Plots show median center line , with interquartile range box , and whiskers show points within 1. Statistical significance analysis of observed changes for each bin and additional bins located at distances further away from LTR5HS is presented in Supplementary file 1. These data suggest that genuine functional differences in regulatory capacity exist within distinct subclasses of HERVK LTR5 elements, and that their regulatory activity is cell type-specific.
Notably, these changes occur in the absence of direct dCas9 binding to the promoters, suggesting that they result from the long-range effects of LTR5HS Figure 5E. Average signals obtained across indicated number of clones are shown. Average expression of each gene across indicated number of clones is shown, measured relative to two housekeeping genes, RPL13A and TBP.
Error bars show standard deviation.
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For all six genes, we observed a significant decrease in expression upon deletion of the nearest LTR5HS. These results demonstrate that long-range effects on gene regulation are directly dependent on LTR5HS DNA sequences and show that a single promoter-distal LTR can provide a very strong contribution to the overall gene activity. These could potentially enable imaging at these loci Chen et al. Given the widespread redundancies in mammalian regulatory landscapes where loss of a single enhancer often has only a minor influence on expression Hay et al.
Considering that other classes of TEs beyond LTR5HS are likely contributing to gene regulation in the early human embryo, these observations are consistent with a pervasive, rather than occasional, role of TEs in transcriptional control. Although the fact that evolutionarily young LTRs such as LTR5HS have been so extensively adapted for enhancer function may seem counterintuitive, it is important to note that preimplantation embryo cells and germ cells may be a privileged environment for such early adaptation, not only due to global DNA hypomethylation in these cells, but because in order to persist through vertical transmission, these ancient retroviruses must have been able to replicate in the germline or early embryonic cells, before the germline has been set aside.
Indeed, OCT4 plays a central role in activating pluripotency network enhancers Boyer et al. It is intriguing to consider whether regulatory repurposing of LTR5HS elements for enhancer function may have contributed to human-specific transcriptome divergence and endowed the early developmental stages of the human embryo with species-specific attributes. We found that both human-specific and older, ape-specific LTR5HS elements contribute to long-range gene regulation, and that some of the genes dependent on them in embryonal carcinoma cells are also expressed in human preimplantation embryos.
Interestingly, we found that transcript levels of genes that are orthologous between human and rhesus macaque and regulated by LTR5HS in human cells are significantly elevated in human blastocysts compared to rhesus blastocysts. Given that rhesus diverged from the human lineage approximately 25 million years ago Rhesus Macaque Genome Sequencing and Analysis Consortium, et al. Although there is no evidence thus far to suggest that the phenotypic consequences of the molecular adaptation of LTR5HS for enhancer function have been beneficial to the host, it is nonetheless tempting to speculate that some LTR5HS-driven changes in gene expression may have measurable phenotypic consequences on early development, endowing it with ape-specific attributes.
All cell lines tested negative for mycoplasma contamination. The twelve guides with lowest off-target rate were selected for the targeting array. Non-targeting guides were taken from Shalem et al. SAM files were converted to BED using bedtools bamtobed function, then the PAM sequence for each alignment was extracted using bedtools getfasta function. Cells were re-selected with puromycin Invivogen to ensure that dCas9-fusions were not lost during second transposition event.
Each condition was also analyzed with at least two independent biological replicates. ChIP assays were performed as described previously Rada-Iglesias et al. Chromatin was sonicated to 0. Total RNA 10 ug from two independent biological replicates was subjected to oligo-dT purification using Dynabeads oligo dT Thermo Fisher Scientific , then fragmented with 10x fragmentation buffer Thermo Fisher Scientific.
Each clone was analyzed at two separate passages. Duplicate reads were removed using the MarkDuplicates function of Picard Tools. Macs2 Zhang et al. Overlaps between ChIP peak calls were performed using bedtools intersect function. Reads from were trimmed of Illumina adapter sequences using cutadapt. For analysis of human non-repeat transcripts, trimmed reads were aligned using hisat2 Kim et al.
