Chromosomes are folded in intricate ways inside cells and their spatial organization is intimately related to regulation of gene expression. Expression of genes can be controlled by regulatory elements that are located at large genomic distances from their target genes (in cis), or even on different chromosomes (in trans). Regulatory elements can act at large genomic distances by engaging in direct physical interactions with their target genes resulting in the formation of chromatin loops. Thus, genes and their regulatory elements come in close spatial proximity irrespective of their relative genomic positions. Analysis of interactions between genes and elements will reveal which elements regulate each gene, and will provide fundamental insights into the spatial organization of chromosomes in general.
Long-range cis- and trans- interactions can be studied at high resolution using the Chromosome Conformation Capture (3C) technology. 3C employs formaldehyde crosslinking to trap physical interactions between loci located throughout the genome. Crosslinked cells are then solubilized and chromatin is digested by a restriction enzyme. After digestion the chromatin is subjected to ligation under very dilute DNA concentrations. These conditions favor intramolecular ligation over intermolecular ligation, and thus result in selective ligation of interacting (and crosslinked) genomic elements. The crosslinks are reversed, the DNA is purified, and interaction frequencies between specific chromosomal loci can be determined by quantifying the amounts of corresponding ligation product that is formed using PCR. This chapter describes detailed protocols for 3C analysis of yeast Saccharomyces cerevisiae and mammalian chromosomes.
DNA; chromatin looping; long-range gene regulation; trans-regulation; formaldehyde crosslinking; spatial organization
Control of gene expression involves the concerted action of multiple regulatory elements some of which can act over large genomic distances. Physical interaction among these elements can lead to looping of the chromatin fiber. Although posttranslational modifications of chromatin are thought to play a role in the conveyance of epigenetic information, it is largely unknown whether higher order chromatin organization such as looping contributes to epigenetic memory. A related unresolved question is whether chromatin loops are the cause or the effect of transcriptional regulation. Recent work from diverse organisms suggests a memory function for long-range chromatin interactions. It is proposed that higher order folding of the chromatin fiber can serve to maintain active and repressed states of gene expression.
Long-range interactions between transcription regulatory elements play an important role in gene activation, epigenetic silencing, and chromatin organization. Transcriptional activation or repression of developmentally regulated genes is often accomplished through tissue specific chromatin architecture and dynamic localization between active transcription factories and repressive Polycomb bodies. However, the mechanisms underlying the structural organization of chromatin and the coordination of physical interactions are not fully understood. Insulators and Polycomb group proteins form highly conserved multi-protein complexes that mediate functional long-range interactions, and have proposed roles in nuclear organization. In this review, we explore recent findings that have broadened our understanding of the function of these proteins and provide an integrative model for the roles of insulators in nuclear organization.
CTCF; Polycomb; TFIIIC; Cohesin; Epigenetics
Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements.
In metazoans, enhancers of gene transcription must often exert their effects over tens of kilobases of DNA. Over the last decade it has become clear that to do this, enhancers come into close proximity with target promoters with the looping away of intervening sequences. In a few cases proteins that are involved in the establishment or maintenance of these loops have been revealed but how the proper gene target is selected remains mysterious. Chromatin insulators had been appreciated as elements that play a role in enhancer fidelity through their enhancer blocking or barrier activity. However, recent work suggests more direct participation of insulators in enhancer-gene interactions. The emerging view begins to incorporate transcription activation by distant enhancers with large scale nuclear architecture and sub-nuclear movement.
RNA polymerases can be shared by a particular group of genes in a transcription “factory” in nuclei, where transcription may be coordinated in concert with the distribution of coexpressed genes in higher-eukaryote genomes. Moreover, gene expression can be modulated by regulatory elements working over a long distance. Here, we compared the conformation of a 130-kb chromatin region containing the mouse α-globin cluster and their flanking housekeeping genes in 14.5-day-postcoitum fetal liver and brain cells. The analysis of chromatin conformation showed that the active α1 and α2 globin genes and upstream regulatory elements are in close spatial proximity, indicating that looping may function in the transcriptional regulation of the mouse α-globin cluster. In fetal liver cells, the active α1 and α2 genes, but not the inactive ζ gene, colocalize with neighboring housekeeping genes C16orf33, C16orf8, MPG, and C16orf35. This is in sharp contrast with the mouse α-globin genes in nonexpressing cells, which are separated from the congregated housekeeping genes. A comparison of RNA polymerase II (Pol II) occupancies showed that active α1 and α2 gene promoters have a much higher RNA Pol II enrichment in liver than in brain. The RNA Pol II occupancy at the ζ gene promoter, which is specifically repressed during development, is much lower than that at the α1 and α2 promoters. Thus, the mouse α-globin gene cluster may be regulated through moving in or out active globin gene promoters and regulatory elements of a preexisting transcription factory in the nucleus, which is maintained by the flanking clustered housekeeping genes, to activate or inactivate α-globin gene expression.
