display both active and repressive chromatin properties. These regions exhibit low levels of gene expression, yet the mechanisms controlling these levels remain unknown. Here, we describe Elongin BC as a binding factor at the promoters of bivalent sites. Biochemical and genome-wide analyses show that Elongin BC is associated with Polycomb Repressive Complex 2 (PRC2) in pluripotent stem cells. Elongin BC is recruited to chromatin by the PRC2-associated factor EPOP (Elongin BC and Polycomb Repressive Complex 2 Associated Protein, also termed C17orf96, esPRC2p48, E130012A19Rik), a protein expressed in the inner cell mass of the mouse blastocyst. Both EPOP and Elongin BC are required to maintain low levels of expression at PRC2 genomic targets. Our results indicate that keeping the balance between activating and repressive cues is a more general feature of chromatin in pluripotent stem cells than previously appreciated.

The Bivalent Genome: Characterization, Structure, and Regulation.

An intricate molecular machinery is at the core of gene expression regulation in every cell. During the initial stages of organismal development, the coordinated activation of diverse transcriptional programs is crucial and must be carefully executed to shape every organ and tissue. Bivalent promoters and poised enhancers are regulatory regions decorated with histone marks that are associated with both positive and negative transcriptional outcomes. These apparently contradictory signals are important for setting bivalent genes in a poised state, which is subsequently resolved during differentiation into either active or repressive states. We discuss the origins of bivalent promoters and the mechanisms implicated in their acquisition and maintenance. We further review how the presence of bivalent marks influences genome architecture. Finally, we highlight the potential link between bivalency and cancer which could drive biomedical research in disease etiology and treatment.

The changing chromatome as a driver of disease: A panoramic view from different methodologies.

Chromatin-bound proteins underlie several fundamental cellular functions, such as control of gene expression and the faithful transmission of genetic and epigenetic information. Components of the chromatin proteome (the "chromatome") are essential in human life, and mutations in chromatin-bound proteins are frequently drivers of human diseases, such as cancer. Proteomic characterization of chromatin and de novo identification of chromatin interactors could, thus, reveal important and perhaps unexpected players implicated in human physiology and disease. Recently, intensive research efforts have focused on developing strategies to characterize the chromatome composition. In this review, we provide an overview of the dynamic composition of the chromatome, highlight the importance of its alterations as a driving force in human disease (and particularly in cancer), and discuss the different approaches to systematically characterize the chromatin-bound proteome in a global manner.

Acute hydroxyurea-induced replication blockade results in replisome components disengagement from nascent DNA without causing fork collapse.

During S phase, replication forks can encounter several obstacles that lead to fork stalling, which if persistent might result in fork collapse. To avoid this collapse and to preserve the competence to restart, cells have developed mechanisms that maintain fork stability upon replication stress. In this study, we aimed to understand the mechanisms involved in fork stability maintenance in non-transformed human cells by performing an isolation of proteins on nascent DNA-mass spectrometry analysis in hTERT-RPE cells under different replication stress conditions. Our results show that acute hydroxyurea-induced replication blockade causes the accumulation of large amounts of single-stranded DNA at the fork. Remarkably, this results in the disengagement of replisome components from nascent DNA without compromising fork restart. Notably, Cdc45-MCM-GINS helicase maintains its integrity and replisome components remain associated with chromatin upon acute hydroxyurea treatment, whereas replisome stability is lost upon a sustained replication stress that compromises the competence to restart.

UHRF1 Licensed Self-Renewal of Active Adult Neural Stem Cells.

Adult neurogenesis in the brain continuously seeds new neurons throughout life, but how homeostasis of adult neural stem cells (NSCs) is maintained is incompletely understood. Here, we demonstrate that the DNA methylation adapter ubiquitin-like, containing PHD and RING finger domains-1 (UHRF1) is expressed in, and regulates proliferation of, the active but not quiescent pool of adult neural progenitor cells. Mice with a neural stem cell-specific deficiency in UHRF1 exhibit a massive depletion of neurogenesis resulting in a collapse of formation of new neurons. In the absence of UHRF1, NSCs unexpectedly remain in the cell cycle but with a 17-fold increased cell cycle length due to a failure of replication phase entry caused by promoter demethylation and derepression of Cdkn1a, which encodes the cyclin-dependent kinase inhibitor p21. UHRF1 does not affect the proportion progenitor cells active within the cell cycle but among these cells, UHRF1 is critical for licensing replication re-entry. Therefore, this study shows that a UHRF1-Cdkn1a axis is essential for the control of stem cell self-renewal and neurogenesis in the adult brain. Stem Cells 2018;36:1736-1751.

New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress.

Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C(Cdh1) is activated upon severe replication stress. Activation of APC/C(Cdh1) prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C(Cdh1) activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C(Cdh1) activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C(Cdh1) function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity.

