Colorectal Cancer Cells Enter a Diapause-like DTP State to Survive Chemotherapy.

Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.

Regulation, functions and transmission of bivalent chromatin during mammalian development.

Cells differentiate and progress through development guided by a dynamic chromatin landscape that mediates gene expression programmes. During development, mammalian cells display a paradoxical chromatin state: histone modifications associated with gene activation (trimethylated histone H3 Lys4 (H3K4me3)) and with gene repression (trimethylated H3 Lys27 (H3K27me3)) co-occur at promoters of developmental genes. This bivalent chromatin modification state is thought to poise important regulatory genes for expression or repression during cell-lineage specification. In this Review, we discuss recent work that has expanded our understanding of the molecular basis of bivalent chromatin and its contributions to mammalian development. We describe the factors that establish bivalency, especially histone-lysine N-methyltransferase 2B (KMT2B) and Polycomb repressive complex 2 (PRC2), and consider evidence indicating that PRC1 shapes bivalency and may contribute to its transmission between generations. We posit that bivalency is a key feature of germline and embryonic stem cells, as well as other types of stem and progenitor cells. Finally, we discuss the relevance of bivalent chromtin to human development and cancer, and outline avenues of future research.

A LINE1-Nucleolin Partnership Regulates Early Development and ESC Identity.

Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.

Systematic identification of barriers to human iPSC generation.

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) holds enormous promise for regenerative medicine. To elucidate endogenous barriers limiting this process, we systematically dissected human cellular reprogramming by combining a genome-wide RNAi screen, innovative computational methods, extensive single-hit validation, and mechanistic investigation of relevant pathways and networks. We identify reprogramming barriers, including genes involved in transcription, chromatin regulation, ubiquitination, dephosphorylation, vesicular transport, and cell adhesion. Specific a disintegrin and metalloproteinase (ADAM) proteins inhibit reprogramming, and the disintegrin domain of ADAM29 is necessary and sufficient for this function. Clathrin-mediated endocytosis can be targeted with small molecules and opposes reprogramming by positively regulating TGF-ß signaling. Genetic interaction studies of endocytosis or ubiquitination reveal that barrier pathways can act in linear, parallel, or feedforward loop architectures to antagonize reprogramming. These results provide a global view of barriers to human cellular reprogramming.

Author Correction: Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3-7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8-10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11-15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.

iPS cells: insights into basic biology.

The discovery that adult somatic cells can be induced to become pluripotent by overexpression of a few key transcription factors provides an exciting new window into the basic biology of pluripotency and differentiation.

Open chromatin in pluripotency and reprogramming.

Pluripotent stem cells can be derived from embryos or induced from adult cells by reprogramming. They are unique among stem cells in that they can give rise to all cell types of the body. Recent findings indicate that a particularly 'open' chromatin state contributes to maintenance of pluripotency. Two principles are emerging: specific factors maintain a globally open chromatin state that is accessible for transcriptional activation; and other chromatin regulators contribute locally to the silencing of lineage-specific genes until differentiation is triggered. These same principles may apply during reacquisition of an open chromatin state upon reprogramming to pluripotency, and during de-differentiation in cancer.

Inhibition of mTOR induces a paused pluripotent state.

Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2-3 days around the time of blastocyst implantation. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent-able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.

What Doesn't Kill You Makes You Stronger: Transposons as Dual Players in Chromatin Regulation and Genomic Variation.

Transposable elements (TEs) are sequences currently or historically mobile, and are present across all eukaryotic genomes. A growing interest in understanding the regulation and function of TEs has revealed seemingly dichotomous roles for these elements in evolution, development, and disease. On the one hand, many gene regulatory networks owe their organization to the spread of cis-elements and DNA binding sites through TE mobilization during evolution. On the other hand, the uncontrolled activity of transposons can generate mutations and contribute to disease, including cancer, while their increased expression may also trigger immune pathways that result in inflammation or senescence. Interestingly, TEs have recently been found to have novel essential functions during mammalian development. Here, the function and regulation of TEs are discussed, with a focus on LINE1 in mammals. It is proposed that LINE1 is a beneficial endogenous dual regulator of gene expression and genomic diversity during mammalian development, and that both of these functions may be detrimental if deregulated in disease contexts.

Sex-specific epigenetic profile of inner cell mass of mice conceived in vivo or by IVF.

