Ruth B. McCole

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide- and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.

Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes

Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

Transcript- and tissue-specific imprinting of a tumour suppressor gene

The Bladder Cancer-Associated Protein gene (BLCAP; previously BC10) is a tumour suppressor that limits cell proliferation and stimulates apoptosis. BLCAP protein or message are downregulated or absent in a variety of human cancers. In mouse and human, the first intron of Blcap/BLCAP contains the distinct Neuronatin (Nnat/NNAT) gene. Nnat is an imprinted gene that is exclusively expressed from the paternally inherited allele. Previous studies found no evidence for imprinting of Blcap in mouse or human. Here we show that Blcap is imprinted in mouse and human brain, but not in other mouse tissues. Moreover, Blcap produces multiple distinct transcripts that exhibit reciprocal allele-specific expression in both mouse and human. We propose that the tissue-specific imprinting of Blcap is due to the particularly high transcriptional activity of Nnat in brain, as has been suggested previously for the similarly organized and imprinted murine Commd1/U2af1-rs1 locus. For Commd1/U2af1-rs1, we show that it too produces distinct transcript variants with reciprocal allele-specific expression. The imprinted expression of BLCAP and its interplay with NNAT at the transcriptional level may be relevant to human carcinogenesis.

Genes with monoallelic expression contribute disproportionately to genetic diversity in humans

An unexpectedly large number of human autosomal genes are subject to monoallelic expression (MAE). Our analysis of 4,227 such genes uncovers surprisingly high genetic variation across human populations. This increased diversity is unlikely to reflect relaxed purifying selection. Remarkably, MAE genes exhibit an elevated recombination rate and an increased density of hypermutable sequence contexts. However, these factors do not fully account for the increased diversity. We find that the elevated nucleotide diversity of MAE genes is also associated with greater allelic age: variants in these genes tend to be older and are enriched in polymorphisms shared by Neanderthals and chimpanzees. Both synonymous and nonsynonymous alleles of MAE genes have elevated average population frequencies. We also observed strong enrichment of the MAE signature among genes reported to evolve under balancing selection. We propose that an important biological function of widespread MAE might be the generation of cell-to-cell heterogeneity; the increased genetic variation contributes to this heterogeneity.

Genome-wide and parental allele-specific analysis of CTCF and cohesin DNA binding in mouse brain reveals a tissue-specific binding pattern and an association with imprinted differentially methylated regions

DNA binding factors are essential for regulating gene expression. CTCF and cohesin are DNA binding factors with central roles in chromatin organization and gene expression. We determined the sites of CTCF and cohesin binding to DNA in mouse brain, genome wide and in an allele-specific manner with high read-depth ChIP-seq. By comparing our results with existing data for mouse liver and embryonic stem (ES) cells, we investigated the tissue specificity of CTCF binding sites. ES cells have fewer unique CTCF binding sites occupied than liver and brain, consistent with a ground-state pattern of CTCF binding that is elaborated during differentiation. CTCF binding sites without the canonical consensus motif were highly tissue specific. In brain, a third of CTCF and cohesin binding sites coincide, consistent with the potential for many interactions between cohesin and CTCF but also many instances of independent action. In the context of genomic imprinting, CTCF and/or cohesin bind to a majority but not all differentially methylated regions, with preferential binding to the unmethylated parental allele. Whether the parental allele-specific methylation was established in the parental germlines or post-fertilization in the embryo is not a determinant in CTCF or cohesin binding. These findings link CTCF and cohesin with the control regions of a subset of imprinted genes, supporting the notion that imprinting control is mechanistically diverse.

Unwitting hosts fall victim to imprinting

Imprinting is a form of epigenetic gene regulation whereby the expression of an allele is dictated by parental origin. This parental legacy is established in the germ-line via heritable epigenetic modifications such as DNA methylation that are maintained throughout the somatic development of offspring. Imprinting is important in aspects of growth and development in mammals, flowering plants and in genetic disease, but in the context of this review, imprinted genes provide a model for dissecting epigenetic mechanisms because the active and silent alleles are in the same cell. In the presence of an identical complement of trans-regulatory factors, allelic differences in transcription are likely to be largely attributable to epigenetic factors. However, imprinting is historically associated with influences on transcription purely at the point of transcript initiation, where maternally and paternally derived alleles of genes are differentially active, allele-specific differences in termination processes have not been previously reported. We discuss how alternative polyadenylation (poly(A)) sites at the imprinted H13 gene are utilized in an allele-specific way and how other imprinted loci behave similarly.

