Judith D. Brown

Gene expression analysis of human induced pluripotent stem cell-derived neurons carrying copy number variants of chromosome 15q11-q13.1

BackgroundDuplications of the chromosome 15q11-q13.1 region are associated with an estimated 1 to 3% of all autism cases, making this copy number variation (CNV) one of the most frequent chromosome abnormalities associated with autism spectrum disorder (ASD). Several genes located within the 15q11-q13.1 duplication region including ubiquitin protein ligase E3A (UBE3A), the gene disrupted in Angelman syndrome (AS), are involved in neural function and may play important roles in the neurobehavioral phenotypes associated with chromosome 15q11-q13.1 duplication (Dup15q) syndrome.MethodsWe have generated induced pluripotent stem cell (iPSC) lines from five different individuals containing CNVs of 15q11-q13.1. The iPSC lines were differentiated into mature, functional neurons. Gene expression across the 15q11-q13.1 locus was compared among the five iPSC lines and corresponding iPSC-derived neurons using quantitative reverse transcription PCR (qRT-PCR). Genome-wide gene expression was compared between neurons derived from three iPSC lines using mRNA-Seq.ResultsAnalysis of 15q11-q13.1 gene expression in neurons derived from Dup15q iPSCs reveals that gene copy number does not consistently predict expression levels in cells with interstitial duplications of 15q11-q13.1. mRNA-Seq experiments show that there is substantial overlap in the genes differentially expressed between 15q11-q13.1 deletion and duplication neurons, Finally, we demonstrate that UBE3A transcripts can be pharmacologically rescued to normal levels in iPSC-derived neurons with a 15q11-q13.1 duplication.ConclusionsChromatin structure may influence gene expression across the 15q11-q13.1 region in neurons. Genome-wide analyses suggest that common neuronal pathways may be disrupted in both the Angelman and Dup15q syndromes. These data demonstrate that our disease-specific stem cell models provide a new tool to decipher the underlying cellular and genetic disease mechanisms of ASD and may also offer a pathway to novel therapeutic intervention in Dup15q syndrome.

Making a long story short: noncoding RNAs and chromosome change

As important as the events that influence selection for specific chromosome types in the derivation of novel karyotypes, are the events that initiate the changes in chromosome number and structure between species, and likewise polymorphisms, variants and disease states within species. Although once thought of as transcriptional ‘noise’, noncoding RNAs (ncRNAs) are now recognized as important mediators of epigenetic regulation and chromosome stability. Here we highlight recent work that illustrates the influence short and long ncRNAs have as participants in the function and stability of chromosome regions such as centromeres, telomeres, evolutionary breakpoints and fragile sites. We summarize recent evidence that ncRNAs can facilitate chromosome change and present mechanisms by which ncRNAs create DNA breaks. Finally, we present hypotheses on how they may create novel karyotypes and thus affect chromosome evolution.

Four core genotypes mouse model: localization of the Sry transgene and bioassay for testicular hormone levels.

BackgroundThe “four core genotypes” (FCG) mouse model has emerged as a major model testing if sex differences in phenotypes are caused by sex chromosome complement (XX vs. XY) or gonadal hormones or both. The model involves deletion of the testis-determining gene Sry from the Y chromosome and insertion of an Sry transgene onto an autosome. It produces XX and XY mice with testes, and XX and XY mice with ovaries, so that XX and XY mice with the same type of gonad can be compared to assess phenotypic effects of sex chromosome complement in cells and tissues.FindingsWe used PCR to amplify the Sry transgene and adjacent genomic sequences, to resolve the location of the Sry transgene to chromosome 3 and confirmed this location by fluorescence in situ hybridization (FISH) of the Sry construct to metaphase chromosomes. Using quantitative PCR, we estimate that 12–14 copies of the transgene were inserted. The anogenital distance (AGD) of FCG pups at 27–29 days after birth was not different in XX vs. XY males, or XX vs. XY females, suggesting that differences between XX and XY mice with the same type of gonad are not caused by difference in prenatal androgen levels.ConclusionThe Sry transgene in FCG mice is present in multiple copies at one locus on chromosome 3, which does not interrupt known genes. XX and XY mice with the same type of gonad do not show evidence of different androgen levels prenatally.

