Science - USA (2020-04-10)

flexibility that would have been an evolution-
ary advantage for Anthropoidea during the
major environmental perturbations of the later
Paleogene, including the global cooling event at
the Eocene-Oligocene boundary.

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ACKNOWLEDGMENTS
The Instituto Geológico, Minero, y Metalúrgico (INGEMMET), Lima,
and particularly C. Chacaltana Budiel and L. Tejada Medina have
facilitated and supported paleontological and geological work at
the Santa Rosa locality since 1995. M. Vilca of INGEMMET and
S. Hynek participated in fieldwork in 2016. Members of the Santa
Rosa indigenous community assisted with fieldwork in 1998 and

2016. I. Mawhinney and M. Shazab helped to process the Santa
      Rosa matrix. T. Jashashvili scanned theUcayalipithecusspecimens.
      M.F.T. is a research associate at the Gothenburg Global Biodiversity
      Centre.Funding:J. Wigmore, W. Rhodes, and R. Seaver helped
      to fund the 1998 expedition that led to the recovery of the
      Ucayalipithecuspartial upper molars. The Leakey Foundation,
      Gordon Getty, and A. Stenger supported fieldwork in 2016. Micro–
      computed tomography scanning was supported by the Keck School of
      Medicine of USC and the U.S. National Science Foundation (BCS-
      1231288).Author contributions:K.E.C. ran the Santa Rosa project.
      K.E.C. collected matrix from Santa Rosa in 1998. J.G.F., F.M.C., and
      K.E.C. collected matrix from Santa Rosa in 2016. M.F.T., N.M.N.,
      and M.B. identified the upper molars ofUcayalipithecusin the collection
      from the 1998 field season. E.R.S. identified the lower molars of
      Ucayalipithecusin the collection from the 2016 field season. E.R.S.,
      M.F.T., J.G.F., N.M.N., M.B., and K.E.C. analyzed the fossils. E.R.S.
      scored the taxa for phylogenetic analysis, ran the phylogenetic and
      biogeographic analyses, and created digital models. J.G.F., K.E.C.,
      D.d.V., and E.R.S. picked the Santa Rosa matrix; D.d.V. created digital
      models. E.R.S. wrote the first draft and created the figures. All authors

read and edited the paper.Competing interests:The authors

declare no competing interests.Data and materials availability:Data

used for the phylogenetic and biogeographic analyses are available

for download on the Dryad Digital Repository ( 31 ). Digital surface

models of theUcayalipithecusspecimens are available for download

on MorphoSource (project 872). Fossils are deposited in the

Colección Paleontológica del INGEMMET (CPI), Lima, Perú.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/368/6487/194/suppl/DC1

Materials and Methods

Supplementary Text

5 November 2019; accepted 19 February 2020

10.1126/science.aba1135

NEUROSCIENCE

Dopaminylation of histone H3 in ventral tegmental

area regulates cocaine seeking

Ashley E. Lepack^1 , Craig T. Werner^2 , Andrew F. Stewart^1 , Sasha L. Fulton^1 , Ping Zhong^3 ,

Lorna A. Farrelly^1 , Alexander C. W. Smith^1 , Aarthi Ramakrishnan^1 , Yang Lyu^1 , Ryan M. Bastle^1 ,

Jennifer A. Martin^2 , Swarup Mitra^2 , Richard M. O’Connor^1 , Zi-Jun Wang^3 , Henrik Molina^4 ,

Gustavo Turecki^5 , Li Shen^1 , Zhen Yan^3 , Erin S. Calipari^6 , David M. Dietz^2 , Paul J. Kenny^1 , Ian Maze1,7*

Vulnerability to relapse during periods of attempted abstinence from cocaine use is

hypothesized to result from the rewiring of brain reward circuitries, particularly ventral

tegmental area (VTA) dopamine neurons. How cocaine exposures act on midbrain dopamine

neurons to precipitate addiction-relevant changes in gene expression is unclear. We found

that histone H3 glutamine 5 dopaminylation (H3Q5dop) plays a critical role in cocaine-induced

transcriptional plasticity in the midbrain. Rats undergoing withdrawal from cocaine showed

an accumulation of H3Q5dop in the VTA. By reducing H3Q5dop in the VTA during withdrawal,

we reversed cocaine-mediated gene expression changes, attenuated dopamine release in the

nucleus accumbens, and reduced cocaine-seeking behavior. These findings establish a

neurotransmission-independent role for nuclear dopamine in relapse-relatedtranscriptional

plasticity in the VTA.

