John M. Levorse

CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.

The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2,3,4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3′ of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.

Dissecting self-renewal in stem cells with RNA interference

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.

The temporal requirement for endothelin receptor-B signalling duringneural crest development

Endothelin receptor B (EDNRB) is a G-protein-coupled receptor with seven transmembrane domains which is required for the development of melanocytes and enteric neurons. Mice that are homozygous for a null mutation in the Ednrb gene are almost completely white and die as juveniles from megacolon. To determine when EDNRB signalling is required during embryogenesis, we have exploited the tetracycline-inducible system to generate strains of mice in which the endogenous Ednrb locus is under the control of the tetracycline-dependant transactivators tTa or rtTA. By using this system to express Ednrb at different stages of embryogenesis, we have determined that EDNRB is required during a restricted period of neural crest development between embryonic days 10 and 12.5. Moreover, our results imply that EDNRB is required for the migration of both melanoblasts and enteric neuroblasts.

CTCF maintains differential methylation at the Igf2 / H19 locus

Genomic imprinting relies on establishing and maintaining the parental-specific methylation of DNA elements that control the differential expression of maternal and paternal alleles. Although the essential DNA methyltransferases have been discovered, proteins that regulate the sequence-specific establishment and maintenance of allelic methylation have not been identified. One candidate regulator of methylation, the zinc-finger protein CTCF, binds to the imprinting control region (ICR) of the genes Igf2 (encoding insulin-like growth factor 2) and H19 (fetal liver mRNA; refs. 1,2). The unmethylated maternal ICR is a chromatin boundary that prevents distant enhancers from activating Igf2 (refs. 3–6). In vitro experiments have suggested that CTCF mediates boundary activity of the maternal ICR, and that methylation of the paternal ICR abolishes this activity by preventing CTCF binding. Using mice with point mutations in all four CTCF sites in the ICR, we show that maternally transmitted mutant ICRs in neonatal mice acquire a substantial but heterogeneous degree of methylation. Mutant ICRs in oocytes and blastocysts are not methylated, however, indicating that binding of CTCF is not required to establish the unmethylated ICR during oogenesis. We also show that the mutant ICR lacks enhancer-blocking activity, as the expression of Igf2 is activated on mutant maternal chromosomes. Conversely, maternal H19 expression is reduced, suggesting a positive role for CTCF in the transcription of that gene. This study constitutes the first in vivo demonstration of the multiple functions of CTCF in an ICR.

The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors.

Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.

Location of enhancers is essential for the imprinting of H19 and Igf2 genes.

Genomic imprinting is the process in mammals by which gamete-specific epigenetic modifications establish the differential expression of the two alleles of a gene. The tightly linked H19 and Igf2 genes are expressed in tissues of endodermal and mesodermal origin, with H19 expressed from the maternal chromosome and Igf2 expressed from the paternal chromosome. A model has been proposed to explain the reciprocal imprinting of these genes; in this model, expression of the genes is governed by competition between their promoters for a common set of enhancers. An extra set of enhancers might be predicted to relieve the competition, thereby eliminating imprinting. Here we tested this prediction by generating mice with a duplication of the endoderm-specific enhancers. The normally silent Igf2 gene on the maternal chromosome was expressed in liver, consistent with relief from competition. We then generated a maternal chromosome containing a single set of enhancers located equidistant from Igf2 and H19; the direction of the imprint was reversed. Thus, the location of the enhancers determines the outcome of competition in liver, and the strength of the H19 promoter is not sufficient to silence Igf2.

Clock Regulatory Elements Control Cyclic Expression of Lunatic fringe during Somitogenesis

Somitogenesis requires a segmentation clock and Notch signaling. Lunatic fringe (Lfng) expression in the presomitic mesoderm (PSM) cycles in the posterior PSM, is refined in the segmenting somite to the rostral compartment, and is required for segmentation. We identify distinct cis-acting regulatory elements for each aspect of Lfng expression. Fringe clock element 1 (FCE1) represents a conserved 110 bp region that is necessary to direct cyclic Lfng RNA expression in the posterior PSM. Mutational analysis of E boxes within FCE1 indicates a potential interplay of positive and negative transcriptional regulation by cyclically expressed bHLH proteins. A separable Lfng regulatory region directs expression to the prospective rostral aspect of the condensing somite. These independent Lfng regulatory cassettes advance a molecular framework for deciphering somite segmentation.

Expression and Functional Analysis of Uch-L3 during Mouse Development

ABSTRACT Mice homozygous for the s1Acrg deletion at the Ednrb locus arrest at embryonic day 8.5. To determine the molecular basis of this defect, we initiated positional cloning of the s1Acrg minimal region. The mouseUch-L3 (ubiquitin C-terminal hydrolase L3) gene was mapped within the s1Acrg minimal region. BecauseUch-L3 transcripts were present in embryonic structures relevant to the s1Acrg phenotype, we created a targeted mutation in Uch-L3 to address its role during development and its possible contribution to thes1Acrg phenotype. Mice homozygous for the mutation Uch-L3Δ3-7 were viable, with no obvious developmental or histological abnormalities. Although high levels of Uch-L3 RNA were detected in testes and thymus,Uch-L3Δ3-7 homozygotes were fertile, and no defect in intrathymic T-cell differentiation was detected. We conclude that the s1Acrg phenotype is either complex and multigenic or due to the loss of another gene within the region. We propose that Uch-L3 may be functionally redundant with its homologue Uch-L1.

Disruption of an imprinted gene cluster by a targeted chromosomal translocation in mice.

Genomic imprinting is an epigenetic process in which the activity of a gene is determined by its parent of origin. Mechanisms governing genomic imprinting are just beginning to be understood. However, the tendency of imprinted genes to exist in chromosomal clusters suggests a sharing of regulatory elements. To better understand imprinted gene clustering, we disrupted a cluster of imprinted genes on mouse distal chromosome 7 using the Cre/loxP recombination system. In mice carrying a site-specific translocation separating Cdkn1c and Kcnq1, imprinting of the genes retained on chromosome 7, including Kcnq1, Kcnq1ot1, Ascl2, H19 and Igf2, is unaffected, demonstrating that these genes are not regulated by elements near or telomeric to Cdkn1c. In contrast, expression and imprinting of the translocated Cdkn1c, Slc22a1l and Tssc3 on chromosome 11 are affected, consistent with the hypothesis that elements regulating both expression and imprinting of these genes lie within or proximal to Kcnq1. These data support the proposal that chromosomal abnormalities, including translocations, within KCNQ1 that are associated with the human disease Beckwith-Wiedemann syndrome (BWS) may disrupt CDKN1C expression. These results underscore the importance of gene clustering for the proper regulation of imprinted genes.

Translation from unconventional 5′ start sites drives tumour initiation

We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5′ untranslated regions in cancer, and expose new targets for therapeutic intervention.