Francesco Ferrari

The Lkb1 metabolic sensor maintains haematopoietic stem cell survival

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.

Comparative analysis of metazoan chromatin organization

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal ‘arms’, and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

Hallmarks of pluripotency

Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.

A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs.

The equivalence of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) remains controversial. Here we use genetically matched hESC and hiPSC lines to assess the contribution of cellular origin (hESC vs. hiPSC), the Sendai virus (SeV) reprogramming method and genetic background to transcriptional and DNA methylation patterns while controlling for cell line clonality and sex. We find that transcriptional and epigenetic variation originating from genetic background dominates over variation due to cellular origin or SeV infection. Moreover, the 49 differentially expressed genes we detect between genetically matched hESCs and hiPSCs neither predict functional outcome nor distinguish an independently derived, larger set of unmatched hESC and hiPSC lines. We conclude that hESCs and hiPSCs are molecularly and functionally equivalent and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional and epigenetic comparisons of pluripotent cell lines, explaining some of the previously observed differences between genetically unmatched hESCs and hiPSCs.

KDM2B promotes pancreatic cancer via Polycomb-dependent and -independent transcriptional programs

Epigenetic mechanisms mediate heritable control of cell identity in normal cells and cancer. We sought to identify epigenetic regulators driving the pathogenesis of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal human cancers. We found that KDM2B (also known as Ndy1, FBXL10, and JHDM1B), an H3K36 histone demethylase implicated in bypass of cellular senescence and somatic cell reprogramming, is markedly overexpressed in human PDAC, with levels increasing with disease grade and stage, and highest expression in metastases. KDM2B silencing abrogated tumorigenicity of PDAC cell lines exhibiting loss of epithelial differentiation, whereas KDM2B overexpression cooperated with KrasG12D to promote PDAC formation in mouse models. Gain- and loss-of-function experiments coupled to genome-wide gene expression and ChIP studies revealed that KDM2B drives tumorigenicity through 2 different transcriptional mechanisms. KDM2B repressed developmental genes through cobinding with Polycomb group (PcG) proteins at transcriptional start sites, whereas it activated a module of metabolic genes, including mediators of protein synthesis and mitochondrial function, cobound by the MYC oncogene and the histone demethylase KDM5A. These results defined epigenetic programs through which KDM2B subverts cellular differentiation and drives the pathogenesis of an aggressive subset of PDAC.

The histone chaperone CAF-1 safeguards somatic cell identity

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

Impact of sequencing depth in ChIP-seq experiments

In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40–50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data.

Transcriptional control of a whole chromosome: emerging models for dosage compensation

Males and females of many animal species differ in their sex-chromosome karyotype, and this creates imbalances between X-chromosome and autosomal gene products that require compensation. Although distinct molecular mechanisms have evolved in three highly studied systems, they all achieve coordinate regulation of an entire chromosome by differential RNA-polymerase occupancy at X-linked genes. High-throughput genome-wide methods have been pivotal in driving the latest progress in the field. Here we review the emerging models for dosage compensation in mammals, flies and nematodes, with a focus on mechanisms affecting RNA polymerase II activity on the X chromosome.

Failure to replicate the STAP cell phenomenon.

