Author: heather michelle betz

Below is a link to the full class’ Wang et al. paper discussion:

https://docs.google.com/document/d/1h94O6QuQykb_NgmFcjnMyrSQCQlkeT56bjHkhDHV3os/edit?usp=sharing

My group focused on Question 4:

Question 4 (Heather, Ana-Maria, Brett, Jamie, Jacob, Josh, Beth)

Consider Figure 3.

a) What was the experiment that lead to those results? (What did the authors do, what did they measure)?

The experiment used a CRISPR-mediated gene KO of the ∆C5 CTCF binding element within the HoxA DNA cluster. Histone methylation marks H3K27me3 and H3K4me3 were measured using ChIP-Seq against two different human foreskin fibroblast cell lines, BJ and HFF. By this method, the authors were able to determine the presence and quantity of the two histone methylation marks at the various HoxA loci, A1-A13. As a final manipulation, the authors also used an siRNA-mediated knockdown of the HOTTIP lncRNA, followed by ChIP-seq, to study the effects of HOTTIP on HoxA locus histone methylation

b) What question were the authors addressing in the experiment?

The authors wanted to investigate how HOTTIP and CTCF are related to HoxA gene expression levels through chromatin modifications (methylation of histone subunits).

c) What additional ChIP control could the authors have included, and what would its purpose have been? [If you have trouble with this question, set it aside and come back to it at the end].

The authors did a HOTTIP knock-down (siRNA) and not a knockout of the HOTTIP locus. Please provide two reasons why a knockout would not have been appropriate.

Because Hox genes are expressed in a collinear manner; i.e. they are expressed in a particular order along the body axis during development, a knockout of the HOTTIP locus may result in disruption of the spatial regulation and the HoxA cluster, whereas siRNA would have no effect on the spatial location of the locus.  

In addition, an RNA knock-down allows temporal control of the experiment, so that the siRNA can be reversed if necessary

Ensures no disruption of the hox cluster structurally or sequence wise

d) What do the data show?

In BJ cells, the H3K4me3 mark across loci A13-A7 were almost identical (capturing ~80% of the input) in terms of presence and quantity between WT and ∆C5 cells; siHOTTIP + ∆C5 cells had between 20 and 40% of the total input capture. Significantly reduced H3K4me3 was seen in WT cells across the A6-A2 locus relative to ∆C5 cells (WT cells captured between 5-20% of the input), and even presence/quantity of the mark across all conditions at the A1 locus.

Also in BJ cells, the H3K27me3 mark presence across loci A1, A2, and A7-13 between the WT, ∆C5 and ∆C5+siHOTTIP cells were fairly identical, though the percentage of input captured varied, with there being a fairly constant gradient from the lowest input covered in A13-10 (~8%) to the highest input covered (~50%) in A1. However, the WT percent capture in A3-6 was on average 2 fold greater than that of the ∆C5 and ∆C5+siHOTTIP knockdowns, hovering around 50% whereas the ∆C5 and ∆C5+siHOTTIP knockdowns decreased over the A3-A6 gradient, from ~30% in A3 to ~20% in A6. This indicates significantly reduced H3K27me3 in those loci in the knockdown experimental groups.

e) Are any of the data surprising? If so, which parts?

Functional lincRNAs are not well characterised, and so although they may be abundant, it is still a surprising/rare phenomenon to come across in gene regulation experiments at the moment

f) What can we directly conclude from the data, and why?

The C5 binding site on the HoxA locus is required for the normal (WT) H3K4me3 (“activating”) and H3K27me3 (“silencing”)  histone modifications levels respective to the HoxA genes. The addition of an siRNA targeting the HOTTIP RNA showed that HOTTIP is necessary for the H3K4me3 histone modifications on the HoxA genes, but not for the H3K27me3 histone modifications in both BJ and HFF cells.

g) In one sentence, what did the authors demonstrate?

The authors demonstrated that HOTTIP in coordination with CTCF binding  is required for maintenance of epigenetic modifications that regulate expression of the HoxA genes in foreskin fibroblasts.

Why was this chosen?

I chose this assignment as one of my top four because it shows how I am now able to interpret complex information, and use it to answer the “big 3” questions (what does the data show, what can one conclude, and what can one infer?). It also shows how much detail can go into a question when one works with a group, showing that group work is important in that it can provide you with unique perspectives that you may not have thought of before now. I enjoyed doing this assignment because at first the paper seemed so complex to me, but after the worksheet it made perfect sense. Now I look back at the paper and really see that looking at data with specific questions in mind makes it much less overwhelming.

1.  I believe that I did learn the first, which was how to be discerning in reading scientific papers and select relevant information. Through having to read many different papers on a wide variety of topics, both for my class and final project, I think that I’ve been able to more easily scan through and identify the most relevant/important information first, and then read more in detail. For the second, I think that I have added to my understanding of how this process works, specifically through gene regulation. I have learned many new methods by which organisms and cells are able to vary their expression throughout development.

2. i) I don’t think that is any circumstance that I have been really attentive to me doing this, though I am pretty sure that I did it as I believe that I was able to successfully regulated my learning.

ii) One specific situation that I was able to resist distractions was when I was writing up the draft of my final project. I think that I did this because I just got so interested in what I was learning/writing, I was actually enjoying it rather than think of other things that I could have been doing.

iii) A circumstance in which I persisted even though the task was difficult would be in creating the group infographic. I didn’t quite understand some of the other papers, as I hadn’t read them myself, but I made sure to ask questions and learn further into what each one was saying, even though at times I did want to just stop and go back to my own paper.

iv) I believe that I responded strategically/adapted flexibly would be when receiving feedback from ComPair for my final project. Some of the suggestions were very vague, such as your writing isn’t clear enough or I didn’t understand this, or the feedback was just “good job”. I adapted to this by asking a few friends to read through my project, and identify for me what exactly wasn’t very clear or what they were having trouble understanding, rather than just get frustrated and keep doing what I wanted to.

3. I think the most challenging part of this course was staying on top of my final project amid demands from many other courses. At times I would let it slide, and have to catch up on it quickly for a deadline (especially for the outline). However, I was able to overcome this challenge by working hard, and writing down important dates in my planner, and trying to work on my project a little bit every day rather than leave it all to the last few days to get it together.

Here’s a copy of our Lonfat et al. article analysis, along with comments from Dr. Kalas:

Lonfat et al Worksheet WITH COMMENTS

I chose to include this because the assignment made me work to figure out data from experiments or analyses that I hadn’t seen before. Because of this, I really had to focus on what the data was showing, and how that could be interpreted in the context of the experiment as a whole. I want to show by including it how I was able to develop more complicated thinking about experiments, and that I now know how to analyze figures and data from many sources, and really think about what it means.