This study analyzed gene expression in fibroblasts from monozygotic twins, one with Down syndrome (T1DS) and one without (T2N). They found domains of differential gene expression (GEDDs) along chromosomes in T1DS. Similar domains were found in a mouse model of Down syndrome. GEDDs correlated with late-replicating domains and histone methylation, but not with lamina-associated domains. Certain genes on chromosome 21 involved in epigenetic regulation, like histone modification and chromatin condensation, may drive the gene expression changes in Down syndrome. Future work could inhibit or overexpress these genes to test their role.

1. Domains of genome-wide gene
expression dysregulation in
Down’s Syndrome.
By Letourneau et al
April 2014
Presented by Heather Parker

2. Terms and Techniques
 Transcriptome=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.

3. Terms
 TIDS=trisomic twin
 T2N=normal twin
 MZ1/2=Normal twins
 Ts65Dn=mouse model for Down’s
Syndrome
 GEDDs=gene expression
dysregulation domains
 LADs=lamina-associated domains
 RPKM=reads per Kb per million

4. Techniques
 RNA 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

5. Techniques
 DamID-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.

6. Tissues
 T1DS 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

7. Overview
 It 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.

8. Difference in Expression of
Genes in T1DS & T2N
 Using 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

9. Comparison Showed
Chromosomal Domains of
expression-Fig 1
Mouse model
Keep in mind,
this is a
comparison
between twins.
FOLD
CHANGE.
Pattern
LADs
Found a total of 337 GEDDs
Never explained why
chrom 3,11, &19?

10. Compared normal twins as a
control
 Compared 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.

11. Fig 2
 Normal twins-dashed lines
 Trisomy Twins solid
 What 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

12. Difference of amplitude
 Trisomy twin
showed
decreased
variation
between high
and low
expression of
genes in all
chromosomes.
Difference of
gene expression
Sup fig 4b

13. Stem Cells
 Turned fibroblasts into induced pluripotent
stem cells (iPS), performed mRNA seq,
and compared gene expression.
• What did they
find?
• Genome-wide
dysregulation of
gene expression.

14. Compare Mouse Model-Fig
4a Performed similar analysis with mouse
model of trisomy 21 (Ts65Dn) and
compared gene expression to normal
littermates (fibroblasts). Results?
Left is mice FOLD
CHANGE
Right human orthologs
SAME PATTERN-
conserved across
species!

15. Where are LADs? Fig5a-c
 Looked 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?

16. Is nuclear lamina interaction
disturbed? Fig 5d
 Used DamID method to find LADs in
discordant twins’ fibroblasts.
What did they find?
Overall, LADs are not
disturbed in T1DS

17. Replication Domains
 Review: 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.

18. 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.

19. 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.

20. What about histone mods??? -
Fig 6b
 Transcription can be affected by
H3K4me3
 Performed 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)

21. Conclusions
Several 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?

22. Future Studies
 Inhibit 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.