Periodic Reporting for period 1 - Pituitary enhancers (Identification and functional validation of novel enhancer sequences involved in pituitary gland development and pathology)
Período documentado: 2020-03-16 hasta 2022-03-15
Regulation of gene transcription is the most common way to control gene expression. Enhancers are DNA sequences that promote transcription. Although they act on genes on the same DNA molecule, they can be located thousands of base pairs away from the site of the gene being regulated. Promoters, in contrast, are DNA sequences that define where transcription of a gene begins and in what direction it proceeds, and they are typically located 25–35 base pairs upstream of where transcription begins. Enhancer-promoter interactions are critical to gene expression. Research in recent years has revealed that the genome is highly organized at a local level into genome units, called topologically associating domains (TADs). These units are insulated by boundaries that facilitate certain gene promoter-enhancer interactions, while decreasing interactions outside the TAD.
This project investigated the over-expression of the GPR101 gene in the pituitary tumours of children with X-linked acrogigantism (X-LAG), believed to be caused by the creation of a new chromatin domain (neo-TAD) where new enhancer-promoter interactions take place. X-LAG is an extremely rare and severe pediatric form of pituitary gigantism that has been described in 40 patients worldwide. It mirrors exactly the disease suffered by the tallest humans ever recorded. Excessive body growth begins during infancy and is caused by enhanced production of growth hormone (GH) from the pituitary gland. This pathology is associated with a genetic defect: duplication of the GPR101 gene. GPR101 specifies for a receptor localized in the outer layer of cells.
Understanding what determines the overexpression of GPR101 will be fundamental in a new therapeutic perspective for patients affected by gigantism. This represents the first step towards the future development of treatments that specifically inhibit the enhancer sequences that interact with GPR101: turning off GPR101’s high expression will reduce the excessive secretion of GH and the associated debilitating symptoms.
The objective of this proposal was to identify the molecular mechanisms underlying GPR101 overexpression in the pituitary tumors of children with GPR101 duplications causing X-LAG. This was tested by:
1. determining the chromatin structure around the GPR101 gene in healthy subjects and patients with X-LAG;
2. characterizing the GPR101 promoter and identifying and functionally characterizing novel pituitary-specific enhancers.
My results identified X-LAG as a new TADopathy; to my knowledge this is the first TADopathy to be described in endocrinology. Xq26.3 duplications disrupt the local chromatin architecture by forming a neo-TAD and this rewiring of GPR101-enhancer interaction causes the marked over-expression of GPR101 in X-LAG pituitary tumors, which in turn drives tumoral GH hypersecretion and gigantism in affected children.
This project investigated the over-expression of the GPR101 gene in the pituitary tumours of children with X-linked acrogigantism (X-LAG), believed to be caused by the creation of a new chromatin domain (neo-TAD) where new enhancer-promoter interactions take place. X-LAG is an extremely rare and severe pediatric form of pituitary gigantism that has been described in 40 patients worldwide. It mirrors exactly the disease suffered by the tallest humans ever recorded. Excessive body growth begins during infancy and is caused by enhanced production of growth hormone (GH) from the pituitary gland. This pathology is associated with a genetic defect: duplication of the GPR101 gene. GPR101 specifies for a receptor localized in the outer layer of cells.
Understanding what determines the overexpression of GPR101 will be fundamental in a new therapeutic perspective for patients affected by gigantism. This represents the first step towards the future development of treatments that specifically inhibit the enhancer sequences that interact with GPR101: turning off GPR101’s high expression will reduce the excessive secretion of GH and the associated debilitating symptoms.
The objective of this proposal was to identify the molecular mechanisms underlying GPR101 overexpression in the pituitary tumors of children with GPR101 duplications causing X-LAG. This was tested by:
1. determining the chromatin structure around the GPR101 gene in healthy subjects and patients with X-LAG;
2. characterizing the GPR101 promoter and identifying and functionally characterizing novel pituitary-specific enhancers.
My results identified X-LAG as a new TADopathy; to my knowledge this is the first TADopathy to be described in endocrinology. Xq26.3 duplications disrupt the local chromatin architecture by forming a neo-TAD and this rewiring of GPR101-enhancer interaction causes the marked over-expression of GPR101 in X-LAG pituitary tumors, which in turn drives tumoral GH hypersecretion and gigantism in affected children.
Using chromosome conformation capture techniques, I showed that GPR101 is normally isolated in its own TAD and insulated from surrounding enhancers by a conserved, strong, and tissue-invariant TAD border. I observed that in X-LAG, genomic duplications disrupt the local TAD structure and create a neo-TAD that places the GPR101 promoter in conjunction with ectopic enhancers. These X-LAG-associated duplications disrupt the TAD border that normally separates GPR101 from nearby genes and regulatory sequences The neo-TAD was consistently seen across samples from six X-LAG individuals with different duplications, indicating the existence of a similar and consistent mechanism of neo-TAD formation. This mechanism of TADs disruption is called TADopathy.
I studied several pituitary active regulatory elements within the neo-TAD. I functionally studied the GPR101 promoter and enhancers using two models, one a human embryonic cell line to provide species and development stage-specificity, and the other an adult rat pituitary tumor cell to study tissue-specificity.
I found that the promoter of GPR101 is active in an embryonic cellular context and is also compatible with adult pituitary tumor cells. Therefore, the GPR101 promoter permits the incorporation of the new regulatory information created by the neo-TAD. Next, I searched for candidate enhancers using publicly available data. Multiple potential enhancers with widespread activity in the pituitary gland were identified. Specifically, I found six enhancers that showed increased interaction frequency with the GPR101 promoter in cells from X-LAG individuals. Then, functional tests in cell lines revealed that one enhancer could increase basal promoter activity in embryonic cells.
