Periodic Reporting for period 3 - PoisedLogic (Dissecting the regulatory logic of poised enhancers)
Reporting period: 2023-09-01 to 2025-02-28
Less than 5% of our genome encodes for proteins. However, the remaining fraction of the human genome is not simply formed by “junked” DNA, as previously thought, but it is actually filled with non-coding sequences, called enhancers, that control when, where and how much genes need to be expressed. Consequently, enhancers play major roles during the establishment of gene expression programs as embryonic development takes place. Furthermore, mutations that directly alter enhancers or the communication with their target genes can cause a large number of human diseases, ranging from multiple types of congenital defects to various types of cancer. However, and despite the major functional and medical relevance of enhancers, we still have a limited understanding of the genetic features and mechanisms that enable enhancers to effectively communicate with their target genes. Therefore, revealing the genetic rules and factors that control the communication and compatibility between genes and enhancers would help us to improve the diagnosis, management and even treatment of many human disorders.
The overall goal of our project is to systematically dissect the genetic factors and proteins that control the function of a group of evolutionary conserved enhancers, known as “poised enhancers”, that regulate the expression of major developmental genes. More specifically, we will investigate which proteins allow “poised enhancers” to physically interact with their target genes through the specific 3D organization of the genome in our cells. Moreover, we will also explore the role of a group of DNA sequences known as CpG islands as facilitators of enhancer-gene communication. Overall, our project should provide important insights into the rules governing enhancer-gene communication, which in turn can help us understanding and predicting the pathological consequences of genetic alterations implicated in human disease.
The overall goal of our project is to systematically dissect the genetic factors and proteins that control the function of a group of evolutionary conserved enhancers, known as “poised enhancers”, that regulate the expression of major developmental genes. More specifically, we will investigate which proteins allow “poised enhancers” to physically interact with their target genes through the specific 3D organization of the genome in our cells. Moreover, we will also explore the role of a group of DNA sequences known as CpG islands as facilitators of enhancer-gene communication. Overall, our project should provide important insights into the rules governing enhancer-gene communication, which in turn can help us understanding and predicting the pathological consequences of genetic alterations implicated in human disease.
1. To evaluate the main factors controlling the physical communication between “poised enhancers” and their target genes.
Enhancers and their target genes can sometimes be located quite far from each other in the genome. However, enhancers need to physically interact with their target genes in order to execute their regulatory function. This is possible through the formation of DNA loops that bring enhancers and their target genes into physical proximity of each other. In our project, we have investigated the role that several proteins have in mediating the physical communication between “poised enhancers” and their target genes through out the genome. In order to do so we have used novel methods that can interrogate the 3D organization of the genome and detect DNA loops between genes and enhancers. By applying these methods to cells in which we eliminated some candidate proteins we uncovered that the combined action of several chromatin regulators (Polycomb, Trithorax, Cohesin, CTCF) is required for the robust “”poised enhancer”-gene communication.
2. To assess the role of CpG islands as tethering elements that mediate the robust communication between “poised enhancers” and their target genes.
Vertebrate genomes contain DNA sequences with high CG content known as CpG islands (CGI). CGI occupy the promoter regions of most genes and it is believed that they provide a permissive environment for gene expression. However, only 50% of the CGI actually occur within promoter regions, while the remaining ones are distally located from genes. These distal CGI have been referred to as orphan CGI (oCGI) and their functional relevance, if any, remains largely unknown. In our project we have shown that oCGI are an essential component of “poised enhancers” that allows these regulatory elements to physically interact with and activate their target genes. Therefore, we have uncovered a novel function for CGI as tethering elements that facilitate enhancer-gene communication and, thus, the robust establishment of gene expression programs. Furthermore, by introducing genetic re-arrangements (i.e. structural variants) close to certain genes, we have shown that this novel function of CGI helps understanding the gene expression changes that structural variants can cause and that can lead to human disease.
Enhancers and their target genes can sometimes be located quite far from each other in the genome. However, enhancers need to physically interact with their target genes in order to execute their regulatory function. This is possible through the formation of DNA loops that bring enhancers and their target genes into physical proximity of each other. In our project, we have investigated the role that several proteins have in mediating the physical communication between “poised enhancers” and their target genes through out the genome. In order to do so we have used novel methods that can interrogate the 3D organization of the genome and detect DNA loops between genes and enhancers. By applying these methods to cells in which we eliminated some candidate proteins we uncovered that the combined action of several chromatin regulators (Polycomb, Trithorax, Cohesin, CTCF) is required for the robust “”poised enhancer”-gene communication.
2. To assess the role of CpG islands as tethering elements that mediate the robust communication between “poised enhancers” and their target genes.
Vertebrate genomes contain DNA sequences with high CG content known as CpG islands (CGI). CGI occupy the promoter regions of most genes and it is believed that they provide a permissive environment for gene expression. However, only 50% of the CGI actually occur within promoter regions, while the remaining ones are distally located from genes. These distal CGI have been referred to as orphan CGI (oCGI) and their functional relevance, if any, remains largely unknown. In our project we have shown that oCGI are an essential component of “poised enhancers” that allows these regulatory elements to physically interact with and activate their target genes. Therefore, we have uncovered a novel function for CGI as tethering elements that facilitate enhancer-gene communication and, thus, the robust establishment of gene expression programs. Furthermore, by introducing genetic re-arrangements (i.e. structural variants) close to certain genes, we have shown that this novel function of CGI helps understanding the gene expression changes that structural variants can cause and that can lead to human disease.
It was previously though that the compatibility between genes and enhancers was preferentially encoded in gene proximal promoter sequences. However, in our project we have uncovered a completely novel function for a group of sequences, called CpG islands, that when present at both promoters and distal enhancers enable their communication and robust gene expression. Importantly, our results indicate that CpG islands achieve this by increasing the physical proximity between genes and enhancers, which in turn facilitates the recruitment of proteins involved in transcription to the target gene promoters. This role of CpG islands as tethering elements that dictate enhancer-gene compatibility has important implications not only for our general understanding of gene regulation but also from a medical point of view, as it can help predicting the pathological consequences of genetic re-arrangements implicated in human disease.
Until the end of the project we are going to keep characterizing the mechanisms that allow CpG islands to act as tethering elements and to boost enhancer activity and target gene expression. Briefly, we will investigate which proteins, when recruited to CpG islands, can act as molecular glues that bring together genes and enhancers. In addition, we will use single-cell methods to investigate whether, as we suspect, CpG islands increase the proportion of cells in which genes are activated by enhancers during a given time-window. Finally, we will also evaluate how the enhancer boosting capacity of the CpG islands is influenced by the linear distance separating enhancers from their target genes. Overall, our project should provide novel insights into the genetic rules governing enhancer-gene compatibility, which can then be incorporated into computational models developed by our group and that aim at predicting the etiological basis of human congenital defects.
Until the end of the project we are going to keep characterizing the mechanisms that allow CpG islands to act as tethering elements and to boost enhancer activity and target gene expression. Briefly, we will investigate which proteins, when recruited to CpG islands, can act as molecular glues that bring together genes and enhancers. In addition, we will use single-cell methods to investigate whether, as we suspect, CpG islands increase the proportion of cells in which genes are activated by enhancers during a given time-window. Finally, we will also evaluate how the enhancer boosting capacity of the CpG islands is influenced by the linear distance separating enhancers from their target genes. Overall, our project should provide novel insights into the genetic rules governing enhancer-gene compatibility, which can then be incorporated into computational models developed by our group and that aim at predicting the etiological basis of human congenital defects.