Identification of a large protein network involved in epigenetic transmission in replicating DNA of embryonic stem cells.

Pluripotency of embryonic stem cells (ESCs) is maintained by transcriptional activities and chromatin modifying complexes highly organized within the chromatin. Although much effort has been focused on identifying genome-binding sites, little is known on their dynamic association with chromatin across cell divisions. Here, we used a modified version of the iPOND (isolation of proteins at nascent DNA) technology to identify a large protein network enriched at nascent DNA in ESCs. This comprehensive and unbiased proteomic characterization in ESCs reveals that, in addition to the core replication machinery, proteins relevant for pluripotency of ESCs are present at DNA replication sites. In particular, we show that the chromatin remodeller HDAC1-NuRD complex is enriched at nascent DNA. Interestingly, an acute block of HDAC1 in ESCs leads to increased acetylation of histone H3 lysine 9 at nascent DNA together with a concomitant loss of methylation. Consistently, in contrast to what has been described in tumour cell lines, these chromatin marks were found to be stable during cell cycle progression of ESCs. Our results are therefore compatible with a rapid deacetylation-coupled methylation mechanism during the replication of DNA in ESCs that may participate in the preservation of pluripotency of ESCs during replication.

Sox2 and Mitf cross-regulatory interactions consolidate progenitor and melanocyte lineages in the cranial neural crest.

The cellular origin and molecular mechanisms regulating pigmentation of head and neck are largely unknown. Melanocyte specification is controlled by the transcriptional activity of Mitf, but no general logic has emerged to explain how Mitf and progenitor transcriptional activities consolidate melanocyte and progenitor cell fates. We show that cranial melanocytes arise from at least two different cellular sources: initially from nerve-associated Schwann cell precursors (SCPs) and later from a cellular source that is independent of nerves. Unlike the midbrain-hindbrain cluster from which melanoblasts arise independently of nerves, a large center of melanocytes in and around cranial nerves IX-X is derived from SCPs, as shown by genetic cell-lineage tracing and analysis of ErbB3-null mutant mice. Conditional gain- and loss-of-function experiments show genetically that cell fates in the neural crest involve both the SRY transcription factor Sox2 and Mitf, which consolidate an SCP progenitor or melanocyte fate by cross-regulatory interactions. A gradual downregulation of Sox2 in progenitors during development permits the differentiation of both neural crest- and SCP-derived progenitors into melanocytes, and an initial small pool of nerve-associated melanoblasts expands in number and disperses under the control of endothelin receptor B (Ednrb) and Wnt5a signaling.

Polycomb function in early mouse development.

Epigenetic factors are crucial for ensuring proper chromatin dynamics during the initial stages of embryo development. Among these factors, the Polycomb group (PcG) of proteins plays a key role in establishing correct transcriptional programmes during mouse embryogenesis. PcG proteins are classified into two complexes: Polycomb repressive complex 1 (PRC1) and PRC2. Both complexes decorate histone proteins with distinct post-translational modifications (PTMs) that are predictive of a silent transcriptional chromatin state. In recent years, a critical adaptation of the classical techniques to analyse chromatin profiles and to study biochemical interactions at low-input resolution has allowed us to deeply explore PcG molecular mechanisms in the very early stages of mouse embryo development- from fertilisation to gastrulation, and from zygotic genome activation (ZGA) to specific lineages differentiation. These advancements provide a foundation for a deeper understanding of the fundamental role Polycomb complexes play in early development and have elucidated the mechanistic dynamics of PRC1 and PRC2. In this review, we discuss the functions and molecular mechanisms of both PRC1 and PRC2 during early mouse embryo development, integrating new studies with existing knowledge. Furthermore, we highlight the molecular functionality of Polycomb complexes from ZGA through gastrulation, with a particular focus on non-canonical imprinted and bivalent genes, and Hox cluster regulation.

The Wnt-dependent master regulator NKX1-2 controls mouse pre-implantation development.

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.

Isolation of Chromatin Proteins by Genome Capture.

Control of gene expression and the faithful transmission of genetic and epigenetic information rely on chromatin-bound proteins. These include the polycomb group of proteins, which can display a remarkable variability in their composition. Alterations in the chromatin-bound protein compositions are relevant for physiology and human disease. Thus, chromatin-bound proteomic profiling can be instrumental for understanding fundamental cellular processes and for identifying therapeutic targets. Inspired by biochemical strategies for the isolation of proteins on nascent DNA (iPOND) and the very similar DNA-mediated chromatin pull-down (Dm-ChP), we described a method for the identification of Protein on Total DNA (iPOTD) for bulk chromatome profiling. Here, we update our iPOTD method and, in particular, detail the experimental procedure for the isolation of chromatin proteins for mass spectrometry-based proteomic analysis.

