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.

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.

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.

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.

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.

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.

Chromatin-Bound Proteome Profiling by Genome Capture.

De novo identification of chromatin interactors can reveal unexpected pathways relevant to physiology and human disease. Inspired by the DNA mediated chromatin pull-down (Dm-ChP) technology (also known as iPOND [isolation of proteins on nascent DNA]) for the proteomic characterization of nascent DNA, we have recently reported a new experimental protocol that allows for the identification of proteins on total DNA (iPOTD) for bulk chromatome profiling and de novo identification of chromatin-bound proteins. Here, we detail a step-by-step protocol to survey the cellular chromatin-bound proteome in a simple, robust, and unbiased manner. For complete details on the use and execution of this protocol, please refer to Aranda et al. (2019).

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.

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.

GATA2 Promotes Hematopoietic Development and Represses Cardiac Differentiation of Human Mesoderm.

In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.

Chromatin capture links the metabolic enzyme AHCY to stem cell proliferation.

Profiling the chromatin-bound proteome (chromatome) in a simple, direct, and reliable manner might be key to uncovering the role of yet uncharacterized chromatin factors in physiology and disease. Here, we have designed an experimental strategy to survey the chromatome of proliferating cells by using the DNA-mediated chromatin pull-down (Dm-ChP) technology. Our approach provides a global view of cellular chromatome under normal physiological conditions and enables the identification of chromatin-bound proteins de novo. Integrating Dm-ChP with genomic and functional data, we have discovered an unexpected chromatin function for adenosylhomocysteinase, a major one-carbon pathway metabolic enzyme, in gene activation. Our study reveals a new regulatory axis between the metabolic state of pluripotent cells, ribosomal protein production, and cell division during the early phase of embryo development, in which the metabolic flux of methylation reactions is favored in a local milieu.

Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate.

Here, we report DNA methylation and hydroxymethylation dynamics at nucleotide resolution using C/EBPα-enhanced reprogramming of B cells into induced pluripotent cells (iPSCs). We observed successive waves of hydroxymethylation at enhancers, concomitant with a decrease in DNA methylation, suggesting active demethylation. Consistent with this finding, ablation of the DNA demethylase Tet2 almost completely abolishes reprogramming. C/EBPα, Klf4, and Tfcp2l1 each interact with Tet2 and recruit the enzyme to specific DNA sites. During reprogramming, some of these sites maintain high levels of 5hmC, and enhancers and promoters of key pluripotency factors become demethylated as early as 1 day after Yamanaka factor induction. Surprisingly, methylation changes precede chromatin opening in distinct chromatin regions, including Klf4 bound sites, revealing a pioneer factor activity associated with alternation in DNA methylation. Rapid changes in hydroxymethylation similar to those in B cells were also observed during compound-accelerated reprogramming of fibroblasts into iPSCs, highlighting the generality of our observations.

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.

NoRC Recruitment by H2A.X Deposition at rRNA Gene Promoter Limits Embryonic Stem Cell Proliferation.

Embryonic stem cells (ESCs) display an abbreviated cell cycle, resulting in a short doubling time and rapid proliferation. The histone variant H2A.X is critical for proliferation of stem cells, although mechanistic insights have remained obscure. Here, we show that H2A.X defines the rate of mouse ESC proliferation independently of the DNA damage response pathway, and it associates with three major chromatin-modifying complexes. Our functional and biochemical analyses demonstrate that H2A.X-associated factors mediate the H2A.X-dependent effect on ESC proliferation and involve the nucleolar remodeling complex (NoRC). A specific H2A.X deposition at rDNA promoters determines the chromatin recruitment of the NoRC, histone modifications, the rRNA transcription, and the rate of proliferation. Collectively, our results suggest that NoRC assembly by H2A.X deposition at rRNA promoters silences transcription, and this represents an important regulatory component for ESC proliferation.