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Olfaction in Mammals Take Home Exam

By Elliot Williams, for Epigenetics with Prof. Holmes

Note that H3K9me3 and H3K20me3 are both markers of heterochromatin formation in development.

(IE by Histone H4K20me3 and HP1 are late heterochromatin markers in development, but present in
undierentiated embryonic stem cells. Cell 2014 ), and by our lectures in the rst third of the course.

Firstly, I would want to start by probing for the possible presence of other histone modications through
ChIP, which would allow you to see if there were any others. This would perhaps provide more information
regarding how the OR gene is randomly chosen, and how the OSN cells prevent more than one OR gene
from being expressed at a time.

DNA Methylation is also associated with the establishment of heterochromatin, so I would be curious to see
the CpG methylation patterns surrounding the 1000 genes on both alleles. In a model in which DNA
methylation completely explained why only one OR gene was activated at a time, you would expect to see
all unexpressed OR genes being methylated, and the one expressed OR gene unmethylated.

One could get at these patterns via bisulte sequencing. The bisulte reaction converts unmethylated
cytosine -> uracil, and so one can sequence the resulting DNA to see methylation sites. One might see DNA
methylation surrounding the unexpressed genes, which would be consistent with the unexpressed ORs
continuing to be silenced in the OSNs.

I would be curious to see if there were any RNAi pathways which redundantly repress any additional OR
genes that might be expressed, not least because the piRNA pathway shows a potential mechanism in
which a protein complex which specically degrades certain types of RNA can also lead to a H3K9me-
induced silencing of the gene which codes for the RNA being degraded.

This is inherently of interest to us, because we observe H3K9me3 markings on the silenced genes; could it
be that the expression of one OR gene causes a PIWI-esque silencing of the other genes through a positive
feedback loop mechanism, which keeps the active OR gene unsilenced, but causes all of the other OR genes
to be targetted by the PIWI-esque mechanism.

In S. pombe, it has been characterized that the RITS complex physically connects siRNAs and
heterochromatin by targeting a lncRNA; this lncRNA becomes a target for dsRNA production, which in turn
gets turned into siRNA by Dicer. It is also characterized that this RITS complex associates with with Clr4 (a
H3K9 HMT), and Swi6 (HPI homologue). This association would result in the "spreading" of H3K9me3 around
the region, ensuring the maintenance of epigenetic silencing.

One could probe for lncRNAs more highly expressed in OSNs vs. regular cells through looking at global
transcriptome data in both cell types, looking for lncRNAs which are more highly expressed in OSNs than
regular cells. Then, one could go about trying to nd RBP partners for the hypothetical lncRNA through
CHIRP-MS, which would allow us to specically identify said partners by immunoblotting and mass spec.

I don't pretend to know the answer to these experiments, but I think what I've proposed is a good way
forward. Assuming that DNA methylation or the histone modications are among the causes of the
silencing, it could be randomly chosen by minor dierences in expression patterns early on in OSN cell
development; if we assume that a trans-acting factor can recruit DNA CpG methylation factors (or H3K9me3
factors, or what have you) for the lesser expressed OR genes, then we could hypothesise that a random,
metastable epigenetic state would occur, where only one OR gene is expressed. This pattern could be
epigenetically inherited because the transmission of nucleosomes between strands is random, and through
spreading would be metastable as it was passed down in an OSN lineage.

The Epigenetic Parental Olfactory study we looked at in class also perhaps suggests a role of DNA
methylation; it shows that parental traumatic exposure to a given odor leads to decreased methylation for
the gene encoding for the receptor for that odor (in the experiment's case, acetophenone) -- which in turn
causes the gene to be expressed in more OSN lineages in the following generations. So, the role of DNA
methylation should be thoroughly investigated; that's probably the angle I would (naively) expect to yield
helpful facts.

Novel idea not necessarily based in classwork:

I would be interested to see what the eect of playing around with the OR promoters would be; if one
modied the promoters in a certain way, could you get two OR genes expressed at once? If so, then it would
implicate the promoter sequences in silencing -- somewhat analogous to the rex sites which DCC binds, in
the example of Dosage Compensation in C. elegans. So, producing various mutants of the promoter regions
surrounding a given OR might yield results that would shed some light.

Note: I tried not to arbitrarily prescribe a model based on the prompt; instead, I mentioned several possible
mechanisms that I came up with based on the studies we looked at in class, and recited the various
methodologies we also discussed, that could be used to assess + quantify the validity of the various
proposals I made.