[show abstract][hide abstract] ABSTRACT: Epigenetics may be broadly defined as the study of processes that produce a heritable phenotype that is not strictly dependent on DNA sequence. The definition has traditionally been restricted to processes that occur in the cell’s nucleus, with the term ‘heritable’ having a loose meaning that can be applied to either the entire organism or single cells. For example, a process that produces a phenotype only in a specific cell type (for instance, chromatin-mediated maintenance of a differentiated state) is usually considered epigenetic even if it is not directly inherited, but instead must be re-established or actively maintained at each cell division. Given this definition, the field of epigenetics has long focused on proteins that affect DNA packaging, and thereby affect the utilization of the genetic information encoded in the DNA template. This focus extends to the enzymatic modification of those proteins, and to the enzymatic modification of the DNA template itself, primarily DNA methylation.
Cytogenetic and Genome Research 02/2006; 114(1):1-15. · 1.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prader-Willi and Angelman syndromes (PWS and AS) typically result from an approximately 4-Mb deletion of human chromosome 15q11-q13, with clustered breakpoints (BP) at either of two proximal sites (BP1 and BP2) and one distal site (BP3). HERC2 and other duplicons map to these BP regions, with the 2-Mb PWS/AS imprinted domain just distal of BP2. Previously, the presence of genes and their imprinted status have not been examined between BP1 and BP2. Here, we identify two known (CYFIP1 and GCP5) and two novel (NIPA1 and NIPA2) genes in this region in human and their orthologs in mouse chromosome 7C. These genes are expressed from a broad range of tissues and are nonimprinted, as they are expressed in cells derived from normal individuals, patients with PWS or AS, and the corresponding mouse models. However, replication-timing studies in the mouse reveal that they are located in a genomic domain showing asynchronous replication, a feature typically ascribed to monoallelically expressed loci. The novel genes NIPA1 and NIPA2 each encode putative polypeptides with nine transmembrane domains, suggesting function as receptors or as transporters. Phylogenetic analyses show that NIPA1 and NIPA2 are highly conserved in vertebrate species, with ancestral members in invertebrates and plants. Intriguingly, evolutionary studies show conservation of the four-gene cassette between BP1 and BP2 in human, including NIPA1/2, CYFIP1, and GCP5, and proximity to the Herc2 gene in both mouse and Fugu. These observations support a model in which duplications of the HERC2 gene at BP3 in primates first flanked the four-gene cassette, with subsequent transposition of these four unique genes by a HERC2 duplicon-mediated process to form the BP1-BP2 region. Duplicons therefore appear to mediate genomic fluidity in both disease and evolutionary processes.
The American Journal of Human Genetics 11/2003; 73(4):898-925. · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prader-Willi syndrome (PWS) results from loss of function of a 1.0- to 1.5-Mb domain of imprinted, paternally expressed genes in human Chromosome (Chr) 15q11-q13. The loss of imprinted gene expression in the homologous region in mouse Chr 7C leads to a similar neonatal PWS phenotype. Several protein-coding genes in the human PWS region are intronless, possibly arising by retrotransposition. Here we present evidence for continued acquisition of genes by the mouse PWS region during evolution. Bioinformatic analyses identified a BAC containing four genes, Mkrn3, Magel2, Ndn, Frat3, and the Atp5l-ps1 pseudogene, the latter two genes derived from recent L1-mediated retrotransposition. Analyses of eight overlapping BACs indicate that these genes are clustered within 120 kb in two inbred strains, in the order tel-Atp5l-ps1-Frat3-Mkrn3-Magel2-Ndn-cen. Imprinting analyses show that Frat3 is differentially methylated and expressed solely from the paternal allele in a transgenic mouse model of Angelman syndrome, with no expression from the maternal allele in a mouse model of PWS. Loss of Frat3 expression may, therefore, contribute to the phenotype of mouse models of PWS. The identification of five intronless genes in a small genomic interval suggests that this region is prone to retroposition in germ cells or their zygotic and embryonic cell precursors, and that it allows the subsequent functional expression of these foreign sequences. The recent evolutionary acquisition of genes that adopt the same imprint as older, flanking genes indicates that the newly acquired genes become 'innocent bystanders' of a primary epigenetic signal causing imprinting in the PWS domain.
[show abstract][hide abstract] ABSTRACT: Experimental data published in recent years showed that up to 10% of all cases of mild to severe idiopathic mental retardation may result from small rearrangements of the subtelomeric regions of human chromosomes. To detect such cryptic translocations, we developed a "telomeric" multiplex fluorescence in situ hybridization (M-FISH) assay, using a set of previously published and commercially available subtelomeric probes. This set of probes includes 41 cosmid/PAC/P1 clones located from less than 100 kilobases to approximately 1 megabase from the end of the chromosomes. Similarly, a published mouse probe set, comprised of BACs hybridizing to the closest known marker toward the centromere and telomere of each mouse chromosome, was used to develop a mouse-specific "telomeric" M-FISH. Three different combinatorial labeling strategies were used to simultaneously detect all human subtelomeric regions on one slide. The simplest approach uses only three fluors and can be performed in laboratories lacking sophisticated imaging equipment or personnel highly trained in cytogenetics. A standard fluorescence microscope equipped with only three filters is sufficient. Fluor-dUTPs and labeled probes can be custom made, thus dramatically reducing costs. Images can be prepared using imaging software (Adobe Photoshop) and analysis performed by simple visual inspection.
[show abstract][hide abstract] ABSTRACT: We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.
