Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements (TEs). Here, we used mouse embryonic stem ...cells (ESCs) to identify an endosiRNA-based mechanism involved in suppression of TE transcription. In ESCs with DNA demethylation induced by acute deletion of Dnmt1, we saw an increase in sense transcription at TEs, resulting in an abundance of sense/antisense transcripts leading to high levels of ARGONAUTE2 (AGO2)-bound small RNAs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response. In vivo, we also found TE-specific endosiRNAs present during primordial germ cell development. Our results suggest that antisense TE transcription is a “trap” that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline.
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•Global DNA demethylation in embryonic stem cells leads to transposon activation•Transposon activation increases the abundance of sense/antisense transcripts•ARGONAUTE2-bound endosiRNAs accumulate at high levels for acute repression•Longer-term transposon repression depends on repressive histone marks
In this issue of Cell Stem Cell, Berrens et al. report the control of transposable elements by endosiRNAs during global DNA demethylation induced in mouse embryonic stem cells. The study uncovered an “immediate” repression of transposons accomplished by endosiRNAs followed by their “chronic/long-term” silencing by repressive histone modifications.
If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies ...are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.
The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic ...models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F–positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2V617F mice develop reduced numbers of lineage−Sca-1+c-Kit+ cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2V617F mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.
Endogenous retroviruses (ERVs), which are responsible for 10% of spontaneous mouse mutations, are kept under control via several epigenetic mechanisms. The H3K9 histone methyltransferase SETDB1 is ...essential for ERV repression in embryonic stem cells (ESCs), with DNA methylation also playing an important role. It has been suggested that SETDB1 protects ERVs from TET-dependent DNA demethylation, but the relevance of this mechanism for ERV expression remains unclear. Moreover, previous studies have been performed in primed ESCs, which are not epigenetically or transcriptionally representative of preimplantation embryos.
We use naïve ESCs to investigate the role of SETDB1 in ERV regulation and its relationship with TET-mediated DNA demethylation. Naïve ESCs show an increased dependency on SETDB1 for ERV silencing when compared to primed ESCs, including at the highly mutagenic intracisternal A particles (IAPs). We find that in the absence of SETDB1, TET2 activates IAP elements in a catalytic-dependent manner. Surprisingly, TET2 does not drive changes in DNA methylation levels at IAPs, suggesting that it regulates these retrotransposons indirectly. Instead, SETDB1 depletion leads to a TET2-dependent loss of H4R3me2s, which is indispensable for IAP silencing during epigenetic reprogramming.
Our results demonstrate a novel and unexpected role for SETDB1 in protecting IAPs from TET2-dependent histone arginine demethylation.
The transcription factor ecotropic viral integration site 1 (Evi1) is associated with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) in patients due to chromosomal aberration of ...chromosome 3. Here we show that Evi1 interacts with the histone methyltransferase SUV39H1. The interaction requires the N-terminal part of Evi1 and the H3-specific histone methyltransferase domain, SET, of SUV39H1 without Evi1 having an inhibitory effect on SUV39H1 methyltransferase activity. Presence of SUV39H1 enhances Evi1 transcriptional repression in a dose dependent manner. In addition, Evi1 also interacts with another histone methyltransferase, G9a, but not with SET9. Our data establish an epigenetic role of Evi1 in cell transformation by recruiting higher order chromatin remodeling complexes.
ETV6 (ets translocation variant gene 6) TEL (translocation ets leukemia), encoding a transcriptional repressor, is involved in various translocations associated with human malignancies. Strikingly, ...the nonrearranged ETV6 allele is often deleted or inactivated in cells harboring these translocations. Although ETV6 translocations are infrequent in acute myeloid leukemia (AML), mutations or deregulated expression of ETV6 may contribute to leukemogenesis. To investigate the involvement of ETV6 in AML, we analysed 300 newly diagnosed patients for mutations in the coding region of the gene. Furthermore, we studied protein expression in 77 patients using two ETV6-specific antibodies. Five somatic heterozygous mutations were detected, which affected either the homodimerization- or the DNA-binding domain of ETV6. The proteins translated from the cDNAs of these mutants were unable to repress transcription and showed dominant-negative effects. In addition, we demonstrate that one-third of AML patients have deficient ETV6 protein expression, which is not related to ETV6 mRNA expression levels. In conclusion, we demonstrate that ETV6 abnormalities are not restricted to translocations and occur more frequently in AML than previously thought. Additional comprehensive studies are required to define the clinical consequence of ETV6 loss of function in AML.
Acute leukaemias are commonly caused by mutations that corrupt the transcriptional circuitry of haematopoietic stem/progenitor cells. However, the mechanisms underlying large-scale transcriptional ...reprogramming remain largely unknown. Here we investigated transcriptional reprogramming at genome-scale in mouse retroviral transplant models of acute myeloid leukaemia (AML) using both gene-expression profiling and ChIP-sequencing. We identified several thousand candidate regulatory regions with altered levels of histone acetylation that were characterised by differential distribution of consensus motifs for key haematopoietic transcription factors including Gata2, Gfi1 and Sfpi1/Pu.1. In particular, downregulation of Gata2 expression was mirrored by abundant GATA motifs in regions of reduced histone acetylation suggesting an important role in leukaemogenic transcriptional reprogramming. Forced re-expression of Gata2 was not compatible with sustained growth of leukaemic cells thus suggesting a previously unrecognised role for Gata2 in downregulation during the development of AML. Additionally, large scale human AML datasets revealed significantly higher expression of GATA2 in CD34+ cells from healthy controls compared with AML blast cells. The integrated genome-scale analysis applied in this study represents a valuable and widely applicable approach to study the transcriptional control of both normal and aberrant haematopoiesis and to identify critical factors responsible for transcriptional reprogramming in human cancer.
The Scl gene encodes a transcription factor essential for haematopoietic development. Scl transcription is regulated by a panel of cis-elements spread over 55 kb with the most distal 3' element being ...located downstream of the neighbouring gene Map17, which is co-regulated with Scl in haematopoietic cells. The Scl/Map17 domain is flanked upstream by the ubiquitously expressed Sil gene and downstream by a cluster of Cyp genes active in liver, but the mechanisms responsible for delineating the domain boundaries remain unclear. Here we report identification of a DNaseI hypersensitive site at the 3' end of the Scl/Map17 domain and 45 kb downstream of the Scl transcription start site. This element is located at the boundary of active and inactive chromatin, does not function as a classical tissue-specific enhancer, binds CTCF and is both necessary and sufficient for insulator function in haematopoietic cells in vitro. Moreover, in a transgenic reporter assay, tissue-specific expression of the Scl promoter in brain was increased by incorporation of 350 bp flanking fragments from the +45 element. Our data suggests that the +45 region functions as a boundary element that separates the Scl/Map17 and Cyp transcriptional domains, and raise the possibility that this element may be useful for improving tissue-specific expression of transgenic constructs.
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