Profiling Epigenetic Modifications in Obesity...Poster presentation at ‘Epigenomics of Common Diseases 2015’, Wellcome trust Conference, Cambridge, UK in November 2015. Oral presentation

Profiling Epigenetic Modifications in Obesity

SAILALITHA BOLLEPALLI

ESR6 WP2

DOCTORAL CANDIDATE

UNIVERSITY OF HELSINKI

EpiTrain Final Meeting

16 th January, 2017.

Supervisors:

Dr. Miina Ollikainen

Prof. Jaakko Kaprio

MAIN OBJECTIVES

3

2

1 To unravel the weight-loss associated gene expression and methylation markers insubcutaneous adipose tissue (SAT).

To elucidate the complex interplay between DNA methylation and histone modificationsusing peripheral blood mononuclear cells (PBMCs).

To assess the usefulness of the leucocyte DNA methylation signatures as potentialbiomarkers to predict obesity.

STUDY 1:

Exploring Expression and Methylation Signatures Reactive to

Weight Loss

AIMS:

To discern the temporal changes in gene expression and DNA methylation

profiles of adipose tissue during weight loss.

To explore the variations in expression and methylation profiles, with respect

to weight loss.

Weight Loss Associated Markers

MATERIAL:

A twelve-month weight loss intervention study

19 healthy obese subjects (mean BMI 34.6 (SE 0.6) Kg/m2) constituting 12 females and 7 males,

35.2±1.8 years .

Very–low-energy diet for first six weeks, followed by weight loss diet.

Counselling sessions and exercise plans.

Three time points: 0, 5 and 12 months

SAT biopsies.

Clinical measurements (body weight, body fat percentage, skeletal muscle percentage,

visceral fat percentage, glucose and insulin levels).


DMPsDEGs Methylation

SAT

Expression

Limma

Intra-individual comparison

Expression ~Fat percentage

Methylation ~Fat percentage

Bonferroni correction

Pearson correlation

Verification Cohort:

SAT from 26 pairs MZ obesity discordant

twins

Pathway analyses


ZERO FIVE TWELVE

12 7

3 3

12 7

49

REGAINERS

CONSTANT LOSERSMETHYLATION EXPRESSIONCLINICAL PARAMETERS

Zero19

DEGs DMPs

Five

Short-term weight loss

Twelve

6

Continuous weight loss

ConstantLosers ConstantLosers

Zero Twelve

Long-term weight loss

RESULTS:

No significant CpGs survived multiple testing correction

RESULTS:

Short-term weight loss has identified 69 DEGs

The most upregulated gene, TMEM100, and the most downregulated gene NQO1

Both associated with weight loss by their increased and decreased expression, respectively.

Pathway analyses revealed enrichment of pathways associated with obesity-related functions.

RESULTS:

Positive correlations for 13 CpG sites and negative correlations for 15 CpG

sites were identified within 21/69 genes.

Long-term weight loss resulted in 35 DEGs

Long-term weight loss -associated gene expression showed broader effects in

pathway level than short-term weight loss.

STUDY 2 :Understanding the Combinatorial Effects of Histone

Modifications and DNA Methylation in Obesity Using Obesity-

discordant MZ Twin Pairs

DNA methylation + Histone modifications Epigenetic Regulation

o Obesity-associated DNA methylation differences are frequently located at regions with histone

modifications marking transcriptionally active sites, promoters and enhancers in leucocytes.

Combinatorial Effects of Histone Modifications and DNA Methylation in Obesity

Histone mark Functional association

H3K4me3 Active promoters

H3K27me3 Inactive chromatin

H3K27ac Active promoters and enhancers

AIMS:

o Optimize ChIP sequencing protocol for H3K4me3, H3K27ac and H3K27me3 from

peripheral blood mononuclear cells (PBMCs).

o Establish ChIP sequencing analysis pipeline for MZ twins.

o Integrate H3K4me3, H3K27me3, H3K27ac and DNA methylation in PBMCs of obesity

discordant MZ twin pairs.