Recent Amplification of the Kangaroo Endogenous Retrovirus, KERV, Limited to the Centromere

ABSTRACT Mammalian retrotransposons, transposable elements that are processed through an RNA intermediate, are categorized as short interspersed elements (SINEs), long interspersed elements (LINEs), and long terminal repeat (LTR) retroelements, which include endogenous retroviruses. The ability of transposable elements to autonomously amplify led to their initial characterization as selfish or junk DNA; however, it is now known that they may acquire specific cellular functions in a genome and are implicated in host defense mechanisms as well as in genome evolution. Interactions between classes of transposable elements may exert a markedly different and potentially more significant effect on a genome than interactions between members of a single class of transposable elements. We examined the genomic structure and evolution of the kangaroo endogenous retrovirus (KERV) in the marsupial genus Macropus. The complete proviral structure of the kangaroo endogenous retrovirus, phylogenetic relationship among relative retroviruses, and expression of this virus in both Macropus rufogriseus and M. eugenii are presented for the first time. In addition, we show the relative copy number and distribution of the kangaroo endogenous retrovirus in the Macropus genus. Our data indicate that amplification of the kangaroo endogenous retrovirus occurred in a lineage-specific fashion, is restricted to the centromeres, and is not correlated with LINE depletion. Finally, analysis of KERV long terminal repeat sequences using massively parallel sequencing indicates that the recent amplification in M. rufogriseus is likely due to duplications and concerted evolution rather than a high number of independent insertion events.

Interspecific hybridization induced amplification of Mdm2 on double minutes in a Mus hybrid

We report a case of interspecific hybridization induced amplification of Chromosome 10 on double minutes (dm) in the karyotype of a hybrid Mus embryo. Stable, non-mosaic dm were previously found in tissues of a 16.5-day Mus musculus × Mus caroli hybrid (Graves, 1984). Dm in tissues of the hybrid was of interest to us because of previous reports of genomic instability in interspecific hybrids (O’Neill et al., 1998) and thus we decided to characterize the dm in the hybrid karyotypes. Whole chromosome painting of the hybrid cell lines showed amplification of Chromosome 10 sequences. Southern analysis with a probe for the candidate gene Mdm2 showed amplification of the paternal allele of this oncogene. Overexpression of Mdm2 was confirmed by a western analysis that also showed an associated inactivation of the tumor suppressor, Trp53. Evidence indicates that the event leading to the instability observed was an early adaptive response to stress on the genome, i.e. interspecific hybridization.

Methylation perturbations in retroelements within the genome of a Mus interspecific hybrid correlate with double minute chromosome formation.

A reduction in the DNA modification of cytosine methylation has been linked directly to chromosome rearrangements concomitant with retroelement amplification in several marsupial hybrid genomes. While phenotypes observed for interspecific eutherian hybrids are suggestive of methylation perturbations and retroelement instability, no link between retroelements, DNA methylation, and chromosome instability has yet been identified. Previous studies in eutherian hybrids, however, have been limited to a gross examination of methylation using methylation-sensitive restriction enzyme analysis or focused on single-copy genes and/or have avoided examination of repetitive DNA. Methylation changes and retroelements are proposed as mechanisms for double minute chromosome formation and oncogene amplification, both present in the genome of a Mus hybrid model, thus making it an ideal system to evaluate methylation status more closely. We have used the PCR-based methodologies methylation-sensitive amplicon subtraction (MS-AS) and methylation-sensitive representational difference analysis (MS-RDA) to detect differentially methylated sequences between three complex genomes and to isolate methylation perturbations in a Mus musculusxMus caroli hybrid. This novel application of MS-AS and MS-RDA resulted in the isolation of differentially methylated retroelements surrounding the locus on Chromosome 10 responsible for double minute chromosome formation within this interspecific eutherian hybrid.

Retroelement Demethylation Associated with Abnormal Placentation in Mus musculus × Mus caroli Hybrids1

ABSTRACT The proper functioning of the placenta requires specific patterns of methylation and the appropriate regulation of retroelements, some of which have been co-opted by the genome for placental-specific gene expression. Our inquiry was initiated to determine the causes of the placental defects observed in crosses between two species of mouse, Mus musculus and Mus caroli. M. musculus × M. caroli fetuses are rarely carried to term, possibly as a result of genomic incompatibility in the placenta. Taking into account that placental dysplasia is observed in Peromyscus and other Mus hybrids, and that endogenous retroviruses are expressed in the placental transcriptome, we hypothesized that these placental defects could result, in part, from failure of the genome defense mechanism, DNA methylation, to regulate the expression of retroelements. Hybrid M. musculus × M. caroli embryos were produced by artificial insemination, and dysplastic placentas were subjected to microarray and methylation screens. Aberrant overexpression of an X-linked Mus retroelement in these hybrid placentas is consistent with local demethylation of this retroelement, concomitant with genome instability, disruption of gene regulatory pathways, and dysgenesis. We propose that the placenta is a specific site of control that is disrupted by demethylation and retroelement activation in interspecific hybridization that occur as a result of species incompatibility of methylation machinery. To our knowledge, the present data provide the first report of retroelement activation linked to decreased methylation in a eutherian hybrid system.