Reads were assigned to gene models using featureCounts Liao et al. For analysis of Repeatmasker transcripts, trimmed reads were aligned using TopHat2 Kim et al. Reads were assigned to repeat models using featureCounts, then RPKM was calculated from these tabulations. For comparison of early embryo single cell RNA-seq, rhesus reads from Wang et al. Ensembl BioMart was used to identify only genes with one-to-one orthology between the two species, and only these were used for further analyses. Transcripts per million TPM was calculated for each gene at each stage in each species.
For chimeric transcript identification, RNA-seq reads were trimmed with skewer Jiang et al. Transcript models were built based on this alignment with StringTie Pertea et al. TPM corresponding to expression level of the known and new transcripts were calculated with separate StringTie run for each library alignment stringtie -e -B -A. All antibodies, primers, and gRNAs used in this study are listed in Supplementary file 2. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses.
A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Thank you for submitting your article "Systematic perturbation of retroviral LTRs reveals widespread long-range effects on human gene regulation" for consideration by eLife.
Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. In this study, the question of the extent to which Transposable Elements and their relics in a genome have been adapted to influence "host" gene regulation is addressed.
The authors examine the effects of this TE modulation on nearby gene expression in human embryonal carcinoma cells derived from a germ-line tumor NCCIT. They go on to look at the range of impact that these LTR5HS elements have is up to kb, suggesting that they might be acting as potential enhancers.
This study is of a great interest for the field as it serves as an important demonstration that expression of hundreds of copies of a specific family of LTR retrotransposons can be efficiently modulated using dCas9 effectors and the recently developed CARGO system and it allows the impact of a whole LTR family on gene expression to be addressed in a systematic way.
Research : Gene Expression Research Group : University of Sussex
Although previous studies Guallar et al. The manuscript is well written, easy to read and the experiments are of high quality. There are however some important issues that the authors will need to address. However, they need to prove this enhancer potential at the endogenous locations. As the study stands they cannot rule out that the elements are acting as alternative promoters, particularly as the three LTR5HS elements that they deleted were in close proximity a few kb to the associated genes.
Several approaches can address this, as outlined in the reviewers' comments. In addition to chromatin state and evolutionary conservation, the detection of eRNAs has been used to identify putative enhancers It remains an open question whether these eRNAs are simply transcriptional noise or are functionally important. Recently, eRNAs produced by enhancers bound by p53 have been found to directly enhance transcription at multiple distant genes ; siRNA knockdown of a number of these eRNAs resulted in a decrease in expression of their target genes, while maintaining existing chromatin interactions.
Other classes of ncRNAs stabilize long-range enhancer:promoter interactions or recruit chromatin remodellers , and eRNAs might perform a similar role. A single promoter can be involved in interactions with a single or multiple enhancers Enhancers can either interact with a specific target gene or with many genes allowing the coordinated regulation of functionally related genes.
Type I tissue-specific PolII promoters are typically controlled by regulatory elements in close proximity 99 , whereas Type III developmental promoters are most often controlled by long-range regulation Evidence suggesting that physical interactions between enhancers and promoters are necessary has been found using a variety of approaches, including fluorescence in situ hybridisation FISH and chromosome conformation capture methods.
By tagging specific sequences with fluorescent probes, FISH allows the identification of regions of the genome that are brought into close spatial proximity Chromosome conformation capture methods — are useful for assessing the frequency of interactions between two genomic loci: either between two pre-selected loci 3C , one locus and the rest of the genome 4C, 3C-seq or all interactions between multiple pre-selected elements 5C. Hi-C allows unbiased and genome-wide investigation of interactions, although currently its resolution is relatively low compared with other techniques , ChIA-PET chromatin interaction assay with paired-end sequencing makes it possible to identify all interactions mediated by a specific protein, allowing investigation of how specific TFs and histone modifications alter the structure of chromatin Currently, the available evidence from 3C-based methods is suggestive of direct physical interactions between enhancers and promoters.
However, recent work has identified that the results from FISH and different 3C-based methods are not always concordant , e. The suggested reasons for these observations are that FISH is a more disruptive method leading to loss of some interactions during cell treatment, or that formaldehyde cross-linking in 3C-based methods does not always reflect average spatial distances between the interacting loci.
