Fátima Santos

Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation

Methylation at the 5′ position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells. Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb, Prdm14, Dppa3, Klf2, Tcl1 and Zfp42) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.

Conservation of methylation reprogramming in mammalian development: Aberrant reprogramming in cloned embryos

Mouse embryos undergo genome-wide methylation reprogramming by demethylation in early preimplantation development, followed by remethylation thereafter. Here we show that genome-wide reprogramming is conserved in several mammalian species and ask whether it also occurs in embryos cloned with the use of highly methylated somatic donor nuclei. Normal bovine, rat, and pig zygotes showed a demethylated paternal genome, suggesting active demethylation. In bovine embryos methylation was further reduced during cleavage up to the eight-cell stage, and this reduction in methylation was followed by de novo methylation by the 16-cell stage. In cloned one-cell embryos there was a reduction in methylation consistent with active demethylation, but no further demethylation occurred subsequently. Instead, de novo methylation and nuclear reorganization of methylation patterns resembling those of differentiated cells occurred precociously in many cloned embryos. Cloned, but not normal, morulae had highly methylated nuclei in all blastomeres that resembled those of the fibroblast donor cells. Our study shows that epigenetic reprogramming occurs aberrantly in most cloned embryos; incomplete reprogramming may contribute to the low efficiency of cloning.

Epigenetic Marking Correlates with Developmental Potential in Cloned Bovine Preimplantation Embryos

During differentiation, somatic nuclei acquire highly specialized DNA and chromatin modifications, which are thought to result in cellular memory of the differentiated state. Upon somatic nuclear transfer into oocytes, the donor nucleus may have to undergo reprogramming of these epigenetic marks in order to achieve totipotency. This may involve changes in epigenetic features similar to those that occur in normal embryos during early development. However, there is accumulating evidence that epigenetic reprogramming is severely deficient in cloned embryos. Several reports reveal inefficient demethylation and inappropriate reestablishment of DNA methylation in quantitative and qualitative patterns on somatic nuclear transfer. Here we examine histone H3 lysine 9 (H3-K9) methylation and acetylation in normal embryos and in those created by somatic nuclear transfer. We find that H3-K9 methylation is reprogrammed in parallel with DNA methylation in normal embryos. However, the majority of cloned embryos exhibit H3-K9 hypermethylation associated with DNA hypermethylation, suggesting a genome-wide failure of reprogramming. Strikingly, the precise epigenotype in cloned embryos depends on the donor cell type, and the proportion of embryos with normal epigenotypes correlates closely with the proportion developing to the blastocyst stage. These results suggest a mechanistic link between DNA and histone methylation in the mammalian embryo and reveal an association between epigenetic marks and developmental potential of cloned embryos.

The Dynamics of Genome-wide DNA Methylation Reprogramming in Mouse Primordial Germ Cells

Summary Genome-wide DNA methylation reprogramming occurs in mouse primordial germ cells (PGCs) and preimplantation embryos, but the precise dynamics and biological outcomes are largely unknown. We have carried out whole-genome bisulfite sequencing (BS-Seq) and RNA-Seq across key stages from E6.5 epiblast to E16.5 PGCs. Global loss of methylation takes place during PGC expansion and migration with evidence for passive demethylation, but sequences that carry long-term epigenetic memory (imprints, CpG islands on the X chromosome, germline-specific genes) only become demethylated upon entry of PGCs into the gonads. The transcriptional profile of PGCs is tightly controlled despite global hypomethylation, with transient expression of the pluripotency network, suggesting that reprogramming and pluripotency are inextricably linked. Our results provide a framework for the understanding of the epigenetic ground state of pluripotency in the germline.

Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human

Summary Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.

Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.

FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency

Summary Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing

Long noncoding RNAs are implicated in a number of regulatory functions in eukaryotic genomes. The paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1 regulates epigenetic gene silencing in an imprinted gene cluster in cis over a distance of 400 kb in the mouse embryo, whereas the silenced region extends over 780 kb in the placenta. Gene silencing by the Kcnq1ot1 RNA involves repressive histone modifications, including H3K9me2 and H3K27me3, which are partly brought about by the G9a and Ezh2 histone methyltransferases. Here, we show that Kcnq1ot1 is transcribed by RNA polymerase II, is unspliced, is relatively stable and is localised in the nucleus. Analysis of conditional Dicer mutants reveals that the RNAi pathway is not involved in gene silencing in the Kcnq1ot1 cluster. Instead, using RNA/DNA FISH we show that the Kcnq1ot1 RNA establishes a nuclear domain within which the genes that are epigenetically inactivated in cis are frequently found, whereas nearby genes that are not regulated by Kcnq1ot1 are localised outside of the domain. The Kcnq1ot1 RNA domain is larger in the placenta than in the embryo, consistent with more genes in the cluster being silenced in the placenta. Our results show for the first time that autosomal long ncRNAs can establish nuclear domains, which might create a repressive environment for epigenetic silencing of adjacent genes. Long ncRNAs in imprinting clusters and the Xist RNA on the inactive X chromosome thus appear to regulate epigenetic gene silencing by similar mechanisms.

Mammalian epigenomics: reprogramming the genome for development and therapy.

Epigenetic modifications of DNA and chromatin are important for genome function during development and in adults. DNA and chromatin modifications have central importance for genomic imprinting and other aspects of epigenetic control of gene expression. In somatic lineages, modifications are generally stably maintained and are characteristic of different specialized tissues. The mammalian genome undergoes major reprogramming of modification patterns in germ cells and in the early embryo. Some of the factors that are involved both in maintenance and in reprogramming, such as methyltransferases, are being identified. Epigenetic reprogramming is deficient in animal cloning, which is a major explanation for the inefficiency of the cloning procedure. Deficiencies in reprogramming are likely to underlie the occurrence of epimutations and of epigenetic inheritance. Environmental factors can alter epigenetic modifications and may thus have long-lasting effects on phenotype. Epigenomics methods are being developed to catalogue genome modifications under normal and pathological conditions. Epigenetic engineering is likely to play an important role in medicine in the future.

Structural differences in centromeric heterochromatin are spatially reconciled on fertilisation in the mouse zygote

In mammals, paternal and maternal pronuclei undergo profound chromatin reorganisation upon fertilisation. How these events are orchestrated within centromeric regions to ensure proper chromosome segregation in the following cellular divisions is unknown. In this study, we followed the dynamic unfolding of the centromeric regions, i.e. the centric and pericentric satellite repeats, by DNA fluorescent in situ hybridization (FISH) during the first cell cycle up to the two-cell stage. The distinct chromatin from female and male gametes both undergo rapid remodelling and reach a zygotic organisation in which the satellites occupy restricted spatial domains surrounding the nucleolar precursor body. A transition from this zygotic to a somatic cell-like organisation takes place during the two-cell stage. Using 3D immuno-FISH, we find that, whereas maternal pericentric regions are marked with H3K9me3, H4K20me3 and HP1β, paternal ones only showed HP1β marking. Thus, despite different chromatin features, male and female pronuclei organise their centromeric regions in the same way within the nuclei to align chromosomes on the metaphase plate and segregate them appropriately. Our findings highlight the importance of ensuring a proper centromere function while preserving the distinction of parental genome origin during the return to totipotency in the zygote.