Single-locus Resolution Research Articles

GuidedNOMe-seq quantifies chromatin states at single allele resolution for hundreds of custom regions in parallel

Since the introduction of next generation sequencing technologies, the field of epigenomics has evolved rapidly. However, most commonly used assays are enrichment-based methods and thus only semi-quantitative. Nucleosome occupancy and methylome sequencing (NOMe-seq) allows for quantitative inference of chromatin states with single locus resolution, but this requires high sequencing depth and is therefore prohibitively expensive to routinely apply to organisms with large genomes. To overcome this limitation, we introduce guidedNOMe-seq, where we combine NOMe profiling with large scale sgRNA synthesis and Cas9-mediated region-of-interest (ROI) liberation. To facilitate quantitative comparisons between multiple samples, we additionally develop an R package to standardize differential analysis of any type of NOMe-seq data. We extensively benchmark guidedNOMe-seq in a proof-of-concept study, dissecting the interplay of ChAHP and CTCF on chromatin. In summary we present a cost-effective, scalable, and customizable target enrichment extension to the existing NOMe-seq protocol allowing genome-scale quantification of nucleosome occupancy and transcription factor binding at single allele resolution.

SCIFER: approach for analysis of LINE-1 mRNA expression in single cells at a single locus resolution

BackgroundEndogenous expression of L1 mRNA is the first step in an L1-initiated mutagenesis event. However, the contribution of individual cell types to patterns of organ-specific L1 mRNA expression remains poorly understood, especially at single-locus resolution. We introduce a method to quantify expression of mobile elements at the single-locus resolution in scRNA-Seq datasets called Single Cell Implementation to Find Expressed Retrotransposons (SCIFER). SCIFER aligns scRNA-Seq reads uniquely to the genome and extracts alignments from single cells by cell-specific barcodes. In contrast to the alignment performed using default parameters, this alignment strategy increases accuracy of L1 locus identification by retaining only reads that are uniquely mapped to individual L1 loci. L1 loci expressed in single cells are unambiguously identified using a list of L1 loci manually validated to be expressed in bulk RNA-Seq datasets generated from the same cell line or organ.ResultsValidation of SCIFER using MCF7 cells determined technical parameters needed for optimal detection of L1 expression in single cells. We show that unsupervised analysis of L1 expression in single cells exponentially inflates both the levels of L1 expression and the number of expressed L1 loci. Application of SCIFER to analysis of scRNA-Seq datasets generated from mouse and human testes identified that mouse Round Spermatids and human Spermatogonia, Spermatocytes, and Round Spermatids express the highest levels of L1 mRNA. Our analysis also determined that similar to mice, human testes from unrelated individuals share as much as 80% of expressed L1 loci. Additionally, SCIFER determined that individual mouse cells co-express different L1 sub-families and different families of transposable elements, experimentally validating their co-existence in the same cell.ConclusionsSCIFER detects mRNA expression of individual L1 loci in single cells. It is compatible with scRNA-Seq datasets prepared using traditional sequencing methods. Validated using a human cancer cell line, SCIFER analysis of mouse and human testes identified key cell types supporting L1 expression in these species. This will further our understanding of differences and similarities in endogenous L1 mRNA expression patterns in mice and humans.

Characterization of Mammalian Regulatory Complexes at Single-Locus Resolution Using TINC.

In mammalian cells, multiprotein complexes form at specific genomic regulatory elements (REs) to control gene expression, which in turn is ultimately responsible for cellular identity. Consequently, insight into the molecular composition of these regulatory complexes is of major importance for our understanding of any physiological or pathological cellular state or transition. However, it remains extremely difficult to identify the protein complex(es) assembled at a specific RE in the mammalian genome using conventional approaches. We therefore developed a novel single locus isolation technique based on Transcription Activator-Like Effector (TALE) proteins termed TALE-mediated isolation of nuclear chromatin (TINC). When coupled with high-resolution mass spectrometry, TINC enables the identification and characterization of protein complexes formed at any RE of interest. Using the Nanog promoter in mouse embryonic stem cells as proof of concept, this chapter describes in detail the novel TINC methodology as well as subsequent mass spectrometric considerations.

Organ-, sex-and age-dependent patterns of endogenous L1 mRNA expression at a single locus resolution.

Expression of L1 mRNA, the first step in the L1 copy-and-paste amplification cycle, is a prerequisite for L1-associated genomic instability. We used a reported stringent bioinformatics method to parse L1 mRNA transcripts and measure the level of L1 mRNA expressed in mouse and rat organs at a locus-specific resolution. This analysis determined that mRNA expression of L1 loci in rodents exhibits striking organ specificity with less than 0.8% of loci shared between organs of the same organism. This organ specificity in L1 mRNA expression is preserved in male and female mice and across age groups. We discovered notable differences in L1 mRNA expression between sexes with only 5% of expressed L1 loci shared between male and female mice. Moreover, we report that the levels of total L1 mRNA expression and the number and spectrum of expressed L1 loci fluctuate with age as independent variables, demonstrating different patterns in different organs and sexes. Overall, our comparisons between organs and sexes and across ages ranging from 2 to 22 months establish previously unforeseen dynamic changes in L1 mRNA expression in vivo. These findings establish the beginning of an atlas of endogenous L1 mRNA expression across a broad range of biological variables that will guide future studies.

