Computational Prediction of Position Effects of Apparently Balanced Human Chromosomal Rearrangements.

Interpretation of variants of uncertain significance, especially chromosomal rearrangements in non-coding regions of the human genome, remains one of the biggest challenges in modern molecular diagnosis. To improve our understanding and interpretation of such variants, we used high-resolution three-dimensional chromosomal structural data and transcriptional regulatory information to predict position effects and their association with pathogenic phenotypes in 17 subjects with apparently balanced chromosomal abnormalities. We found that the rearrangements predict disruption of long-range chromatin interactions between several enhancers and genes whose annotated clinical features are strongly associated with the subjects' phenotypes. We confirm gene-expression changes for a couple of candidate genes to exemplify the utility of our analysis of position effect. These results highlight the important interplay between chromosomal structure and disease and demonstrate the need to utilize chromatin conformational data for the prediction of position effects in the clinical interpretation of non-coding chromosomal rearrangements.

Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance.

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome.

Selective nucleolus organizer inactivation in Arabidopsis is a chromosome position-effect phenomenon.

Nucleolus organizer regions (NORs) are chromosomal loci where hundreds of rRNA genes are clustered. Despite being nearly identical in sequence, specific rRNA genes are selected for silencing during development via choice mechanism(s) that remain unclear. In Arabidopsis thaliana, rRNA gene subtypes that are silenced during development were recently mapped to the NOR on chromosome 2, NOR2, whereas active rRNA genes map to NOR4, on chromosome 4. In a mutant line deficient for ATXR5 or ATXR6-dependent histone H3 lysine 27 (H3K27) monomethylation, we show that millions of base pairs of chromosome 4, including the telomere, TEL4N, and much of NOR4, have been converted to the corresponding sequences of chromosome 2. This genomic change places rRNA genes of NOR2, which are normally silenced, at the position on chromosome 4 where active rRNA genes are normally located. At their new location, NOR2-derived rRNA genes escape silencing, independent of the atxr mutations, indicating that selective rRNA gene silencing is chromosome 2-specific. The chromosome 2 position effect is not explained by the NOR2-associated telomere, TEL2N, which remains linked to the translocated NOR, implicating centromere-proximal sequences in silencing.

Regulated Gene Expression as a Tool for Analysis of Heterochromatin Position Effect in Drosophila.

Position effect variegation (PEV) is a perturbation of genes expression resulting from the changes in their chromatin organization due to the abnormal juxtaposition with heterochromatin. The exact molecular mechanisms of PEV remain enigmatic in spite of the long history of PEV studies. Here, we developed a genetic model consisting of PEV-inducing chromosome rearrangement and a reporter gene under control of the UAS regulatory element. Expression of the reporter gene could be regulated by adjustment of the GAL4 transactivator activity. Two UAS-based systems of expression control were tested - with thermosensitive GAL4 repressor GAL80ts and GAL4-based artificial transactivator GeneSwitch. Both systems were able to regulate the expression of the UAS-controlled reporter gene over a wide range, but GAL80ts repressed the reporter gene more efficiently. Measurements of the heterochromatin-mediated repression of the reporter gene in the GAL4+GAL80ts system point to the existence of a threshold level of expression, above which no PEV is observed.

Targeted transgene integration overcomes variability of position effects in zebrafish.

Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish.

Copy number variants analysis in a cohort of isolated and syndromic developmental delay/intellectual disability reveals novel genomic disorders, position effects and candidate disease genes.

BACKGROUND: Array-comparative genomic hybridization (array-CGH) is a widely used technique to detect copy number variants (CNVs) associated with developmental delay/intellectual disability (DD/ID). AIMS: Identification of genomic disorders in DD/ID. MATERIALS AND METHODS: We performed a comprehensive array-CGH investigation of 1,015 consecutive cases with DD/ID and combined literature mining, genetic evidence, evolutionary constraint scores, and functional information in order to assess the pathogenicity of the CNVs. RESULTS: We identified non-benign CNVs in 29% of patients. Amongst the pathogenic variants (11%), detected with a yield consistent with the literature, we found rare genomic disorders and CNVs spanning known disease genes. We further identified and discussed 51 cases with likely pathogenic CNVs spanning novel candidate genes, including genes encoding synaptic components and/or proteins involved in corticogenesis. Additionally, we identified two deletions spanning potential Topological Associated Domain (TAD) boundaries probably affecting the regulatory landscape. DISCUSSION AND CONCLUSION: We show how phenotypic and genetic analyses of array-CGH data allow unraveling complex cases, identifying rare disease genes, and revealing unexpected position effects.