Molecular characterization and evolution of X and Y-borne ATRX homologues in American marsupials.

In eutherians, the sex-reversing ATRX gene on the X has no homologue on the Y chromosome. However, testis-specific and ubiquitously expressed X-borne genes have been identified in Australian marsupials. We studied nucleotide sequence and chromosomal location of ATRX homologues in two American marsupials, the opossums Didelphis virginiana and Monodelphis domestica. A PCR fragment of M. domestica ATRX was used to probe Southern blots and to screen male genomic libraries. Southern analysis demonstrated ATRX homologues on both X and Y in D. virginiana, and two clones were isolated which hybridized to a single position on the Y chromosome in male-derived cells but to multiple sites of the X in female cells. In M. domestica, there was a single clone that mapped to the X but not to the Y, suggesting that it represents the M. domestica ATRX. However a male-specific band was detected in Southern blots probed with the D. virginiana ATRY and with a mouse ATRX clone, which implies that the Y copy in M. domestica has diverged further from other ATRX homologues. Thus there appears to be a Y-borne copy of ATRY in American, as well as Australian marsupials, although it has diverged in sequence, as have other Y genes that are testis-specific in both eutherian and marsupial lineages.

The repetitive DNA element BncDNA, enriched in the B chromosome of the cichlid fish Astatotilapia latifasciata , transcribes a potentially noncoding RNA

Supernumerary chromosomes have been studied in many species of eukaryotes, including the cichlid fish, Astatotilapia latifasciata. However, there are many unanswered questions about the maintenance, inheritance, and functional aspects of supernumerary chromosomes. The cichlid family has been highlighted as a model for evolutionary studies, including those that focus on mechanisms of chromosome evolution. Individuals of A. latifasciata are known to carry up to two B heterochromatic isochromosomes that are enriched in repetitive DNA and contain few intact gene sequences. We isolated and characterized a transcriptionally active repeated DNA, called B chromosome noncoding DNA (BncDNA), highly represented across all B chromosomes of A. latifasciata. BncDNA transcripts are differentially processed among six different tissues, including the production of smaller transcripts, indicating transcriptional variation may be linked to B chromosome presence and sexual phenotype. The transcript lengths and lack of similarity with known protein/gene sequences indicate BncRNA might represent a novel long noncoding RNA family (lncRNA). The potential for interaction between BncRNA and known miRNAs were computationally predicted, resulting in the identification of possible binding of this sequence in upregulated miRNAs related to the presence of B chromosomes. In conclusion, Bnc is a transcriptionally active repetitive DNA enriched in B chromosomes with potential action over B chromosome maintenance in somatic cells and meiotic drive in gametic cells.

