DUX is a non-essential synchronizer of zygotic genome activation.

Some of the earliest transcripts produced in fertilized human and mouse oocytes code for DUX, a double homeodomain protein that promotes embryonic genome activation (EGA). Deleting Dux by genome editing at the one- to two-cell stage in the mouse impairs EGA and blastocyst maturation. Here, we demonstrate that mice carrying homozygous Dux deletions display markedly reduced expression of DUX target genes and defects in both pre- and post-implantation development, with, notably, a disruption of the pace of the first few cell divisions and significant rates of late embryonic mortality. However, some Dux-/- embryos give rise to viable pups, indicating that DUX is important but not strictly essential for embryogenesis.

De novo DNA methylation of endogenous retroviruses is shaped by KRAB-ZFPs/KAP1 and ESET.

Endogenous retroviruses (ERVs) undergo de novo DNA methylation during the first few days of mammalian embryogenesis, although the factors that control the targeting of this process are largely unknown. We asked whether KAP1 (KRAB-associated protein 1) is involved in this mechanism because of its previously defined role in maintaining the silencing of ERVs through the histone methyltransferase ESET and histone H3 lysine 9 trimethylation. Here, we demonstrate that introduced ERV sequences are sufficient to direct rapid de novo methylation of a flanked promoter in embryonic stem (ES) cells. This mechanism requires the presence of an ERV sequence-recognizing KRAB zinc-finger protein (ZFP) and both KAP1 and ESET. Furthermore, this process can also take place on a strong cellular promoter and leads to methylation signatures that are subsequently maintained in vivo throughout embryogenesis. Finally, we show that methylation of ERVs residing in the genome is affected by knockout of KAP1 in early embryos. KRAB-ZFPs, KAP1 and ESET are thus likely to be responsible for the early embryonic instatement of stable epigenetic marks at ERV-containing loci.

KAP1 controls endogenous retroviruses in embryonic stem cells.

More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5' untranslated region (5'UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5'UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.

MicroRNA-124 is a subventricular zone neuronal fate determinant.

New neurons are continuously generated from neural stem cells with astrocyte properties, which reside in close proximity to the ventricle in the postnatal and adult brain. In this study we found that microRNA-124 (miR-124) dictates postnatal neurogenesis in the mouse subventricular zone. Using a transgenic reporter mouse we show that miR-124 expression is initiated in the rapid amplifying progenitors and remains expressed in the resulting neurons. When we stably inhibited miR-124 in vivo, neurogenesis was blocked, leading to the appearance of ectopic cells with astrocyte characteristics in the olfactory bulb. Conversely, when we overexpressed miR-124, neural stem cells were not maintained in the subventricular zone and neurogenesis was lost. In summary, our results demonstrate that miR-124 is a neuronal fate determinant in the subventricular zone.

The Krüppel-associated box repressor domain can induce reversible heterochromatization of a mouse locus in vivo.

The study of chromatin and its regulators is key to understanding and manipulating transcription. We previously exploited the Krüppel-associated box (KRAB) transcriptional repressor domain, present in hundreds of vertebrate-specific zinc finger proteins, to assess the effect of its binding to gene bodies. These experiments revealed that the ectopic and doxycycline (dox)-controlled tet repressor KRAB fusion protein (tTRKRAB) can induce reversible and long-range silencing of cellular promoters. Here, we extend this system to in vivo applications and use tTRKRAB to achieve externally controllable repression of an endogenous mouse locus. We employed lentiviral-mediated transgenesis with promoterless TetO-containing gene traps to engineer a mouse line where the endogenous kinesin family member 2A (Kif2A) promoter drives a YFP reporter gene. When these mice were crossed to animals expressing the TetO-binding tTRKRAB repressor, this regulator was recruited to the Kif2A locus, and YFP expression was reduced. This effect was reversed when dox was given to embryos or adult mice, demonstrating that the cellular Kif2A promoter was only silenced upon repressor binding. Molecular analyses confirmed that tTRKRAB induced transcriptional repression through the spread of H3K9me3-containing heterochromatin, without DNA methylation of the trapped Kif2A promoter. Therefore, we demonstrate that targeting of tTRKRAB to a gene body in vivo results in reversible transcriptional repression through the spreading of facultative heterochromatin. This finding not only sheds light on KRAB-mediated transcriptional processes, but also suggests approaches for the externally controllable and reversible modulation of chromatin and transcription in vivo.

