lincRNAs Act in the
Circuitry Controlling
Pluripotency and
Andrea Hand
BIOL 303
linc RNA
• Large Intergenic Non-Coding RNAs
• Intergenic Non-Coding: transcribed from
a region of DNA between two genes that
are used to make proteins but itself does
not code for a protein
• Determine the function of lincRNA in
mouse embryonic stem cells.
• The intergenic regions they come from
are enhancer regions for the genes that
surround them, and are byproducts only.
• They act in cis to activate transcription
(for the genes right around them)
• They act in trans to repress transcription
(in genes not localized around where
they were transcribed)
What makes Embryonic
Stem (ES) Cells So Special?
• They are pluripotent- can change into multiple
types of cells, specifically…
• Ectoderm- external layer of cells that become skin
• Neuroectoderm- type of ectoderm that
becomes the nervous system
• Endoderm- forms digestive, endocrine,
auditory, and respiratory systems
• Mesoderm- forms muscles, including cardiac
• Trophectoderm- differentiates into placenta
What makes ES cells
• They self-renew in culture.
• The methods through which they signal,
transcribe, and regulate chromatin to
control pluripotency is well known.
They used short hairpin RNA (shRNA)
targeting lincRNA sections of DNA that
were snuck in by a lentivirus to knockdown
effectiveness of lincRNA (if of course they
have an effect).
They put one shRNA into one set of cells.
How shRNA work in
When a section of DNA that has the
reversely transcribed shRNA in it is
transcribed, the RNA from the shRNA
section will bend and bond to itself as
shRNA contains 2 sections of RNA that are
can bond to itself and so forms a hairpin
shape. The shRNA give the lincRNA a
different shape and thus makes them
unsuitable for its previous function.
Methods Continued
They made 5 shRNA for each of the 226
lincRNA that have been identified in ES cells.
They found out which of the 5 shRNA was
most effective and specific for knocking down
the lincRNA through multiple trials and used
the best in the final experiment. Second most
effective gave similar results.
The shRNA suppressed 147 different lincRNA
by ~75% from their amounts in uninfected ES
Positive Control
They targeted shRNA on sections of DNA
that code for proteins that regulate
pluripotency, knowing that this should
affect pluripotency.
They targeted ~50 proteins and 40 of the
proteins were affected by the shRNA.
Negative Control
They made 27 shRNA that would not be
able to be spliced into any known section of
DNA and spliced them into lentiviruses that
infected ES cells. Knowing the shRNA
should not affect the DNA and therefore
not affect pluripotency.
To determine how the
knockdown of lincRNA affected
ES cells…
They disregarded any effects that the
negative control had on the ES cells in their
assessment of how lincRNA knockdown
affected the ES cells.
This should have prevented their being a
reaction attributed to lincRNA knockdown
that was really due to lentivirus infection.
Results Compared to byproduct
In 93% of the cultures with changed
lincRNA there was a significant change in
gene expression with around 175 proteincoding transcripts being affected on
average for each lincRNA knockdown.
This was very similar to the positive control
which had results that 95% of the cultures
with disabled proteins relevant to
pluripotency affected on average 207 genes
Results vs. Byproduct
Although a few lincRNA only repress
genes- and therefore in this experiment
they were overexpressed, the knockdown
of lincRNA activated and repressed genes
relatively evenly.
Therefore, lincRNA are not just byproducts
but are functional parts of ES cells.
Results in Relation to Cis
versus Trans Theories
While a few lincRNAs do act in cis, the
knockdown of lincRNA only affected
neighbouring genes for 2 lincRNA. Only 13
affected genes closer than 10 genes from
The genes the lincRNA affected were
similar distances from their locus as the
distance at which proteins affect gene
Results Compared to Cis
vs. Trans
These results suggest the locations of the
genes that lincRNAs affect is just as random
as any other proteins or RNA.
Therefore while some lincRNA affect close
by gene expression levels, they mostly
affect genes in Trans.