The Mysteries of Chromosome Evolution in Gibbons: Methylation Is a Prime Suspect

Dobzhansky and Sturtevant provided the first view of the molecular basis of species identity in their 1938 seminal study classifying the chromosome rearrangements that distinguish two Drosophila species [1]. Decades of study of genome architecture from an evolutionary perspective then followed, enriching our knowledge of developmental genetics, gene regulation, human genetic disorders, and cancer, while greatly contributing to the neo-Darwinian view of the divergence of species. The view that has emerged over the last decade, with a sharp acceleration since the publication of the human genome sequence, is of a fluid genomic landscape that is dotted with evidence of both large- and fine-scale chromosome rearrangements. What has remained a mystery are the mechanisms responsible for chromosome rearrangements that karyotypically define species. In this issue of PLoS Genetics, Lucia Carbone et al. [2] use the northern white-cheeked gibbon (Nomascus leucogenys leucogenys) to address a fascinating problem in evolutionary biology: why are some groups of organisms characterized by a high frequency of chromosome change while others are karyotypically stable? Gibbons are members of the Hominoidea superfamily, which includes humans and great apes, but they are unique among Hominoidea, and indeed rare among mammals, in having experienced an extraordinarily high rate of karyotypic change [2]. Carrying a remarkable number of lineage-specific breaks of synteny, the four genera of gibbons separated from their common hominoid ancestor with humans between 15 and 20 million years ago [3]. Gibbons carry a broad array of chromosome rearrangements, including pericentric and paracentric inversions, fissions, fusions, and Robertsonian and reciprocal translocations, placing this group of endangered mammals among the most karyotypically diverse within primates. Building on their previous work defining the synteny map for the northern white-cheeked gibbon with respect to its human cousin, these authors used a comparative genomics approach to analyze sequences spanning breaks of synteny for repeat distribution and genomic signatures that would lend some insight into the mechanism of interchromosomal rearrangement. Corroborating data from other studies on a smaller set of gibbon breakpoints [4],[5], this analysis of 57 breakpoints found a correlation between segmental duplications and breaks of synteny. While there is clearly a tight association between segmental duplications and chromosomal breaks in many primate lineages (including humans), it is apparent from these studies that many segmental duplications in gibbons are specific to the gibbon lineage and are thus not a contributor to the initial cascade of events responsible for the rearrangements themselves, but rather are a result of the double-strand break events at these rearrangement sites [2],[4],[5]. Rather than simply quantifying the repeat classes at the gibbon-specific breaks of synteny, Carbone et al. took this study one step further by asking whether the epigenetic signatures of specific repeat classes may be an important distinguishing feature in highly divergent genomes. Previous work has shown that gibbon Alu elements are more active than their human counterparts [6]. Taken with the observation from Carbone et al. that the Alu elements found at gibbon breaks of synteny carry a higher CpG content, the control of mobile element activity by DNA methylation stands out as a potential epigenetic signature at these breakpoints. The epigenetic alteration of genomic sequences by DNA methylation is appreciated as a major regulatory force in the evolution of genome structure and expression, and is known as a potent regulator of mobile DNA activity. Through bisulfite sequence analysis, the authors show that the gibbon Alus are undermethylated compared to their human orthologues. The authors suggest these epigenetic differences between human and gibbon as a possible mechanism to account for the disparity in the number of chromosome rearrangements between the gibbons and old world primates. The proposal that mobile DNA itself participates in DNA rearrangement is not new to biology. Mobile elements, such as transposons and retrotransposons, were first implicated in DNA rearrangements in studies of maize by McClintock [7]. Their mobility is known to alter chromosome structure as well as gene expression, and may promote the genetic variability necessary for rapid evolution. Others have proposed that chromosomal rearrangements can promote reproductive isolation between species and may lead to rapid speciation [8],[9]. Hybridization between these two populations could then lead to mobilization of transposable elements that could cause the dysgenesis of hybrids. The novelty in this study is that there is hypomethylation of the gibbon Alus at evolutionary breakpoints, and thus the epigenetic architecture of these regions may have facilitated the rearrangements in the gibbon karyotype. The lower levels of methylation in these repeats may lead to an open chromatin configuration that increases the opportunity for double-strand breakage and repair mechanisms such as intrachromosomal non-allelic homologous recombination and non-homologous end joining (Figure 1). However, many of the gibbon breakpoints do not carry a signature (such as microhomology or Alu-Alu recombination events) that easily implicates any particular mechanism of rearrangement. Thus it is intriguing to consider the possibility that the epigenetic state of specific elements may have been disrupted at some point during the evolution of this gibbon species, which in turn increased the frequency for such elements to participate in rearrangement. Figure 1 Schematic of epigenetic state of Alu elements at gibbon and human orthologous evolutionary breakpoints. McClintock first implicated transposable elements in the speciation process when she stated that “species crosses are…a potent source of genomic modification” and that “major restructuring of chromosome components may arise in a hybrid” [10]. She added species crosses to the growing list of genomic stresses that could cause the activation of mobile elements. Given the suggestion that gibbons may have experienced hybridization events sometime in the last 15 million years [11], hybridization-induced perturbation of mobile element methylation and stability [12],[13] may be one process through which these mobile elements participate in genome shuffling [2]. Exciting advances in sequencing technology will now afford full genome-scale methylation studies (i.e., characterization of the full methylome) that can offer insight into the diversity of elements that may be differentially methylated between gibbons and humans, and whether Alus are the sole target. Additionally, testing for a similar association between mobile DNA and methylation state at breaks of synteny in other species groups that have experienced rapid karyotypic change (such as mice, dogs, horses, and kangaroos) are exciting areas of future work that may finally shed light on the mechanisms responsible for the chromosome diversity observed in a broad range of species.