Nevertheless, these technologies have provided insights into how promoters and enhancers communicate in 3D space and into the effects of chromosomal conformation on gene expression These chromatin structures depend on sequence-specific TFs bound to both the enhancer and promoter and thus can explain the specificity observed in enhancer:promoter interactions. Interactions between regulatory elements separated by large genomic distances have been observed at high frequencies at other loci, leading to the proposition that chromatin looping generally results in the formation of hub-like structures.
These active chromatin hubs ACH are responsible for bringing enhancers and promoters into close spatial proximity as well as providing an environment that is transcriptionally permissive Figure 3 a. At the Myb locus during erythoid proliferation, intergenic enhancers, the Myb promoter and its first intron are brought together to form an ACH The first intron of this gene contains a site for regulating transcriptional elongation.
Interactions between this element and distal enhancers lead to the generation of full-length transcripts. This structure is lost when cells terminally differentiate, coincident with the loss of expression of Myb and a reduction in TF binding at regulatory elements. Chromatin looping is responsible for forming higher-order hub-like structures within the nucleus. Interactions between promoters and enhancers and represented by dashed lines. This structure potentially prevents enhancers from communicating with their cognate promoter by looping them out and preventing them from interacting.
This structure may also restrict the amount of PolII from binding to gene promoters. This movement allows enhancers to interact with targets that were previously held in the repressive domain. Chromatin hub-like structures have also been found to play a role in repressing gene expression Studies of the GATA-4 locus in undifferentiated Tera-2 cells have revealed the existence of a 3D structure consisting of multiple chromatin loops Figure 3 b , termed a pre-repressive chromatin hub pre-RCH , which is lost when these cells differentiate.
This structure was found to comprise several H3K27me3-enriched elements and was maintained by Polycomb, suggesting that Polycomb may repress genes through the formation and maintenance of these structures. In undifferentiated cells, this structure results in GATA-4 being held in a poised state, and its loss during differentiation was accompanied with an increase in the expression of GATA This structure may function by either preventing enhancer:promoter interactions or by restricting the access of PolII to the GATA-4 promoter.
In vertebrates, Hox genes are typically organized into clusters and are transcribed sequentially; with the first gene in the cluster being expressed in the anterior part of the organism. This collinear expression pattern results from changes in both the histone modification patterns within a Hox cluster and its overall 3D conformation Within a Hox cluster, active and inactive genes are separated into distinct domains labelled by distinct histone modifications, H3K4me3 and H3K27me3, respectively. It is thought that Hox genes are expressed sequentially because of their highly regulated movement out of chromosome territories and the decondensation of associated chromatin Studies of the HoxD locus using 4C have found that when all genes within a cluster are not expressed, these genes form a single 3D structure that represses transcription.
This led the authors to propose a model whereby as genes are expressed they progressively migrate from this structure and cluster into a transcriptionally active structure, leading to a bimodal organization of chromatin at a single Hox cluster Figure 3 c and d. This movement is associated with changes in histone modifications and suggests that the observed collinear expression may be the result of a stepwise movement of genes from an RCH-like structure to an ACH-like structure.
An examination of the HoxD locus, important for limb development, using chromosome conformation capture techniques has revealed that this locus has a tissue-specific conformation involving interactions between genes and enhancers located within the adjacent gene deserts The active section of gene cluster seems to be in contact with several enhancers concurrently, each of which seem to be important for some aspect of limb development.
The concept of a regulatory archipelago proposed by Montavon et al. These findings suggests that the 3D conformation of loci changes during differentiation as a result of interactions between promoters and enhancers, resulting in the creation of cell-type—specific patterns of gene expression and organization. Indeed, the most recent results suggest that the 3D conformation at developmental loci is both the most dynamic and most divergent across mammals Hi-C has provided further evidence for the presence of CTs and found that eukaryotic genomes are organized into functional domains A and B compartments that are important for controlling DNA transcription By applying a hidden Markov model to Hi-C data, Dixon et al.