Maternal depletion of Piwi, a component of the RNAi system, impacts heterochromatin formation in Drosophila.

A persistent question in epigenetics is how heterochromatin is targeted for assembly at specific domains, and how that chromatin state is faithfully transmitted. Stable heterochromatin is necessary to silence transposable elements (TEs) and maintain genome integrity. Both the RNAi system and heterochromatin components HP1 (Swi6) and H3K9me2/3 are required for initial establishment of heterochromatin structures in S. pombe. Here we utilize both loss of function alleles and the newly developed Drosophila melanogaster transgenic shRNA lines to deplete proteins of interest at specific development stages to dissect their roles in heterochromatin assembly in early zygotes and in maintenance of the silencing chromatin state during development. Using reporters subject to Position Effect Variegation (PEV), we find that depletion of key proteins in the early embryo can lead to loss of silencing assayed at adult stages. The piRNA component Piwi is required in the early embryo for reporter silencing in non-gonadal somatic cells, but knock-down during larval stages has no impact. This implies that Piwi is involved in targeting HP1a when heterochromatin is established at the late blastoderm stage and possibly also during embryogenesis, but that the silent chromatin state created is transmitted through cell division independent of the piRNA system. In contrast, heterochromatin structural protein HP1a is required for both initial heterochromatin assembly and the following mitotic inheritance. HP1a profiles in piwi mutant animals confirm that Piwi depletion leads to decreased HP1a levels in pericentric heterochromatin, particularly in TEs. The results suggest that the major role of the piRNA system in assembly of heterochromatin in non-gonadal somatic cells occurs in the early embryo during heterochromatin formation, and further demonstrate that failure of heterochromatin formation in the early embryo impacts the phenotype of the adult.

TRIP through the chromatin: a high throughput exploration of enhancer regulatory landscapes.

Enhancers are regulatory elements that promote gene expression in a spatio-temporal way and are involved in a wide range of developmental and disease processes. Both the identification and subsequent functional dissection of enhancers are key steps in understanding these processes. Several high-throughput approaches were recently developed for these purposes; however, in almost all cases enhancers are being tested outside their native chromatin context. Until recently, the analysis of enhancer activities at their native genomic locations was low throughput, laborious and time-consuming. Here, we discuss the potential of a powerful approach, TRIP, to study the functioning of enhancers in their native chromatin environments by introducing sensor constructs directly in the genome. TRIP allows for simultaneously analyzing the quantitative readout of numerous sensor constructs integrated at random locations in the genome. The high-throughput and flexible nature of TRIP opens up potential to study different aspects of enhancer biology at an unprecedented level.

[Organization of the Drosophila melanogaster SF1 insulator and its role in transcription regulation in transgenic lines].

The SF1 insulator was found to contain a polyadenylation signal, which corresponded to the functional polyadenylation signal in embryonic S2 cells and the transgenic lines of Drosophila and bi-directional promoter that functioned in S2 cells. The studies performed did not confirm the ability of the SF1 insulator to protect expression of reporter gene white from the chromosome position effect in transgenic lines.

Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice.

We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn't fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.

Poised transcription factories prime silent uPA gene prior to activation.

