Domains of genome-wide gene expression dysregulation in

Down’s Syndrome.By Letourneau et al

April 2014

Presented by Heather Parker

Terms and TechniquesTranscriptome=all RNA’s in the cell

including mRNA, tRNA, rRNA and ncRNA. Unlike the genome, it can change with the environment. Reflects genes being ACTIVELY expressed.

Monozygotic twins=same as identical. One zygote from one sperm/egg split, so twins have identical genome and same sex.

TermsTIDS=trisomic twinT2N=normal twinMZ1/2=Normal twinsTs65Dn=mouse model for Down’s

SyndromeGEDDs=gene expression

dysregulation domainsLADs=lamina-associated domainsRPKM=reads per Kb per million

TechniquesRNA isolation using TRIzol reagant (Life

technologies) after 10-13 passages of twin fibroblasts.

To isolate mRNA, use bead w polyT oligos to bind poly A tails. Then reverse transcribe.

Messenger RNA sequencing-Illumina kit-paired end sequencing. These were mapped against human or mouse genomes using GEM (Genome Multi-tool) mapper. Independent mapping by TopHat.

EdgeR used to evaluate gene expression differences: counts # of reads for each gene

TechniquesDamID-very cool! Used to map binding

sites of DNA-binding proteins. In eukaryotes adenine not normally methylated, but it is in bacteria. Use e.coli Adenine methyltransferase (Dam) which methylates GA*TC, and fuse it to protein of interest, like a TF. Ptn binds DNA, and DAM methylates nearby adenines, leaving a footprint.

TissuesT1DS and T2N samples: Forearm

primary fetal skin fibroblasts collected post mortem (wonder what happened-miscarriage?)

MZ1 & MZ2-normal twins for comparison: primary fibroblasts from umbilical cord tissue.

MEFs-Mouse embryo fibroblasts: from 14.5 day old embryos

OverviewIt is generally thought that Down’s is due to

gene expression disturbances due to extra copy

Studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins, one with Down’s Syndrome.

Differential expression was found in DOMAINS along chromosomes, or GEDDs.

Found similar results in the mouse model of Down’s Syndrome

GEDDs correlate with LADs, which are not altered in Down’s twins.

Difference in Expression of Genes in T1DS & T2NUsing mRNA seq, and GEM mapping, found

182 genes that were significantly different in expression levels.

Gene ontology analysis (bioinformatics that connects gene and gene product) showed reduced expression of proteins involved in signaling, specifically cytokine-cytokine receptor interaction pathways and inflammatory response.

Point: Defective cell signaling may contribute to impairment of immune system in Down’s

Comparison Showed Chromosomal Domains of expression-Fig 1

Mouse model

Keep in mind, this is a comparison between twins. FOLD CHANGE.PatternLADs

Found a total of 337 GEDDs

Never explained why chrom 3,11, &19?

Compared normal twins as a controlCompared expression levels

between MZ1/2 fibroblasts-no domains. Must be due to trisomy 21.

They looked at expression levels within the domains. They classified genes as low, med, high and looked at position along chrom.

Fig 2Normal twins-dashed linesTrisomy Twins solidWhat does this show?Top=Low-shouldn’t be much

change, but in discordant twins there is a lot in both directions, more upregulation overall.

Middle=Med- and Bottom=High- both show downregulation of genes in T1DS

Difference of amplitudeTrisomy twin

showed decreased variation between high and low expression of genes in all chromosomes. Difference of gene expression amplitude.

Sup fig 4b

Stem CellsTurned fibroblasts into induced

pluripotent stem cells (iPS), performed mRNA seq, and compared gene expression.

• What did they find?

• Genome-wide dysregulation of gene expression.

Compare Mouse Model-Fig 4aPerformed similar analysis with mouse

model of trisomy 21 (Ts65Dn) and compared gene expression to normal littermates (fibroblasts). Results?

Left is mice FOLD CHANGERight human orthologsSAME PATTERN-conserved across species!

Where are LADs? Fig5a-cLooked at correlations between LADs and expression

domains (GEDDs). LADs are normally areas of repression.T1Ds/T2N-genes within LADs were ON AVERAGE OE in

T1DS relative to T2N. But def. more change in LADs, right?Same pattern in mice.Healthy twins-no change…less change??? More even dist.Point: TIDS LADs are not being down regulated….are the

LADs there? Have they moved?

Is nuclear lamina interaction disturbed? Fig 5dUsed DamID method to find LADs

in discordant twins’ fibroblasts.

What did they find?

Overall, LADs are not disturbed in T1DS

Replication DomainsReview: In S phase, DNA replicates via

replication domains.

Early replication domains (RD) correlate with LADs, contain active genes, and localize to the center of the nucleus. Late RDs contain less active genes, and tend to localize at the nucleus periphery.

Comparison of LADs and RDs

Some are more convincing than others, but domains that are upregulated in T1DS correlate with Late replication, and areas that are downregulated, are correlated with early replication.

If it isn’t LADs, maybe Epigenetic Mod?Performed Reduced

Representation Bisulphite Sequencing (RRBS) (last week) on discordant twin DNA to see if there were differences in DNA methylation at CpG’s.

Sup fig 10a,b –not much to see.No pattern between methylation

and gene expression changes.

What about histone mods??? -Fig 6bTranscription can be affected by H3K4me3Performed chromatin immunoprecipitation

and sequencing (ChIP-seq) in discordant twins. Found enriched regions.

Compared to GEDDs. What did they find?

There is a definite correlation between histone methylation and changes in gene expression (GEDDs)

ConclusionsSeveral genes on chromosome 21 that potentially may be responsible for gene expression changes:HLCS-also triplicated in mouse model and is

involved in chromatin condensation and gene repression. Also assoc. w nuclear lamina and phys. Interacts w chromatin-modifying ptns.

HMGN1-influences chromatin via histone mods. An imp modulator of gene expression.

DYRK1QA, BRWD1,RUNX1-all influence epigenetic architecture in nucleus.

Also mentions extended cell cycle in Down’s. Prolonged chromatin access time leading to increased transcription?

Future StudiesInhibit H3K4me3 modification (?) in

trisomy 21cells/mice and see if it changes gene expression patterns

OE HSA21 genes in question in a normal cells/mouse and see if you get a Down’s phenotype

KO HAS21 genes in trisomy 21 cells/mouse and see if normal phenotype

They mention other trisomies may have same GEDD patterns due to extra chromosomal material-need to test.