Cohesin is a multiprotein ringed complex that is most well known for its role in stabilizing the association of sister chromatids between S phase and M. More recently cohesin was found to be associated with transcriptional insulators, elements that are associated with the organization of chromatin into regulatory domains. The human major histocompatibility complex class II (MHC-II) locuscontains ten intergenic elements, termed MHC-II insulators, which bind the transcriptional insulator protein CCCTC transcription factor (CTCF). MHC-II insulators interact with each other forming a base architecture of discrete loops and potential regulatory domains. When MHC-II genes are expressed, their proximal promoter regulatory regions reorganize to the foci established by the interacting MHC-II insulators. MHC-II insulators also bind cohesin, but the functional role of cohesin in regulating this system is not known. Here we show that the binding of cohesin to MHC-II insulators occurred irrespective of MHC-II expression but was required for optimal expression of the HLA-DR and HLA-DQ genes. In a DNA dependent manner, cohesin subunits interacted with CTCF and the MHC-II specific transcription factors RFX and CIITA. Intriguingly, cohesin subunits were important for DNA looping interactions between the HLA-DRA promoter region and a 5’ MHC-II insulator but were not required for interactions between the MHC-II insulators themselves. This latter observation introduces cohesin as a regulator of MHC-II expression by initiating or stabilizing MHC-II promoter regulatory element interactions with the MHC-II insulator elements; events which are required for maximal MHC-II transcription.
When placed between an enhancer and promoter, certain DNA sequence elements inhibit enhancer-stimulated gene expression. The best characterized of these enhancer blocking insulators, gypsy in Drosophila and the CTCF-binding element in vertebrates and flies, stabilize contacts between distant genomic regulatory sites leading to the formation of loop domains. Current results show that CTCF mediates long-range contacts in the mouse β-globin locus and at the Igf2/H19 imprinted locus. Recently described active chromatin hubs and transcription factories also involve long-range interactions; it is likely that CTCF interferes with their formation when acting as an insulator. The properties of CTCF, and its newly described genomic distribution, suggest that it may play an important role in large scale nuclear architecture, perhaps mediated by the co-factors with which it interacts in vivo.
Long-range interactions between regulatory DNA elements such as enhancers, insulators and promoters play an important role in regulating transcription. As chromatin contacts have been found throughout the human genome and in different cell types, spatial transcriptional control is now viewed as a general mechanism of gene expression regulation. Chromosome Conformation Capture Carbon Copy (5C) and its variant Hi-C are techniques used to measure the interaction frequency (IF) between specific regions of the genome. Our goal is to use the IF data generated by these experiments to computationally model and analyze three-dimensional chromatin organization.
We formulate a probabilistic model linking 5C/Hi-C data to physical distances and describe a Markov chain Monte Carlo (MCMC) approach called MCMC5C to generate a representative sample from the posterior distribution over structures from IF data. Structures produced from parallel MCMC runs on the same dataset demonstrate that our MCMC method mixes quickly and is able to sample from the posterior distribution of structures and find subclasses of structures. Structural properties (base looping, condensation, and local density) were defined and their distribution measured across the ensembles of structures generated. We applied these methods to a biological model of human myelomonocyte cellular differentiation and identified distinct chromatin conformation signatures (CCSs) corresponding to each of the cellular states. We also demonstrate the ability of our method to run on Hi-C data and produce a model of human chromosome 14 at 1Mb resolution that is consistent with previously observed structural properties as measured by 3D-FISH.