Quantitative Comparison of Multiple Chromatin Immunoprecipitation-Sequencing (ChIP-seq) Experiments with spikChIP.

The chromatin immunoprecipitation coupled with the next-generation sequencing (ChIP-seq) is a powerful technique that enables to characterize the genomic distribution of chromatin-associated proteins, histone posttranslational modifications, and histone variants. However, in the absence of a reference control for monitoring experimental and biological variations, the standard ChIP-seq scheme is unable to accurately assess changes in the abundance of chromatin targets across different experimental samples. To overcome this limitation, the combination of external spike-in material with the experimental chromatin is offered as an effective solution for quantitative comparison of ChIP-seq data across different conditions. Here, we detail (i) the experimental protocol for preparing quality control spike-in chromatin from Drosophila melanogaster cells and (ii) the computational protocol to compare ChIP-seq samples with spike-in based on the use of the spikChIP software.

DYRK family of protein kinases: evolutionary relationships, biochemical properties, and functional roles.

Dual-specificity tyrosine-regulated kinases (DYRKs) comprise a family of protein kinases within the CMGC group of the eukaryotic kinome. Members of the DYRK family are found in 4 (animalia, plantae, fungi, and protista) of the 5 main taxa or kingdoms, and all DYRK proteins studied to date share common structural, biochemical, and functional properties with their ancestors in yeast. Recent work on DYRK proteins indicates that they participate in several signaling pathways critical for developmental processes and cell homeostasis. In this review, we focus on the DYRK family of proteins from an evolutionary, biochemical, and functional point of view and discuss the most recent, relevant, and controversial contributions to the study of these kinases.

Functional and Pathological Roles of AHCY.

Adenosylhomocysteinase (AHCY) is a unique enzyme and one of the most conserved proteins in living organisms. AHCY catalyzes the reversible break of S-adenosylhomocysteine (SAH), the by-product and a potent inhibitor of methyltransferases activity. In mammals, AHCY is the only enzyme capable of performing this reaction. Controlled subcellular localization of AHCY is believed to facilitate local transmethylation reactions, by removing excess of SAH. Accordingly, AHCY is recruited to chromatin during replication and active transcription, correlating with increasing demands for DNA, RNA, and histone methylation. AHCY deletion is embryonic lethal in many organisms (from plants to mammals). In humans, AHCY deficiency is associated with an incurable rare recessive disorder in methionine metabolism. In this review, we focus on the AHCY protein from an evolutionary, biochemical, and functional point of view, and we discuss the most recent, relevant, and controversial contributions to the study of this enzyme.

SpikChIP: a novel computational methodology to compare multiple ChIP-seq using spike-in chromatin.

In order to evaluate cell- and disease-specific changes in the interacting strength of chromatin targets, ChIP-seq signal across multiple conditions must undergo robust normalization. However, this is not possible using the standard ChIP-seq scheme, which lacks a reference for the control of biological and experimental variabilities. While several studies have recently proposed different solutions to circumvent this problem, substantial analytical differences among methodologies could hamper the experimental reproducibility and quantitative accuracy. Here, we propose a computational method to accurately compare ChIP-seq experiments, with exogenous spike-in chromatin, across samples in a genome-wide manner by using a local regression strategy (spikChIP). In contrast to the previous methodologies, spikChIP reduces the influence of sequencing noise of spike-in material during ChIP-seq normalization, while minimizes the overcorrection of non-occupied genomic regions in the experimental ChIP-seq. We demonstrate the utility of spikChIP with both histone and non-histone chromatin protein, allowing us to monitor for experimental reproducibility and the accurate ChIP-seq comparison of distinct experimental schemes. spikChIP software is available on GitHub (https://github.com/eblancoga/spikChIP).

Polycomb Factor PHF19 Controls Cell Growth and Differentiation Toward Erythroid Pathway in Chronic Myeloid Leukemia Cells.

Polycomb group (PcG) of proteins are a group of highly conserved epigenetic regulators involved in many biological functions, such as embryonic development, cell proliferation, and adult stem cell determination. PHD finger protein 19 (PHF19) is an associated factor of Polycomb repressor complex 2 (PRC2), often upregulated in human cancers. In particular, myeloid leukemia cell lines show increased levels of PHF19, yet little is known about its function. Here, we have characterized the role of PHF19 in myeloid leukemia cells. We demonstrated that PHF19 depletion decreases cell proliferation and promotes chronic myeloid leukemia (CML) differentiation. Mechanistically, we have shown how PHF19 regulates the proliferation of CML through a direct regulation of the cell cycle inhibitor p21. Furthermore, we observed that MTF2, a PHF19 homolog, partially compensates for PHF19 depletion in a subset of target genes, instructing specific erythroid differentiation. Taken together, our results show that PHF19 is a key transcriptional regulator for cell fate determination and could be a potential therapeutic target for myeloid leukemia treatment.