QUESTIONS FOR MIDTERM 2-BIOL463 2012
Assigned paper:
Wang et al. (2011). A long noncoding RNA maintains active chromatin to coordinate
homeotic gene expression.
Information about the exam
Three of these questions will make up your midterm exam; each of the three question will be
worth 10 points. The exam will be an individual, open-book effort. You are encouraged to
work on the questions with your classmates, but each student needs to have his/her own
answers (the overall content may be the same, but each student needs to express it in his/her
own words).
One of the best ways to ensure that you are not writing the same thing as your classmates is
to take notes while you discuss, then write your full answers on your own, without consulting
the notes at first. Once you have completed your answers to the best of your ability, you’ll go
back to the notes to add details or relevant information.
Keep in mind that you will have 45 minutes to write your midterm. Practice writing your
answers and make sure that you can complete them in the allotted time. If you feel that 45
minutes is too short a time, then you are likely including too many non-essential or irrelevant
details.
You will see that some questions are longer than others (this is to “make up” for the
differences in difficulty and/or length of the answers). Note that your answers to all the
questions that won’t be asked in the midterm would make an excellent extra piece for your
individual portfolio!
Question 1: general background about the HoxA cluster and its regulation
a) The authors are studying some aspects of the HoxA cluster regulation. What are the
general structure and organization of the HoxA gene cluster at the DNA level?
HoxA cluster is flanked by two lincRNA elements: HOTAIRM1 at the 3’ end, HOTTIP at
the 5’ end. Lower numbered genes (HoxA1, HoxA2, etc.) are expressed at 3’end while
higher numbered genes (HoxA13, Hox11, etc.) are expressed at the 5’end. Higher
order structure of cluster depends on anatomical position of cell.
b) What is the typical expression pattern of the various HoxA genes in time and space, as
well as in terms of expression, during early embryonic development? How does it relate
to the organization of the HoxA cluster?
Different HoxA genes are activated depending on anatomical position of cell.
Typically, 3’ to 5’ genomic position of genes parallel the anterior-posterior and distalproximal pattern of expression in animal. For HoxA, 3’ end HoxA genes are expressed
more proximally in terms of the anatomy while 5’ end HoxA genes are expressed more
distally (Fig. 1b). 3’ end genes are expressed first in proximal cells, then 5’ end genes
are expressed later in distal cells. This expression pattern is conserved from
development to adulthood (Fig. 1c).
c) The authors of the paper discovered an additional level of “collinearity” between the
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structure of the HoxA cluster and the (human) body plan. Briefly describe it in your own
words.
In proximal parts of the body, 3’end HoxA genes are expressed (lots of chromatin
interactions, compact and looped conformation) while 5’ end HoxA genes are silent
(linear chromatin). In distal parts of the body, 5’ end HoxA genes are expressed (lots of
chromatin interactions, compact and looped conformation) while 3’ end HoxA genes
are silent (Fig.1A).
d) Where is the HOTTIP gene located in the human genome, what does it code for, and
how many copies of it are present in a primary human fibroblast (at G1)?
HOTTIP gene flanks 5’ end of HoxA cluster. It encodes a long intergenic non-coding
RNA. A primary human fibroblast has 0.3 copies of HOTTIP (Fig. S2).
e) Based on its genomic location, where and when do you expect HOTTIP to be
expressed? Provide at least two specific examples, explain your rationale, and list any
evidence presented in the paper that confirms or supports your prediction.
Since HOTTIP is located on 5’ end of HoxA cluster and 5’end genes are expressed in
distal parts of the body, HOTTIP should be expressed during development of distal parts
and in distal cells. For example, foreskin fibroblast is a distal cell therefore 5’end HoxA
genes should be expressed in these cells including HOTTIP. Another example would be
foot fibroblasts because they too are distal cells so they should also express HOTTIP. The
study shows that HOTTIP is expressed in distal/posterior anatomic sites (Fig. 1b) and in
expressed in distal areas of developing embryo (Fig. 1c).
Question 2: the chromatin structure of the HoxA cluster
a) What question were the authors addressing by generating the data that gave rise to
the top part of Figure 1A? (This would be the part with two big squares with some red
pixels inside (“heat maps”) and the title “proximal cells and “distal cells”).
What and where is the chromatin interaction state for proximal and distal cells and is
there any long-range chromatin interaction.
b) Explain/present the top part of Figure 1A. In your answer include what the stick
diagrams represent, how one should “read” the figure, what the red pixels indicate,
and two specific examples that illustrate/demonstrate how to interpret the figure.
