Substance-specific and shared transcription and
epigenetic changes in the human hippocampus
chronically exposed to cocaine and alcohol
Zhifeng Zhoua, Qiaoping Yuana, Deborah C. Mashb, and David Goldmana,1
aLaboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20849; andbDepartment of
Neurology, University of Miami School of Medicine, Miami, FL 33146
Edited by Leslie Lars Iversen, University of Oxford, Oxford, United Kingdom, and approved March 5, 2011 (received for review December 20, 2010)
The hippocampus is a key brain region involved in both short- and
long-term memory processes and may play critical roles in drug-
of mRNA transcripts (RNA-Seq) and immunoprecipitation-enriched
genomic DNA (ChIP-Seq) coupled with histone H3 lysine 4 trime-
thylation (H3K4me3), we found extensive hippocampal gene ex-
pression changes common to both cocaine-addicted and alcoholic
individuals that may reflect neuronal adaptations common to both
addictions. However, we also observed functional changes that
were related only to long-term cocaine exposure, particularly the
inhibition of mitochondrial inner membrane functions related to
oxidative phosphorylation and energy metabolism, which has also
been observed previously in neurodegenerative diseases. Cocaine-
and alcohol-related histone H3K4me3 changes highly overlapped,
but greater effects were detected under cocaine exposure. There
was no direct correlation, however, between either cocaine- or
alcohol- related histone H3k4me3 and gene expression changes at
an individual gene level, indicating that transcriptional regulation
as well as drug-related gene expression changes are outcomes of
a complex gene-regulatory process that includes multifaceted
drug addiction|histone methylation
derlying addiction to cocaine and alcohol has primarily been
gained from animal models of acute and chronic drug exposure,
from which specific molecular pathways to addiction have been
identified (1, 2). Studies on human postmortem brain (3) have
yielded data that are complementary and tend to validate animal
models of exposure. Addiction-associated molecular alterations
observed in animal and human studies are diverse. Clearly, cel-
lular and molecular responses to addictive drugs depend on the
type and timing of exposure, the timing of observations within
the progression to addiction, and the brain regions and cells in
which observations are made. Specific changes in signal trans-
duction pathways, including in the transcription factor ΔFosB and
the cAMP-response element binding protein (CREB), are likely
to be more prominent in early stages of drug-induced neuro-
adaptation (4). Longstanding adaptations may be marked by
changes in expression of genes involved in the regulation of cel-
lular functions including ion transport, chromosome remodeling,
stress and immune response, cell adhesion, cell cycle, apoptosis,
protein and lipid metabolism, and mitochondrial functions (3).
Later changes may be more closely relevant to addictive behav-
iors and long-lasting vulnerability to relapse.
The hippocampus is also a brain region critically involved in
addiction. Most studies have focused on the mesolimbic system,
in which medium-sized spiny neurons in the dorsal striatum and
nucleus accumbens mediate dopaminergic, glutamatergic, and
GABAergic neurotransmission (5) and are key in drug-reward
and drug-seeking behavior. However, hippocampal functions
related to short- and long-term memory processes and involving
synaptic plasticity are necessary for drug-associated learning and
memory. The hippocampus directly projects excitatory efferents
nowledge of the cellular and molecular adaptations un-
to the nucleus accumbens in the mesolimbic system and can also
activate dopaminergic neurons of the ventral tegmental area,
explaining its involvement in cue-conditioned dopamine release.
Cocaine modulates hippocampal functions such as long-term
potentiation (LTP) (6), raising the possibility that long-term drug
exposure may impair adaptive plasticity relevant to learning.
By using a massively parallel sequencing approach, which
enables whole genome views of RNA transcription and epige-
netic state, we surveyed genome-wide changes in gene expression
and histone methylation modification in postmortem hippo-
campal tissue from individuals chronically exposed to cocaine or
alcohol, and from carefully matched drug-free controls. Based
on their mechanisms of action, and, to some extent, published
literature, we hypothesized that this approach would reveal
changes that were substance-specific and other changes that were
common to general processes of addiction.
Global Change in mRNA Expression in Chronic Cocaine and Alcohol
Exposure.The hippocampal mRNA transcriptome was sequenced
in 24 age-, ethnicity-, and postmortem interval-matched men:
eight chronic cocaine addicts, eight alcoholics, and eight drug-
free control subjects (SI Appendix, Table S1). RNA expression
was detected for 16,008 RefSeq genes by using the sequencing
reads pooled from all individuals. The expression of each gene
was thereby quantified for each individual within a consistent
physical framework, defined by pooled analysis of expression sig-
nals and by National Center for Biotechnology Information gene
annotations that allowed signals from multiple exons to be com-
bined into a measure of average sequencing read counts for each
gene. Gene expression was highly correlated among individual
samples (SI Appendix, Fig. S1A), indicating reproducibility and
quality of RNA-Seq results. The signal distribution (SI Appendix,
Fig. S1B) of all expressed transcripts was also consistent with
previous studies of gene expression in the brain (7). However,
the depth of sequencing coverage enabled the detection, and
quantification of more transcripts of low-abundance compared
with microarray-based analysis.