The preimplantation stage of development is exquisitely sensitive to environmental stresses, and changes occurring during this developmental phase may have long-term health effects. Animal studies indicate that IVF offspring display metabolic alterations, including hypertension, glucose intolerance and cardiac hypertrophy, often in a sexual dimorphic fashion. The detailed nature of epigenetic changes following in-vitro culture is, however, unknown. This study was performed to evaluate the epigenetic (using whole-genome bisulfite sequencing (WGBS) and assay for transposase-accessible chromatin using sequencing (ATAC-seq)) and transcriptomic changes (using RNA-seq) occurring in the inner cell mass (ICM) of male or female mouse embryos generated in vivo or by IVF. We found that the ICM of IVF embryos, compared to the in-vivo ICM, differed in 3% of differentially methylated regions (DMRs), of which 0.1% were located on CpG islands. ATAC-seq revealed that 293 regions were more accessible and 101 were less accessible in IVF embryos, while RNA-seq revealed that 21 genes were differentially regulated in IVF embryos. Functional enrichment analysis revealed that stress signalling (STAT and NF-kB signalling), developmental processes and cardiac hypertrophy signalling showed consistent changes in WGBS and ATAC-seq platforms. In contrast, male and female embryos showed minimal changes. Male ICM had an increased number of significantly hyper-methylated DMRs, while only 27 regions showed different chromatin accessibility and only one gene was differentially expressed. In summary, this study provides the first comprehensive analysis of DNA methylation, chromatin accessibility and RNA expression changes induced by IVF in male and female ICMs. This dataset can be of value to all researchers interested in the developmental origin of health and disease (DOHaD) hypothesis and might lead to a better understanding of how early embryonic manipulation may affect adult health.

Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells.

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(II) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

Chd1 is essential for the high transcriptional output and rapid growth of the mouse epiblast.

The pluripotent mammalian epiblast undergoes unusually fast cell proliferation. This rapid growth is expected to generate a high transcriptional demand, but the underlying mechanisms remain unknown. We show here that the chromatin remodeler Chd1 is required for transcriptional output and development of the mouse epiblast. Chd1(-/-) embryos exhibit proliferation defects and increased apoptosis, are smaller than controls by E5.5 and fail to grow, to become patterned or to gastrulate. Removal of p53 allows progression of Chd1(-/-) mutants only to E7.0-8.0, highlighting the crucial requirement for Chd1 during early post-implantation development. Chd1(-/-) embryonic stem cells (ESCs) have a self-renewal defect and a genome-wide reduction in transcriptional output at both known mRNAs and intergenic transcripts. These transcriptional defects were only uncovered when cell number-normalized approaches were used, and correlate with a lower engagement of RNAP II with transcribed genes in Chd1(-/-) ESCs. We further show that Chd1 directly binds to ribosomal DNA, and that both Chd1(-/-) epiblast cells in vivo and ESCs in vitro express significantly lower levels of ribosomal RNA. In agreement with these findings, mutant cells in vivo and in vitro exhibit smaller and more elongated nucleoli. Thus, the RNA output by both Pol I and II is reduced in Chd1(-/-) cells. Our data indicate that Chd1 promotes a globally elevated transcriptional output required to sustain the distinctly rapid growth of the mouse epiblast.

Emergence of hematopoietic stem and progenitor cells involves a Chd1-dependent increase in total nascent transcription.

Lineage specification during development involves reprogramming of transcriptional states, but little is known about how this is regulated in vivo. The chromatin remodeler chomodomain helicase DNA-binding protein 1 (Chd1) promotes an elevated transcriptional output in mouse embryonic stem cells. Here we report that endothelial-specific deletion of Chd1 leads to loss of definitive hematopoietic progenitors, anemia, and lethality by embryonic day (E)15.5. Mutant embryos contain normal numbers of E10.5 intraaortic hematopoietic clusters that express Runx1 and Kit, but these clusters undergo apoptosis and fail to mature into blood lineages in vivo and in vitro. Hematopoietic progenitors emerging from the aorta have an elevated transcriptional output relative to structural endothelium, and this elevation is Chd1-dependent. In contrast, hematopoietic-specific deletion of Chd1 using Vav-Cre has no apparent phenotype. Our results reveal a new paradigm of regulation of a developmental transition by elevation of global transcriptional output that is critical for hemogenesis and may play roles in other contexts.

HiTSelect: a comprehensive tool for high-complexity-pooled screen analysis.

Genetic screens of an unprecedented scale have recently been made possible by the availability of high-complexity libraries of synthetic oligonucleotides designed to mediate either gene knockdown or gene knockout, coupled with next-generation sequencing. However, several sources of random noise and statistical biases complicate the interpretation of the resulting high-throughput data. We developed HiTSelect, a comprehensive analysis pipeline for rigorously selecting screen hits and identifying functionally relevant genes and pathways by addressing off-target effects, controlling for variance in both gene silencing efficiency and sequencing depth of coverage and integrating relevant metadata. We document the superior performance of HiTSelect using data from both genome-wide RNAi and CRISPR/Cas9 screens. HiTSelect is implemented as an open-source package, with a user-friendly interface for data visualization and pathway exploration. Binary executables are available at http://sourceforge.net/projects/hitselect/, and the source code is available at https://github.com/diazlab/HiTSelect.

Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development.