C

ocaine increases dopamine neurotrans-

mission from the ventral tegmental area

(VTA) to reward-relevant brain regions.

This action is central to its addictive

properties. Non-neurotransmission roles

for dopamine in cocaine dependency have not

been considered. Our laboratory has recent-

ly described a role for serotonin (5-HT) in de-

veloping 5-HTergic neurons, whereby 5-HT

located in the nucleus of these neurons was

shown to covalently attach to histone proteins—

specifically on H3 glutamine 5 (H3Q5)—to regu-

late gene expression through a process called

serotonylation ( 1 , 2 ). We had hypothesized

that this mechanism may generalize to other

monoamines in brain, such as dopamine. If

true, this process could potentially play a role

in the addiction-relevant actions of drugs that

stimulate dopaminergic transmission.

Drug addictions are defined by pathological

drug-seeking behavior that persists despite ad-

verse consequences. Prolonged vulnerability to

relapse is hypothesized to reflect the function-

al rewiring of brain reward circuitries ( 3 , 4 ).

This is precipitated, at least in part, by drug-

induced transcriptional plasticity in midbrain

dopamine neurons ( 5 , 6 ). Histone mechanisms

that control chromatin structures, and conse-

quently gene expression, regulate addiction-

relevant behaviors ( 7 , 8 ). Given that histone

H3canbemodifiedbymonoaminesinre-

sponse to fluctuations in intracellular availa-

bility, we assessed whether dopamine, like

5-HT, can be transferred to the H3 N-terminal

tail. We performed targeted, peptide-based liq-

uid chromatography–tandem mass spectrome-

try (LC-MS/MS) after in vitro transglutaminase 2

(TGM2) ( 1 , 2 , 9 ) enzymatic assays with dopa-

mine. Peptide LC-MS/MS analyses (fig. S1, A to

D) revealed Q5 as a reactive substrate for the

dopaminyl mark [H3 glutamine 5 dopaminyla-

tion (H3Q5dop)]. Given that the serotonyl modi-

fication can exist both in isolation (H3Q5ser)

and in combination with H3 lysine 4 trimethyl-

ation (H3K4me3Q5ser), we examined the effect

of K4me3 on TGM2-mediated dopaminylation

in vitro. Unmodified versus methylated mono-

nucleosomes were subjected to TGM2 dopa-

minylation assays. Using an antibody against

H3Q5dop, vide infra, we found that TGM2

equally dopaminylates unmodified and K4me3

SCIENCEsciencemag.org 10 APRIL 2020•VOL 368 ISSUE 6487 197

(^1) Department of Neuroscience, Friedman Brain Institute,
Icahn School of Medicine at Mount Sinai, New York, NY
10029, USA.^2 Department of Pharmacology and Toxicology,
Program in Neuroscience, State University of New York at
Buffalo, Buffalo, NY 14214, USA.^3 Department of Physiology
and Biophysics, Jacobs School of Medicine and Biomedical
Sciences, State University of New York at Buffalo, Buffalo,
NY 14203, USA.^4 Proteomics Resource Center, The
Rockefeller University, New York, NY 10065, USA.
(^5) Department of Psychiatry, McGill University, Montreal, QC
H3A 1A1, Canada.^6 Department of Pharmacology, Center for
Addiction Research, Vanderbilt University School of
Medicine, Nashville, TN 37232, USA.^7 Department of
Pharmacological Sciences, Icahn School of Medicine at
Mount Sinai, New York, NY 10029, USA.
*Corresponding author. Email: [email protected]
RESEARCH | REPORTS