Long-range Gene Regulation Research Articles

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation.

Copy number variants in patients with intellectual disability affect the regulation of ARX transcription factor gene

Protein-coding mutations in the transcription factor-encoding gene ARX cause various forms of intellectual disability (ID) and epilepsy. In contrast, variations in surrounding non-coding sequences are correlated with milder forms of non-syndromic ID and autism and had suggested the importance of ARX gene regulation in the etiology of these disorders. We compile data on several novel and some already identified patients with or without ID that carry duplications of ARX genomic region and consider likely genetic mechanisms underlying the neurodevelopmental defects. We establish the long-range regulatory domain of ARX and identify its brain region-specific autoregulation. We conclude that neurodevelopmental disturbances in the patients may not simply arise from increased dosage due to ARX duplication. This is further exemplified by a small duplication involving a non-functional ARX copy, but with duplicated enhancers. ARX enhancers are located within a 504-kb region and regulate expression specifically in the forebrain in developing and adult zebrafish. Transgenic enhancer-reporter lines were used as in vivo tools to delineate a brain region-specific negative and positive autoregulation of ARX. We find autorepression of ARX in the telencephalon and autoactivation in the ventral thalamus. Fluorescently labeled brain regions in the transgenic lines facilitated the identification of neuronal outgrowth and pathfinding disturbances in the ventral thalamus and telencephalon that occur when arxa dosage is diminished. In summary, we have established a model for how breakpoints in long-range gene regulation alter the expression levels of a target gene brain region-specifically, and how this can cause subtle neuronal phenotypes relating to the etiology of associated neuropsychiatric disease.

Disruption of long-range gene regulation in human genetic disease: a kaleidoscope of general principles, diverse mechanisms and unique phenotypic consequences

The precise control of gene expression programs is crucial for the establishment of the diverse gene activity patterns required for the correct development, patterning and differentiation of the myriad of cell types within an organism. The crucial importance of non-coding regions of the genome in the control of gene regulation is well established and depends on a diverse group of sequence fragments called cis-regulatory elements that reside in these regions. Advances in novel genome-wide techniques have greatly increased the ability to identify potential regulatory elements. In contrast, their functional characterisation and the determination of their diverse modes of action remain a major bottleneck. Greater knowledge of gene expression control is of major importance for human health as disruption of gene regulation has become recognised as a significant cause of human disease. Appreciation of the role of cis-regulatory polymorphism in natural variation and susceptibility to common disease is also growing. While novel techniques such as GWAS and NGS provide the ability to collect large genomic datasets, the challenge for the twenty-first century will be to extract the relevant sequences and how to investigate the functional consequences of disease-associated changes. Here, we review how studies of transcriptional control at selected paradigm disease gene loci have revealed general principles of cis-regulatory logic and regulatory genome organisation, yet also demonstrate how the variety of mechanisms can combine to result in unique phenotypic outcomes. Integration of these principles with the emerging wealth of genome-wide data will provide enhanced insight into the workings of our regulatory genome.

H4K16 acetylation marks active genes and enhancers of embryonic stem cells, but does not alter chromatin compaction

Compared with histone H3, acetylation of H4 tails has not been well studied, especially in mammalian cells. Yet, H4K16 acetylation is of particular interest because of its ability to decompact nucleosomes in vitro and its involvement in dosage compensation in flies. Here we show that, surprisingly, loss of H4K16 acetylation does not alter higher-order chromatin compaction in vivo in mouse embryonic stem cells (ESCs). As well as peaks of acetylated H4K16 and KAT8 histone acetyltransferase at the transcription start sites of expressed genes, we report that acetylation of H4K16 is a new marker of active enhancers in ESCs and that some enhancers are marked by H3K4me1, KAT8, and H4K16ac, but not by acetylated H3K27 or EP300, suggesting that they are novel EP300 independent regulatory elements. Our data suggest a broad role for different histone acetylation marks and for different histone acetyltransferases in long-range gene regulation.