Rescue of a severe mouse model for spinal muscular atrophy by U7 snRNA-mediated splicing modulation.

In spinal muscular atrophy (SMA), the leading genetic cause of early childhood death, the survival motor neuron 1 gene (SMN1) is deleted or inactivated. The nearly identical SMN2 gene has a silent mutation that impairs the utilization of exon 7 and the production of functional protein. It has been hypothesized that therapies boosting SMN2 exon 7 inclusion might prevent or cure SMA. Exon 7 inclusion can be stimulated in cell culture by oligonucleotides or intracellularly expressed RNAs, but evidence for an in vivo improvement of SMA symptoms is lacking. Here, we unambiguously confirm the above hypothesis by showing that a bifunctional U7 snRNA that stimulates exon 7 inclusion, when introduced by germline transgenesis, can efficiently complement the most severe mouse SMA model. These results are significant for the development of a somatic SMA therapy, but may also provide new means to study pathophysiological aspects of this devastating disease.

Inducible gene and shRNA expression in resident hematopoietic stem cells in vivo.

Hematopoietic stem cells (HSC) are probably the best understood somatic stem cells and often serve as a paradigm for other stem cells. Nevertheless, most current techniques to genetically manipulate them in vivo are either constitutive and/or induced in settings of hematopoietic stress such as after irradiation. Here, we present a conditional expression system that allows for externally controllable transgenesis and knockdown in resident HSCs, based on a lentiviral vector containing a tet-O sequence and a transgenic mouse line expressing a doxycyclin-regulated tTR-KRAB repressor protein. HSCs harvested from tTR-KRAB mice are transduced with the lentiviral vector containing a cDNA (i.e., Green Fluorescent Protein (GFP)) and/or shRNA (i.e., p53) of interest and then transplanted into lethally irradiated recipients. While the vector is effectively repressed by tTR-KRAB during homing and engraftment, robust GFP/shp53 expression is induced on doxycyclin treatment in HSCs and their progeny. Doxycylin-controllable transcription is maintained on serial transplantation, indicating that repopulating HSCs are stably modified by this approach. In summary, this easy to implement conditional system provides inducible and reversible overexpression or knock down of genes in resident HSCs in vivo using a drug devoid of toxic or activating effects.

Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice.

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT(1A)), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT(1A) leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.

Regulation of episomal gene expression by KRAB/KAP1-mediated histone modifications.

KAP1 is an essential cofactor of KRAB zinc finger proteins, a family of vertebrate-specific epigenetic repressors of largely unknown functions encoded in the hundreds by the mouse and human genomes. So far, KRAB/KAP1-mediated gene regulation has been studied within the environment of chromosomal DNA. Here we demonstrate that KRAB/KAP1 regulation is fully functional within the context of episomal DNA, such as adeno-associated viral and nonintegrated lentiviral vectors, and is correlated with histone modifications typically associated with this epigenetic regulator.

APOBEC3-independent interferon-induced viral clearance in hepatitis B virus transgenic mice.

Interferon (IFN) has been part of the standard treatment of chronic hepatitis B infection for more than 2 decades, yet the mechanism of action of this antiviral remains poorly understood. It was recently observed that members of the human APOBEC family of cytidine deaminases endowed with anti-hepatitis B virus (HBV) activity are upregulated by type I and II IFNs. However, we demonstrated that, in tissue culture, these cellular enzymes are not essential effectors of the anti-HBV action of these cytokines. Here, we show that murine APOBEC3 (muA3) can also block HBV replication. While expressed at low levels in the mouse liver at baseline, muA3 is upregulated upon IFN induction. However, in HBV-transgenic muA3 knockout mice, IFN induction blocked HBV DNA production as efficiently as in control HBV-transgenic muA3-competent animals. We conclude that APOBEC3 is not an essential mediator of the IFN-mediated inhibition of HBV in vivo.

DUX-family transcription factors regulate zygotic genome activation in placental mammals.