TINC— A Method to Dissect Regulatory Complexes at Single-Locus Resolution— Reveals an Extensive Protein Complex at the Nanog Promoter

SummaryCellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.

Comparative analysis on the expression of L1 loci using various RNA-Seq preparations

BackgroundRetrotransposons are one of the oldest evolutionary forces shaping mammalian genomes, with the ability to mobilize from one genomic location to another. This mobilization is also a significant factor in human disease. The only autonomous human retroelement, L1, has propagated to make up 17% of the human genome, accumulating over 500,000 copies. The majority of these loci are truncated or defective with only a few reported to remain capable of retrotransposition. We have previously published a strand-specific RNA-Seq bioinformatics approach to stringently identify at the locus-specific level the few expressed full-length L1s using cytoplasmic RNA. With growing repositories of RNA-Seq data, there is potential to mine these datasets to identify and study expressed L1s at single-locus resolution, although many datasets are not strand-specific or not generated from cytoplasmic RNA.ResultsWe developed whole-cell, cytoplasmic and nuclear RNA-Seq datasets from 22Rv1 prostate cancer cells to test the influence of different preparations on the quality and effort needed to measure L1 expression. We found that there was minimal data loss in the identification of full-length expressed L1 s using whole cell, strand-specific RNA-Seq data compared to cytoplasmic, strand-specific RNA-Seq data. However, this was only possible with an increased amount of manual curation of the bioinformatics output to eliminate increased background. About half of the data was lost when the sequenced datasets were non-strand specific.ConclusionsThe results of these studies demonstrate that with rigorous manual curation the utilization of stranded RNA-Seq datasets allow identification of expressed L1 loci from either cytoplasmic or whole-cell RNA-Seq datasets.

First molecular approach to the octopus fauna from the southern Caribbean.

The octopus fauna from the southern Caribbean is an understudied field. However, recent taxonomic work in the Colombian Caribbean has led to the discovery of several new species in the family Octopodidae. To provide molecular evidence for recent descriptions in the area (i.e., Octopus taganga, O. tayrona and Macrotritopus beatrixi) and contribute to the systematics of the family, we reconstructed the first molecular phylogenies of the family including Colombian Caribbean octopus species. Using cytochrome c oxidase subunit I and rhodopsin sequences from specimens collected in three sites (Santa Marta, Old Providence and San Andrés Islands) we inferred maximum-likelihood trees and delimited species with PTP. Our mitochondrial analysis supported the monophyly of species found in the area (i.e., O. taganga, O. hummelincki and O. briareus). The genetic distinction of the species O. tayrona and O. insularis was not resolved, as these were found in one clade together with Caribbean O. vulgaris and O. aff. tayrona species (O. spB) and delimited as a single species. Additionally, our results suggest a distant relationship of the Type I O. vulgaris group (Caribbean region) from the other forms of the species complex (Old World and Brazil). Lastly, the third newly described species M. beatrixi emerged as an independent lineage and was delimited as a single species. However, its relationship to other species of its genus remains unknown due to the lack of sequences in databases. Altogether, our molecular approach to the octopus fauna from the southern Caribbean adds on information to the relationship of Octopodidae species world-wide by providing sequences from recently described species from an understudied region. Further studies employing higher taxon sampling and more molecular information are needed to fill taxonomic gaps in the area and account for single-locus resolution on the systematics of this group.

Identification of RNA Polymerase III-Transcribed SINEs at Single-Locus Resolution from RNA Sequencing Data.

Short Interspersed Element (SINE) retrotransposons are one of the most abundant DNA repeat elements in the human genome. They have been found to impact the expression of protein-coding genes, but the possible roles in cell physiology of their noncoding RNAs, generated by RNA polymerase (Pol) III, are just starting to be elucidated. For this reason, Short Interspersed Element (SINE) expression profiling is becoming mandatory to obtain a comprehensive picture of their regulatory roles. However, their repeated nature and frequent location within Pol II-transcribed genes represent a serious obstacle to the identification and quantification of genuine, Pol III-derived SINE transcripts at single-locus resolution on a genomic scale. Among the recent Next Generation Sequencing technologies, only RNA sequencing (RNA-Seq) holds the potential to solve these issues, even though both technical and biological matters need to be taken into account. A bioinformatic pipeline has been recently set up that, by exploiting RNA-seq features and knowledge of SINE transcription mechanisms, allows for easy identification and profiling of transcriptionally active genomic loci which are a source of genuine Pol III SINE transcripts.

Whole-genome expression analysis of mammalian-wide interspersed repeat elements in human cell lines.

