John Wen 1

SUMMARY
In the paper Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral
tegmental area, Koo et al. asked whether epigenetic modifications mediates chronic morphine-induced
suppression of Bdnf expression1. First, they used qPCR to measure levels of mRNA for Bdnf exon IX, the
protein-coding region shared by all BDNF transcripts. They found that in human heroin addicts, rats that
self-administered heroin, rats chronically injected with morphine, and mice injected with subchronic
morphine followed by conditional place preference (CPP), the levels of Bdnf exon IX transcripts were
downregulated in the ventral tegmental area (VTA). Next, the authors asked whether chronic morphine
might be decreasing Bdnf exon IX mRNA by stalling polymerases. qChIP indicated that chronic morphine
leads to enhanced binding of phospho-Ser5-Pol II, the form of Pol II associated with stalled activity at
promoters, at Bdnf promoters 2, 4, and 6, and decreased binding of phospho-Ser2-Pol II, the form of Pol
II associated with stalled activity at exons, at exons 2, 4, and 6. Next, to probe for epigenetic alterations at
Bdnf promoters, they performed qChIP on a number of histone markers, finding most prominently an
increase in the levels of histone markers of gene repression H3K4me3 and H3K27me3 at promoter 2.
Complementing the increase in histone repressive marks was the increase in mSIN3a and ING2 binding
to promoters, two elements of repressive complexes. Furthermore, chronic morphine enhanced binding of
SUZ12 and EZH2, two components of the PRC2 complex, at promoter 2, consistent with the increased
H3K27me3 levels at that promoter. Interestingly, viral-mediated EZH2 overexpression in the VTA
decreased Bdnf mRNA and increased morphine CPP. Conversely, viral-mediated Ezh2 repressed Ezh2
mRNA and consequently prevented decreases in Bdnf transcripts after chronic morphine treatment.
Similarly, overexpression of CREB rescued Bdnf mRNA while knocking down CREB decreased Bdnf
mRNA, suggesting a role for CREB in Bdnf gene expression regulation. Finally, the authors found
decreased levels of NURR1 binding to promoters 1 and 2 of Bdnf, decreased total Nurr1 mRNA, and
increased H3K27me3 at Nurr1 promoters. Conditional overexpression of NURR1 in VTA blocked a
chronic morphine-induced behavior, but not in mice lacking BDNF. Altogether, their data suggests that
epigenetic alterations may be responsible for suppression of Bdnf after chronic morphine.

John Wen 2
CRITICISM
While the paper by Koo and colleagues is impressive in its comprehensiveness, there are many
crucial questions they didnt address. Their previous work showed the effects of chronic morphine on
Bdnf gene expression specifically in dopaminergic neurons in the VTA2, but here, they make no effort to
determine whether its dopaminergic or non-dopaminergic cells that are responsible for the decrease in
Bdnf mRNA. Furthermore, while they show that Bdnf mRNA levels are altered, they never confirm that
protein levels BDNF are also altered. Additionally, the authors seemed to have sacrificed depth for
breadth. While they collected a lot of evidence that points to an epigenetic mechanism underlying
suppression of Bdnf, most of their evidence is correlative. Enhanced binding of mSIN3a, ING2, and
SUZ12 to Bdnf promoters does not conclusively establish their roles in Bdnf suppression. Furthermore,
their experiments involving alterations in EZH2 levels are somewhat leaky since EZH2 can alter the
expression of many genes. Consequently, EZH2 overexpression or knockout may have indirect effects on
Bdnf expression independent of epigenetic state. It would have been nice if they performed qChIP of
histone markers at Bdnf promoters after chronic morphine in animals with EZH2 conditionally
overexpressed or knocked out. Aside from these major concerns, there is also the minor issue of switching
between rat and mouse models throughout the paper. Overall, their paper provides preliminary evidence
for an epigenetic role in morphine-induced Bdnf suppression, but there is a lot of follow-up work that
needs to be done.
FUTURE EXPERIMENTS
1. Determining the molecular mechanisms by which chronic morphine increases H3K27me3
The increase in H3K27me3 at certain Bdnf promoters following chronic morphine can be due to
either an increase in methylation or decrease in demethylation. Fortunately, only a few enzymes are
responsible for methylation and demethylation of H3K27me3. In particular, EZH2 is the only known
histone methyltransferase for H3K27me3, and UTX and JMJD3 are the only known histone demethylases
for H3K27me33,4.