A Case-by-Case Evolutionary Analysis of Four Imprinted Retrogenes

Retroposition is a widespread phenomenon resulting in the generation of new genes that are initially related to a parent gene via very high coding sequence similarity. We examine the evolutionary fate of four retrogenes generated by such an event; mouseu2002Inpp5f_v2, Mcts2, Nap1l5, andu2002U2af1‐rs1.u2002These genes are all subject to the epigenetic phenomenon of parental imprinting. We first provide new data on the age of these retrogene insertions. Using codon‐based models of sequence evolution, we show these retrogenes have diverse evolutionary trajectories, including divergence from the parent coding sequence under positive selection pressure, purifying selection pressure maintaining parent‐retrogene similarity, and neutral evolution. Examination of the expression pattern of retrogenes shows an atypical, broad pattern across multiple tissues. Protein 3D structure modeling reveals that a positively selected residue inu2002U2af1‐rs1, not shared by its parent, may influence protein conformation. Our case‐by‐case analysis of the evolution of four imprinted retrogenes reveals that this interesting class of imprinted genes, while similar in regulation and sequence characteristics, follow very varied evolutionary paths.

Abnormal dosage of ultraconserved elements is highly disfavored in healthy cells but not cancer cells.

Ultraconserved elements (UCEs) are strongly depleted from segmental duplications and copy number variations (CNVs) in the human genome, suggesting that deletion or duplication of a UCE can be deleterious to the mammalian cell. Here we address the process by which CNVs become depleted of UCEs. We begin by showing that depletion for UCEs characterizes the most recent large-scale human CNV datasets and then find that even newly formed de novo CNVs, which have passed through meiosis at most once, are significantly depleted for UCEs. In striking contrast, CNVs arising specifically in cancer cells are, as a rule, not depleted for UCEs and can even become significantly enriched. This observation raises the possibility that CNVs that arise somatically and are relatively newly formed are less likely to have established a CNV profile that is depleted for UCEs. Alternatively, lack of depletion for UCEs from cancer CNVs may reflect the diseased state. In support of this latter explanation, somatic CNVs that are not associated with disease are depleted for UCEs. Finally, we show that it is possible to observe the CNVs of induced pluripotent stem (iPS) cells become depleted of UCEs over time, suggesting that depletion may be established through selection against UCE-disrupting CNVs without the requirement for meiotic divisions.

Short Interspersed Element (SINE) Depletion and Long Interspersed Element (LINE) Abundance Are Not Features Universally Required for Imprinting

Genomic imprinting is a form of gene dosage regulation in which a gene is expressed from only one of the alleles, in a manner dependent on the parent of origin. The mechanisms governing imprinted gene expression have been investigated in detail and have greatly contributed to our understanding of genome regulation in general. Both DNA sequence features, such as CpG islands, and epigenetic features, such as DNA methylation and non-coding RNAs, play important roles in achieving imprinted expression. However, the relative importance of these factors varies depending on the locus in question. Defining the minimal features that are absolutely required for imprinting would help us to understand how imprinting has evolved mechanistically. Imprinted retrogenes are a subset of imprinted loci that are relatively simple in their genomic organisation, being distinct from large imprinting clusters, and have the potential to be used as tools to address this question. Here, we compare the repeat element content of imprinted retrogene loci with non-imprinted controls that have a similar locus organisation. We observe no significant differences that are conserved between mouse and human, suggesting that the paucity of SINEs and relative abundance of LINEs at imprinted loci reported by others is not a sequence feature universally required for imprinting.

Oligopaints: highly efficient, bioinformatically designed probes for fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful tool to study chromosome structure, positioning, and gene expression on a cell-by-cell basis. We have developed Oligopaints [1], a PCR-based method for generating highly efficient FISH probes from complex DNA libraries. Our method can visualize genomic regions ranging in size from tens of kilobases to megabases with the same basic protocol and gives researchers precise control over the location and patterning of each probe set. We have mined the reference genomes of C. elegans, D. melanogaster, A. thaliana, M. musculus, and humans for genomically unique 32-base sequences with thermodynamically desirable hybridization properties, and have made these sequences available on the Oligopaints website [http://genetics.med.harvard.edu] along with a suite of scripts and documentation that will assist researchers with probe set design and allow our technology to be extended to any organism whose genome has been sequenced. Oligopaints robustly label chromosomes both in tissue culture cells and whole-mount tissue preparations and can be generated using standard molecular biology techniques and equipment at a price well below the cost of commercial FISH probes. The flexibility offered by our bioinformatic design platform has allowed us to perform complicated hybridizations, such as the simultaneous targeting of RNA and the genomic DNA flanking its site of transcription. Thus, we anticipate that Oligopaints will be a valuable tool for the study of nuclear architecture and the relationship between chromosome positioning and gene expression.