New Research on the Link Between Chromosome Topology and Regulation of Gene Expression
August 22, 2019
The link between nucleosome organization (and epigenetic modifications of histones) and gene expressions is solidly established, but the functions of higher-order chromatin structures are still not completely understood. Transcription is both cis-regulated by elements close to the promoter and trans-regulated by distal elements located far away (Spitz F. & Furlong EE, 2012). A better understanding of how these long-distance elements are able to regulate gene expression will teach us a lot about biology in general as well as about the mechanisms involved in many human diseases.
Chromosome topology (3-dimensional DNA conformation) in the nucleus has already been shown to result in spatial proximity between distal trans-acting elements and promoters, and is therefore involved in regulating gene expression (Jin F. et al, 2013). Chromosome topology has also been implicated in mechanisms involved in DNA replication and DNA repair. It is thought that in general, chromosome topology contributes to its regulatory mechanisms by coordinating and concentrating the right factors with the right DNA region.
In the nucleus, the genome is distributed into topologically associated domains (TADs), which are high frequency self-interacting genomic regions that facilitate the access to the appropriate enhancers and prevent promoters from being ectopically activated (Rao SS. et al, 2014). However, their importance in gene expression regulation and their precise functions are not fully understood.
Eileen Furlong and her team at the Genome Biology Unit in the European Molecular Biology Laboratory bravely took up the challenge of deciphering the interplay between genome topology and gene expression in detail by cleverly integrating Hi-C, Capture-C, and RNA-Seq data in a Drosophila model of balancer chromosomes (Ghavi-Helm Y. et al, 2019).
How is the Genome Organized in the Nucleus?
Chromosomes are often organized in territories in the nucleus. In eukaryotic cells, heterochromatin is concentrated at the periphery of the nucleus and around the nucleolus. In contrast, transcriptionally active genes and associated regulatory regions occupy a central position within the nucleus. Researchers have observed different chromosomal structures within the nucleus: the lamina associated domains (LADs), TADs, and local DNA loops (Cremer T. & Cremer C., 2006).
The nuclear envelope is composed of the inner and outer nuclear membranes (INM, ONM). The nuclear lamina is located just inside the INM and is also connected to the nucleoskeleton. The nuclear lamina is composed of the proteins Lamin A, B1, B2, and C. Lamins can contact DNA either directly or indirectly through binding proteins and thus actively participate in chromatin organization within the nucleus (Dechat T. et al, 2009).
Heterochromatin is primarily located close to the nuclear lamina and regions of heterochromatin are often flanked by CTCF-binding sites. Epigenetic mechanisms are involved in the establishment and the maintenance of heterochromatin distribution. For instance, methylation of histone H3 on lysine 9 (H3K9me) and CEC-4 (chromodomain protein) facilitates heterochromatin binding to the nuclear membrane in C. elegans (Gonzalez-Sandoval A. et al, 2015).
Lamin A can also play the role of transcriptional repressor in human cells when binding to a promoter. LADs are enriched in heterochromatin but recently, in mouse, Lamin A/C and B1 were shown to bind to euchromatin. LAD also affect the 3-D organization of the nucleus by modifying interactions among TADs (Zheng X. et al, 2018). Indeed, chromatin is a dynamic structure, and upon activation, genes can be relocated from LADs to TADs.
TADs are mega-based sized regions where DNA self-interacts more often than with another region of DNA. On one hand, TADs can create a favorable environment for transcription factors and enhancers, and on the other hand, they can also isolate promoters from unrequired or unwanted activation.
TADs are composed of loop domains that are stabilized by CTCF. CTCF is also found at TAD boundaries. These structures are dynamic, and local chromatin decondensation and looping are induced for protein accessibility. This mechanism is partly regulated by CTCF and cohesin complex. Cohesin complex forms the motor that is able to force out the DNA loop. Another mechanism independent of the cohesion complex is also able to extrude the DNA loop. Finally, TADs are also involved in the development of chromosome compartments.
Chromosome Conformation Can Influence Gene Expression
Chromosome conformation evolves with development and aging, is altered in some diseases, and can influence gene expression. The organization and structure of the genome within the nucleus is dynamic and conformation changes actively participate in the regulation of gene transcription.
In humans, various cis-alterations in the TAD spanning the Wnt6/Ihh/Epha4/Pax3 loci induce limb malformation. TAD modification provokes a misplacement of Eph4 enhancer leading to the expression of the Wnt6. (Lupianez DG. et al, 2015). The same kind of modification is also observed in cancer. In women patients with lung cancer, two SNPs were associated with an alteration in ANGPT1 expression, and ANGPT1 downregulation was correlated with increased lung cancer risk (Yao S. et al., 2019).
