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Identification and functional validation of novel enhancer sequences involved in pituitary gland development and pathology

Project description

Promoting and enhancing understanding of pituitary tumours affecting growth in children

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. The EU-funded Pituitary enhancers project is investigating the over-expression of a gene in the pituitary tumours of children with X-linked acrogigantism, believed to be caused by the creation of a new chromatin domain where de novo enhancer-promoter interactions take place. Understanding those mechanisms could point to therapies that enhance outcomes for paediatric patients.

Objective

There is a fundamental gap in understanding how the GPR101 gene regulates human growth in physiological and pathological conditions. Children’s growth is remarkably clinical relevant and is an important indicator of their health and general well-being. The specific objective of this proposal is to identify the molecular mechanisms underlying GPR101 overexpression in the pituitary tumors of children with GPR101 duplications causing X-linked acrogigantism (X-LAG).
My central hypothesis is that GPR101 duplications disrupt the structure of the local chromatin, leading to the creation of a new chromatin domain where de novo enhancer-promoter interactions take place, causing abnormal GPR101 expression. This hypothesis will be tested by pursuing three specific aims:
1. Elucidate the transcriptional regulation of GPR101 in normal and pathological conditions
2. Identify and functionally characterize novel pituitary-specific enhancer sequences
3. Investigate these regulatory sequences in patients with different pituitary pathologies.
To achieve aim 1) I will perform an in vitro functional characterization of GPR101 promoter: promoter activity will be studied by luciferase-based reporter assays, by conducting a methylation analysis, and by determining its accessibility to transcription factors.
To achieve aim 2) I will validate my preliminary results showing the formation of a novel chromatin domain by 4C-Seq. Four putative enhancer sequences located within the duplicated GPR10 region and identified in silico will be functionally evaluated in vitro to establish their impact on transcriptional activity. To identify novel pituitary-specific enhancers, a whole-genome profile of enhancer-specific histone marks will be performed in normal and tumoral pituitary cells by ChIP-Seq.
To achieve aim 3) I will screen patients with different pituitary disorders for mutations (Sanger sequencing) and structural variations (CNV assays) in the functionally-verified enhancers.

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MSCA-IF-EF-RI - RI – Reintegration panel

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(opens in new window) H2020-MSCA-IF-2018

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Coordinator

HUMANITAS MIRASOLE SPA
Net EU contribution

Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.

€ 183 473,28
Address
VIA MANZONI 56
20100 Rozzano (Mi)
Italy

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Region
Nord-Ovest Lombardia Milano
Activity type
Private for-profit entities (excluding Higher or Secondary Education Establishments)
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Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

€ 183 473,28
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