In animal embryos, transcription is mostly silent for several cell divisions, until the release of the first major wave of embryonic transcripts through so-called zygotic genome activation (ZGA). Maternally provided ZGA-triggering factors have been identified in Drosophila melanogaster and Danio rerio, but their mammalian homologs are still undefined. Here, we provide evidence that the DUX family of transcription factors is essential to this process in mice and potentially in humans. First, human DUX4 and mouse Dux are both expressed before ZGA in their respective species. Second, both orthologous proteins bind the promoters of ZGA-associated genes and activate their transcription. Third, Dux knockout in mouse embryonic stem cells (mESCs) prevents the cells from cycling through a 2-cell-like state. Finally, zygotic depletion of Dux leads to impaired early embryonic development and defective ZGA. We conclude that DUX-family proteins are key inducers of zygotic genome activation in placental mammals.

A KRAB/KAP1-miRNA cascade regulates erythropoiesis through stage-specific control of mitophagy.

During hematopoiesis, lineage- and stage-specific transcription factors work in concert with chromatin modifiers to direct the differentiation of all blood cells. We explored the role of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor KAP1 in this process. In mice, hematopoietic-restricted deletion of Kap1 resulted in severe hypoproliferative anemia. Kap1-deleted erythroblasts failed to induce mitophagy-associated genes and retained mitochondria. This was due to persistent expression of microRNAs (miRNAs) targeting mitophagy transcripts, itself secondary to a lack of repression by stage-specific KRAB-ZFPs. The KRAB/KAP1-miRNA regulatory cascade is evolutionarily conserved, as it also controls mitophagy during human erythropoiesis. Thus, a multilayered transcription regulatory system is present, in which protein- and RNA-based repressors are superimposed in combinatorial fashion to govern the timely triggering of an important differentiation event.

Lentiviral Vector Mediated Transgenesis.

The genetic manipulation of rodents through the generation of fully transgenic animals or via the modification of selective cells or organs is a procedure of paramount importance for biomedical research, either to address fundamental questions or to develop preclinical models of human diseases. Lentiviral vectors occupy the front stage in this scene, as they can mediate the integration and stable expression of transgenes both in vitro and in vivo. Widely used to modify a variety of cells, including re-implantable somatic and embryonic stem cells, lentiviral vectors can also be directly administered in vivo, for instance in the brain. However, perhaps their most spectacular research application is in the generation of transgenic animals. Compared with the three-decade-old DNA pronuclear injection technique, lentivector-mediated transgenesis is simple, cheap, and highly efficient. Furthermore, it can take full advantage of the great diversity of lentiviral vectors developed for other applications, and thus allows for ubiquitous or tissue-specific or constitutive or externally controllable transgene expression, as well as RNAi-mediated gene knockdown. Curr. Protoc. Mouse Biol. 1:169-184. © 2011 by John Wiley & Sons, Inc.

Measuring in vivo protein half-life.

Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Here, we demonstrate how chemical labeling of SNAP-tag fusion proteins can be exploited to measure the half-life of resident intracellular and extracellular proteins in living mice. First, we demonstrate that SNAP-tag substrates have wide bioavailability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then apply near-infrared probes to perform noninvasive imaging of in vivo-labeled tumors. Finally, we use SNAP-mediated chemical pulse-chase labeling to perform measurement of the in vivo half-life of different extra- and intracellular proteins. These results open broad perspectives for studying protein function in living animals.

Chromosome conformation capture uncovers potential genome-wide interactions between human conserved non-coding sequences.

Comparative analyses of various mammalian genomes have identified numerous conserved non-coding (CNC) DNA elements that display striking conservation among species, suggesting that they have maintained specific functions throughout evolution. CNC function remains poorly understood, although recent studies have identified a role in gene regulation. We hypothesized that the identification of genomic loci that interact physically with CNCs would provide information on their functions. We have used circular chromosome conformation capture (4C) to characterize interactions of 10 CNCs from human chromosome 21 in K562 cells. The data provide evidence that CNCs are capable of interacting with loci that are enriched for CNCs. The number of trans interactions varies among CNCs; some show interactions with many loci, while others interact with few. Some of the tested CNCs are capable of driving the expression of a reporter gene in the mouse embryo, and associate with the oligodendrocyte genes OLIG1 and OLIG2. Our results underscore the power of chromosome conformation capture for the identification of targets of functional DNA elements and raise the possibility that CNCs exert their functions by physical association with defined genomic regions enriched in CNCs. These CNC-CNC interactions may in part explain their stringent conservation as a group of regulatory sequences.

A systematic enhancer screen using lentivector transgenesis identifies conserved and non-conserved functional elements at the Olig1 and Olig2 locus.

Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers.