Animal Science

ABSTRACT

Background:
Human organism develops from one cell type into more than 250 types. During these processes, the nuclear genome remains mostly unchanged in somatic cells. The phenome, such as body form evolves and progresses. However, a large part of genetic causes for phenotypic variability and complex traits such as obesity is still unknown; termed “missing heritability”. It was hypothesized that alternative transcripts, or RNA variants are key to facilitate information transfer from genome to phenome in response to various environments. In order to determine transcriptome diversity, whole transcriptome start site sequencing (WTSS-seq) and whole transcriptome termini site sequencing (WTTS-seq) were developed to profile 5’- and 3’-ends of transcripts. WTTS-seq and WTSS-seq have shown a potential to identify novel associations between genome and phenome and have demonstrated advantages in comparison to the conventional RNA-seq to study RNA variants.

Aim of the study:
The aim of this proposal is to use WTSS-seq and WTTS-seq methods to analyze to what extent alternative promoter use and alternative polyadenylation drive divergent fat deposition in a mouse model. Unique Fat and Lean lines developed by 60 generations of divergent selection on fatness will be employed. These two models present globally unique animal models suitable for the identification of genes and mechanisms of healthy leanness and obesity in human. The main objective will be to examine how long term divergent selection in high and low body fat changed the genome-wide mechanisms and architecture of alternative promoter use or alternative polyadenylation site regulation.

Materials and Methods:
Mouse lines selected for fatness for more than 60 generations will be used and divided into four groups: 1. fat content (fat/lean), 2. diet (high/low fat), 3. age (young/adult), and 4. sex. RNA will be extracted from white fat. Alternative 5’ transcriptional start site and poly(A) site usages will be analyzed using WTSS-seq and WTTS-seq methods followed by validation using in silico / dry (genomics database) and wet lab (qPCR) methods. Sequencing will be performed on Ion Torrent Personal Genome Machine. Update of the Obesity gene atlas in mouse will be performed using available databases and bioinformatics tools. Associations between genetic variants, RNA variants, gain/loss of miRNA binding sites and gain/loss of protein interactions will be performed using synthesis of multi-omics data.

Expected results:
We expect to identify novel candidate loci via RNA variants associated with fat content in mouse polygenic model. New candidate loci will then be combined with other, previously reported genomics loci associated with analyzed traits to enable multi-omics view of molecular mechanisms responsible for phenotype variability. Results could contribute to identification of genes and mechanisms of healthy leanness and obesity and novel targets for therapy.

THE PHASES OF THE PROJECT AND THEIR REALIZATION

• mouse breeding
• tissue sampling
• RNA extraction
• sequencing (WTTS-seq, WTSS-seq)
• analysis of sequencing data
• experimental and computational validation
• statistics and bioinformatics analysis
• multispecies comparative analysis
• update of the obesity gene atlas in mouse
• data integration and multi-omics analysis 

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