A transcriptome analysis conducted by RNA-seq in X-LAG tumors and normal pituitary tissues further supported an effect on gene expression that was focused on GPR101. GPR101 was markedly upregulated while other duplicated genes at the X-LAG locus were unaltered or remained expressed at low levels. A differential gene expression analysis showed that GPR101 was by far the most significantly dysregulated gene overall in X-LAG tumors vs. normal pituitary. Specific genes for hormones secreted by other pituitary cell types were highly downregulated in X-LAG pituitary.
The announcement of the project and its main findings have been published on the Host Institution’s website dedicated to informing the general public about the research and pathologies studied by its researchers. The news is available in Italian (https://tinyurl.com/mr3c4972(se abrirá en una nueva ventana)) and https://tinyurl.com/2p8m6axd(se abrirá en una nueva ventana) and in English (https://tinyurl.com/3968mu2f(se abrirá en una nueva ventana) and https://tinyurl.com/4j97h4j9(se abrirá en una nueva ventana)).
I studied several pituitary active regulatory elements within the neo-TAD. I functionally studied the GPR101 promoter and enhancers using two models, one a human embryonic cell line to provide species and development stage-specificity, and the other an adult rat pituitary tumor cell to study tissue-specificity.
I found that the promoter of GPR101 is active in an embryonic cellular context and is also compatible with adult pituitary tumor cells. Therefore, the GPR101 promoter permits the incorporation of the new regulatory information created by the neo-TAD. Next, I searched for candidate enhancers using publicly available data. Multiple potential enhancers with widespread activity in the pituitary gland were identified. Specifically, I found six enhancers that showed increased interaction frequency with the GPR101 promoter in cells from X-LAG individuals. Then, functional tests in cell lines revealed that one enhancer could increase basal promoter activity in embryonic cells.
A transcriptome analysis conducted by RNA-seq in X-LAG tumors and normal pituitary tissues further supported an effect on gene expression that was focused on GPR101. GPR101 was markedly upregulated while other duplicated genes at the X-LAG locus were unaltered or remained expressed at low levels. A differential gene expression analysis showed that GPR101 was by far the most significantly dysregulated gene overall in X-LAG tumors vs. normal pituitary. Specific genes for hormones secreted by other pituitary cell types were highly downregulated in X-LAG pituitary.
The announcement of the project and its main findings have been published on the Host Institution’s website dedicated to informing the general public about the research and pathologies studied by its researchers. The news is available in Italian (https://tinyurl.com/mr3c4972(se abrirá en una nueva ventana)) and https://tinyurl.com/2p8m6axd(se abrirá en una nueva ventana) and in English (https://tinyurl.com/3968mu2f(se abrirá en una nueva ventana) and https://tinyurl.com/4j97h4j9(se abrirá en una nueva ventana)).
For this project I employed reporter assays for the functional evaluation of predicted enhancers. While these assays are widely-used to characterize promoters/enhancers, their chromatin does not necessarily reflect the endogenous state. Therefore, I am now creating new vectors that integrate into the DNA and will be used for experiments in zebrafish embryos. Using such an approach, I expect to identify the anatomical regions in which a functional enhancer is active.
Moreover, limited biochemical annotations are publicly available to aid in the identification of candidate enhancers that are active in the human pituitary. This paucity of data could have led to our missing the identification of other enhancers. Therefore, I am currently identifying novel pituitary enhancers by conducting a whole-genome profile of enhancer-specific marks using normal human and tumoral pituitary cells. These analyses will generate a great amount of genome-wide data that will serve as the basis for future studies going beyond the scope of this project.
I am also using the RNA-seq data to identify differentially expressed transcription factors active in pituitary development that may bind the identified enhancers and participate in the pathological process.
Altogether, this project identified DNA sequences that have the potential to regulate the expression of genes involved in the development of the pituitary gland. This discovery has implications that go beyond our comprehension of the aetiology of X-linked acrogigantism (X-LAG). Indeed, it provides a framework to predict the phenotypic outcome in patients harbouring similar genomic variations involving the X-LAG locus. Moreover, it has the potential to lead to a diagnosis in patients with defects of pituitary development (prevalence 1:8000). More than three quarters of these patients are without a known genetic diagnosis to date.
Moreover, limited biochemical annotations are publicly available to aid in the identification of candidate enhancers that are active in the human pituitary. This paucity of data could have led to our missing the identification of other enhancers. Therefore, I am currently identifying novel pituitary enhancers by conducting a whole-genome profile of enhancer-specific marks using normal human and tumoral pituitary cells. These analyses will generate a great amount of genome-wide data that will serve as the basis for future studies going beyond the scope of this project.
I am also using the RNA-seq data to identify differentially expressed transcription factors active in pituitary development that may bind the identified enhancers and participate in the pathological process.
Altogether, this project identified DNA sequences that have the potential to regulate the expression of genes involved in the development of the pituitary gland. This discovery has implications that go beyond our comprehension of the aetiology of X-linked acrogigantism (X-LAG). Indeed, it provides a framework to predict the phenotypic outcome in patients harbouring similar genomic variations involving the X-LAG locus. Moreover, it has the potential to lead to a diagnosis in patients with defects of pituitary development (prevalence 1:8000). More than three quarters of these patients are without a known genetic diagnosis to date.