With more than 500,000 copies, mammalian-wide interspersed repeats (MIRs), a sub-group of SINEs, represent ∼2.5% of the human genome and one of the most numerous family of potential targets for the RNA polymerase (Pol) III transcription machinery. Since MIR elements ceased to amplify ∼130 myr ago, previous studies primarily focused on their genomic impact, while the issue of their expression has not been extensively addressed. We applied a dedicated bioinformatic pipeline to ENCODE RNA-Seq datasets of seven human cell lines and, for the first time, we were able to define the Pol III-driven MIR transcriptome at single-locus resolution. While the majority of Pol III-transcribed MIR elements are cell-specific, we discovered a small set of ubiquitously transcribed MIRs mapping within Pol II-transcribed genes in antisense orientation that could influence the expression of the overlapping gene. We also identified novel Pol III-transcribed ncRNAs, deriving from transcription of annotated MIR fragments flanked by unique MIR-unrelated sequences, and confirmed the role of Pol III-specific internal promoter elements in MIR transcription. Besides demonstrating widespread transcription at these retrotranspositionally inactive elements in human cells, the ability to profile MIR expression at single-locus resolution will facilitate their study in different cell types and states including pathological alterations.

Alu expression profiles as a novel RNA signature in biology and disease

SINE retrotransposons of the Alu subfamily are the most numerous active mobile DNA elements in the human genome. Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, but activated in response to viral infection, genotoxic anticancer agents and other stimuli, through uncharacterized epigenetic switches interspersed throughout the genome. The elucidation of Alu RNA roles in cell biology and pathology has long been hampered by difficulties in their profiling at single-locus resolution, due to their repetitive nature. We recently found how to overcome this limitation by computational screening of RNA-seq data, thus opening the way to Alu transcriptome profiling as a novel tool to explore disease-related epigenome alterations.

Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data.

Of the ∼1.3 million Alu elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which Alu transcripts originate is complicated by their highly repetitive nature. By exploiting RNA-Seq data sets and unique Alu DNA sequences, we devised a bioinformatic pipeline allowing us to identify Pol III-dependent transcripts of individual Alu elements. When applied to ENCODE transcriptomes of seven human cell lines, this search strategy identified ∼1300 Alu loci corresponding to detectable transcripts, with ∼120 of them expressed in at least three cell lines. In vitro transcription of selected Alus did not reflect their in vivo expression properties, and required the native 5′-flanking region in addition to internal promoter. We also identified a cluster of expressed AluYa5-derived transcription units, juxtaposed to snaR genes on chromosome 19, formed by a promoter-containing left monomer fused to an Alu-unrelated downstream moiety. Autonomous Pol III transcription was also revealed for Alus nested within Pol II-transcribed genes. The ability to investigate Alu transcriptomes at single-locus resolution will facilitate both the identification of novel biologically relevant Alu RNAs and the assessment of Alu expression alteration under pathological conditions.

Epigenetic Reprogramming in Primordial Germ Cells in Mice

Abstract: Germline cells are the sole source of the transmission of genetic and epigenetic information to the next generation. Epigenetic information is reprogrammed during germ cell development to reacquire cellular totipotency and prevent the accumulation of epimutations. In this review, we summarize epigenetic reprogramming, in particular, DNA demethylation in developing primordial germ cells (PGCs). The recent development of next-generation sequencing, and the discovery of 5-methylcytosine oxidation are major breakthroughs in the study of epigenetic reprogramming in PGCs. DNA methylation analysis with high-throughput sequencing has uncovered the dynamics of DNA methylation erasure at single-locus resolution, which has revealed the global loss of DNA methylation in migrating PGCs, and locus-specific DNA demethylation in gonadal PGCs. The disruption of ten-eleven translocation genes shows that they are required for DNA demethylation at germline-specific genes in gonadal PGCs. These findings indicate tha...

Estimation and assessment of raw copy numbers at the single locus level

Although copy-number aberrations are known to contribute to the diversity of the human DNA and cause various diseases, many aberrations and their phenotypes are still to be explored. The recent development of single-nucleotide polymorphism (SNP) arrays provides researchers with tools for calling genotypes and identifying chromosomal aberrations at an order-of-magnitude greater resolution than possible a few years ago. The fundamental problem in array-based copy-number (CN) analysis is to obtain CN estimates at a single-locus resolution with high accuracy and precision such that downstream segmentation methods are more likely to succeed. We propose a preprocessing method for estimating raw CNs from Affymetrix SNP arrays. Its core utilizes a multichip probe-level model analogous to that for high-density oligonucleotide expression arrays. We extend this model by adding an adjustment for sequence-specific allelic imbalances such as cross-hybridization between allele A and allele B probes. We focus on total CN estimates, which allows us to further constrain the probe-level model to increase the signal-to-noise ratio of CN estimates. Further improvement is obtained by controlling for PCR effects. Each part of the model is fitted robustly. The performance is assessed by quantifying how well raw CNs alone differentiate between one and two copies on Chromosome X (ChrX) at a single-locus resolution (27kb) up to a 200kb resolution. The evaluation is done with publicly available HapMap data. The proposed method is available as part of an open-source R package named aroma.affymetrix. Because it is a bounded-memory algorithm, any number of arrays can be analyzed.