Elements within these domains preferentially interacted with other elements in the same domain. The boundaries of these domains were found to be associated with known elements displaying barrier activity and correlated well with known CTCF-binding sites. Hi-C has enabled the calculation of the probability that two randomly chosen loci interact This contact probability follows a power-law distribution and suggests that chromatin is organized globally in a fractal globule structure.
However, the exponent that describes this distribution has been found to vary , and recent studies have found that the contact probability plateaus as the genomic distance increases These findings suggest that this model cannot adequately describe the observed patterns of chromatin folding.
The strings and binders model has been proposed, which not only recapitulates this structure but is also more related to the known underlying biology of protein-mediated chromatin folding.
By examining 5C data from three different cell lines, Sanyal et al. Long-range interactions between enhancer:promoter pairs were found in domains enriched for both H3K9ac and H3K27ac. ChIA-PET has revealed insights into how these interactions are mediated by TFs, allowing the generation of genome-wide chromatin interactomes. As expected, most interactions occur between elements located on the same chromosome, rather than interchromosomally.
A positive association was found between chromatin interactions and the activation of genes involved in those interactions. In addition to identifying interactions between distal sites and promoters, a number of interactions were found between distal sites, hinting at the possibility of widespread enhancer:enhancer interactions. Li et al. Expression of genes regulated by promoters involved in interactions with other promoters was found to be highly correlated.
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This may explain how transcription of tissue-specific and housekeeping genes is co-ordinated. Long-range interactions between distal elements and promoters were found to be highly cell-type specific, and promoters were involved in interactions at different distances depending on the cell line under investigation. To investigate enhancer:promoter interactions, Chepelev et al. Promoters that were found to interact with the same enhancer showed evidence of tissue-specific co-expression. The interactions between CTCF-bound sites and promoters seem to be more cell-type invariant when compared with enhancer:promoter interactions, as is CTCF binding.
As such, insulators involved in enhancer-blocking may function to mediate cell-type—specific long-range interactions. However, recently a number of looping interactions were found to skip sites bound by both CTCF and cohesin, which may suggest that additional factors are required This form of interaction data can be easily represented as a network, with an edge present between two nodes each of which corresponds to a region of chromatin , when they have been found to interact. This network exhibits a hierarchical structure and has a scale-free degree distribution.
Investigation of communities of strongly connected elements within this network revealed that these were involved in functional compartmentalization. The majority of these chromatin communities were conserved between cell lines, indicating that cell-type specificity may be defined by long-range transient interactions or by small differences in the content and organization of these communities.
The identification of the different factors and histone modifications involved in long-range regulation will help elucidate how specific enhancers target specific promoters without binding to intervening promoters. It does not seem that there are any features specific to enhancers involved in long-range regulation compared with those involved with regulation over shorter distances.
Indeed, it seems that the ability to respond to long-range interactions depends on the promoter architecture of a gene and the state of intervening insulators. The development of new genome-wide techniques has provided a way to systematically identify regions that are targeted by specific proteins and co-localize in nuclear space.
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The results from ChIP-seq and chromosome conformation capture assays are, however, averaged over a cell population, and they do not provide any information on the temporal dynamics and cell-to-cell variation of long-range interactions. Improvements in the temporal resolution of experiment techniques should help determine how chromatin moves during the cell cycle and how this affects and which genes are upregulated and downregulated at specific stages. Studies of long-range interactions are also limited by the relatively low resolution of Hi-C.
As this improves, we expect that new findings will follow regarding the global patterns of interactions between individual regulatory elements. Examination of this in multiple cell types and species will enable us to understand how the constraints on 3D conformation affect the arrangement and conservation of regulatory elements. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Article Navigation.
Close mobile search navigation Article Navigation. Volume Article Contents. Chromatin and epigenetic features of long-range gene regulation Nathan Harmston. Oxford Academic. Google Scholar. Boris Lenhard. Cite Citation. Permissions Icon Permissions.
Abstract The precise regulation of gene transcription during metazoan development is controlled by a complex system of interactions between transcription factors, histone modifications and modifying enzymes and chromatin conformation. Figure 1. Open in new tab Download slide. Figure 2. Figure 3. Search ADS. Genomic regulatory blocks encompass multiple neighboring genes and maintain conserved synteny in vertebrates. Metazoan promoters: emerging characteristics and insights into transcriptional regulation. Classification of human genomic regions based on experimentally determined binding sites of more than transcription-related factors.
Histone modifications at human enhancers reflect global cell-type-specific gene expression. An integrated holo-enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape. A transcription factor collective defines cardiac cell fate and reflects lineage history. Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer.
Defining a genomic radius for long-range enhancer action: duplicated conserved non-coding elements hold the key. Retroviral enhancer detection insertions in zebrafish combined with comparative genomics reveal genomic regulatory blocks - a fundamental feature of vertebrate genomes. Human GLI3 intragenic conserved non-coding sequences are tissue-specific enhancers. A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly.
An ultraconserved Hox-Pbx responsive element resides in the coding sequence of Hoxa2 and is active in rhombomere 4. Exonic remnants of whole-genome duplication reveal cis-regulatory function of coding exons. Ancient duplicated conserved noncoding elements in vertebrates: a genomic and functional analysis. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Looping and interaction between hypersensitive sites in the active beta-globin locus.
Arrays of ultraconserved non-coding regions span the loci of key developmental genes in vertebrate genomes. Highly conserved non-coding sequences are associated with vertebrate development. Close sequence comparisons are sufficient to identify human cis-regulatory elements. Ancora: a web resource for exploring highly conserved noncoding elements and their association with developmental regulatory genes. Metrics of sequence constraint overlook regulatory sequences in an exhaustive analysis at phox2b.
Conservation of RET regulatory function from human to zebrafish without sequence similarity. Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation.
Genomic regulatory blocks underlie extensive microsynteny conservation in insects. Conserved noncoding sequences highlight shared components of regulatory networks in dicotyledonous plants. Highly conserved regulatory elements around the SHH gene may contribute to the maintenance of conserved synteny across human chromosome 7q Extensive conservation of ancient microsynteny across metazoans due to cis-regulatory constraints. An ancient genomic regulatory block conserved across bilaterians and its dismantling in tetrapods by retrogene replacement. Disease-causing 7. An 8q24 gene desert variant associated with prostate cancer risk confers differential in vivo activity to a MYC enhancer.
A common variant associated with dyslexia reduces expression of the KIAA gene. Systematic localization of common disease-associated variation in regulatory DNA. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo. Combinatorial complexity in chromatin structure and function: revisiting the histone code.
Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. FoxA1 translates epigenetic signatures into enhancer-driven lineage-specific transcription. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Combinatorial patterns of histone acetylations and methylations in the human genome. Understanding these mechanisms will lead to both insights into the disease process and a better grasp of the normal regulatory machinery.
We use approaches that combine mouse molecular genetics, chromosomal engineering, transcriptome analysis and biochemistry to gain insights into the signalling and transcriptional networks that orchestrate vertebrate organogenesis. We investigate the embryonic mechanisms that organise the overall design of the developing organ systems. These mechanisms define the structures and their final arrangement in composing the embryonic anatomy. Our aims are to investigate developmental and gene regulatory processes that participate in organogenesis and understand how genetic inaccuracies lead to human congenital abnormalities.
We aim to identify the transcriptional network of genes that specify cellular identity in the well-established and highly accessible genetic system of the developing limb bud and how these organise the development of the skeleton. The relationship of enhancer structure to function is poorly understood and our work aims to understand how enhancers are activated during development, and once activated, how enhancers situated a long distances from their target promoters up to a million bases away conveys this information to regulate accurate temporal and spatial gene expression.
Sonic hedgehog Shh is a key signalling molecule required for normal development of many of the organ systems in the embryo including head and face, brain, neural tube, lung, gut and skeleton. Within these tissues the developmental role that Shh plays is dependent on complex gene regulation and like many developmental genes requires long-range regulatory mechanisms for its full spatiotemporal pattern of expression.
With the Shh coding region lying adjacent to a large gene desert, the expression is controlled by a group of cis-regulators that lie upstream of the gene with the regulatory domain spanning nearly a million base pairs and which together control the complex gene expression pattern seen in the embryo.