MATERIAL:

oFinnTwin12 and Older Twin Cohort of Finnish Twins 40 obesity-discordant MZ twin pairs

oIntensive metabolic study protocol and assessment of behavioral traits .

oPBMC DNA methylation data (450K) has been generated from all pairs.

oPBMC histone modification data is being sequenced.


Secondment at Diagenode

o Optimised ChIP protocol for PBMCs from obesity

discordant twins.

o PBMCs from 11 MZ twin pairs discordant for obesity

o Two histone marks; H3K4me3 as active mark and

H3K27me3 as inactive mark.

Dr. Teodora Ribarska (ER)

Additional 29 pairs.

H3K27ac mark.


Burrows-Wheeler Aligner 0.7.13: alignment algorithm

Samtools: conversion of .sam file into .bam file, sorting and indexing

the bam files

FastQC: quality check on reads

Peak calling: SICER

Binning: GenomicRanges, Rsamtools, GenomicAlignments

Visualisation : Integrative Genomics Viewer (IGV)

METHODS:



Differential binding

analysesBin size: 10000 bp


Awaiting for the sequencing data from the additional samples, and also data

from the H3K27ac mark.

DNA methylation data will be integrated with the ChIP sequencing data:

• Identification of regulatory networks disrupted in obesity

• Refined DNA methylaiton signatures to be used in prognosis and

prediction of obesity.

CURRENT STATUS:

• Genome-wide DNA methylation and gene expression differences in SAT of 26 BMI discordant MZ twin pairs.

• 17 novel obesity-associated genes that were differentially methylated. Nine of them were also differentially

expressed.

• Pathway analyses indicated that dysregulation of SAT in obesity includes a paradoxical downregulation of

lipo/adipogenesis and upregulation of inflammation and extracellular matrix remodeling.

• Reanalyzed the gene expression data from human pre-adiopocytes by Mikkelsen et al to address the possible

role of the nine differentially methylated and expressed genes in adipogenesis.

Genome-wide Methylation anaylses in Adipose tissue

GWAS on Coffee Preference

• To identify genes related to coffee consumption/preference. Finnish Twin data.

• Additive model of GEMMA (Genome-wide efficient mixed -model association).

• We do not know the status of this work.

Alcohol consumption associatedmethylation profiling in twins.

• 450K data from whole blood tissues of discordant twin pairs for alcohol consumption (latent class and binge drinking).

• Performed preprocessing and normalization followed by batch correction.

• I am currently performing EWAS and discordant pair analyses.

Menopause & Menarche (CHARGE consortia)

• Whole blood DNA methylationprofiles of female twins wereused to study the association ofDNA methylation with multiplephenotypes.

• Under meta-analysis by consortia.

CONSORTIA PROJECTS

Replication for UK Twins Ygen Genetics of DNA Methylation

Consortium (GODMC)

• Tobacco smoking induced DNA methylation changes in adipose tissue.

• 36 CpG significant CpG sites using methylation data with adipose tissue (n=91).

• Manuscript under preparation.

• First international consortium to assess the influence of Y- chromosome variation on complex traits.

• 69 SNPs included in the Y- chromosome.

• Under meta-analysis by consortia.

• Systematic identification of methylation quantitative trait loci with complex traits, (BMI, height, cell counts, smoking and ageing).

• Performed Cis- and trans-meQTLanalysis, Cis- and trans-var-meQTLanalysis and EWAS on height and BMI.

• Awaiting for the phase II analyses to start.

CONSORTIA PROJECTS

Accepted publications: KH Pietiläinen, K Ismail, E Järvinen , S Heinonen , M Tummers , S Bollepalli , R Lyle , M Muniandy, E

Moilanen , A Hakkarainen, J Lundbom, N Lundbom , A Rissanen , J Kaprio, M Ollikainen. DNA methylation and gene expression

patterns in adipose tissue differ significantly within young adult monozygotic BMI-discordant twin pairs. International

Journal of Obesity, 2015.

