Abstract The relationship between cell cycle length and differentiation competence has been studied in developmental biology, particularly in embryonic stem cells, which show a very short G1 phase required to maintain pluripotency. How and whether this principle operates in cancer— where the cell cycle is deregulated and G1 is frequently shortened — and whether it can be pharmacologically exploited for therapy, has not been explored. Here we show that the duration of G1 is a causal determinant of chromatin state in cancer cells and that extending G1 creates a therapeutic window for epigenetic drugs. Using acute myeloid leukemia (AML) as a model, we demonstrate that low-dose palbociclib — at concentrations well below those required for cytostatic arrest — extends G1 without halting proliferation. This modest prolongation reshapes the histone modification landscape: repressive marks (H3K9me2/3, H4K20me2/3) increase while acetylation decreases, and chromatin accessibility rises broadly in the euchromatic compartment. Naturally slow-cycling AML lines share this epigenetic signature regardless of their oncogenic driver mutations, and pharmacologically extending G1 in fast-cycling cells recapitulates it — establishing G1 length as a causal regulator of the cancer epigenome rather than a passive correlate. To identify epigenetic vulnerabilities created by G1 extension, we performed complementary drug and CRISPR-Cas9 screens in G1-extended AML cells. Both approaches converged on the histone demethylase LSD1 (KDM1A): slow-cycling AML cells are intrinsically sensitive to LSD1 inhibition, while fast-cycling cells become sensitive when G1 is prolonged. The combination of low-dose palbociclib and LSD1 inhibition triggers differentiation and significantly prolongs survival in AML xenograft models. p21 (CDKN1A) emerges as the central molecular determinant of this response. In slow-cycling AML cells, p21 is highly expressed and its knockdown abolishes LSD1 inhibitor sensitivity. Structure-function analysis using p21 mutants separates the two known activities of p21: the CDK-inhibitory function (which extends G1) is required for sensitization, whereas the PCNA-binding function is dispensable. Three pharmacological routes converge on the same endpoint — CDK inhibition, G1 extension, and a differentiation-competent chromatin state: direct CDK4/6 inhibition by palbociclib, p21 overexpression, and p21 induction through treatment with HDAC or EZH1/2 inhibitors. Palbociclib bypasses the requirement for p21 entirely, confirming that G1 length itself — not p21 as a protein — is the critical variable. Mechanistically, the combination of G1 extension and LSD1 inhibition yield a qualitatively distinct chromatin state rather than an additive one. ATAC-seq reveals thousands of combination-exclusive accessible regions, enriched for footprints of myeloid differentiation transcription factors including SPI1/PU.1, IRF1, and STAT1/2. A double-lock principle governs this remodeling: palbociclib drives the removal of repressive marks (H3K9me3 and H3K27me3), while LSD1 inhibition installs active marks at the newly accessible regions. The ncBAF chromatin remodeling complex, identified in our CRISPR screen and validated by knockout of its essential subunits BRD9 and SMARCD1, is specifically required for this response. Loss of ncBAF abolishes the combination-induced chromatin remodeling and differentiation program but does not affect the initial G1 extension or retinoic acid–induced differentiation, indicating that ncBAF specifically couples cell-cycle modulation to chromatin remodeling rather than acting as a general differentiation factor. The principle generalizes beyond AML. In melanoma, breast cancer, and small-cell lung cancer (SCLC), sensitivity to LSD1 inhibition tracks with p21 expression and cycling speed. Primary melanoma samples stratified by p21 expression recapitulate the same pattern: p21-high, slow-cycling cells are sensitive; p21-low, fast-cycling cells are resistant but can be sensitized by palbociclib cotreatment. Cisplatin-induced drug-tolerant persister (DTP) cells — which emerge as a slow-cycling, chemo-resistant population and upregulate both p21 and LSD1 — become vulnerable to LSD1 inhibition and are eradicated by the combination. In melanoma patient-derived xenograft (PDX) models, p21-high tumors respond to LSD1 inhibitor monotherapy, while p21-low tumors are sensitized by palbociclib cotreatment, with p21 knockdown abolishing the response. Together, these findings establish cell-cycle duration as a tunable regulator of the cancer epigenome and demonstrate that pharmacological G1 extension converts cytostatic CDK4/6 inhibition into an epigenetic sensitization strategy. Both fast-proliferating and slow-cycling tumor compartments — including drug-resistant persister cells — can be targeted by matching the epigenomic state to the appropriate combination of cell-cycle modulators and epigenetic drugs. p21 emerges as a candidate biomarker for patient stratification. More broadly, our work repositions the cell cycle from a passive conduit for proliferation signals to an active, druggable regulator of chromatin fate, with implications that extend from cancer therapy to stem cell biology and regenerative medicine.

Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment in mice, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. By profiling the chromatin-bound proteome (chromatome) through genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos leads to developmental arrest, reduction of H3K9me3, and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early 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.

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.

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.

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.

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.

Human naive pluripotent stem cells are generally believed to possess an unrestricted capacity to differentiate into both embryonic and extraembryonic lineages. However, two new studies now uncover a role for the Polycomb repressive complex 2 (PRC2) as a lineage gatekeeper that shields the potency of these 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.

Abstract 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).

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.

During mammalian development the pluripotency appears in cells within the inner cell mass (ICM) of the blastocyst and disappears during gastrulation, corresponding to a 4- to 5-day period in mice and approximately 2 weeks in humans. During this period the pluripotent tissue, the epiblast, expands from about 10 homogeneous cells to several hundred. This rapid increase in cell number correlates with the transcriptional and epigenetic rewiring of pluripotent escapees that differentiate during gastrulation. The differentiation processes have long been attributed mainly as a coordinated response to extrinsic signaling cues. However, in the last years, the cell cycle has emerged as an intrinsic determinant that contributes to cell fate decision-making. In this review, we discuss the recent discoveries in this area and, in particular, how the cell cycle is regulated and impacts in the biology of the pluripotent cells.

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-bystep 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).

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.

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.

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 technical and analytical differences among methodologies could hamper the experimental reproducibility and the quantitative accuracy. Here we propose a local regression strategy to accurately normalize ChIP-seq data in a genome-wide manner. Overall, our proposed experimental and computational standard for comparative ChIP-seq (Comp-ChIP-seq) will increase experimental reproducibility, thereby reducing this major confounding factor in interpreting ChIP-seq results.

ABSTRACT Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is a pivotal technique for understanding the functionality of the chromatin-bound factors and for mapping the functional elements of the genome. 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 technical and analytical differences among methodologies could hamper the experimental reproducibility. Here we provide a practical binary decision-making process to experimentally implement a normalizing method for comparative ChIP-seq across different samples. In addition, we evaluate side-by-side the current computational approaches for normalizing using a reference internal genome. Finally, we propose a local regression strategy to accurately normalize ChIP-seq data in a genome-wide manner. Overall, our proposed experimental and computational standard for comparative ChIP-seq (Comp-ChIP-seq) will increase experimental reproducibility, thereby reducing this major confounding factor in interpreting ChIP-seq results.

Two reports examine how a protein complex that adds transcription-repressing marks to histone proteins is potently inhibited by the protein EZHIP during the maturation of sperm and egg cells and in a type of brain cancer.

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.

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 con tradictory 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.