Alterations to the remote control ofShhgene expression cause congenital abnormalities

Multi-species conserved non-coding elements occur in the vertebrate genome and are clustered in the vicinity of developmentally regulated genes. Many are known to act as cis-regulators of transcription and may reside at long distances from the genes they regulate. However, the relationship of conserved sequence to encoded regulatory information and indeed, the mechanism by which these contribute to long-range transcriptional regulation is not well understood. The ZRS, a highly conserved cis-regulator, is a paradigm for such long-range gene regulation. The ZRS acts over approximately 1 Mb to control spatio-temporal expression of Shh in the limb bud and mutations within it result in a number of limb abnormalities, including polydactyly, tibial hypoplasia and syndactyly. We describe the activity of this developmental regulator and discuss a number of mechanisms by which regulatory mutations in this enhancer function to cause congenital abnormalities.

Chromatin and epigenetic features of long-range gene regulation

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. Developments in chromosome conformation capture technologies have revealed that interactions between regions of chromatin are pervasive and highly cell-type specific. The movement of enhancers and promoters in and out of higher-order chromatin structures within the nucleus are associated with changes in expression and histone modifications. However, the factors responsible for mediating these changes and determining enhancer:promoter specificity are still not completely known. In this review, we summarize what is known about the patterns of epigenetic and chromatin features characteristic of elements involved in long-range interactions. In addition, we review the insights into both local and global patterns of chromatin interactions that have been revealed by the latest experimental and computational methods.

Enhancer-like long-range transcriptional activation by λ CI-mediated DNA looping.

How distant enhancer elements regulate the assembly of a transcription complex at a promoter remains poorly understood. Here, we use long-range gene regulation by the bacteriophage λ CI protein as a powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed by CI bridging its binding sites at OR and OL, is known already to enhance repression at the lysogenic promoter PRM, located at OR. Here, we show that CI looping also activates PRM by allowing the C-terminal domain of the α subunit of the RNA polymerase bound at PRM to contact a DNA site adjacent to the distal CI sites at OL. Our results establish OL as a multifaceted enhancer element, able to activate transcription from long distances independently of orientation and position. We develop a physicochemical model of our in vivo data and use it to show that the observed activation is consistent with a simple recruitment mechanism, where the α-C-terminal domain to DNA contact need only provide ∼2.7 kcal/mol of additional binding energy for RNA polymerase. Structural modeling of this complete enhancer-promoter complex reveals how the contact is achieved and regulated, and suggests that distal enhancer elements, once appropriately positioned at the promoter, can function in essentially the same way as proximal promoter elements.

Impact of copy number variations (CNVs) on long-range gene regulation at the HoxD locus

Copy number variations are genomic structural variants that are frequently associated with human diseases. Among these copy number variations, duplications of DNA segments are often assumed to lead to dosage effects by increasing the copy number of either genes or their regulatory elements. We produced a series of large targeted duplications within a conserved gene desert upstream of the murine HoxD locus. This DNA region, syntenic to human 2q31-32, contains a range of regulatory elements required for Hoxd gene transcription, and it is often disrupted and/or reorganized in human genetic conditions collectively known as the 2q31 syndrome. Unexpectedly, one such duplication led to a transcriptional down-regulation in developing digits by impairing physical interactions between the target genes and their upstream regulatory elements, thus phenocopying the effect obtained when these enhancer sequences are deleted. These results illustrate the detrimental consequences of interrupting highly conserved regulatory landscapes and reveal a mechanism where genomic duplications lead to partial loss of function of nearby located genes.

DNA Binding by GATA Transcription Factor Suggests Mechanisms of DNA Looping and Long-Range Gene Regulation

GATA transcription factors regulate transcription during development and differentiation by recognizing distinct GATA sites with a tandem of two conserved zinc fingers, and by mediating long-range DNA looping. However, the molecular basis of these processes is not well understood. Here, we determined three crystal structures of the full DNA-binding domain (DBD) of human GATA3 protein, which contains both zinc fingers, in complex with different DNA sites. In one structure, both zinc fingers wrap around a palindromic GATA site, cooperatively enhancing the binding affinity and kinetic stability. Strikingly, in the other two structures, the two fingers of GATA DBD bind GATA sites on different DNA molecules, thereby bridging two separate DNA fragments. This was confirmed in solution by an in-gel fluorescence resonance energy transfer analysis. These findings not only provide insights into the structure and function of GATA proteins but also shed light on the molecular basis of long-range gene regulation.