We believe that tools like MCMC5C are essential for the reliable analysis of data from the 3C-derived techniques such as 5C and Hi-C. By integrating complex, high-dimensional and noisy datasets into an easy to interpret ensemble of three-dimensional conformations, MCMC5C allows researchers to reliably interpret the result of their assay and contrast conformations under different conditions.
Recent studies on transcriptional control of gene expression have pinpointed the importance of long-range interactions and three-dimensional organization of chromatins within the nucleus. Distal regulatory elements such as enhancers may activate transcription over long distances; hence, their action must be restricted within appropriate boundaries to prevent illegitimate activation of non-target genes. Insulators are DNA elements with enhancer-blocking and/or chromatin-bordering functions. In vertebrates, the versatile transcription regulator CCCTC-binding factor (CTCF) is the only identified trans-acting factor that confers enhancer-blocking insulator activity. CTCF-binding sites were found to be commonly distributed along the vertebrate genomes. We have constructed a CTCF-binding site database (CTCFBSDB) to characterize experimentally identified and computationally predicted CTCF-binding sties. Biological knowledge and data from multiple resources have been integrated into the database, including sequence data, genetic polymorphisms, function annotations, histone methylation profiles, gene expression profiles and comparative genomic information. A web-based user interface was implemented for data retrieval, analysis and visualization. In silico prediction of CTCF-binding motifs is provided to facilitate the identification of candidate insulators in the query sequences submitted by users. The database can be accessed at http://insulatordb.utmem.edu/
The mammalian genome is packed tightly in the nucleus of the cell. This packing is primarily facilitated by histone proteins and results in an ordered organization of the genome in chromosome territories that can be roughly divided in heterochromatic and euchromatic domains. On top of this organization several distinct gene regulatory elements on the same chromosome or other chromosomes are thought to dynamically communicate via chromatin looping. Advances in genome-wide technologies have revealed the existence of a plethora of these regulatory elements in various eukaryotic genomes. These regulatory elements are defined by particular in vitro assays as promoters, enhancers, insulators, and boundary elements. However, recent studies indicate that the in vivo distinction between these elements is often less strict. Regulatory elements are bound by a mixture of common and lineage-specific transcription factors which mediate the long-range interactions between these elements. Inappropriate modulation of the binding of these transcription factors can alter the interactions between regulatory elements, which in turn leads to aberrant gene expression with disease as an ultimate consequence. Here we discuss the bi-modal behavior of regulatory elements that act in cis (with a focus on enhancers), how their activity is modulated by transcription factor binding and the effect this has on gene regulation.
enhancer; transcription factor; chromatin looping; transcription; cis-regulation
Spatial organization of chromatin plays an important role at multiple levels of genome regulation. On a global scale its function is evident in processes like metaphase and chromosome segregation. On a detailed level, long range interactions between regulatory elements and promoters are essential for proper gene regulation. Microscopic techniques like FISH can detect chromatin contacts, although the resolution is generally low making detection of enhancer-promoter interaction difficult. The 3C methodology allows for high-resolution analysis of chromatin interactions. 3C is now widely used and has revealed that long-range looping interactions between genomic elements is widespread. However, studying chromatin interactions in large genomic regions by 3C is very labor intensive. This limitation is overcome by the 5C technology. 5C is an adaptation of 3C, in which the concurrent use of thousands of primers permits the simultaneous detection of millions of chromatin contacts. The design of the 5C primers is critical, since this will determine which and how many chromatin interactions will be examined in the assay. Starting material for 5C is a 3C template. To make a 3C template, chromatin interactions in living cells are crosslinked using formaldehyde. Next, chromatin is digested and subsequently ligated under conditions favoring ligation events between crosslinked fragments. This yields a genome-wide 3C library of ligation products representing all chromatin interactions in vivo. 5C then employs multiplex ligation mediated amplification to detect, in a single assay, up to millions of unique ligation products present in the 3C library. The resulting 5C library can be analyzed by microarray analysis or deep sequencing. The observed abundance of a 5C product is a measure of the interaction frequency between the two corresponding chromatin fragments. The power of the 5C technique described in this chapter is the high-throughput, high-resolution and quantitative way in which the spatial organization of chromatin can be examined.
