Chromosomal Contact Permits Transcription between Coregulated Genes2013-10-24 12:12:21
Cell; 2013 Oct 24; 155(3):606-20
Stephanie Fanucchi, Youtaro Shibayama, Shaun Burd, Marc S. Weinberg, Musa M. Mhlanga
- Transcription of genes in a multigene complex can be asymmetric
- Site-specific nucleases can discretely disrupt chromatin loops
- Disrupting chromatin contacts influences cotranscription of interacting genes
- Chromatin looping in multigene complexes may be governed by hierarchical regulation
Transcription of coregulated genes occurs in the context of long-range chromosomal contacts that form multigene complexes. Such contacts and transcription are lost in knockout studies of transcription factors and structural chromatin proteins. To ask whether chromosomal contacts are required for cotranscription in multigene complexes, we devised a strategy using TALENs to cleave and disrupt gene loops in a well-characterized multigene complex. Monitoring this disruption using RNA FISH and immunofluorescence microscopy revealed that perturbing the site of contact had a direct effect on transcription of other interacting genes. Unexpectedly, this effect on cotranscription was hierarchical, with dominant and subordinate members of the multigene complex engaged in both intra- and interchromosomal contact. This observation reveals the profound influence of these chromosomal contacts on the transcription of coregulated genes in a multigene complex.
Transcription is replete with proximal and distal chromatin looping interactions whose formation represents the basic organizing principle of nuclear architecture and gene activity (Miele and Dekker, 2008; Palstra et al., 2008; Tan-Wong et al., 2012). Loop-mediated chromosomal contacts are usually identified
on a genome-wide scale using population-based ‘‘chromosome conformation capture’’ (3C) technologies (Dekker et al., 2002; Fullwood et al., 2009; Lieberman-Aiden et al., 2009; Li et al., 2012). Analyses of 3C-based data reveal a large heterogeneity in global chromatin interactions (Fullwood et al., 2009; Noordermeer
et al., 2011; Li et al., 2012). Therefore, interacting DNA elements identified by 3C-based technologies are verified at the single-cell level using fluorescent in situ hybridization (FISH) assays (Schoenfelder et al., 2010; Papantonis et al., 2012). These highly sensitive assays can target either DNA or nascent mRNA and have revealed the colocalization between FISH foci in a fraction of the population (Schoenfelder et al., 2010; Papantonis et al., 2010). This suggests that a subset of cells within the population may be enriched for specific chromosomal interactions. Chromosomes are large and constrained in their ability to roam the entire nuclear volume (Strickfaden et al., 2010; Barbieri et al., 2013). Thus, it is reasonable to surmise that the topological arrangements after each cellular
division shuffle chromosomal proximities such that their threedimensional (3D) arrangements are altered in one-dimensional (1D) space (Gibcus and Dekker, 2013). This may lead to every cell in the population possessing unique spatial arrangements of its chromosomes (Orlova et al., 2012).
Enhancer-promoter interactions utilize chromatin looping to trigger dynamic changes in transcription initiation (Osborne et al., 2004; Deng et al., 2012). An example of this is the wellestablished model between the locus control region (LCR) and the promoter of the b-globin gene (Osborne et al., 2004). In a tissue-
specific manner, the LCR has been shown to physically contact the promoter of the b-globin gene and initiate transcription (Deng et al., 2012). These LCR-mediated chromosomal interactions have been shown to result in variability in b-globin gene transcript levels or variegated gene expression across the population (Noordermeer et al., 2011). In an otherwise identical population of cells, presumably through chromosomal interactions, such ‘‘jackpot’’ cells display higher levels of b-globin transcription (Noordermeer et al., 2011). Accordingly, the specific set of chromosomal interactions (and consequent gene expression
that may depend on LCR-mediated interactions) will vary between cells across the population (Strickfaden et al., 2010). This heterogeneity reveals the absolute requirement of singlecell analysis in global interactome and gene loop studies.
