Title
- A little bit too vague; I would suggest something more specific that addresses the
research topic! – Though I’m guessing that wasn’t going to be your final title
anyways haha
- See the section title Evidence based research question for further elaboration
Introduction
- “It is known that ncRNA is a highly functional class that possesses enzymatic
activity, structural roles and transcriptional functions”
- “Some ncRNA is transcribed from the DNA sequence of enhancer regions and
such transcripts are referred to as enhancer RNAs (eRNAs)”
- “To bring the enhancer and promoter in close enough proximity to interact, a
chromatin loop structure is formed”
- Just a random point: When you refer to figures in your paper, capitalize them and
abbreviate them - (Fig. 1) as opposed to (figure 1)
- “eRNAs that contribute to enhancer activity are likely to interact with other
effector molecules, and whether this happens via a cis or trans mechanism is
unclear” – perhaps some eRNAs act in cis while others act in trans, which is
something to consider
- “eRNA may help facilitate or maintain the spatial interaction between enhancer
and promoter that is established during chromosomal looping”
- On the whole, I think you have included enough information in your introduction
to substantiate your research, but the organization of the info is a little haphazard
- I would suggest including the -globin locus as an example of enhancer
function prior to defining eRNAs, since the transition from eRNAs to the
locus is a little awkward
- Then, perhaps try and relate your discussion of eRNAs back to the -globin locus
immediately because as it stands you bring up eRNAs, then bring up the locus
then go back to talking about eRNAs without relating it back to the locus at all (in
the introduction I mean)
- For example, you could say something along these lines after the third paragraph
of the introduction (that ends in the word cohesin): “It is, however, unknown how
these DNA-interacting and effector proteins are recruited and maintained within
the chromatin loop formation. One possible mechanism to have been proposed
involves the use of so called enhancer RNAs (eRNAs). These are noncoding RNAs
(ncRNAs) that are transcribed from the DNA sequence of enhancer regions … “
Evidence-based research question
- Weird title haha I think just “Proposed research question” will do – I don’t think
any research questions aren’t based on evidence hahah
- “… and this indicates potential eRNA involvement in the process of chromatin
loop formation”
- “Knockdown of eRNAs results in reduced enhancer-promoter interaction,
decreased transcription of protein-coding genes, and decreased loop
formation between enhancers and genes”
- No paragraph break should appear here
- “This further supports a direct role of eRNA in loop formation between
enhancers and target genes and leads naturally to the question: How is eRNA
involved in chromatin loop formation”
- I don’t think you need to include the research question as a title on the second
page – in fact this is probably a perfectly sufficient title for your proposal that you
could include on your first page in place of “A possible new addition to the ever
expanding puzzle”
o Either that or something like “Elucidating the role of eRNA in
chromatin loop formation”
Relevance
- eRNA is RNA so it must be transcribed so the phrasing of the first sentence is a
little strange. I think a better first sentence would be something like “Though
eRNA was discovered x (I have no idea how long ago!) years ago, its functional
role in gene regulation and necessity in enhancer-promoter loop formation
has only been known for several years”
- The second sentence and third sentence can be combined as follows: “In
elucidating the precise role of eRNA in chromatin loop formation, this study
will improve our understanding of the regulation of gene expression in
homeostasis and development, and, more specifically, will contribute to the
expansion of treatments for various enhancer-related diseases.”
Impact
- I would recommend fusing this with the above section, since, once again, the
transition is a little awkward
- The first sentence of the new paragraph (i.e. right after “… will improve current
disease models”) could be something along the lines of: “For example, in
prostate cancer cells, the silencing of eRNA transcribed from enhancers of a
gene regulated by the androgen receptor (AR) causes a modest negative effect
on the proliferation of such cells” – the facts about breast cancer and prostate
cancer prior to the this are a little unnecessary I think!
- “Knowledge of the … diseases, known as enhanceropathies”
- The last two sentences in this section are unnecessary I think!
