Rosa Castanon

Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

Abnormalities in human pluripotent cells due to reprogramming mechanisms

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.

Molecular Criteria for Defining the Naive Human Pluripotent State

Summary Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.

A transcription factor hierarchy defines an environmental stress response network

Complex transcription factor interactions To respond to environmental changes, such as drought, plants must regulate numerous cellular processes. Working in the model plant Arabidopsis, Song et al. profiled the binding of 21 transcription factors to chromatin and mapped the complex gene regulatory networks involved in the response to the plant hormone abscisic acid. The work provides a framework for understanding and modulating plant responses to stress. Science, this issue p. 598 The response to the abscisic acid hormone in the model plant Arabidopsis involves complex transcription factor dynamics. INTRODUCTION Transcription factors (TFs) are often studied one by one or in clusters of a few related factors. However, the integration and networks of transcriptional changes to response to environmental stresses often involve many related TFs. In many organisms, such as plants, overlapping functions can make it difficult to understand how a biologically relevant end result can be achieved via the complex signaling networks controlled by these TFs. To better understand how the reference plant Arabidopsis deals with the stresses incurred by water limitation via the hormone abscisic acid (ABA), we characterized all DNA sequences that bind to the 21 ABA-related TFs in vivo. RATIONALE There have been limited systematic studies of stress-responsive TF networks in multicellular organisms. We chose ABA, an essential plant hormone that is required for both development and responses to osmotic stress, as an elicitor to investigate complex gene regulatory networks under stress. Combining differential binding (DB) of 21 ABA-related TFs at a single time point measured by chromatin immunoprecipitation sequencing (ChIP-seq) with differentially expressed genes from a time-series RNA sequencing (RNA-seq) data set, we analyzed the relationship between DB of TFs and differential expression (DE) of target genes, the determinants of DB, and the combinatorial effects of multi-TF binding. These data sets also provide a framework to construct an ABA TF network and to predict genes and cis-regulatory elements important to ABA responses and related environmental stresses. RESULTS We found that, in general, DNA binding is correlated with transcript and protein levels of TFs. Most TFs in our study are induced by ABA and gain binding sites (termed “peaks”) after the hormone treatment. ABA also increases the binding of the TFs at most peaks. However, in some peaks, TF binding may be static or even decrease after ABA exposure, revealing the complexity of locus-specific gene regulation. De novo motif discovery enabled us to identify distinct, primary motifs often centrally localized in the ChIP-seq peaks for most TFs. However, it is not uncommon to find motifs, such as the G-box, that are shared by peaks from multiple TFs and may contribute to binding dynamics at these sites. DB of multiple TFs is a robust predictor of both the DE and ABA-related functions of the target genes. Using the DB and DE data, we constructed a network of TFs and canonical ABA pathway genes and demonstrated a regulatory hierarchy of TFs and extensive feedback of ABA responses. On the basis of a “guilt-by-association” paradigm, we further predicted genes whose functions were previously not linked to ABA responses, and we thus functionally characterized a new family of transcriptional regulators. CONCLUSION These data sets will provide the plant community with a roadmap of ABA-elicited transcriptional regulation by 21 ABA-related TFs. We propose that dynamic, multi-TF binding could be a criterion for prioritizing the characterization of TF binding events, cis-regulatory elements, and functionally unknown genes in both plants and other species. In our proof-of-principle experiments, ectopic expression of the transcriptional regulators ranked highly in our model results in altered sensitivity to both ABA and high salinity. Together with the fact that our modeling recovered genes related to seed development and osmotic stresses, we believe such predictions are likely applicable to a broad range of developmental stages and osmotic stresses. Transcriptional landscape of the ABA response. ABA response pathway gene targets were identified by large-scale ChIP-seq and time-series RNA-seq experiments. A network model was built to reveal the hierarchy of TFs and the impact of multi-TF dynamic binding on gene expression. A new family of transcriptional regulators was predicted by the model and was functionally tested to evaluate the role of these regulators in osmotic stress in plants. Environmental stresses are universally encountered by microbes, plants, and animals. Yet systematic studies of stress-responsive transcription factor (TF) networks in multicellular organisms have been limited. The phytohormone abscisic acid (ABA) influences the expression of thousands of genes, allowing us to characterize complex stress-responsive regulatory networks. Using chromatin immunoprecipitation sequencing, we identified genome-wide targets of 21 ABA-related TFs to construct a comprehensive regulatory network in Arabidopsis thaliana. Determinants of dynamic TF binding and a hierarchy among TFs were defined, illuminating the relationship between differential gene expression patterns and ABA pathway feedback regulation. By extrapolating regulatory characteristics of observed canonical ABA pathway components, we identified a new family of transcriptional regulators modulating ABA and salt responsiveness and demonstrated their utility to modulate plant resilience to osmotic stress.

Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex

Methylation and the single neuronal cell The presence or absence of methylation on chromosomal DNA can drive or repress gene expression. Now, a comprehensive map of methylation variation in neuronal cell populations, including a between-species comparison, illustrates how epigenetic diversity plays important roles in neuronal development. Luo et al. examined how DNA methylation is both similar and different within neurons at the single-nucleus level in humans and mice. They identified 16 mouse and 21 human neuronal clusters, with greater complexity of excitatory neurons in deep brain layers than in superficial layers. Science, this issue p. 600 Single-nucleus methylomes distinguish neuron types and predict conserved gene regulatory elements in mice and humans. The mammalian brain contains diverse neuronal types, yet we lack single-cell epigenomic assays that are able to identify and characterize them. DNA methylation is a stable epigenetic mark that distinguishes cell types and marks regulatory elements. We generated >6000 methylomes from single neuronal nuclei and used them to identify 16 mouse and 21 human neuronal subpopulations in the frontal cortex. CG and non-CG methylation exhibited cell type–specific distributions, and we identified regulatory elements with differential methylation across neuron types. Methylation signatures identified a layer 6 excitatory neuron subtype and a unique human parvalbumin-expressing inhibitory neuron subtype. We observed stronger cross-species conservation of regulatory elements in inhibitory neurons than in excitatory neurons. Single-nucleus methylomes expand the atlas of brain cell types and identify regulatory elements that drive conserved brain cell diversity.

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

Significance In mammals, when and where a gene is transcribed are primarily regulated by the activity of regulatory DNA elements, or enhancers. Genetic mutation disrupting enhancer function is emerging as one of the major causes of human diseases. However, our knowledge remains limited about the location and activity of enhancers in the numerous and distinct cell types and tissues. Here, we develop a computational approach, regulatory element prediction based on tissue-specific local epigenetic marks (REPTILE), to precisely locate enhancers based on genome-wide DNA methylation and histone modification profiling. We systematically tested REPTILE on a variety of human and mouse cell types and tissues. Compared with existing methods, we found that enhancer predictions from REPTILE are more likely to be active in vivo and the predicted locations are more accurate. Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulatory element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared with existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types. REPTILE is available at https://github.com/yupenghe/REPTILE/.

Dynamic DNA methylation reconfiguration during seed development and germination

BackgroundUnlike animals, plants can pause their life cycle as dormant seeds. In both plants and animals, DNA methylation is involved in the regulation of gene expression and genome integrity. In animals, reprogramming erases and re-establishes DNA methylation during development. However, knowledge of reprogramming or reconfiguration in plants has been limited to pollen and the central cell. To better understand epigenetic reconfiguration in the embryo, which forms the plant body, we compared time-series methylomes of dry and germinating seeds to publicly available seed development methylomes.ResultsTime-series whole genome bisulfite sequencing reveals extensive gain of CHH methylation during seed development and drastic loss of CHH methylation during germination. These dynamic changes in methylation mainly occur within transposable elements. Active DNA methylation during seed development depends on both RNA-directed DNA methylation and heterochromatin formation pathways, whereas global demethylation during germination occurs in a passive manner. However, an active DNA demethylation pathway is initiated during late seed development.ConclusionsThis study provides new insights into dynamic DNA methylation reprogramming events during seed development and germination and suggests possible mechanisms of regulation. The observed sequential methylation/demethylation cycle suggests an important role of DNA methylation in seed dormancy.

CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.

Broad-scale protein–protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq′s improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

Functional Human Oocytes Generated by Transfer of Polar Body Genomes

Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.

Allele-specific non-CG DNA methylation marks domains of active chromatin in female mouse brain

Significance Mammalian cells contain two copies of the genome inherited from the two parents. Although most genes are expressed using both, a small but critical part of the genome has different levels of expression from each copy. These parts include the X chromosome in females and imprinted genes in both genders, which play key roles in brain development and cognition. We measured gene expression and DNA methylation, an epigenetic modification of the genome, in the brains of mice using a technique that allowed us to analyze the maternal and paternal copies of the genome separately. Our findings show that a brain-specific form of DNA methylation called non-CG methylation marks regions of active transcription within the inactive X chromosome. DNA methylation at gene promoters in a CG context is associated with transcriptional repression, including at genes silenced on the inactive X chromosome in females. Non-CG methylation (mCH) is a distinct feature of the neuronal epigenome that is differentially distributed between males and females on the X chromosome. However, little is known about differences in mCH on the active (Xa) and inactive (Xi) X chromosomes because stochastic X-chromosome inactivation (XCI) confounds allele-specific epigenomic profiling. We used whole-genome bisulfite sequencing in a mouse model with nonrandom XCI to examine allele-specific DNA methylation in frontal cortex. Xi was largely devoid of mCH, whereas Xa contained abundant mCH similar to the male X chromosome and the autosomes. In contrast to the repressive association of DNA methylation at CG dinucleotides (mCG), mCH accumulates on Xi in domains with transcriptional activity, including the bodies of most genes that escape XCI and at the X-inactivation center, validating this epigenetic mark as a signature of transcriptional activity. Escape genes showing CH hypermethylation were the only genes with CG-hypomethylated promoters on Xi, a well-known mark of active transcription. Finally, we found extensive allele-specific mCH and mCG at autosomal imprinted regions, some with a negative correlation between methylation in the two contexts, further supporting their distinct functions. Our findings show that neuronal mCH functions independently of mCG and is a highly dynamic epigenomic correlate of allele-specific gene regulation.