Joanne L. Thorvaldsen

Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice.

The serine-threonine kinase Akt, also known as protein kinase B (PKB), is an important effector for phosphatidylinositol 3-kinase signaling initiated by numerous growth factors and hormones. Akt2/PKBβ, one of three known mammalian isoforms of Akt/PKB, has been demonstrated recently to be required for at least some of the metabolic actions of insulin (Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science292, 1728–1731). Here we show that mice deficient in another closely related isoform of the kinase, Akt1/PKBα, display a conspicuous impairment in organismal growth. Akt1 −/− mice demonstrated defects in both fetal and postnatal growth, and these persisted into adulthood. However, in striking contrast to Akt2/PKBβ null mice, Akt1/PKBα-deficient mice are normal with regard to glucose tolerance and insulin-stimulated disposal of blood glucose. Thus, the characterization of the Akt1 knockout mice and its comparison to the previously reported Akt2 deficiency phenotype reveals the non-redundant functions of Akt1 andAkt2 genes with respect to organismal growth and insulin-regulated glucose metabolism.

Maternal imprinting at the H19–Igf2 locus maintains adult haematopoietic stem cell quiescence

The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19–Igf2 locus, in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele. In addition, H19 serves as a source of miR-675, which restricts Igf1r expression. We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2–Igfr1 pathway, as shown by the translocation of phosphorylated FoxO3 (an inactive form) from nucleus to cytoplasm and the release of FoxO3-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19–Igf2 locus in maintaining adult stem cells.

Analysis of Sequence Upstream of the Endogenous H19 Gene Reveals Elements Both Essential and Dispensable for Imprinting

ABSTRACT Imprinting of the linked and oppositely expressed mouse H19 and Igf2 genes requires a 2-kb differentially methylated domain (DMD) that is located 2 kb upstream of H19. This element is postulated to function as a methylation-sensitive insulator. Here we test whether an additional sequence 5′ of H19 is required for H19 and Igf2 imprinting. Because repetitive elements have been suggested to be important for genomic imprinting, the requirement of a G-rich repetitive element that is located immediately 3′ to the DMD was first tested in two targeted deletions: a 2.9-kb deletion (ΔDMDΔG) that removes the DMD and G-rich repeat and a 1.3-kb deletion (ΔG) removing only the latter. There are also four 21-bp GC-rich repetitive elements within the DMD that bind the insulator-associated CTCF (CCCTC-binding factor) protein and are implicated in mediating methylation-sensitive insulator activity. As three of the four repeats of the 2-kb DMD were deleted in the initial 1.6-kb ΔDMD allele, we analyzed a 3.8-kb targeted allele (Δ3.8kb-5′H19), which deletes the entire DMD, to test the function of the fourth repeat. Comparative analysis of the 5′ deletion alleles reveals that (i) the G-rich repeat element is dispensable for imprinting, (ii) the ΔDMD and ΔDMDΔG alleles exhibit slightly more methylation upon paternal transmission, (iii) removal of the 5′ CTCF site does not further perturb H19 and Igf2 imprinting, suggesting that one CTCF-binding site is insufficient to generate insulator activity in vivo, (iv) the DMD sequence is required for full activation of H19 and Igf2, and (v) deletion of the DMD disrupts H19 and Igf2 expression in a tissue-specific manner.

Three-dimensional conformation at the H19/Igf2 locus supports a model of enhancer tracking

Insight into how the mammalian genome is structured in vivo is key to understanding transcriptional regulation. This is especially true in complex domains in which genes are coordinately regulated by long-range interactions between cis-regulatory elements. The regulation of the H19/Igf2 imprinted region depends on the presence of several cis-acting sequences, including a methylation-sensitive insulator between Igf2 and H19 and shared enhancers downstream of H19. Each parental allele has a distinct expression pattern. We used chromosome conformation capture to assay the native three-dimensional organization of the H19/Igf2 locus on each parental copy. Furthermore, we compared wild-type chromosomes to several mutations that affect the insulator. Our results show that promoters and enhancers reproducibly co-localize at transcriptionally active genes, i.e. the endodermal enhancers contact the maternal H19 and the paternal Igf2 genes. The active insulator blocks traffic of the enhancers along the chromosome, restricting them to the H19 promoter. Conversely, the methylated inactive insulator allows the enhancers to contact the upstream regions, including Igf2. Mutations that either remove or inhibit insulator activity allow unrestricted access of the enhancers to the whole region. A mutation that allows establishment of an enhancer-blocker on the normally inactive paternal copy diminishes the contact of the enhancer with the Igf2 gene. Based on our results, we propose that physical proximity of cis-acting DNA elements is vital for their activity in vivo. We suggest that enhancers track along the chromosome until they find a suitable promoter sequence to interact with and that insulator elements block further tracking of enhancers.