Histone variants can replace canonical histones in the nucleosome and modify chromatin structure and gene expression. The histone variant H3.3 preferentially associates with active chromatin and has been implicated in the regulation of a diverse range of developmental processes. However, the mechanisms by which H3.3 may regulate gene activity are unclear and gene duplication has hampered an analysis of H3.3 function in mouse. Here, we report that the specific knockdown of H3.3 in fertilized mouse zygotes leads to developmental arrest at the morula stage. This phenotype can be rescued by exogenous H3.3 but not by canonical H3.1 mRNA. Loss of H3.3 leads to over-condensation and mis-segregation of chromosomes as early as the two-cell stage, with corresponding high levels of aneuploidy, but does not appear to affect zygotic gene activation at the two-cell stage or lineage gene transcription at the morula stage. H3.3-deficient embryos have significantly reduced levels of markers of open chromatin, such as H3K36me2 and H4K16Ac. Importantly, a mutation in H3.3K36 that disrupts H3K36 methylation (H3.3K36R) does not rescue the H3.3 knockdown (KD) phenotype. In addition, H3.3 KD embryos have increased incorporation of linker H1. Knockdown of Mof (Kat8), an acetyltransferase specific for H4K16, similarly leads to excessive H1 incorporation. Remarkably, pan-H1 RNA interference (RNAi) partially rescues the chromosome condensation of H3.3 KD embryos and allows development to the blastocyst stage. These results reveal that H3.3 mediates a balance between open and condensed chromatin that is crucial for the fidelity of chromosome segregation during early mouse development.

Chd1 regulates open chromatin and pluripotency of embryonic stem cells.

An open chromatin largely devoid of heterochromatin is a hallmark of stem cells. It remains unknown whether an open chromatin is necessary for the differentiation potential of stem cells, and which molecules are needed to maintain open chromatin. Here we show that the chromatin remodelling factor Chd1 is required to maintain the open chromatin of pluripotent mouse embryonic stem cells. Chd1 is a euchromatin protein that associates with the promoters of active genes, and downregulation of Chd1 leads to accumulation of heterochromatin. Chd1-deficient embryonic stem cells are no longer pluripotent, because they are incapable of giving rise to primitive endoderm and have a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. Our results indicate that Chd1 is essential for open chromatin and pluripotency of embryonic stem cells, and for somatic cell reprogramming to the pluripotent state.

Polycomb-like 3 promotes polycomb repressive complex 2 binding to CpG islands and embryonic stem cell self-renewal.

Polycomb repressive complex 2 (PRC2) trimethylates lysine 27 of histone H3 (H3K27me3) to regulate gene expression during diverse biological transitions in development, embryonic stem cell (ESC) differentiation, and cancer. Here, we show that Polycomb-like 3 (Pcl3) is a component of PRC2 that promotes ESC self-renewal. Using mass spectrometry, we identified Pcl3 as a Suz12 binding partner and confirmed Pcl3 interactions with core PRC2 components by co-immunoprecipitation. Knockdown of Pcl3 in ESCs increases spontaneous differentiation, yet does not affect early differentiation decisions as assessed in teratomas and embryoid bodies, indicating that Pcl3 has a specific role in regulating ESC self-renewal. Consistent with Pcl3 promoting PRC2 function, decreasing Pcl3 levels reduces H3K27me3 levels while overexpressing Pcl3 increases H3K27me3 levels. Furthermore, chromatin immunoprecipitation and sequencing (ChIP-seq) reveal that Pcl3 co-localizes with PRC2 core component, Suz12, and depletion of Pcl3 decreases Suz12 binding at over 60% of PRC2 targets. Mutation of conserved residues within the Pcl3 Tudor domain, a domain implicated in recognizing methylated histones, compromises H3K27me3 formation, suggesting that the Tudor domain of Pcl3 is essential for function. We also show that Pcl3 and its paralog, Pcl2, exist in different PRC2 complexes but bind many of the same PRC2 targets, particularly CpG islands regulated by Pcl3. Thus, Pcl3 is a component of PRC2 critical for ESC self-renewal, histone methylation, and recruitment of PRC2 to a subset of its genomic sites.

Smad2 is essential for maintenance of the human and mouse primed pluripotent stem cell state.

Human embryonic stem cells and mouse epiblast stem cells represent a primed pluripotent stem cell state that requires TGF-ß/activin signaling. TGF-ß and/or activin are commonly thought to regulate transcription through both Smad2 and Smad3. However, the different contributions of these two Smads to primed pluripotency and the downstream events that they may regulate remain poorly understood. We addressed the individual roles of Smad2 and Smad3 in the maintenance of primed pluripotency. We found that Smad2, but not Smad3, is required to maintain the undifferentiated pluripotent state. We defined a Smad2 regulatory circuit in human embryonic stem cells and mouse epiblast stem cells, in which Smad2 acts through binding to regulatory promoter sequences to activate Nanog expression while in parallel repressing autocrine bone morphogenetic protein signaling. Increased autocrine bone morphogenetic protein signaling caused by Smad2 down-regulation leads to cell differentiation toward the trophectoderm, mesoderm, and germ cell lineages. Additionally, induction of Cdx2 expression, as a result of decreased Smad2 expression, leads to repression of Oct4 expression, which, together with the decreased Nanog expression, accelerates the loss of pluripotency. These findings reveal that Smad2 is a unique integrator of transcription and signaling events and is essential for the maintenance of the mouse and human primed pluripotent stem cell state.