Beyond the Theories
They next tried to find a correlation
between the affects of the lack of lincRNA
and the regulation of the Embryonic Stem
Cell state.
This regulation encompasses maintaining
pluripotency and repressing differentiation.
LincRNA affects on
Maintenance of Pluripotent
To further test lincRNA affects, they
infected cells with a mutation for a reporter
gene (indicates if a gene is being used) from
the Nanog promoter with shRNA targeted
at lincRNA.
Nanog is a key transcription factor that
marks and is required for a cell to be
Results with Nanog Reporter
Excluded results when shRNA infection
caused a reduction in cell numbers.
26 lincRNA knockdowns had similar affects
on Nanog levels as the knockdown of genes
that code for protein regulators of
pluripotency- for example Oct4 and Nanog
This affect means lincRNA contribute to the
maintenance of the pluripotent state.
Just to make Sure!
They knocked down lincRNAs in wild-type ES cells
then measured multiple pluripotency markers
after 8 days.
Those markers were Oct4, Sox2, Nanog, Klf4, and
For all 26 lincRNA there was a significant
reduction in multiple markers, >90% significantly
decreased Oct4 and Nanog levels.
Using an alternative hairpin shRNA for 15 lincRNA
there were similar results in the reduction of
The Conclusion for Nanog Exp.
“>90% of lincRNA knockdowns affecting
Nanog reporter levels led to loss of ES cell
morphology” (Page 2).
In other words, 90% of the 26 lincRNA
knockdowns led to markers of the
differentiation from embryonic stem cell
form to a different one.
This means that lincRNA do in fact
contribute in a significant way to the
maintenance of the pluripotent state in ES
Do they Repress
They compared the final state of cells after
knockdowns to what lincRNA were repressed with
Nanog repression and Oct4 repression as
controls. The control proteins differentiated to
the expected state and so lincRNA knockdown
affects were accepted as results of their absence
not other affects of infection.
Repressing Continued
They discovered that specific lincRNA do
suppress specific differentiated state traits,
and so upon their knockdown the cells
showed early signs of differentiation, but
did not actually cause differentiation. So in
the absence of lincRNA we see markers we
use to determine what a cell is
differentiating to.
How do they fit into our
They Experimented and determined that
75% of the lincRNA promoters- were bound
by at least one of 9 transcription factors
that were already associated with
pluripotency .
Most commonly they were bound by 3 of
the pluripotency factors.
When those pluripotency transcription
factors were knocked down, 50% of those
lincRNA affected by these factors exhibited
expression changes.
Do they affect Chromatin Proteins?
Specifically, do lincRNA associate with
polycomb repressive complexes physically?
These are proteins that reshape chromatin
and are significant to ES cell regulation.
~10% of the lincRNA in the ES cells
associated with the 2 polycomb
components Suz12 and Ezh2.
It was also determined that ~30% of the
lincRNA interact with one or more of the
twelve chromatin complexes they tested.
• lincRNA are not just byproducts
• They affect genes far away from their
transcription location
• They help to maintain the pluripotent
• The repress differentiation pathways
• Pluripotency associated transcription
factors aid in the transcription of lincRNA
• lincRNA interact directly with chromatin
Work Cited
Works Cited
Guttman, Mitchell, Julie Donaghey, Bryce W. Carey,
Manuel Garber, Jennifer K. Grenier, Glen Munson, Geneva
Young, Anne B. Lucas, Robert Ach, Laurakay Bruhn,
Xiaoping Yang, Ido Amit, Alexander Meissner, Aviv Regev,
John L. Rinn, David E. Root, and Eric S. Lander. "LinRNAs
Act in the Circuitry Controlling Pluripotency and
Differentiation." Nature 000.00 (2011).
Nature Publishing Group, 28 Aug. 2011. Web. 28 Aug.
"Stem-loop." Wikipedia, the Free Encyclopedia. Web. 29
Aug. 2011. <>.

similar documents