Developmental Profile of H19 Differentially Methylated Domain (DMD) Deletion Alleles Reveals Multiple Roles of the DMD in Regulating Allelic Expression and DNA Methylation at the Imprinted H19/Igf2 Locus

ABSTRACT The differentially methylated domain (DMD) of the mouse H19 gene is a methylation-sensitive insulator that blocks access of the Igf2 gene to shared enhancers on the maternal allele and inactivates H19 expression on the methylated paternal allele. By analyzing H19 DMD deletion alleles H19ΔDMD and H19Δ3.8kb-5′H19 in pre- and postimplantation embryos, we show that the DMD exhibits positive transcriptional activity and is required for H19 expression in blastocysts and full activation of H19 during subsequent development. We also show that the DMD is required to establish Igf2 imprinting by blocking access to shared enhancers when Igf2 monoallelic expression is initiated in postimplantation embryos and that the single remaining CTCF site of the H19ΔDMD allele is unable to provide this function. Furthermore, our data demonstrate that sequence outside of the DMD can attract some paternal-allele-specific CpG methylation 5′ of H19 in preimplantation embryos, although this methylation is not maintained during postimplantation in the absence of the DMD. Finally, we report a conditional allele floxing the 1.6-kb sequence deleted from the H19ΔDMD allele and demonstrate that the DMD is required to maintain repression of the maternal Igf2 allele and the full activity of the paternal Igf2 allele in neonatal liver.

The Transcriptional Status but Not the Imprinting Control Region Determines Allele-Specific Histone Modifications at the Imprinted H19 Locus

ABSTRACT Genomic imprinting governs allele-specific gene expression in an epigenetically heritable manner. The characterization of histone modifications at imprinted gene loci is incomplete, and whether specific histone marks determine transcription or are dependent on it is not understood. Using chromatin immunoprecipitations, we examined in multiple cell types and in an allele-specific manner the active and repressive histone marks of several imprinted loci, including H19, KvDMR1, Snrpn promoter/exon 1, and IG-DMR imprinting control regions. Expressed alleles are enriched for specific actively modified histones, including H3 di- and trimethylated at Lys4 and acetylated histones H3 and H4, while their silent counterparts are associated with repressive marks such as H3 trimethylated at Lys9 alone or in combination with H3 trimethylated at Lys27 and H4/H2A symmetrically dimethylated at Arg3. At H19, allele-specific histone modifications occur throughout the entire locus, including nontranscribed regions such as the differentially methylated domain (DMD) as well as sequences in the H19 gene body that are not differentially methylated. Significantly, the presence of active marks at H19 depends on transcriptional activity and occurs even in the absence of the DMD. These findings suggest that histone modifications are dependent on the transcriptional status of imprinted alleles and illuminate epigenetic mechanisms of genomic imprinting.

SnapShot: Imprinted Gene Clusters

The majority of imprinted genes are found in conserved clusters in the mammalian genome. Shown are mouse imprinted genes that are part of larger imprinting clusters (variations in hum cluster has a prominent gene indicated in the left most column and one or more noncoding (nc)RNAs, which in many cases are critical for the domain-wide regulation of the cluster. A associated imprinted genes that typically are jointly regulated through a common imprinting control region (ICR). Single imprinted gene loci are not included in this table and can be foun ics Unit, Genomic Imprinting: http://www.mgu.har.mrc.ac.uk/research/imprinting; University of Otago, Catalogue of Parent of Origin Effects: http://igc.otago.ac.nz/home.html. The offi c parentheses. 1The prominent protein-coding gene within each cluster is listed in this column. The exception to this is Xist, which encodes a long ncRNA that coats the inactive X chromosome in female 2In most cases these associated genes are jointly regulated through the linked ICR. 3ICR indicates that the DMR has been validated as an imprinting control region by gene targeting studies in mice: deletion or mutation of the DMR results in loss of imprinting of at least o 4The mechanism of imprinting is unknown for most of the clusters (designated as not determined, ND) but currently two types of clusters have been identifi ed. In one type of cluster a C expression. In the second type of cluster imprinting requires transcription of a long ncRNA that is usually initiated from a hypomethylated DMR/promoter. A question mark indicates that t 5UCSC Mouse build February 2006 and Human build March 2006. 6Deletion, mutation, or aberrant expression of the genes in the cluster results in the listed human disease. Additionally, ICR deletions and methylation abnormalities can cause these s disomies are associated with distinct abnormal phenotypes. Only proven associations are shown. 7Tissue-specifi c imprinting. 8Not imprinted in human. 9Imprinting status in human is unknown or confl icting. 10All short ncRNAs at this locus may be part of a longer ncRNA. 11The ICR regulating the paternally expressed genes has been identifi ed for mouse and human (PWS-IC) whereas the ICR that regulates the maternally expressed genes has been identifi e appears to be important for regulating both paternal and maternal genes at this locus. 12Loss of multiple paternally expressed genes is responsible for Prader-Willi syndrome (PWS) and loss of Ube3a expression is responsible for Angelman syndrome (AS). 13Mutations in SGCE are found in patients with Myoclonus dystonia syndrome. 14No human ortholog present at locus. S ee oline vsion or refences nd aknow ed em ets. 58

Novel cis-regulatory function in ICR-mediated imprinted repression of H19.