The stick diagram represents the HoxA cluster and where the lincRNA and HoxA genes
are located. The stick and arrow represent the promoter and direction of gene
transcription. The HoxA cluster stick diagrams form the X- and Y-axis and together
make up a Cartesian coordinate map. The heat map shows the intensity of interaction
between two points (X,Y) on the HoxA cluster. The more intense the colour is, the
greater the interaction. The diagonal represents cis interaction while deviations from it
represents long range looping interactions.
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For example, the box with coordinates of HoxA11 on X-axis and HoxA11 on Y-axis in
distal cells is bright red, indicating there is strong interaction within the area of HoxA11.
It is on the diagonal, which confirms its cis-regulation. On the other hand, the box with
coordinates of HoxA11 on X-axis and HoxA7 on the Y-axis in distal cells is pale red. This
shows that there is less interaction between HoxA11 and HoxA7. The box is also off the
diagonal showing that there is some long range activity.
c) What can be concluded from the top part of Figure 1A? In your answer include (but
do not limit yourself to) a comparison between the findings in proximal and distal cells.
Higher order chromosome structure depends on position of cells, where distal cells
have more chromatin interaction at 5’ end of HoxA cluster and proximal cells have
more chromatin interaction at 3’ end of HoxA cluster.
d) Briefly describe, and compare the chromatin “composition” along the HoxA cluster
(include also the HOTTIP locus) in proximal vs. distal wild-type fibroblasts, and relate it to
its transcriptional state (e.g. silenced, transcriptionally competent, etc.).
NOTE: include as much information as you can find in the paper, and cite which
figure/table the information was extracted from. Your answer may be structured as a
summary table or point form list.
Position of cell
Chromatin
composition
Chromosome
structure
Transcriptional state
Distal cells
HOTTIP locus: few
H3K27me3, lots of
H3K4me3 (Fig.1a,
Fig.3a)
5’HoxA: abundant
chromatin
interaction, looped
and compact
conformation (Fig.1A)
5’HoxA:
transcriptionally
active
5’HoxA: lots of
H3K4me3 (Fig.1a)
3’HoxA: lots of
H3K27me3 (Fig.1a)
Proximal cells
HOTTIP locus:
H3K27me3 and
H3K4me3 (Fig.1a,
S10b)
5’HoxA: lots of
H3K27me3 (Fig.1a,
S10b)
3’ HoxA:
transcriptionally silent
3’HoxA: no long
range interaction,
largely linear (Fig.1A)
3’HoxA: abundant
chromatin
interaction, looped
and compact
conformation (Fig.1A)
3’HoxA:
transcriptionally
active
5’ HoxA:
transcriptionally silent
5’HoxA: no long
range interaction,
largely linear (Fig.1A)
3’HoxA: lots of
H3K27me3 (Fig.1a,
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S10b)
Question 3: 3C, 4C and 5C
a) What are the techniques known as 3C (Chromosome/chromatin Conformation
Capture), 4C and 5C generally used for?
Chromosome conformation capture is used to analyze the organization of
chromosome structure and examine structural properties and spatial organization of
chromosome. It identifies interactions within chromosome.
b) Describe the different steps necessary to perform 3C, and indicate the purpose of
each step. Include all the critical information, but not list unessential details.
Cross-linking: keep interaction on DNA (protein-DNA, DNA-DNA, protein-protein) stable
so that we don’t lose any of it during manipulations
Restriction digest: separate and isolate cross-linked DNA to uncross-linked DNA
Intramolecular ligation: ligate the ends of cross-linked fragments together so that one
fragment is formed
Reverse cross-link: remove cross-links so fragment is linear and read for processing
Quantification: quantify and measure fragments that interact with each other
c) How is 5C (used in the paper) different from 3C?
3C uses regular primers to amplify the cross-linked fragments into 3C library, which is
then quantified through PCR. 5C uses multiplexed ligation mediated amplification
where special primers with universal RNA sequence ligate to fragment ends. It copies
then amplifies the 3C library. The new 5C library is capable of being analyzed on a
microarray with the universal sequence. Microarray analysis allows for large scale
detection of chromosome interactions.
d) Why did the authors use 5C and not simply ChIP? Be very specific.
ChIP depends on protein-DNA interaction. It uses antibodies to pull down proteins and
its associated DNA. For the purpose of this study, they are looking at DNA-DNA
interaction, not protein-DNA interaction (lincRNA is not a protein, it can’t be
immunoprecipitated). ChIP would not work because it wouldn’t be able to pull down
any DNA-DNA interactions. 5C can pick up lots of different interactions including DNADNA interactions.
e) Could the authors, theoretically, have obtained the data they did by using 3C instead
of 5C? Explain.