The position of genes in the interphase nucleus and their association with functional landmarks correlate with active and/or silent states of expression. Gene activation can induce chromatin looping from chromosome territories (CTs) and is thought to require de novo association with transcription factories. We identify two types of factory: "poised transcription factories," containing RNA polymerase II phosphorylated on Ser5, but not Ser2, residues, which differ from "active factories" associated with phosphorylation on both residues. Using the urokinase-type plasminogen activator (uPA) gene as a model system, we find that this inducible gene is predominantly associated with poised (S5p(+)S2p(-)) factories prior to activation and localized at the CT interior. Shortly after induction, the uPA locus is found associated with active (S5p(+)S2p(+)) factories and loops out from its CT. However, the levels of gene association with poised or active transcription factories, before and after activation, are independent of locus positioning relative to its CT. RNA-FISH analyses show that, after activation, the uPA gene is transcribed with the same frequency at each CT position. Unexpectedly, prior to activation, the uPA loci internal to the CT are seldom transcriptionally active, while the smaller number of uPA loci found outside their CT are transcribed as frequently as after induction. The association of inducible genes with poised transcription factories prior to activation is likely to contribute to the rapid and robust induction of gene expression in response to external stimuli, whereas gene positioning at the CT interior may be important to reinforce silencing mechanisms prior to induction.

[Correlation on a cellular level of gene transcriptional silencing and heterochromatin compartment dragging in case of PEV-producing eu-heterochromatin rearrangement in Drosophila melanogaster].

Eu-heterochromatic rearrangements transfer genes into the heterochromatin and cause their variegated inactivation (PEV). Genes affected by PEV often demonstrate association with heterochromatic nuclear compartment (a distinct area composed of heterochromatin sequences like satellite DNA and enriched in specific chromatin proteins e.g. HP1). Here, we investigate the nuclear localization and the expression levels of the genes subjected to PEV caused by chromosome inversion, In(2)A4. We demonstrate that the degree of PEV-caused gene inactivation depends on a developmental stage, and the maximum of repression corresponds to the gene expression activation period. In the case of In(2)A4 rearrangement we detect the dragging of affected euchromatic region into heterochromatic nuclear compartment and the increase in HP1 occupancy in this region. We developed a protocol of simultaneous RNA-DNA-protein staining to demonstrate firstly in a single cell a strong correlation between transcriptional activity of affected gene and its distance from chromosome 2 satellite DNA.

Position effect variegation and epigenetic modification of a transgene in a pig model.

Sequences proximal to transgene integration sites are able to regulate transgene expression, resulting in complex position effect variegation. Position effect variegation can cause differences in epigenetic modifications, such as DNA methylation and histone acetylation. However, it is not known which factor, position effect or epigenetic modification, plays a more important role in the regulation of transgene expression. We analyzed transgene expression patterns and epigenetic modifications of transgenic pigs expressing green fluorescent protein, driven by the cytomegalovirus (CMV) promoter. DNA hypermethylation and loss of acetylation of specific histone H3 and H4 lysines, except H4K16 acetylation in the CMV promoter, were consistent with a low level of transgene expression. Moreover, the degree of DNA methylation and histone H3/H4 acetylation in the promoter region depended on the integration site; consequently, position effect variegation caused variations in epigenetic modifications. The transgenic pig fibroblast cell lines were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. Transgene expression was promoted by reversing the DNA hypermethylation and histone hypoacetylation status. The differences in DNA methylation and histone acetylation in the CMV promoter region in these cell lines were not significant; however, significant differences in transgene expression were detected, demonstrating that variegation of transgene expression is affected by the integration site. We conclude that in this pig model, position effect variegation affects transgene expression.

[Nuclear envelope attachment sites of interphase chromosomes: barrier elements but not insulators].

A neDNA fragment that protects a transgene from position effect variegation when flanking it was tested for insulator properties. The fragment did not act as an insulator. A similarity of neDNA and various barrier elements was examined, and Drosophila melanogaster and Mus musculus chromosome DNA regions homologous to neDNA were analyzed. Additional conserved sites and DNA duplex destabilization sites were found in the neDNA sequence, and DNA conformational specifics were assumed for the chromosomal neighborhood of neDNA sites of the D. melanogaster and M. musculus genomes.

Chromosomal position effects are linked to sir2-mediated variation in transcriptional burst size.