Molecular and Cellular Biology 02/2001; 21(1):298-309. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Expression of a construct integrated at different genomic locations often varies because of position effects that have been subcategorized as stable (decreased level of expression) and variegating (decreased proportion of expressing cells). It is well established that locus control regions (LCRs) generally overcome position effects in transgenes. However, whether stable and variegated position effects are equally overcome by an intact LCR has not been determined. We report that single-copy yeast artificial chromosome transgenes containing an unmodified human beta -globin locus were not subject to detectable stable position effects but did undergo mild to severe variegating position effects at three of the four non-centromeric integration sites tested. We also find that, at a given integration site, the distance and the orientation of the LCR relative to the regulated gene contributes to the likelihood of variegating position effects, and can affect the magnitude of its transcriptional enhancement. DNase I hypersensitive site (HSS) formation varies with the proportion of expressing cells, not the level of gene expression, suggesting that silencing of the transgene is associated with a lack of HSS formation in the LCR region. We conclude that transcriptional enhancement and variegating position effects are caused by fundamentally different but inter-dependent mechanisms.
Human Molecular Genetics 04/2000; 9(4):631-6. · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nuclear matrix binding assays (NMBAs) define certain DNA sequences as matrix attachment regions (MARs), which often have cis-acting epigenetic regulatory functions. We used NMBAs to analyze the functionally important 15q11-q13 imprinting center (IC). We find that the IC is composed of an unusually high density of MARs, located in close proximity to the germ line elements that are proposed to direct imprint switching in this region. Moreover, we find that the organization of MARs is the same at the homologous mouse locus, despite extensive divergence of DNA sequence. MARs of this size are not usually associated with genes but rather with heterochromatin-forming areas of the genome. In contrast, the 15q11-q13 region contains multiple transcribed genes and is unusual for being subject to genomic imprinting, causing the maternal chromosome to be more transcriptionally silent, methylated, and late replicating than the paternal chromosome. We suggest that the extensive MAR sequences at the IC are organized as heterochromatin during oogenesis, an organization disrupted during spermatogenesis. Consistent with this model, multicolor fluorescence in situ hybridization to halo nuclei demonstrates a strong matrix association of the maternal IC, whereas the paternal IC is more decondensed, extending into the nuclear halo. This model also provides a mechanism for spreading of the imprinting signal, because heterochromatin at the IC on the maternal chromosome may exert a suppressive position effect in cis. We propose that the germ line elements at the 15q11-q13 IC mediate their effects through the candidate heterochromatin-forming DNA identified in this study.
Proceedings of the National Academy of Sciences 01/2000; 96(25):14430-5. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genes are recognized as undergoing genomic imprinting when they are capable of being expressed only from the paternal or only from the maternal chromosome. The process can occur coordinately within large physical domains in mammalian chromosomes. One interesting facet of the study of genomic imprinting is that it offers insight into the regulation of large chromosomal regions. Understanding this regulation involves elucidating the cis-acting regulators of gene expression and defining the elements that maintain chromatin insulation, both required for understanding more practically applicable areas of biological research, such as efficient transgene production. This review is focused on the regulation of the imprinted domain of human chromosome 11p15.5, responsible for Beckwith-Wiedemann syndrome (BWS). Recent findings indicate that the maintenance of imprinting within this domain is critically dependent on the stable maintenance of chromatin insulation.
[show abstract][hide abstract] ABSTRACT: Genes subject to genomic imprinting generally occur in clusters of hundreds of kilobases. These domains exhibit several gamete of origin-dependent manifestations, including a pattern of asynchronous replication when studied by fluorescence in situ hybridization (FISH). We find a transition from asynchronous replication at the imprinted mouse H19 gene to synchronous replication at the downstream Rpl23 gene, the human homologue of which appears to be non-imprinted. Two-colour FISH demonstrates that this transition is due solely to a difference in replication timing between the upstream and downstream chromatin on the later-replicating (maternal) chromosome. This difference is lost in mice deleted for the H19 gene body and 9.9 kb of upstream DNA when this deletion is maternally inherited, with synchronous replication patterns extending over 110 kb upstream from the deleted area. No effect is seen when the deletion is paternally inherited. The presence of a boundary element in this region has been suggested by observations of position-independent expression of H19 -containing transgenes and the blocking of accessibility of downstream enhancers to the upstream Igf2 and Ins2 genes on the maternal chromosome. The FISH studies presented here demonstrate the insulation of replication patterns within the imprinted domain from downstream, non-imprinted chromatin, mediated by an element at the H19 locus which is subject to genomic imprinting.
Human Molecular Genetics 02/1998; 7(1):91-5. · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have mapped the matrix-attachment regions (MARs) in 200 kilobases of the mouse Chromosome (Chr) 7F imprinted domain. MARs are genetic elements known to have effects in cis on methylation at nonimprinted loci. The imprinting of the Igf2 and Ins2 genes is dependent on the transcription of the downstream H19 gene. The transcription of H19 is dependent in turn on its methylation status. The cis-acting regulators of methylation at this site are not known. As MARs are potential regulators not only of methylation but also other elements of genomic imprinting, we mapped the MARs within the 200 kilobases around H19. This report describes the mapping of four MARs from this region.
[show abstract][hide abstract] ABSTRACT: We are currently in an era of increased interest in the role of the epigenome in normal cellular physiology and its role in
human disease. Part of this increased interest is driven by new technologies that have allowed us to gain insights never previously
possible. Our view of cytosine methylation is expanding not only in terms of how much of the genome we can study at a time,
but also in terms of what we think cytosine methylation might be doing functionally. While DNA methylation in mammalian cells
has been studied for more than 45 years at this point (srinivasan1962), new insights are revealing the sobering reality that
we understand much less about its functional consequences than we may have believed. In this review, the insights gained from
new technologies to study cytosine methylation are examined so that we can redefine the paths for further exploration of this
intriguing molecular regulator.