Manuscript under preparation:

Adipose tissue gene expression and DNA methylation respond to both short- and long-term weight loss

PUBLICATIONS

Poster presentation at ‘Epigenomics of Common Diseases 2014’, Wellcome trust Conference, Cambridge, UK in October 2014.

Poster presentation at the ‘Mining Data in Complex Disease Genetics by NIASC’ course held at University of Helsinki in

January 2015.

Oral presentation at ‘Pre-congress of International Twins Conference ’, Budapest, Hungary in November 2014.

Oral presentations at the Departmental work group (Kaprio) meetings, held biweekly at the FIMM (Presented four times).

Poster presentation at ‘Epigenomics of Common Diseases 2015’, Wellcome trust Conference, Cambridge, UK in November

2015.

Oral presentation at ‘Society for Research on Nicotine and Tobacco Europe Chapter (SRNT) Europe 2016’, Prague, Czech

Republic (September 10th 2016).

Poster presentation at ‘Epigenomics of Common Diseases 2016’, Wellcome trust Conference, Cambridge, UK (November

2016).

Poster presentation at ‘7th Annual FIMM Poster Session, Helsinki’, (November 2016).

Poster presentation at ‘Society for Research on Nicotine & Tobacco (SRNT) 23rd Annual Meeting’, to be held in Florence,

Italy (Upcoming March 2017).

Invited lecture on ChIP sequencing at University of Helsinki, Finland.

PRESENTATIONS

CONFERENCES &MEETINGS Epigenomics of Common Diseases 2013, 2014, 2015 &2016 (Cambridge, UK).

International Twin Conference 2014 (Budapest, Hungary)

EpiTrain Kick-off meeting 2013 & Annual meeting(2014) , Cambridge,UK.

NIASC (The Nordic Information for Action eScience Center) meeting, Helsinki, Finland.(2015)

Kaprio Group Meetings, Department of Public Health, Helsinki, Finland.

1. Organised by EpiTrain:

• Research Ethics, Cambridge, UK (Nov '13)

• Public Speaking and Drug Development, University College London, UK (May '15).

• Novel technologies in Epigenetics, Technology Transfer and Entrepreneurship, IDIBELL, Spain (Oct

'15).

2. The 2014 International Workshop on Statistical genetic methods for human complex traits, University of

Colorado, USA (Mar '14).

3. Analysis of Twin Data in Health Research, University of Southern Denmark, Denmark (May '14).

4. Next-Generation Sequencing Applications and Data Analysis, EMBL, Germany (Oct '14).

5. HumanMethylation450 Array Analysis Workshop, Queen Mary University, UK (Apr '15).

6. Analysis of High-throughput sequencing data, University of Cambridge, UK (Jun '15).

International Courses

• Doctoral Programme in Public Health, University of Helsinki (Two year grant starting from January 2017).

• Chancellor’s Travel Grant for doctoral candidates (800 Euros, November 2016).• University of Helsinki Funds Travel Grant (750 Euros, April 2016).

GRANTS & AWARDS

• Attained 46.2 credits from University of Helsinki.• About 20 credits from international courses, workshops, conferences and internship.

COURSE WORK

ACKNOWLEDGEMENTS

• MSc Khadeeja Ismail

• Dr. Teodora Ribarska

• Dr. Kirsi Pietiläinen

• Dr. Simon Anders

• Prof. Jaakko Kaprio

• Dr. Miina Ollikainen

Thanks to all the participants in the studies.

Documents

Profiling Epigenetic Modifications in Obesity...Poster presentation at ‘Epigenomics of Common Diseases 2015’, Wellcome trust Conference, Cambridge, UK in November 2015. Oral presentation