Gene expression noise varies with genomic position and is a driving force in the evolution of chromosome organization. Nevertheless, position effects remain poorly characterized. Here, we present a systematic analysis of chromosomal position effects by characterizing single-cell gene expression from euchromatic positions spanning the length of a eukaryotic chromosome. We demonstrate that position affects gene expression by modulating the size of transcriptional bursts, rather than their frequency, and that the histone deacetylase Sir2 plays a role in this process across the chromosome.

The winged-helix transcription factor JUMU is a haplo-suppressor/triplo-enhancer of PEV in various tissues but exhibits reverse PEV effects in the brain of Drosophila melanogaster.

Gene expression goes along with changes in chromatin structure and is regulated by chromatin-modifying factors. If genes are transposed from their euchromatic position to the vicinity of heterochromatin, their expression can underly a position effect variegation (PEV). In Drosophila melanogaster a few genes are known that function in a gene dose-dependent manner as haplo-suppressors and triplo-enhancers of PEV or vice versa. The gene jumeaux (jumu) encodes a winged-helix transcription factor of multiple regulatory functions. A novel PEV test system for Drosophila melanogaster reveals that JUMU behaves as a haplo-suppressor/triplo-enhancer in different larval and adult tissues, but surprisingly behaves in the reverse manner as a haplo-enhancer/triplo-suppressor in larval and adult brains. Like jumu, the Su(var)3-9 gene also behaves as a haplo-suppressor/triplo-enhancer, but in our test system does not show any PEV effect in the brains.

Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position.

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.

Drosophila Histone Demethylase KDM4A Has Enzymatic and Non-enzymatic Roles in Controlling Heterochromatin Integrity.

Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. We demonstrate that dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. We conclude that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions.

upSET, the Drosophila homologue of SET3, Is Required for Viability and the Proper Balance of Active and Repressive Chromatin Marks.

Chromatin plays a critical role in faithful implementation of gene expression programs. Different post-translational modifications (PTMs) of histone proteins reflect the underlying state of gene activity, and many chromatin proteins write, erase, bind, or are repelled by, these histone marks. One such protein is UpSET, the Drosophila homolog of yeast Set3 and mammalian KMT2E (MLL5). Here, we show that UpSET is necessary for the proper balance between active and repressed states. Using CRISPR/Cas-9 editing, we generated S2 cells that are mutant for upSET We found that loss of UpSET is tolerated in S2 cells, but that heterochromatin is misregulated, as evidenced by a strong decrease in H3K9me2 levels assessed by bulk histone PTM quantification. To test whether this finding was consistent in the whole organism, we deleted the upSET coding sequence using CRISPR/Cas-9, which we found to be lethal in both sexes in flies. We were able to rescue this lethality using a tagged upSET transgene, and found that UpSET protein localizes to transcriptional start sites (TSS) of active genes throughout the genome. Misregulated heterochromatin is apparent by suppressed position effect variegation of the wm4 allele in heterozygous upSET-deleted flies. Using nascent-RNA sequencing in the upSET-mutant S2 lines, we show that this result applies to heterochromatin genes generally. Our findings support a critical role for UpSET in maintaining heterochromatin, perhaps by delimiting the active chromatin environment.

Using TRIP for genome-wide position effect analysis in cultured cells.

The influence of local chromatin context on gene expression can be explored by integrating a transcription reporter at different locations in the genome as a sensor. Here we provide a detailed protocol for analyzing thousands of reporters integrated in parallel (TRIP) at a genome-wide level. TRIP is based on tagging each reporter with a unique barcode, which is used for independent reporter expression analysis and integration site mapping. Compared with previous methods for studying position effects, TRIP offers a 100-1,000-fold higher throughput in a faster and less-labor-intensive manner. The entire experimental protocol takes ∼42 d to complete, with high-throughput sequencing and data analysis requiring an additional ∼11 d. TRIP was developed by using transcription reporters in mouse embryonic stem (mES) cells, but because of its flexibility the method can be used to probe the influence of chromatin context on a variety of molecular processes in any transfectable cell line.