Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. The paternally expressed gene insulin-like growth-factor 2 (Igf2) is separated by approximately 100 kb from the maternally expressed noncoding gene H19 on mouse distal chromosome 7. Differentially methylated regions in Igf2 and H19 contain chromatin boundaries, silencers and activators and regulate the reciprocal expression of the two genes in a methylation-sensitive manner by allowing them exclusive access to a shared set of enhancers. Various chromatin models have been proposed that separate Igf2 and H19 into active and silent domains. Here we used a GAL4 knock-in approach as well as the chromosome conformation capture technique to show that the differentially methylated regions in the imprinted genes Igf2 and H19 interact in mice. These interactions are epigenetically regulated and partition maternal and paternal chromatin into distinct loops. This generates a simple epigenetic switch for Igf2 through which it moves between an active and a silent chromatin domain.     

Natural variation in cardiac metabolism and gene expression in Fundulus heteroclitus

Individual variation in gene expression is important for evolutionary adaptation and susceptibility to diseases and pathologies. In this study, we address the functional importance of this variation by comparing cardiac metabolism to patterns of mRNA expression using microarrays. There is extensive variation in both cardiac metabolism and the expression of metabolic genes among individuals of the teleost fish Fundulus heteroclitus from natural outbred populations raised in a common environment: metabolism differed among individuals by a factor of more than 2, and expression levels of 94% of genes were significantly different (P < 0.01) between individuals in a population. This unexpectedly high variation in metabolic gene expression explains much of the variation in metabolism, suggesting that it is biologically relevant. The patterns of gene expression that are most important in explaining cardiac metabolism differ between groups of individuals. Apparently, the variation in metabolism seems to be related to different patterns of gene expression in the different groups of individuals. The magnitude of differences in gene expression in these groups is not important; large changes in expression have no greater predictive value than small changes. These data suggest that variation in physiological performance is related to the subtle variation in gene expression and that this relationship differs among individuals.     

Stochastic switching as a survival strategy in fluctuating environments

A classic problem in population and evolutionary biology is to understand how a population optimizes its fitness in fluctuating environments. A population might enhance its fitness by allowing individual cells to stochastically transition among multiple phenotypes, thus ensuring that some cells are always prepared for an unforeseen environmental fluctuation. Here we experimentally explore how switching affects population growth by using the galactose utilization network of Saccharomyces cerevisiae. We engineered a strain that randomly transitions between two phenotypes as a result of stochastic gene expression. Each phenotype was designed to confer a growth advantage over the other phenotype in a certain environment. When we compared the growth of two populations with different switching rates, we found that fast-switching populations outgrow slow switchers when the environment fluctuates rapidly, whereas slow-switching phenotypes outgrow fast switchers when the environment changes rarely. These results suggest that cells may tune inter-phenotype switching rates to the frequency of environmental changes.     

An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo

The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from E(mu)-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53-/- lymphomagenesis in the E(mu)-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo.     

Action of BTN1, the yeast orthologue of the gene mutated in Batten disease.

Neuronal ceroid-lipofuscinoses (NCL) are autosomal recessive disorders that form the most common group of progressive neurodegenerative diseases in children, with an incidence as high as 1 in 12,500 live births, and with approximately 440,000 carriers in the United States. Disease progression is characterized by a decline in mental abilities, increased severity of untreatable seizures, blindness, loss of motor skills and premature death. The CLN3 gene, which is responsible for Batten disease, has been positionally cloned. The yeast gene, denoted BTN1, encodes a non-essential protein that is 39% identical and 59% similar to human CLN3. Strains lacking Btn1p, btn1-delta, are resistant to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP) in a pH-dependent manner. This phenotype was complemented by expression of human CLN3, demonstrating that yeast Btn1p and human CLN3 share the same function. Here, we report that btn1-delta yeast strains have an abnormally acidic vacuolar pH in the early phases of growth. Furthermore, DNA microarray analysis of BTN1 and btn1-delta strains revealed differential expression of two genes, with at least one, HSP30, involved in pH control. Because Btn1p is located in the vacuole, we suggest that Batten disease is caused by a defect in vacuolar (lysosomal) pH control. Our findings draw parallels between fundamental biological processes in yeast and previously observed characteristics of neurodegeneration in humans.     

Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse

The genetic bases underlying prostate tumorigenesis are poorly understood. Inactivation of the tumor-suppressor gene PTEN and lack of p27(KIP1) expression have been detected in most advanced prostate cancers. But mice deficient for Cdkn1b (encoding p27(Kip1)) do not develop prostate cancer. PTEN activity leads to the induction of p27(KIP1) expression, which in turn can negatively regulate the transition through the cell cycle. Thus, the inactivation of p27(KIP1) may be epistatic to PTEN in the control of the cell cycle. Here we show that the concomitant inactivation of one Pten allele and one or both Cdkn1b alleles accelerates spontaneous neoplastic transformation and incidence of tumors of various histological origins. Cell proliferation, but not cell survival, is increased in Pten(+/-)/Cdkn1b(-/-) mice. Moreover, Pten(+/-)/Cdkn1b(-/-) mice develop prostate carcinoma at complete penetrance within three months from birth. These cancers recapitulate the natural history and pathological features of human prostate cancer. Our findings reveal the crucial relevance of the combined tumor-suppressive activity of Pten and p27(Kip1) through the control of cell-cycle progression.     

The peripheral myelin gene PMP-22/GAS-3 is duplicated in Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a DNA duplication at chromosome 17p11.2. In view of the point mutation in the gene for peripheral myelin protein pmp-22/gas-3 in Trembler mice, a murine model for CMT1A, we have analysed whether this gene is altered in CMT1A. Here we show that the human homologue of the murine pmp-22 gene is located within the CMT1A DNA duplication, which is a direct repeat and does not interrupt the coding region of PMP-22. Expression of PMP-22 in CMT1A fibroblasts is similar to expression in control fibroblasts. Increased gene dosage or altered PMP-22 expression in the peripheral nervous system are therefore possible mechanisms by which PMP-22 is involved in CMT1A.     

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.     

Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.