Looping also brings distal genes into close proximity, enabling chromosomal contact in ‘‘multigene complexes’’ at a single focus of multiple RNA polymerases (Schoenfelder et al., 2010; Papantonis et al., 2012; Li et al., 2012). Numerous studies have demonstrated that the formation of loop-mediated contact coincides
with alterations in gene expression (Spilianakis et al., 2005; Apostolou and Thanos, 2008; Fullwood et al., 2009; Schoenfelder et al., 2010). Indeed, chromosomal contacts in multigene complexes appear to be the main modality of transcription in metazoan cells, as they are associated with >95% of transcriptional activity (Li et al., 2012; Sandhu et al., 2012). In a comparable manner to enhancer-promoter interactions, specific chromatin interactions in multigene complexes are detected in a subset of cells within the population (Schoenfelder et al., 2010; Papantonis et al., 2010). Genome-wide chromatin interaction analysis with paired end tags (ChIA-PET) uncovered a multigene complex, including the GREB1 locus and three other genes (Li et al., 2012). Of the four interacting genes, only GREB1 transcription is activated by the estrogen receptor-a (ERa) (Li et al., 2012). Intriguingly, despite the fact that this multigene complex may not assemble in every cell in the population, siRNAs targeting ERa disrupted all four interacting genes (Li et al., 2012). Therefore, even though these chromosomal contacts may only occur in a fraction of the population, they clearly play a significant role in gene regulation. Moreover, this data supports a model of synergistic transcription in which chromosomal contact influences the transcription of the interacting genes. This would connote that the topological framework for transcriptional regulation is physical contact via chromosomal looping in multigene complexes.
Current siRNA and 3C-based experimental approaches cannot be applied to multigene complexes in which all interacting genes are activated by the same transcription factor. Tumor necrosis factor alpha (TNFa) has been shown to induce the formation of such multigene complexes in which all interacting genes are activated by NF-kB (Papantonis et al., 2012). Ten minutes after TNFa stimulation, the promoters of genes located on the same chromosome (SAMD4A and TNFAIP2) and on a different chromosome (SLC6A5) associate to form part of a NF-kB-dependent multigene complex (Papantonis et al., 2010). RNA FISH assays targeting the approximate sites of interaction identified by 3C suggest an association between the formation of this NF-kB-regulated multigene complex and the cotranscription
of interacting genes (Papantonis et al., 2010). However, both 3C and FISH approaches fail to reveal the necessity of chromosomal contacts for cotranscription of these interacting genes. Therefore, to accurately interrogate a model of synergistic regulation, a discrete perturbation of a single site within a gene loop while monitoring the transcriptional status of other members of the multigene complex is required. Importantly, owing to variegated gene expression (Noordermeer et al., 2011), this can only be achieved with a single-cell approach.
Here, we devise a single-cell strategy using TALE nucleases (TALENs) to discretely perturb sites within gene loops that are established to engage in chromosomal contact in the wellcharacterized NF-kB-regulated multigene complex (Papantonis et al., 2010). This enabled us to address the long-standing question
of the requirement of loop-mediated contact for transcriptional coregulation in a multigene complex. Using RNA FISH and immunofluorescence (IF), we imaged the site of the disrupted loop simultaneously with the transcriptional activity of other interacting genes in the NF-kB-regulated multigene complex. This unique single-cell perspective revealed that perturbing loop-mediated contact between the NF-kB-regulated genes altered the transcriptional status of interacting genes. In addition, this effect on cotranscription was hierarchical, with dominant and subordinate members of the multigene complex engaged in intrachromosomal contact at distances >48 Mbp, as well as interchromosomal interactions. Furthermore, restoration of a disrupted gene loop re-established both chromosomal contacts and transcription of interacting genes in a sequence independent manner. The unexpected hierarchical organization within the TNFa-induced multigene complex reveals the unprecedented level of influence of these chromosomal contacts on the transcription of coregulated genes in a multigene complex.
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