So just to clarify thus far, the information you have included is good, but the
organization of it is a little weird at times. I think the separation of the
introduction into various subsections like relevance, impact and the question
to be addressed, makes transitions a little awkward and is never really found
in scientific articles so I would probably avoid this kind of structure. Secondly,
there are a number of times where you have repeated the same thing over
again or are a little too vague in what the research can accomplish, which
should not happen in a proposal! 
Hypothesis
- I think you should mention that the hypothesis pertains specifically to the globin locus and not to eRNA in general since this is a little misleading
- Also, once again, try and link the hypothesis to the rest of the information you
have included thus far to make the transition smoother. For example “Having
established the relevance of determining the role of eRNA in chromatinloop formation, we now propose the mechanism by which this occurs: by
linking proteins between the enhancer and target promoter during chromosomal
looping”
Prediction
- “If this hypothesis is correct, it will be possible to isolate …”
- I am a little confused by the next sentence. What exactly do you mean by the
transcriptional stage of locus activation? If this is relevant to your study, you may
want to include theses stages in the introduction when you talk about the globin locus
- Whoops I see in the next section you explain what you mean, but this information
may be useful to include before your prediction
Experimental strategy
Cell line and transcriptional stages
- “Since long-range … are very well studied …”
- “At the end of HMBA treatment of the induced cells, the protocol by Ule et al.
(2005) will be applied …”
The CLIP experiment
- “UV-irradiation at 254 nm does not result in the cross-linking of proteinprotein interactions …”
The proteins
- “These proteins, listed below, will be subjected to immunoprecipitation in the
CLIP experiment” – there is no need to say that the proteins have different names
and abbreviations!
- “The Mediator complex, which interacts …, and CTCF, which mediates …”
- “… and it has been shown that cohesin can interact with several eRNAs not
associated with the -globin locus”
The antibodies and eRNA evaluation
- “… and pre-attached to beads before being added to the lysates”
- “The use of polyclonal Abs is preferred since the proteins of interest will be
involved in multi-protein complexes formed during chromatin looping
o I have no idea what polyclonal antibodies are so you may want to
explain this concept? Haha if it is prevalent in the literature though
you obviously don’t need to explain it
- “Secondary Abs conjugated to an enzyme are then added …”
- “It is assumed that … do not affect Ab specificity
o While this may be true, I don’t think it would hurt to also test Ab
specificity at these intermediate stages. This will help confirm the
results of previous experiments and, so, add to the legitimacy of your
study
On the whole, I think your experimental design is really well done, which is
clear from the fact that most of the errors I found were grammatical haha.
However, I am a little confused as to why you need to add linkers to the RNA
for reverse transcription. I thought the eRNA sequences were known, which
would make the use of linkers redundant. If we do not know the eRNA
sequences, then how can we be sure that the RNA amplified in this manner is
not coming from some other source, say rRNA from the ribosome, which will
inevitably be in the vicinity if transcription is taking place. Of course other
ncRNAs, not transcribed from enhancers could also be in this vicinity
interacting with the effector proteins, but we are not interested in those
either.
Possible outcomes
Positive result
- “… but this does not rule out a global functional role”
- “RNAi knockdown experiments … could be traced elsewhere in the genome”
- You may also want to include which transcriptional stage(s) you suspect will
show eRNA cross-linking
o For example, if eRNA is necessary for transcriptional activation, then
you would expect eRNA cross-linking after 24 hours (and potentially
at 48 hours and 72 hours depending on whether it is just required to
bring proteins together or whether it is also required for maintaining
the chromatin loop formation)
Negative result
- “If no eRNA is detectable when CLIP experiments are performed, then we may
conclude that eRNA does not interact with proteins involved in chromatin
looping during induced active transcription of the -globin major gene”
- Remove the next sentence since it is redundant!
- “In this case, it is possible that this eRNA may still contribute to enhancer
function of …”
As I mentioned above, I think you can expand on the “Possible outcomes”
section by specifying what the presence of eRNA cross-linking at each of the
stages of transcription implies about the role of eRNA rather than just stating
your broad expectations.