Among the 16,008 hippocampal expressed genes, at an un-
corrected P value lower than 0.05, a total of 1,994 were differen-
tially expressed in chronic cocaine-exposed individuals, and 1,275
were differentially expressed in the chronic alcohol-exposed
individuals. However, to compensate for multiple testing leading
to false-positive findings at any significance threshold, we calcu-
Author contributions: Z.Z., Q.Y., D.C.M., and D.G. designed research; Z.Z., Q.Y., D.C.M.,
and D.G. performed research; Z.Z., Q.Y., and D.C.M. contributed new reagents/analytic
tools; Z.Z., Q.Y., and D.G. analyzed data; and Z.Z. and D.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the
GenBank database (accession nos. SRA029279 and SRA029275).
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
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relies on postmortem brain tissue from individuals with pro-
longed heavy exposures. It is likely that, in the early stages of
adaptation to cocaine exposure, brain region and signal trans-
duction-specific changes are more visible. In the late stages of
the addiction, long-term allostatic processes may become
prominent. One well known transcript induced by cocaine and
other drugs is CART (cocaine- and amphetamine-regulated
transcript; gene symbol CARTPT). After acute exposure to co-
caine and other addictive drugs, CART mRNA levels were
usually found to be increased in the striatum (24, 25), and the
transcript is induced through dopaminergic transmission (26).
However, we did not observe significant changes of the CART
transcript in the hippocampus of chronic cocaine addicts. Some
other molecules identified in animal studies and considered to be
important in mediating cocaine-induced changes are CREB1,
Δ-FosB (FOSB), two NAD-dependent deacetylases, namely sir-
tuin1 (Sirt1) and sirtuin 2 (Sirt2) (1, 4), and DNMT3a, a DNA
methyltransferase recently found to play key roles in regulating
cocaine response and spine plasticity (14). We observed some
consistent changes in our chronic cocaine abusers. These include
elevated levels of the DNMT3a transcript (Table 4). We also
observed altered expression of CREB1 (uncorrected P < 0.05)
and the histone deacetylases HDAC2 (P = 0.0007, FDR = 0.09
in cocaine addicts and P = 0.007 in alcoholics), and HDAC4
(uncorrected P < 0.05 in both cocaine and alcohol addicts).
However, FOSB, SIRT1, and SIRT2 expression were not altered
in the hippocampus of the chronic cocaine abusers. Those dif-
ferences from previous observations can be stage-specific and
also brain region-specific. For example, the cocaine-induced
Δ-FosB and sirtuin elevation was observed in the striatum (1).
We profiled the genome-wide distribution of histone H3K4me3
modifications in the human hippocampus and the cocaine and
alcohol-induced changes in H3K4me3 distribution. The obser-
vation that the majority of the H3K4me3 peaks are located near
known gene promoters and the correlation between H3K4me3
modification and gene activation are consistent with previous
findings (20). However, it is of great interest to note that many
H3K4me3 peaks (47%) were located away from promoters of
known protein-coding genes. Although we cannot exclude the
possibility that some non-H3K4me3 signals resulted from cross-
reactivity, given that, in the known gene regions, H3K4me3
peaks are always positioned near a TSS, the level of cross-
reactivity was low in the H3K4me3 ChIP we performed. There-
fore, it is likely that many of the peaks that are apparently located
far from a known TSS mark promoters of unannotated genes,
including noncoding RNA genes. As H3K4me3 is a relatively well
studied histone modification known to be linked to gene acti-
vation, we intended to examine the effect of cocaine and alcohol
addiction on H3K4me3 modification and its relation to changes
in gene expression. Although we cannot exclude the possibility
that sensitivity of the ChIP assay may be a factor, it appears that
histone H3 lysine 4 trimethylation is relatively stable following
cocaine and, especially, alcohol addiction. However, we did ob-
serve significant overlap between cocaine- and alcohol-related
changes, reflecting shared epigenetic alteration related to chro-
matin remodeling in the brain. It is also apparent that in the
majority of the expressed genes, the changes of H3K4me3
modification were not directly associated with gene expression
changes. This points to the complexity of gene regulation by
multifaceted histone modification (20, 27) and the limited effect
of one type of chromatin modification on global gene expression.
Similarly, studies have also shown a lack of locus overlap be-
tween cocaine-induced methylation and acetylation changes in
mice (1). In addition to histone modifications, gene expression is
also regulated by many components of the complex transcrip-
tional machinery and also involves other mechanisms such as
DNA methylation. Nonetheless, our results reveal genome-wide
alteration of histone H3K4 trimethylation resulting from long-
term cocaine and alcohol exposure, and accompanying large-
scale changes in gene expression that implicate several functional
pathways in substance-shared and substance-specific fashion.
Materials and Methods
The materials and methods used in the present study are described in detail
in SI Appendix, SI Materials and Methods. Postmortem brain tissue was
provided by the University of Miami Brain Bank. Double-stranded cDNA li-
braries were synthesized from fragmented mRNA. Histone H3K4me3-specific
chromatin DNA was enriched by ChIP assays. Sequencing was performed on
the Illumina Genome Analyzer IIx. Details of data analysis are described in SI
Appendix, SI Materials and Methods.
ACKNOWLEDGMENTS. We thank M.-A. Enoch for critical reading of the
manuscript, and E. Moore for technical support. The acquisition of brain
specimens was supported by Public Health Service Grant DA06227-18.
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