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3C uses PCR to detect individual chromatin interactions, which works best for smallscale studies where set candidates interactions are known. 5C uses microarrays and
DNA sequencing, which is good for large-scale detection of chromatin interactions
throughout genome. Technically, the data could be obtained from 3C as long as
specific primers for HoxA clusters were used to see which regions interacted with
which.
Question 4: the roles and functions of HOTTIP
a) The authors use siRNA to knock down HOTTIP. What is the advantage of using siRNA
instead of a genomic knockout (not including the fact that a knockout is more labour
intensive and costly)?
SiRNA degrade RNA products and doesn’t affect the transcription of lincRNA, which
may be the mechanism by which it works to activate genes. Genomic knockout will
eliminate the gene altogether, which rules out everything regarding how it works. This is
useful to distinguish the function of HOTTIP RNA from its corresponding DNA element.
b) Propose an alternative way/strategy to “remove” HOTTIP from a cell without losign the
advantage listed in part a).
MicroRNA for HOTTIP can remove HOTTIP RNA in a non-specific way.
c) What evidence do the authors provide to show that the siRNA treatment successfully
knocked down HOTTIP?
The authors measured HOTTIP RNA expression level with RT-PCR after siRNA treatment.
They observed decreased level of HOTTIP RNA (Fig.2A).
d) What can be concluded from the HOTTIP knockdown about its role in the expression of
the Hox genes? Make sure to provide a complete answer and include a reference to
the figures/tables that provide each piece of information that you mention.
HOTTIP is necessary to coordinate activation of 5’HoxA genes (Fig.2A) in distal parts of
the body in vivo (Fig.2E). Furthermore, HOTTIP activity strength is dependent on
distance from HoxA genes, where HOTTIP activity decreases as HoxA gene is further
away from HOTTIP locus (Fig.2A). HOTTIP is not necessary for regulation of HoxD or BID
genes (Fig.2B).
e) What is the effect of HOTTIP knockdown on the higher-order chromatin architecture of
the HoxA clutser? Briefly describe the evidence on which you based your answer.
HOTTIP knockdown has no effect on higher order chromatin architecture of HoxA
cluster. The authors showed that knockdown of HOTTIP showed little change in
structure (Fig. S6A), which demonstrates that it is not necessary in chromatin structure
configuration.
f) What is the effect of HOTTIP knockdown on the chromatin “composition” along the
HoxA cluster? (Careful: there are lots of figures that have information about this).
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HOTTIP knockdown changed the chromatin composition along HoxA cluster. It led to
loss of H3K4me3 and H3K4me2 across HoxA locus (specifically 5’HoxA and HOTTIP
gene, Fig. 3A, S6B, S7). It also resulted in increase of H3K27me3 at HOTTIP gene (Fig.3A).
g) What is the purpose of having “siGFP” data/samples in Figure 3a and Supplementary
Figure 6? Couldn’t the authors just compare their siHOTTIP data to those in Figure 1?
Explain.
siGFP is a control. In this experiment, the authors are introducing a siRNA into the
system. To make sure the results observed were due specifically to HOTTIP knockdown
and not because of siRNA addition, the authors need to compare siHOTTIP data to
siGFP, which also has a siRNA addition but no HOTTIP knockdown.
h) In Supplementary Figure 6 the authors show ChIP data labeled “Histone 3”. What
antibody did they use in this set of ChIPs, and what is the benefit of having those data?
They used an antibody against Histone 3. The data shows that HOTTIP manipulation
doesn’t affect the overall H3 distribution, which supports the thought that higher order
chromatin structure is independent of HOTTIP. Knockdown of HOTTIP only affects
histone modification (i.e. methylation status) and the occupancy of H3. It is specific to
H3K4 and H3K27.
i)
Is HOTTIP sufficient to cause expression of the more distal HoxA genes (HoxA7-13)? List
the evidence presented in the paper.
HOTTIP is not sufficient to cause expression of distal HoxA genes. Ectopic expression of
HOTTIP does not activate 5’ HoxA genes proximal fibroblasts (Fig. S10). No changes to
H3K4me3 or H3K27me3 pattern were observed (Fig. S10). Furthermore, it can’t activate
5’ HoxA genes in distal fibroblasts or rescue phenotype of HOTTIP-depleted distal
fibroblasts (Fig. S11).
Question 5: HOTTIP and MLL & Co.
a) The authors say that MLL1 is required to maintain Hox activation state. Based on this
information, and on the function of MLL1, what would we see in a developing mouse
where MLL1 is completely removed?
If MLL1 is required for maintenance of Hox activation, LOF of MLL1 will result in no
maintained activation of all Hox genes. The developing mouse would not display
anterior-posterior and distal-proximal patterns.
b) Predict where you would find them located in proximally-derived fibroblasts? Briefly
explain your rationale.