John Wen 3
To test whether the increase in H3K27me3 is due to an increase in methylation or a decrease in
demethylation, one could treat animals chronically or acutely with morphine or saline. It would be nice to
use animals whose dopaminergic neurons in the VTA are specifically labeled with a fluorophore. Next,
after performing the locomoter assay described in the paper by Koo et al., the animals would undergo
micro-dissection to obtain the VTA. Since the dopaminergic neurons would be labeled with a fluorophore,
one could use fluorescence-activated cell sorting to determine the effects of chronic morphine on a
specific population of cells.
After obtaining only the dopaminergic neurons from the VTA of morphine or saline-treated
animals, one could run a simple western blot and probe for EZH2, UTX, and JMJD3. One would expect
to see an increase in EZH2 levels or a decrease in UTX or JMJD3 levels, which could begin to explain the
increased H3K27me3 levels. However, if total levels of any of these proteins are not altered, it is possible
that their activity is altered. Like most enzymes, EZH2, UTX, and JMJD3, are regulated in part by posttranslational modifications. Thus, one could specifically probe for various phosphorylated states of the
enzymes to detect active and inactive forms of the enzymes. A more direct way to test their activity levels
after morphine treatment would be to purify the proteins from lysates and run activity assays. One would
expect that the overall activity of EZH2 is increased and/or the activity of UTX and JMJD3 is decreased.
If none of the experiments mentioned above yield any positive data, it is still possible that the
EZH2, UTX, and JMJD3 are partially responsible for the increase in H3K27me3. This is because the
abundance or activity of recruiters for these enzymes to Bdnf promoters might be altered. To start
determining the genes that might be responsible for recruiting the enzymes to the Bdnf promoters, one
could do a mass spectrometry-based immunoprecipitation to determine the binding partners of the
enzymes. Particularly useful would be to obtain these data from animals treated with either morphine or
saline. Since mass spectrometry-based immunoprecipitation can also quantify the abundance of specific
partners of a protein of interest, this experiment could identify whether chronic morphine alters the
binding of other proteins to BDNF.

John Wen 4
After identifying candidate genes that may be responsible for recruiting the enzymes to Bdnf
promoters, one could use chemical and genetic approaches to overexpress them or to knock them out in
the presence or absence of chronic morphine. For all of the experiments, any effects on H3K27me3 state
would be confirmed by doing qPCR of Bdnf transcripts as well as western blot to probe for total BDNF
protein levels.
2. Determining the role of NURR1 in chronic morphine-induced molecular and behavioral adaptations.
The paper by Koo et al. established that NURR1, a transcription factor, might also be responsible
for the reduction in Bdnf and ultimately lead to morphine-induced molecular and behavioral adaptations.
While NURR1 overexpression can rescue behavioral adaptations, their paper shows that mice lacking
BDNF (conditionally knocked out) have no rescue. Thus, it is likely that NURR1 acts through BDNF to
cause behavioral changes after chronic morphine.
However, further experiments should be done to confirm that NURR1 does not act through other
pathways. First, a specific way to approach determine whether NURR1 acts through BDNF would be to
perform an in vitro experiment in which the BDNF promoter is mutated such that NURR1 cannot bind it.
It would be best to use dopaminergic neurons from VTA and then to treat them with either morphine or
vehicle. Finally, the cells would be lysed and total BDNF would be quantified through western blot. We
would expect that cells treated with morphine would result in lower total BDNF compared to vehicletreated cells. If NURR1 plays a crucial role in BDNF expression, then mutating the NURR1-binding site
should lead to no difference between cells treated with vehicle or morphine.
Koo and colleagues propose that morphine decreases pCREB to decrease binding to Nurr1
promoters and ultimately decrease Bdnf gene expression. To test this proposed pathway, one could first
design an animal model with a conditional phosphomimetic CREB. Chronic morphine paired with the
expression of phosphomimetic CREB should result in increased NURR1 levels and rescue Bdnf transcript
and protein levels. Crucially, it would be wise to also pair conditional phosphomimetic CREB expression
with NURR1 deletion to ensure that any effects on Bdnf mRNA and protein are mediated through
NURR1.

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Finally, since NURR1 is a transcription factor and likely binds multiple genes, the reduction in
total NURR1 may lead to behavioral adaptations through a BDNF-independent manner. To test this, one
might first do ChIP-sequencing to determine what genes NURR1 binds. After assembling a list of
candidate genes that it binds, one could design primers for against those genes. Next, animals would be
treated with chronic morphine or saline and the VTA would be analyzed with ChIP followed by qPCR to
determine whether any of the candidate genes are altered by morphine. Any of the candidate genes that
are altered after chronic morphine could then be overexpressed or knocked out to determine whether they
also play a role in morphine-induced molecular and behavioral adaptations. In all animal experiments, the
same locomoter test or some other behavioral assay could be used.
CONCLUSIONS
In 2012, Koo and coworkers found an exciting difference between opiates and other drugs of
abuse on the expression of BDNF in the VTA. Whereas cocaine increases Bdnf gene expression,
morphine actually decreases it. Since the publication of their 2012 paper, Koo et al. have started probing
the mechanisms underlying the decrease in Bdnf expression in the VTA following chronic morphine
treatment. Of course, further work needs to be done to elucidate the mechanisms by which BDNF levels
are altered, but their paper provides direction for future experiments. The epigenetic alterations they
found are particularly exciting since epigenetic changes are often long-lasting and thus may play a role in
the long-term effects of chronic drug abuse. Determining the mechanisms by which Bdnf is altered might
result in novel drugs to block and maybe even reverse the long-term effects of chronic morphine use.

REFERENCES
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Koo, J.W., et al. Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral
tegmental area. Nat Neurosci 18, 415-422 (2015).
Koo, J.W., et al. BDNF is a negative modulator of morphine action. Science 338, 124-128 (2012).
Kooistra, S.M. & Helin, K. Molecular mechanisms and potential functions of histone
demethylases. Nat Rev Mol Cell Biol 13, 297-311 (2012).
Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P. & Reinberg, D. Histone
methyltransferase activity associated with a human multiprotein complex containing the
Enhancer of Zeste protein. Genes Dev 16, 2893-2905 (2002).

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