Chromosome conformation capture; chromatin looping; chromatin structure; long-range gene regulation; high-throughput
Knockdown of the insulator factor CCCTC binding factor (CTCF), which binds XL9, an intergenic element located between HLA-DRB1 and HLA-DQA1, was found to diminish expression of these genes. The mechanism involved interactions between CTCF and class II transactivator (CIITA), the master regulator of major histocompatibility complex class II (MHC-II) gene expression, and the formation of long-distance chromatin loops between XL9 and the proximal promoter regions of these MHC-II genes. The interactions were inducible and dependent on the activity of CIITA, regulatory factor X, and CTCF. RNA fluorescence in situ hybridizations show that both genes can be expressed simultaneously from the same chromosome. Collectively, the results suggest a model whereby both HLA-DRB1 and HLA-DQA1 loci can interact simultaneously with XL9, and describe a new regulatory mechanism for these MHC-II genes involving the alteration of the general chromatin conformation of the region and their regulation by CTCF.
Enhancers can regulate designate promoters over long distances by forming chromatin loops. Whether chromatin loops are lost or reconfigured during gene repression is largely unexplored. We examined the chromosome conformation of the Kit gene that is expressed during early erythropoiesis, but is downregulated upon cell maturation. Kit expression is controlled by sequential occupancy of two GATA family transcription factors. In immature cells, a distal enhancer bound by GATA-2 is in physical proximity with the active Kit promoter. Upon cell maturation, GATA-1 displaces GATA-2 and triggers a loss of the enhancer/promoter interaction. Moreover, GATA-1 reciprocally increases the proximity in nuclear space among distinct downstream GATA elements. GATA-1-induced transitions in chromatin conformation are not simply the consequence of transcription inhibition and require the cofactor FOG-1. This work shows that a GATA factor exchange reconfigures higher order chromatin organization and suggests that de novo chromatin loop formation is employed by nuclear factors to specify repressive outcomes.
chromatin; chromatin loops; GATA-1; GATA-2; gene expression; hematopoiesis; erythropoiesis; Kit
There are many examples within gene complexes of transcriptional enhancers interacting with only a subset of target promoters. A number of molecular mechanisms including promoter competition, insulators and chromatin looping are thought to play a role in regulating these interactions. At the Drosophila bithorax complex (BX-C), the IAB5 enhancer specifically drives gene expression only from the Abdominal-B (Abd-B) promoter, even though the enhancer and promoter are 55 kb apart and are separated by at least three insulators. In previous studies, we discovered that a 255 bp cis-regulatory module, the promoter tethering element (PTE), located 5′ of the Abd-B transcriptional start site is able to tether IAB5 to the Abd-B promoter in transgenic embryo assays. In this study we examine the functional role of the PTE at the endogenous BX-C using transposon-mediated mutagenesis. Disruption of the PTE by P element insertion results in a loss of enhancer-directed Abd-B expression during embryonic development and a homeotic transformation of abdominal segments. A partial deletion of the PTE and neighboring upstream genomic sequences by imprecise excision of the P element also results in a similar loss of Abd-B expression in embryos. These results demonstrate that the PTE is an essential component of the regulatory network at the BX-C and is required in vivo to mediate specific long-range enhancer-promoter interactions.
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.
DNA sequences known as chromatin insulator or barrier elements are considered key components of genome organization as they can establish boundaries between transcriptionally permissive and repressive chromatin domains. Here we address the hypothesis that barrier elements in vertebrates can protect genes from transcriptional silencing that is marked by DNA methylation. We have found that the HS4 insulator element from the β-globin gene locus can protect a gene promoter from DNA methylation. Protection from DNA methylation is separable from other insulator activities and is mapped to three transcription factor binding sites occupied by the zinc finger protein VEZF1, a novel chromatin barrier protein. VEZF1 is a candidate factor for the protection of promoters from DNA methylation. We found that VEZF1-specific binding sites are sufficient to mediate demethylation and protection of the APRT gene promoter from DNA methylation. We propose that barrier elements in vertebrates must be capable of preventing DNA methylation in addition to blocking the propagation of silencing histone modifications, as either process is sufficient to direct the establishment of an inactive chromatin state.