Graph rigidity reveals well-constrained regions of chromosome conformation embeddings

BackgroundChromosome conformation capture experiments result in pairwise proximity measurements between chromosome locations in a genome, and they have been used to construct three-dimensional models of genomic regions, chromosomes, and entire genomes. These models can be used to understand long-range gene regulation, chromosome rearrangements, and the relationships between sequence and spatial location. However, it is unclear whether these pairwise distance constraints provide sufficient information to embed chromatin in three dimensions. A priori, it is possible that an infinite number of embeddings are consistent with the measurements due to a lack of constraints between some regions. It is therefore necessary to separate regions of the chromatin structure that are sufficiently constrained from regions with measurements that do not provide enough information to reconstruct the embedding.ResultsWe present a new method based on graph rigidity to assess the suitability of experiments for constructing plausible three-dimensional models of chromatin structure. Underlying this analysis is a new, efficient, and accurate algorithm for finding sufficiently constrained (rigid) collections of constraints in three dimensions, a problem for which there is no known efficient algorithm. Applying the method to four recent chromosome conformation experiments, we find that, for even stringently filtered constraints, a large rigid component spans most of the measured region. Filtering highlights higher-confidence regions, and we find that the organization of these regions depends crucially on short-range interactions.ConclusionsWithout performing an embedding or creating a frequency-to-distance mapping, our proposed approach establishes which substructures are supported by a sufficient framework of interactions. It also establishes that interactions from recent highly filtered genome-wide chromosome conformation experiments provide an adequate set of constraints for embedding. Pre-processing experimentally observed interactions with this method before relating chromatin structure to biological phenomena will ensure that hypothesized correlations are not driven by the arbitrary choice of a particular unconstrained embedding. The software for identifying rigid components is GPL-Licensed and available for download at http://cbcb.umd.edu/kingsford-group/starfish.

Integrated analysis identifies a class of androgen-responsive genes regulated by short combinatorial long-range mechanism facilitated by CTCF

Recently, much attention has been given to elucidate how long-range gene regulation comes into play and how histone modifications and distal transcription factor binding contribute toward this mechanism. Androgen receptor (AR), a key regulator of prostate cancer, has been shown to regulate its target genes via distal enhancers, leading to the hypothesis of global long-range gene regulation. However, despite numerous flows of newly generated data, the precise mechanism with respect to AR-mediated long-range gene regulation is still largely unknown. In this study, we carried out an integrated analysis combining several types of high-throughput data, including genome-wide distribution data of H3K4 di-methylation (H3K4me2), CCCTC binding factor (CTCF), AR and FoxA1 cistrome data as well as androgen-regulated gene expression data. We found that a subset of androgen-responsive genes was significantly enriched near AR/H3K4me2 overlapping regions and FoxA1 binding sites within the same CTCF block. Importantly, genes in this class were enriched in cancer-related pathways and were downregulated in clinical metastatic versus localized prostate cancer. Our results suggest a relatively short combinatorial long-range regulation mechanism facilitated by CTCF blocking. Under such a mechanism, H3K4me2, AR and FoxA1 within the same CTCF block combinatorially regulate a subset of distally located androgen-responsive genes involved in prostate carcinogenesis.

Long-range gene regulation by retinoic acid response elements in the mouse HoxB cluster

Long-range gene regulation by retinoic acid response elements in the mouse HoxB cluster

Enhancers: From Developmental Genetics to the Genetics of Common Human Disease

In mammals, long-range gene regulation became apparent through simple Mendelian disease genetics in human and developmental genetics in the mouse. Can the insights into gene control, provided by the study of these enhancers, help us understand the functional significance of sequence variation associated with common/complex human disease and quantitative traits?

