In the olfactory epithelium (OE) the detection of volatile compounds (odors) is accomplished by a large family of olfactory receptors (ORs), located on the surface of the cilia of olfactory sensory neurons (OSNs). These represent the major sensory component of the OE and reside in the nasal cavity. The extraordinary chemical diversity of olfactory ligands is matched in the mouse genome by a collection of more than 1200 mouse and 350 human active OR genes encoding for G-protein-coupled receptors (GPCRs). Each mature OSN in the OE is thought to express only one allele of a single OR gene (monoallelic and monogenic expression). A given OR gene is expressed in a mosaic or punctate pattern of OSNs within a characteristic zone of the OE. The transcriptional mechanisms that underlie this extraordinarily tight regulation of gene expression remain unclear. I hypothesize that OR expression choice can be influenced by somatic LINE-1- associated genomic variations. Indeed, it is now well established that active LINE-1s can create genomic rearrangements at insertional and post-insertional stages. Besides promoting genome plasticity and diversification during evolution, somatic variations can contribute to gene expression regulation for those genes that are characterized by a stochastic and monoallelic expression.Under this hypothesis, I expect the genomic sequence around the expressed ORs to be different with respect to that around the same ORs in non-expressing cells, for the presence of variations able to activate chromatin and promote ORs transcription. I first showed high LINE-1 expression and retrotransposition in OE. Then I investigated the presence and involvement of LINE-1-associated variations with OR expression, comparing the genomic sequence around an active and an inactive OR locus. In particular, I analyzed a genomic region of 50 kb around the Olfr2 TSS taking advantage of a GFP knock-in mouse. In these mice, the OSNs naturally expressing Olfr2 co-express also GFP. Targeted sequencing of Olfr2 locus revealed hundreds of heterozygous structural variants (insertions, deletions, inversions and duplications) in the vicinity of the locus. Deletions were the most abundant variation category.By end point PCR I validated six LINE-1 associated deletions potentially involved in Olfr2 expression. Nevertheless, functional validation experiments in vivo will be performed to prove their effective role in Olfr2 choice. Looking at the putative mechanisms supporting the deletions, I started investigating a possible involvement of DSBs. With this aim, I performed a chromatin immunoprecipitation and sequencing (ChIP-Seq) analysis for endogenous gamma-H2AX (an early response marker for DNA-DSBs) in mouse OE and liver. I performed a general characterization of endogenous gamma-H2AX in normal tissues. In both tissues analyzed, gamma- H2AX signal was not randomly distributed in the genome but preferentially localized within transcribed and regulatory regions. Overall, gamma-H2AX peaks were depleted in the OR clusters. Interestingly, an exception was given by a peak located within the Olfr2 locus, in close proximity to two validated deleted regions.
|Titolo:||The scent of genome complexity: exploring genomic instability in mouse Olfactory Epithelium|
|Data di pubblicazione:||26-gen-2017|
|Appare nelle tipologie:||8.1 PhD thesis|