Recent years have seen unprecedented characterization of mammalian chromatin thanks to advances in chromatin assays, antibody development and genomics. Genome-wide maps of chromatin state can now be readily acquired using microarrays or next-generation sequencing technologies. These datasets reveal local and long-range chromatin patterns that offer insight into the locations and functions of underlying regulatory elements and genes. These patterns are dynamic across developmental stages and lineages. Global studies of chromatin in embryonic stem cells have led to intriguing hypotheses regarding Polycomb/trithorax and RNA polymerase roles in ‘poising’ transcription. Chromatin state maps thus provide a rich resource for understanding chromatin at a ‘systems level’, and a starting point for mechanistic studies aimed at defining epigenetic controls that underlie development.
Enhancers are regulatory DNA sequences that activate transcription over long distances. Recent studies revealed a widespread role of distant activation in eukaryotic gene regulation and in development of various human diseases, including cancer. Genomic and gene-targeted studies of enhancer action revealed novel mechanisms of transcriptional activation over a distance. They include formation of stable, inactive DNA-protein complexes at the enhancer and target promoter before activation, facilitated distant communication by looping of the spacer chromatin-covered DNA, and promoter activation by mechanisms that are different from classic recruiting. These studies suggest the similarity between the looping mechanisms involved in enhancer action on DNA in bacteria and in chromatin of higher organisms.
The functional output of the genome is closely dependent on its organization within the nucleus, which ranges from the 10 nm chromatin fiber to the three-dimensional arrangement of this fiber in the nuclear space. Recent observations suggest that intra-and inter-chromosomal interactions between distant sequences underlie several aspects of transcription regulatory processes. These contacts can bring enhancers close to their target genes, or prevent inappropriate interactions between regulatory sequences via insulators. In addition, intra- and inter-chromosomal interactions can bring co-activated or co-repressed genes to the same nuclear location. Recent technological advances have made it possible to map long-range cis and trans interactions at relatively high resolution. This information is being used to develop three-dimensional maps of the arrangement of the genome in the nucleus and to understand causal relationships between nuclear structure and function.
Chromatin; Epigenetics; Transcription; Nucleus
Chromatin folding inside the interphase nucleus of eukaryotic cells is done on multiple scales of length and time. Despite recent progress in understanding the folding motifs of chromatin, the higher-order structure still remains elusive. Various experimental studies reveal a tight connection between genome folding and function. Chromosomes fold into a confined subspace of the nucleus and form distinct territories. Chromatin looping seems to play a dominant role both in transcriptional regulation as well as in chromatin organization and has been assumed to be mediated by long-range interactions in many polymer models. However, it remains a crucial question which mechanisms are necessary to make two chromatin regions become co-located, i.e. have them in spatial proximity. We demonstrate that the formation of loops can be accomplished solely on the basis of diffusional motion. The probabilistic nature of temporary contacts mimics the effects of proteins, e.g. transcription factors, in the solvent. We establish testable quantitative predictions by deriving scale-independent measures for comparison to experimental data. In this Dynamic Loop (DL) model, the co-localization probability of distant elements is strongly increased compared to linear non-looping chains. The model correctly describes folding into a confined space as well as the experimentally observed cell-to-cell variation. Most importantly, at biological densities, model chromosomes occupy distinct territories showing less inter-chromosomal contacts than linear chains. Thus, dynamic diffusion-based looping, i.e. gene co-localization, provides a consistent framework for chromatin organization in eukaryotic interphase nuclei.
The major histocompatibility complex class II (MHC-II) genes are regulated at the level of transcription. Recent studies have shown that chromatin modification is critical for efficient transcription of these genes, and a number of chromatin modifying complexes recruited to MHC-II genes have been described. The MHC-II genes are segregated from each other by a series of chromatin elements, termed MHC-II insulators. Interactions between MHC-insulators and the promoters of MHC-II genes are mediated by the insulator factor CCCTC-binding protein and are critical for efficient expression. This regulatory mechanism provides a novel view of how the entire MHC-II locus is assembled architecturally and can be coordinately controlled.