Expression of coregulated imprinted genes, H19 and Igf2, is monoallelic and parent-of-origin-dependent. Like most imprinted genes, H19 and Igf2 are regulated by a differentially methylated imprinting control region (ICR). CTCF binding sites and DNA methylation at the ICR have previously been identified as key cis-acting elements required for proper H19/Igf2 imprinting. Here, we use mouse models to elucidate further the mechanism of ICR-mediated gene regulation. We specifically address the question of whether sequences outside of CTCF sites at the ICR are required for paternal H19 repression. To this end, we generated two types of mutant ICRs in the mouse: (i) deletion of intervening sequence between CTCF sites (H19(ICR∆IVS)), which changes size and CpG content at the ICR; and (ii) CpG depletion outside of CTCF sites (H19(ICR-8nrCG)), which only changes CpG content at the ICR. Individually, both mutant alleles (H19(ICR∆IVS) and H19(ICR-8nrCG)) show loss of imprinted repression of paternal H19. Interestingly, this loss of repression does not coincide with a detectable change in methylation at the H19 ICR or promoter. Thus, neither intact CTCF sites nor hypermethylation at the ICR is sufficient for maintaining the fully repressed state of the paternal H19 allele. Our findings demonstrate, for the first time in vivo, that sequence outside of CTCF sites at the ICR is required in cis for ICR-mediated imprinted repression at the H19/Igf2 locus. In addition, these results strongly implicate a novel role of ICR size and CpG density in paternal H19 repression.

Tissue specific insulator function at H19/Igf2 revealed by deletions at the imprinting control region

Parent-of-origin-specific expression at imprinted genes is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). This mechanism of gene regulation, where one element controls allelic expression of multiple genes, is not fully understood. Furthermore, the mechanism of gene dysregulation through ICR epimutations, such as loss or gain of DNA methylation, remains a mystery. We have used genetic mouse models to dissect ICR-mediated genetic and epigenetic regulation of imprinted gene expression. The H19/insulin-like growth factor 2 (Igf2) ICR has a multifunctional role including insulation, activation and repression. Microdeletions at the human H19/IGF2 ICR (IC1) are proposed to be responsible for IC1 epimutations associated with imprinting disorders such as Beckwith-Wiedemann syndrome (BWS). Here, we have generated and characterized a mouse model that mimics BWS microdeletions to define the role of the deleted sequence in establishing and maintaining epigenetic marks and imprinted expression at the H19/IGF2 locus. These mice carry a 1.3 kb deletion at the H19/Igf2 ICR [Δ2,3] removing two of four CCCTC-binding factor (CTCF) sites and the intervening sequence, ∼75% of the ICR. Surprisingly, the Δ2,3 deletion does not perturb DNA methylation at the ICR; however, it does disrupt imprinted expression. While repressive functions of the ICR are compromised by the deletion regardless of tissue type, insulator function is only disrupted in tissues of mesodermal origin where a significant amount of CTCF is poly(ADP-ribosyl)ated. These findings suggest that insulator activity of the H19/Igf2 ICR varies by cell type and may depend on cell-specific enhancers as well as posttranslational modifications of the insulator protein CTCF.

Nonrandom X Chromosome Inactivation Is Influenced by Multiple Regions on the Murine X Chromosome

During the development of female mammals, one of the two X chromosomes is inactivated, serving as a dosage-compensation mechanism to equalize the expression of X-linked genes in females and males. While the choice of which X chromosome to inactivate is normally random, X chromosome inactivation can be skewed in F1 hybrid mice, as determined by alleles at the X chromosome controlling element (Xce), a locus defined genetically by Cattanach over 40 years ago. Four Xce alleles have been defined in inbred mice in order of the tendency of the X chromosome to remain active: Xcea < Xceb < Xcec < Xced. While the identity of the Xce locus remains unknown, previous efforts to map sequences responsible for the Xce effect in hybrid mice have localized the Xce to candidate regions that overlap the X chromosome inactivation center (Xic), which includes the Xist and Tsix genes. Here, we have intercrossed 129S1/SvImJ, which carries the Xcea allele, and Mus musculus castaneus EiJ, which carries the Xcec allele, to generate recombinant lines with single or double recombinant breakpoints near or within the Xce candidate region. In female progeny of 129S1/SvImJ females mated to recombinant males, we have measured the X chromosome inactivation ratio using allele-specific expression assays of genes on the X chromosome. We have identified regions, both proximal and distal to Xist/Tsix, that contribute to the choice of which X chromosome to inactivate, indicating that multiple elements on the X chromosome contribute to the Xce.