Correction for Ragvin et al., Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX , SOX4 , and IRX3

Correction for Ragvin et al., Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to <i>HHEX</i> , <i>SOX4</i> , and <i>IRX3</i>

Epigenetic domains found in mouse embryonic stem cells via a hidden Markov model

BackgroundEpigenetics is an important layer of transcriptional control necessary for cell-type specific gene regulation. Recent studies have shown significant epigenetic patterns associated with developmental stages and diseases. However, previous studies have been mostly limited to focal epigenetic patterns, whereas methods for analyzing large-scale organizations are still lacking.ResultsWe developed a hidden Markov model (HMM) approach for detecting the types and locations of epigenetic domains from multiple histone modifications. We used this method to analyze a published ChIP-seq dataset of five histone modification marks (H3K4me2, H3K4me3, H3K27me3, H3K9me3, and H3K36me3) in mouse embryonic stem (ES) cells. We identified three types of domains, corresponding to active, non-active, and null states. In total, our three-state HMM identified 258 domains in the mouse genome containing 9.6 genes on average. These domains were validated by a number of criteria. The largest domains correspond to olfactory receptor (OR) gene clusters. Each Hox gene cluster also forms a separate epigenetic domain. We found that each type of domain is associated with distinct biological functions and structural changes during early cell differentiation.ConclusionsThe HMM approach successfully detects domains of consistent epigenetic patterns from ChIP-seq data, providing new insights into the role of epigenetics in long-range gene regulation.

Mandibulofacial dysostosis, microtia, and limb anomalies in a newborn: a new form of acrofacial dysostosis syndrome?

The acrofacial dysostoses (AFDs) are a heterogeneous group of disorders involving craniofacial dysostosis and limb anomalies. Depending on the type of limb defects, two major groups have been defined: Nager syndrome with predominant preaxial anomalies and Miller syndrome with postaxial malformations. Genomic copy number variation, a common type of genomic variability, can influence gene expression by disrupting coding sequences, perturbing long-range gene regulation, or altering gene dosage, and these effects could contribute to phenotypic variations or disease risk. We present a distinct AFD case with mandibulofacial dysostosis, microtia and limb malformations but without limb defects, which may represent a new form of AFD. To investigate the etiology of the phenotype, whole genomic high-resolution array comparative genomic hybridization analysis was carried out, revealing two cryptic duplications, 1p36.33 and 1q21.3-q22 duplications. Two genes, VWA1 and PYGO2, contained in the two duplications, respectively, are likely to be the candidate genes for the phenotype of our patient.

VEZF1 Elements Mediate Protection from DNA Methylation

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.

Functional intronic polymorphisms: Buried treasure awaiting discovery within our genes

Functional intronic polymorphisms: Buried treasure awaiting discovery within our genes

Chromosome looping at the human α-globin locus is mediated via the major upstream regulatory element (HS −40)

AbstractPrevious studies in the mouse have shown that high levels of α-globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human α cluster and their interactions with the α-globin promoter. All of these elements interact with the α-globin gene in an erythroid-specific manner. These results were confirmed in a mouse model of human α globin expression in which the human cluster replaces the mouse cluster in situ (humanized mouse). We have also shown that expression and all of the long-range interactions depend largely on just one of these elements; removal of the previously characterized major regulatory element (called HS −40) results in loss of all the interactions and α-globin expression. Reinsertion of this element at an ectopic location restores both expression and the intralocus interactions. In contrast to other more complex systems involving multiple upstream elements and promoters, analysis of the human α-globin cluster during erythropoiesis provides a simple and tractable model to understand the mechanisms underlying long-range gene regulation.

Web-based tools and approaches to study long-range gene regulation in Metazoa

Regulatory elements can affect specific genes from megabase distances, often from within or beyond unrelated neighbouring genes. The task of computational charting of regulatory inputs in the genome can be approached from several directions. Typically, computational identification of putative regulatory elements for a gene of interest requires tools that will aid in estimating the extent of the (potentially vast) genomic region around the gene that is likely to contain regulatory elements, as well as tools for the identification and characterization of individual elements. Conversely, starting from a putative regulatory element or a regulatory variation in a non-coding region, one often wants to associate the regulatory element with the correct target gene(s). The design of tools for these purposes relies on the remarkably high level of sequence conservation of thousands of regulatory enhancers, their strong tendency to cluster around their target genes, as well as a constrained range of functional categories of the corresponding target genes, many of which are developmental regulators. Additional evolutionary information, such as conservation of synteny, and a growing body of functional genomic and epigenomic data are being rapidly added to established and emerging tools for studying developmental regulation and cross-species conservation to provide new functional insights into the roles of these regions. In this article, we give an overview of the functionality available in general purpose and new/specialized web tools for the above tasks, and discuss current and future developments in the field.