Spatial chromatin organization is emerging as an important mechanism to regulate the expression of genes. However, very little is known about genome architecture at high-resolution in vivo. Here, we mapped the three-dimensional organization of the human Hox clusters with chromosome conformation capture (3C) technology. We show that computational modeling of 3C data sets can identify candidate regulatory proteins of chromatin architecture and gene expression. Hox genes encode evolutionarily conserved master regulators of development which strict control has fascinated biologists for over 25 years. Proper transcriptional silencing is key to Hox function since premature expression can lead to developmental defects or human disease. We now show that the HoxA cluster is organized into multiple chromatin loops that are dependent on transcription activity. Long-range contacts were found in all four silent clusters but looping patterns were specific to each cluster. In contrast to the Drosophila homeotic bithorax complex (BX-C), we found that Polycomb proteins are only modestly required for human cluster looping and silencing. However, computational three-dimensional Hox cluster modeling identified the insulator-binding protein CTCF as a likely candidate mediating DNA loops in all clusters. Our data suggest that Hox cluster looping may represent an evolutionarily conserved structural mechanism of transcription regulation.
Transcriptional insulators are specialized cis-acting elements that protect promoters from inappropriate activation by distal enhancers. The H19 imprinting control region (ICR) functions as a CTCF-dependent, methylation-sensitive transcriptional insulator. We analyzed several insertional mutations and demonstrate that the ICR can function as a methylation-regulated maternal chromosome-specific insulator in novel chromosomal contexts. We used chromosome conformation capture and chromatin immunoprecipitation assays to investigate the configuration of cis-acting elements at these several insertion sites. By comparing maternal and paternal organizations on wild-type and mutant chromosomes, we hoped to identify mechanisms for ICR insulator function. We found that promoter and enhancer elements invariably associate to form DNA loop domains at transcriptionally active loci. Conversely, active insulators always prevent these promoter-enhancer interactions. Instead, the ICR insulator forms novel loop domains by associating with the blocked promoters and enhancers. We propose that these associations are fundamental to insulator function.
Barrier elements that are able to block the propagation of transcriptional silencing in yeast are functionally similar to chromatin boundary/insulator elements in metazoans that delimit functional chromosomal domains. We show that the upstream activating sequences of many highly expressed ribosome protein genes and glycolytic genes exhibit barrier activity. Analyses of these barriers indicate that binding sites for transcriptional regulators Rap1p, Abf1p, Reb1p, Adr1p and Gcn4p may participate in barrier function. We also present evidence suggesting that Rap1p is directly involved in barrier activity, and its barrier function correlates with local changes in chromatin structure. We further demonstrate that tethering the transcriptional activation domain of Rap1p to DNA is sufficient to recapitulate barrier activity. Moreover, targeting the activation domain of Adr1p or Gcn4p also establishes a barrier to silencing. These results support the notion that transcriptional regulators could also participate in delimiting functional domains in the genome.
Recent advances are now providing novel insights into the mechanisms that underlie how cellular complexity, diversity, and connectivity are encoded within the genome. The repressor element-1 silencing transcription factor / neuron-restrictive silencing factor (REST/NRSF) and non-coding RNAs (ncRNAs) are emerging as key regulators that seem to orchestrate almost every aspect of nervous system development, homeostasis, and plasticity. REST and its primary cofactor, CoREST, dynamically recruit highly malleable macromolecular complexes to widely distributed genomic regulatory sequences, including the repressor element 1 / neuron restrictive silencer element (RE1/NRSE). Through epigenetic mechanisms, such as site-specific targeting and higher-order chromatin remodeling, REST and CoREST can mediate cell type- and developmental stage-specific gene repression, gene activation, and long-term gene silencing for protein-coding genes and for several classes of ncRNAs (e.g. microRNAs [miRNAs] and long ncRNAs). In turn, these ncRNAs have similarly been implicated in the regulation of chromatin architecture and dynamics, transcription, post-transcriptional processing, and RNA editing and trafficking. In addition, REST and CoREST expression and function are tightly regulated by context-specific transcriptional and post-transcriptional mechanisms including bidirectional feedback loops with various ncRNAs. Not surprisingly, deregulation of REST and ncRNAs are both implicated in the molecular pathophysiology underlying diverse disorders that range from brain cancer and stroke to neurodevelopmental and neurodegenerative diseases. This review summarizes emerging aspects of the complex mechanistic relationships between these intricately interlaced control systems for neural gene expression and function.
repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF); CoREST; neural stem cell; oligodendrocyte; glia; neuron; epigenetic; non-coding RNA (ncRNA); microRNA (miRNA)