Although the reports that stress (such as exposure to acid) can coax somatic cells into a novel state of pluripotency have been retracted, the validity of stimulus-triggered acquisition of pluripotency (STAP) remains unclear (http://dx.doi.org/10.1038/protex. 2014.008 and Supplementary Information). Here we describe the efforts of seven laboratories to replicate STAP, including experiments performed within the laboratory where STAP first originated, as well as re-analysis of the sequencing data from the STAP reports. Neonatal cells treated with two STAP protocols exhibited artefactual autofluoresence rather than bona fide reactivation of an Oct4 (also known as Pou5f1) and green fluorescent protein (GFP) transgene reporter, did not reactivate pluripotency markers towards embryonic stem (ES)-cell-like levels, and failed to generate teratomas or chimaerize blastocysts. Re-analysis of the original RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data identified discrepancies in the sex and genetic composition of parental donor cells and converted stem cells, and revealed a STAP-derived cell line to be a mixture containing trophoblast stem cells, attesting to the importance of validating the properties and provenance of pluripotent stem cells using a wide range of criteria. To assess the reprogramming capacity of STAP protocols, we used a transgenic Oct4-GFP reporter, which shows GFP reactivation during Oct4/Sox2/Klf4 reprogramming, in established induced pluripotent stem (iPS) cells and in the gonads of mid-gestation ‘all iPS cell’ embryos generated by tetraploid complementation (Extended Data Figs 1 and 2a). Working within the Vacanti laboratory where the concept of STAP cells originated, and assisted by a co-author of the STAP papers, a Daley laboratory member (A.D.L.A.) attempted to replicate two reported STAP protocols: (1) mechanical trituration and acid treatment of mouse lung cells (Brigham and Women’s Hospital (BWH) protocol; see Supplementary Information), and (2) acid treatment of mouse splenocytes (RIKEN protocol; Methods and Extended Data Fig. 2b). Seventy-two hours after stress treatment of lung cells, floating spheres appeared amidst cellular debris. Fluorescence microscopy revealed that both Oct4-GFP and wild-type spheres emitted lowlevel broad spectrum fluorescence detectable within both green and red filters, indicating autofluorescence (Fig. 1a). Untreated Oct4-GFP ES cells did not emit the same low-level broad spectrum fluorescence as STAP-treated cells. STAP-treated splenocytes formed spheres with lower efficiency, but also appeared autofluorescent. Flow cytometry indicated STAP-treated Oct4-GFP cells did not exhibit Oct4-GFP reactivation at levels comparable to control Oct4GFP mouse ES cells, and were indistinguishable from stressed wildtype controls (Fig. 1b). Absence of ES-cell-like levels of Oct4, Sox2 and Nanog transcripts and nonspecific immunofluorescence corroborated flow cytometry data (Extended Data Fig. 2c, d). Rare pluripotent cells should generate teratomas in immunocompromised mice, but STAP cells could not, unlike control ES cells (Extended Data Fig. 2e, f). Replication of the poly-L-glycolic acid (PLGA)-based teratoma production method described in the original STAP reports with GFP cells to distinguish host and donor contribution produced distinct masses of connective tissue, muscle and scar, with minimal GFP content, indicating primarily host origin (Fig. 1c, d and Extended Data Fig. 2g). Rare GFP-positive clusters did not form differentiated tissues characteristic of ES-cell-derived teratomas (Fig. 1d). Autofluorescent spheres failed to enter development after morula aggregation or blastocyst injection (Fig. 1e and Extended Data Fig. 2h–j). Therefore, pluripotency was undetectable in STAP experiments. Six other laboratories (Deng, Hanna, Hochedlinger, Jaenisch, Pei and Wernig) also attempted to generate STAP cells (Table 1) and made the following observations. First, autofluorescent sphere-like aggregates after STAP treatment were universally seen. Second, transgenic reporters used by Obokata and colleagues (GOF18-Oct4-GFP, containing the 18-kilobase genomic Oct4 fragment (GOF18)) and by the Daley, Pei and Hanna laboratories (GOF18-Oct4DPE-GFP, lacking the Oct4 proximal enhancer (PE) element) both exhibit activity in pre-implantation embryos, early post-implantation epiblast cells (embryonic day (E) 5.5), germ cells, and mouse ES/iPS cells; however, differential activity in late post-implantation epiblast (E6.5) and early passage mouse epiblast-derived stem cells has been ascribed to the Oct4 proximal enhancer. Using the same reporter as Obokata and colleagues, the Deng laboratory observed that the GFP signal in chemical iPS cells was easily distinguishable from the autofluorescence of STAP-treated cells (Extended Data Fig. 2k). The Jaenisch, Wernig and Hochedlinger laboratories failed to observe GFP reactivation with Oct4 or Nanog knock-in reporters, excluding a scenario of uncoupling between GFP and endogenous pluripotency expression. Despite a range of tested reporters, no group documented authentic Oct4/Nanog reporter activation that resembled bona fide ES cells. Third, the Deng laboratory failed to observe Oct4, Sox2 and Nanog induction 3 and 7 days after STAP treatment, reducing the likelihood that pluripotency was transiently activated and silenced by day 7 (Extended Data Fig. 2l). Finally, the Hanna, Wernig and Hochedlinger laboratories failed to generate stem-cell lines by culturing STAP-treated cells in leukaemia inhibitory factor (LIF) and adrenocorticotropic hormone (ACTH)-supplemented medium. In summary, 133 replicate attempts failed to document generation of ES-cell-like cells, corroborating and extending a recent report. We re-examined the high-throughput sequencing data from the STAP reports to investigate the genetic provenance of parental CD45 cells and converted STAP cells, STAP stem cells and Fgf4-induced stem cells (FI-SCs) (Fig. 1f). Comparative genomic hybridization array data mentioned in the original paper were not publicly released. Copy number variation (CNV) analysis conducted using ChIP-seq input samples revealed a discrepancy in sex across samples as well as chromosomal aberrations (Fig. 1g). In the original STAP reports, the authors stated that they mixed CD45 cells from male and female mice owing to the small number of CD45 cells retrieved from individual neonatal spleens. However, our analysis indicates that CD45 cells were female, whereas the derived cells (STAP cells, STAP stem cells and FI-SCs) were all male, a clear inconsistency. We note that control ES cells were also male (Fig. 1g). FI-SCs possessed trisomy 8, which renders mouse ES cells germline-incompetent (Fig. 1g). Inferred single nucleotide variants (SNVs) from RNA-seq data allowed classification of samples as genetically similar or dissimilar (Fig. 1h). Control ES cells, parental donor female CD45 cells, STAP cells, and STAP stem cells all possessed similar SNV profiles, consistent with their derivation from a first generation hybrid of C57BL6/129 strains, the reported genotype (Fig. 1h and Extended Data Fig. 3). By contrast, FI-SCs had an SNV profile that matched a single nucleotide polymorphism (SNP) profile of C57BL6 strain origin, indicating

Comment on “Drosophila Dosage Compensation Involves Enhanced Pol II Recruitment to Male X-Linked Promoters”

Conrad et al. (Reports, 10 August 2012, p. 742) reported a doubling of RNA polymerase II (Pol II) occupancy at X-linked promoters to support 5′ recruitment as the key mechanism for dosage compensation in Drosophila. However, they employed an erroneous data-processing step, overestimating Pol II differences. Reanalysis of the data fails to support the authors’ model for dosage compensation.