MLL is required for maintenance of Hox activation. In proximal fibroblasts, 3’HoxA
genes are activated, therefore MLL1 would be required at 3’end of HoxA cluster to
keep those genes activated.
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c) Which GST-fusion proteins were observed to bind to HOTTIP RNA in vitro?
WDR5 (Fig.4A, 4B).
d) What were the authors testing in Figure 4c?
Testing to see if HOTTIP and WDR5 bind in different cell lines (or all cell lines) and
whether theirs interaction are specific to each other.
e) Why (in Figure 4c) did they choose to do IPs with each of WDR5, SIRT6 and IgG? Explain
what the purpose of each IP was.
WDR5 is the only molecule that specifically binds HOTTIP (Fig. 4A, 4B). The authors want
to confirm that HOTTIP binds only to WDR5 and not just any random molecule or one
that is in proximity. They confirmed so by doing IP with a molecule is everywhere in cell
(IgG) and another molecule that is associated with chromatin (SIRT6).
f) What is the purpose of quantifying the amount of U1 RNA in the IP’ed material?
It’s a negative control used to show that there is a significant interaction between
HOTTIP and WDR5, and that WDR5 doesn’t just bind every RNA molecule to the same
extent as it does with HOTTIP.
g) What is the purpose of using two different cell lines for the IPs?
To show that HOTTIP and WDR5 binding occurs in general and is not specific to a
particular cell line.
h) What could be an issue with using those two particular cell lines? Suggest a cell line, or
a series of cell lines, that would make the experiment in Figure 4c a stronger and more
complete one.
These two cell lines are cancer cell lines. Cancer cell lines are different from normal
cells, they display abnormal proportions of certain proteins. This could be true for
WDR5, therefore it would be best to use non-cancer cell lines. For example foot or
foreskin fibroblasts would suffice, as they are distal cells and they express HOTTIP.
i)
Draw a bar graph, using the same format as in Figure 4c, that represents the expected
results for a set of IPs using an antibody against MLL.
Question 6: overall conclusions discussion and integration
a) What pieces of evidence do the authors generate to show that WDR5 is essential for
proper regulation of the HoxA genes? Do you think it is fair to extrapolate from the
piece of evidence to say that all HoxA genes, in all cells, require WDR5 for proper
regulation? Explain your answer.
The authors show that there is a mutual interdependence between WDR5 and HOTTIP,
where knockdown of WDR5 inhibited expression of 5’ HoxA genes and decreased
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transcription of HOTTIP (Fig. 4d). I don’t think it is fair to say all HoxA genes require
WDR5, because in the study, they only show the effect of WDR5 knockdown on HoxA713. There is no evidence that shows WDR5 is required for the rest of genes on HoxA
cluster.
b) A biologist proposes the hypothesis that histone modifications are responsible for
directing and regulating higher-order chromatin structure by facilitating or hindering
long-range interactions. Explain how the data presented in the paper support, fail to
support, or clash with this hypothesis. Be specific.
The data presented in the paper clash with this hypothesis. The authors show that
knockdown of HOTTIP mRNA shows little change in the higher order chromosomal
structure (Fig. S6a) but does change the proportion of chromatin structures (loss of
H3K4me3 and gain of H3K27me3, Fig. 3a). In addition, siHOTTIP doesn’t affect H3
distribution (Fig. S6b). This shows that even if gene expression and histone modification
change, it won’t affect chromosomal looping or higher order structures because that
occurs upstream of those events.
c) What could be a reason why HOTTIP is not required for proper expression of the HoxD
genes? Propose a hypothesis/model that highlights the difference between the
regulation of the two clusters.
HOTTIP flanks 5’ end of HoxA gene cluster only. It is capable of activating 5’HoxA
genes because the chromosomal looping puts it in proximity to those genes. Maybe
HOTTIP can’t regulate HoxD genes because it is not close to that cluster and it can only
works in cis. HoxD could have its own lincRNA.
d) Predict the phenotype resulting from the addition of HOTTIP RNA, MLL and WDR5 into
proximal areas of a developing mouse embryo. Include phenotypes at the molecular,
cellular, and overall organismal levels.
Proximal areas of developing mouse embryo do not express HOTTIP. Addition of HOTTIP
RNA, MLL and WDR5 would not change the phenotype of the embryo at any level.
The authours showed that ectopic expression of HOTTIP did not induce 5’HoxA genes
in neither cells that expressed endogenous HOTTIP (and thus express MLL and WDR5)
nor cells that didn’t (Fig. S10a and S11). HOTTIP needs to be positioned near 5’HoxA for
control, therefore randomly scattered ectopic HOTTIP RNA will not turn on 5’HoxA
genes of proximal cells even if MLL and WDR5 are present.
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