Todd S. Macfarlan

Embryonic stem cell potency fluctuates with endogenous retrovirus activity

Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.

Transposable elements as genetic regulatory substrates in early development

The abundance and ancient origins of transposable elements (TEs) in eukaryotic genomes has spawned research into the potential symbiotic relationship between these elements and their hosts. In this review, we introduce the diversity of TEs, discuss how distinct classes are uniquely regulated in development, and describe how they appear to have been coopted for the purposes of gene regulation and the orchestration of a number of processes during early embryonic development. Although young, active TEs play an important role in somatic tissues and evolution, we focus mostly on the contributions of the older, fixed elements in mammalian genomes. We also discuss major challenges inherent in the study of TEs and contemplate future experimental approaches to further investigate how they coordinate developmental processes.

TRIM28 repression of retrotransposon-based enhancers is necessary to preserve transcriptional dynamics in embryonic stem cells

TRIM28 is critical for the silencing of endogenous retroviruses (ERVs) in embryonic stem (ES) cells. Here, we reveal that an essential impact of this process is the protection of cellular gene expression in early embryos from perturbation by cis-acting activators contained within these retroelements. In TRIM28-depleted ES cells, repressive chromatin marks at ERVs are replaced by histone modifications typical of active enhancers, stimulating transcription of nearby cellular genes, notably those harboring bivalent promoters. Correspondingly, ERV-derived sequences can repress or enhance expression from an adjacent promoter in transgenic embryos depending on their TRIM28 sensitivity in ES cells. TRIM28-mediated control of ERVs is therefore crucial not just to prevent retrotransposition, but more broadly to safeguard the transcriptional dynamics of early embryos.

The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses

Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Krüppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.

LSD1n is an H4K20 demethylase regulating memory formation via transcriptional elongation control

We found that a neuron-specific isoform of LSD1, LSD1n, which results from an alternative splicing event, acquires a new substrate specificity, targeting histone H4 Lys20 methylation, both in vitro and in vivo. Selective genetic ablation of LSD1n led to deficits in spatial learning and memory, revealing the functional importance of LSD1n in neuronal activity–regulated transcription that is necessary for long-term memory formation. LSD1n occupied neuronal gene enhancers, promoters and transcribed coding regions, and was required for transcription initiation and elongation steps in response to neuronal activity, indicating the crucial role of H4K20 methylation in coordinating gene transcription with neuronal function. Our results indicate that this alternative splicing of LSD1 in neurons, which was associated with altered substrate specificity, serves as a mechanism acquired by neurons to achieve more precise control of gene expression in the complex processes underlying learning and memory.

Spotting the enemy within: Targeted silencing of foreign DNA in mammalian genomes by the Krüppel-associated box zinc finger protein family

Tandem C2H2-type zinc finger proteins (ZFPs) constitute the largest transcription factor family in animals. Tandem-ZFPs bind DNA in a sequence-specific manner through arrays of multiple zinc finger domains that allow high flexibility and specificity in target recognition. In tetrapods, a large proportion of tandem-ZFPs contain Krüppel-associated-box (KRAB) repression domains, which are able to induce epigenetic silencing through the KAP1 corepressor. The KRAB-ZFP family continuously amplified in tetrapods through segmental gene duplications, often accompanied by deletions, duplications, and mutations of the zinc finger domains. As a result, tetrapod genomes contain unique sets of KRAB-ZFP genes, consisting of ancient and recently evolved family members. Although several hundred human and mouse KRAB-ZFPs have been identified or predicted, the biological functions of most KRAB-ZFP family members have gone unexplored. Furthermore, the evolutionary forces driving the extraordinary KRAB-ZFP expansion and diversification have remained mysterious for decades. In this review, we highlight recent studies that associate KRAB-ZFPs with the repression of parasitic DNA elements in the mammalian germ line and discuss the hypothesis that the KRAB-ZFP family primarily evolved as an adaptive genomic surveillance system against foreign DNA. Finally, we comment on the computational, genetic, and biochemical challenges of studying KRAB-ZFPs and attempt to predict how these challenges may be soon overcome.

Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition

Significance Transcriptional enhancers control cell-identity gene expression and thus determine cell identity. Enhancers are primed by histone H3K4 mono-/di-methyltransferase MLL4 before they are activated by histone H3K27 acetyltransferase p300. Here, we show that MLL4 is dispensable for cell-identity maintenance but essential for cell fate transition using several model systems including embryonic stem cell (ESC) differentiation toward somatic cells and somatic cell reprogramming into ESC-like cells. Mechanistically, MLL4 is dispensable for maintaining p300 binding on active enhancers of cell-identity genes but is required for p300 binding on enhancers activated during cell fate transition. These results indicate that, although enhancer priming by MLL4 is dispensable for cell-identity maintenance, it controls cell fate transition by orchestrating p300-mediated enhancer activation. Transcriptional enhancers control cell-type–specific gene expression. Primed enhancers are marked by histone H3 lysine 4 (H3K4) mono/di-methylation (H3K4me1/2). Active enhancers are further marked by H3K27 acetylation (H3K27ac). Mixed-lineage leukemia 4 (MLL4/KMT2D) is a major enhancer H3K4me1/2 methyltransferase with functional redundancy with MLL3 (KMT2C). However, its role in cell fate maintenance and transition is poorly understood. Here, we show in mouse embryonic stem cells (ESCs) that MLL4 associates with, but is surprisingly dispensable for the maintenance of, active enhancers of cell-identity genes. As a result, MLL4 is dispensable for cell-identity gene expression and self-renewal in ESCs. In contrast, MLL4 is required for enhancer-binding of H3K27 acetyltransferase p300, enhancer activation, and induction of cell-identity genes during ESC differentiation. MLL4 protein, rather than MLL4-mediated H3K4 methylation, controls p300 recruitment to enhancers. We also show that, in somatic cells, MLL4 is dispensable for maintaining cell identity but essential for reprogramming into induced pluripotent stem cells. These results indicate that, although enhancer priming by MLL4 is dispensable for cell-identity maintenance, it controls cell fate transition by orchestrating p300-mediated enhancer activation.

Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

Somatic cell nuclear transfer has established that the oocyte contains maternal factors with epigenetic reprogramming capacity. Yet the identity and function of these maternal factors during the gamete to embryo transition remains poorly understood. In C. elegans, LSD1/KDM1A enables this transition by removing H3K4me2 and preventing the transgenerational inheritance of transcription patterns. Here we show that loss of maternal LSD1/KDM1A in mice results in embryonic arrest at the 1-2 cell stage, with arrested embryos failing to undergo the maternal-to-zygotic transition. This suggests that LSD1/KDM1A maternal reprogramming is conserved. Moreover, partial loss of maternal LSD1/KDM1A results in striking phenotypes weeks after fertilization; including perinatal lethality and abnormal behavior in surviving adults. These maternal effect hypomorphic phenotypes are associated with alterations in DNA methylation and expression at imprinted genes. These results establish a novel mammalian paradigm where defects in early epigenetic reprogramming can lead to defects that manifest later in development. DOI: http://dx.doi.org/10.7554/eLife.08848.001

Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells

Limiting potential for totipotency Biological roles for microRNAs are not limited to RNA silencing and posttranscriptional regulation; they have now been shown to also regulate cell pluripotency. Choi et al. eliminated miR-34a from mouse embryonic stem cells and found that the cells exhibited a bidirectional cell fate potential, generating both embryonic and extraembryonic lineages (see the Perspective by Hasuwa and Siomi). During miR-34a deficiency, an endogenous retrovirus was induced, at least in part through Gata2-dependent transcriptional activation. Thus, the interplay of protein-coding genes, noncoding RNAs, and endogenous retroviruses can change cell fate plasticity and the developmental potential of pluripotent stem cells. Science, this issue p. eaag1927; see also p. 581 In mouse pluripotent stem cells, miR-34a deficiency expands developmental potential to both embryonic and extraembryonic lineages. INTRODUCTION Mouse zygotes and early blastomeres have totipotent cell fate potential, generating both embryonic and extraembryonic cell lineages during normal development. This totipotent potential is gradually restricted during development, with the first cell fate specification event being completed by the blastocyst stage. Mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) exhibit a pluripotent potential similar to that of the epiblast in blastocysts, efficiently generating all embryonic cell types but rarely contributing to extraembryonic lineages in the placenta and yolk sac. Experimentally, ESCs and iPSCs can be induced into cells with expanded developmental potential, albeit with low efficiency. Such pluripotent stem cells are characterized by their bidirectional developmental potential (contributing to both embryonic and extraembryonic lineages) and their strong induction of the MuERV-L (MERVL) endogenous retroviruses (ERVs), both of which are features of totipotent two-cell (2C) blastomeres. The low efficiency in generating bipotential ESCs reflects the existence of multiple cellular and molecular impediments that restrict the pluripotent cell fate potential. RATIONALE We identified miR-34a microRNA (miRNA) as a potent regulator that restricts the cell fate potential of ESCs and iPSCs to a pluripotent state. miR-34a−/− pluripotent stem cells exhibit a bidirectional cell fate potential, generating both embryonic and extraembryonic cell lineages in multiple functional assays. Hence, the miR-34a−/− pluripotent stem cells provide a powerful experimental system to dissect the molecular mechanisms that restrict cell fate potential in ESCs and iPSCs. RESULTS miR-34a−/− ESCs and iPSCs exhibited an expanded cell fate potential, generating both embryonic and extraembryonic lineages in teratomas, embryoid bodies, and chimeric embryos. In particular, a single miR-34a−/− ESC injected into a recipient morula could yield progenies in both inner cell mass (ICM) and trophectoderm. Expression profiling studies comparing wild-type and miR-34a−/− pluripotent stem cells revealed a strong and specific induction of the MERVL ERVs, together with many MERVL-proximal genes, in miR-34a−/− ESCs and iPSCs. Whereas wild-type ESCs and iPSCs almost exclusively expressed Oct4, miR-34a−/− ESCs and iPSCs were heterogeneous, containing mutually exclusive populations with either Oct4 expression or MERVL induction. Because MERVL is a specific and highly expressed molecular marker for totipotent 2C blastomeres and for bipotential ESCs, we investigated the mechanism by which miR-34a regulates MERVL expression. We demonstrated that MERVL induction in miR-34a–deficient pluripotent stem cells is regulated at the transcriptional level, at least in part because of an increase of the transcription factor Gata2, a direct target of miR-34a. Knockdown of gata2 in miR-34a–deficient pluripotent stem cells phenocopied miR-34a overexpression, not only down-regulating the expression of MERVL but also abolishing their bipotential cell fate. Thus, miR-34a restricts cell fate potential and represses MERVL induction in pluripotent stem cells, at least in part through down-regulation of Gata2. CONCLUSION We have identified miR-34a as a noncoding RNA that restricts the cell fate potential of ESCs and iPSCs to a pluripotent state. The miR-34a/gata2/MERVL axis plays an essential role in modulating the transition between pluripotent stem cells and bipotential stem cells in culture. Thus, an intricate network of protein-coding genes, noncoding RNAs, and endogenous retroviruses could act cooperatively to define cell fate plasticity and developmental potential in pluripotent stem cells. The miR-34a/Gata2 pathway restricts the cell fate potential of ESCs and iPSCs to a pluripotent state. Pluripotent stem cell cultures contain mutually exclusive populations of pluripotent MERVLlow/Oct4high cells and bipotent MERVLhigh/Oct4low cells. In wild-type ESC and iPSC culture, this equilibrium strongly favors the MERVLlow/Oct4high population at the expense of the MERVLhigh/Oct4low population. miR-34a deficiency increases Gata2-dependent transcription of MERVL endogenous retroviruses, shifting the equilibrium to enable more cells to acquire a bipotential cell fate that yields both embryonic and extraembryonic cell lineages. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) efficiently generate all embryonic cell lineages but rarely generate extraembryonic cell types. We found that microRNA miR-34a deficiency expands the developmental potential of mouse pluripotent stem cells, yielding both embryonic and extraembryonic lineages and strongly inducing MuERV-L (MERVL) endogenous retroviruses, similar to what is seen with features of totipotent two-cell blastomeres. miR-34a restricts the acquisition of expanded cell fate potential in pluripotent stem cells, and it represses MERVL expression through transcriptional regulation, at least in part by targeting the transcription factor Gata2. Our studies reveal a complex molecular network that defines and restricts pluripotent developmental potential in cultured ESCs and iPSCs.

Local compartment changes and regulatory landscape alterations in histone H1-depleted cells

BackgroundLinker histone H1 is a core chromatin component that binds to nucleosome core particles and the linker DNA between nucleosomes. It has been implicated in chromatin compaction and gene regulation and is anticipated to play a role in higher-order genome structure. Here we have used a combination of genome-wide approaches including DNA methylation, histone modification and DNase I hypersensitivity profiling as well as Hi-C to investigate the impact of reduced cellular levels of histone H1 in embryonic stem cells on chromatin folding and function.ResultsWe find that depletion of histone H1 changes the epigenetic signature of thousands of potential regulatory sites across the genome. Many of them show cooperative loss or gain of multiple chromatin marks. Epigenetic alterations cluster to gene-dense topologically associating domains (TADs) that already showed a high density of corresponding chromatin features. Genome organization at the three-dimensional level is largely intact, but we find changes in the structural segmentation of chromosomes specifically for the epigenetically most modified TADs.ConclusionsOur data show that cells require normal histone H1 levels to expose their proper regulatory landscape. Reducing the levels of histone H1 results in massive epigenetic changes and altered topological organization particularly at the most active chromosomal domains. Changes in TAD configuration coincide with epigenetic landscape changes but not with transcriptional output changes, supporting the emerging concept that transcriptional control and nuclear positioning of TADs are not causally related but independently controlled by the locally associated trans-acting factors.