A more serious example of chromosome conformation remodeling leading to serious consequences is progeria disease. In progeria, the gene encoding Lamin A is mutated and instead of producing Lamin A that supports the nuclear envelop and the genome conformation, the cells produce progerin. Progerin alters histone methylation patterns (H3K27me3 and H3K9me3) and induces a decrease of peripheral heterochromatin and altered CpG island methylation (Scaffidi P. & Misteli T., 2006). Globally, accumulation of progerin affects the nuclear shape, nuclear lamina function, and the structure of LADs.
However, although genome conformation changes can have strong effects on gene expression, not all modifications have such an important role. Deletion of TAD boundaries in the mouse HoxD locus has few effects on limb bud development. A deletion of 40 kb or larger is necessary to observe abnormal interaction in the TAD and altered gene expression (Rodríguez-Carballo E. et al., 2017).
Taken together, the results described in these previous publications show that the link between genome 3-D conformation and gene expression regulation is complex and not completely understood.
Changes in Chromosome Topology Do Not Directly Alter Gene Regulation
In order to deeply understand the connection between genome topology and gene transcription, Ghavi-Helm et al. used the Drosophila model of balancer chromosomes in their recent publication.
Balancer chromosomes are genetic tools involving chromosomes that are highly rearranged with sequence inversions to prevent recombination with their homologs, but that are homologous lethal. Furlong’s team used balancer chromosomes 2 and 3, which correspond to ~75% of the Drosophila genome. They worked on Drosophila embryos heterozygotic for balancer chromosomes and analyzed the genome conformation using Hi-C and Capture-C and analyzed gene expression using RNA-Seq.
Hi-C and Capture-C technologies are methods to analyze long-range chromatin interactions. After crosslinking the chromatin, the DNA is fragmented and ligated with NGS adapters, followed by next-generation sequencing. The difference between Hi-C and Capture-C is that Hi-C is a genome-wide method whereas Capture-C focuses on promoter sequences.
This is what the researchers discovered:
- Balancer chromosomes contain more structural variants in regulatory and coding regions than their homologs.
- Despite wide rearrangement of balancer topography, only 9.6% of genes present in balancer chromosomes exhibited altered expression.
- Only 12% of TAD boundaries were lost in balancer chromosomes. In these regions, 10% of genes were differentially expressed and these genes were the closest to the breakpoints.
- Inactive genes were less impacted by TADs modifications. Overall, the majority of genes located in shuffled TADs presented no difference in expression.
- By looking more closely at the details of intra-TAD contacts for genes differentially expressed, the authors discovered a concordance between promoter/promoter contact and the corresponding gene expression alteration. However, changes in genome topology at promoters were not sufficient to predict gene expression variations.
- Focusing on long-range inter-TAD loops, the scientists realized that even with the increased distance between genes, in some cases, the loop is maintained, suggesting that the mechanism drawing distal loci closer is still functional. When the loop disappears because of the sequence inversion, they couldn’t see any effect on the expression of interacting genes.
Taken together, this study showed that global modifications of genome topology did not globally affect gene expression. However, in specific circumstances, when TADs were disrupted or intra-TAD contacts were changed, gene expression can be deregulated.
To follow up on a previous study in which CTCF and cohesin knockdowns drastically reduced the number of TADs but only modestly disturbed the expression of a few genes (Nora EP. at al, 2018), we can now investigate whether TADs play a critical in regulating gene expression and determine the differences between the genes and their environment that make them sensitive or not to TAD modifications.
Summary: We Still Have a Lot to Learn About Chromosome Organization
Previous studies focusing on specific gene expression have shown that TAD disruption induces gene expression alterations, suggesting that TADs and genome conformation in general actively regulate gene transcription. However, publications that investigated the effect of genome conformation on global modification, including large deletions or CTCF depletion (Nora EP. et al, 2018), showed slight, or even no, effects on gene expression.
Using a balancer chromosomes in experiments with Drosophila embryos, Ghavi-Helm et al. demonstrated that on a global level, the modification of TAD boundaries and increasing the distance between genes did not greatly affect gene expression, suggesting that TADs have only a moderate impact on the regulation of gene transcription in this organism and tissue type.
However, when gene expression was altered, the team observed a correlation with changes in intra-TAD contact, suggesting that in certain circumstances deregulation of TADs can alter gene regulation.
In addition to rendering chromatin available for enhancer interactions and favoring long-distance DNA interactions, TADs seem to have a more subtle mechanism of gene regulation that is still not well understood.
Reference: Ghavi-Helm, Y. et al. Highly rearranged chromosomes reveal uncoupling between genome topology and gene expression. Nature Genetics 51(8): 1272-1282. (2019)