mecp2 co-ordinates liver lipid metabolism with the ncor1/hdac3

O R I G I N A L A R T I C L E

MeCP2 co-ordinates liver lipid metabolism with

the NCoR1HDAC3 corepressor complexStephanie M Kyle12 Pradip K Saha3 Hannah M Brown2daggerLawrence C Chan3 and Monica J Justice1241Genetics and Genome Biology Program The Hospital for Sick Children The Peter Gilgan Centre for Researchand Learning Toronto ON M5G 0A4 Canada 2Department of Molecular and Human Genetics 3Department ofMedicine Division of Diabetes Endocrinology and Metabolism Baylor College of Medicine Houston TX 77030USA and 4Department of Molecular Genetics University of Toronto Toronto ON M5S 1A1 Canada

To whom correspondence should be addressed at Tel thorn416-813-7654 Fax 416-813-4931 Email monicajusticesickkidsca

AbstractRett syndrome (RTT OMIM 312750) a progressive neurological disorder is caused by mutations in methyl-CpG-binding pro-tein 2 (MECP2 OMIM 300005) a ubiquitously expressed factor A genetic suppressor screen designed to identify therapeutictargets surprisingly revealed that downregulation of the cholesterol biosynthesis pathway improves neurological phenotypesin Mecp2 mutant mice Here we show that MeCP2 plays a direct role in regulating lipid metabolism Mecp2 deletion in miceresults in a host of severe metabolic defects caused by lipid accumulation including insulin resistance fatty liver perturbedenergy utilization and adipose inflammation by macrophage infiltration We show that MeCP2 regulates lipid homeostasisby anchoring the repressor complex containing NCoR1 and HDAC3 to its lipogenesis targets in hepatocytes Consistently wefind that liver targeted deletion of Mecp2 causes fatty liver disease and dyslipidemia similar to HDAC3 liver-specific deletionThese findings position MeCP2 as a novel component in metabolic homeostasis Rett syndrome patients also show signs ofperipheral dyslipidemia thus together these data suggest that RTT should be classified as a neurological disorder withsystemic metabolic components We previously showed that treatment of Mecp2 mice with statin drugs alleviated motorsymptoms and improved health and longevity Lipid metabolism is a highly treatable target therefore our results shed lighton new metabolic pathways for treatment of Rett syndrome

IntroductionMutations in the X-linked gene encoding methyl-CpG-bindingprotein 2 (MECP2) are the primary cause of the neurological con-dition Rett syndrome (1) Females with loss-of-function muta-tions in MECP2 achieve developmental milestones during thefirst 6ndash18 months of life followed by progressive loss of acquiredlinguistic and motor skills stereotypic hand movements

difficulty walking irregular breathing and seizures MECP2hemizygous males typically die perinatally of encephalopathyHowever Mecp2 hemizygous (Mecp2Y) male mice survivethrough birth and develop Rett syndrome (RTT)-like symptomsbeginning at 4 weeks of age that progressively worsen untildeath occurs between 6 and 12 weeks Mecp2Y mice are the pri-mary model of RTT their study has been vital towards under-standing the etiology of the disease as Mecp2 heterozygous

daggerPresent address Robinson Research Institute Centre for Nanoscale Biophotonics University of Adelaide North Adelaide South Australia 5006 AustraliaReceived February 19 2016 Revised May 2 2016 Accepted May 18 2016

VC The Author 2016 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc40) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly citedFor commercial re-use please contact journalspermissionsoupcom

3029

Human Molecular Genetics 2016 Vol 25 No 14 3029ndash3041

doi 101093hmgddw156Advance Access Publication Date 10 June 2016Original Article

Downloaded from httpsacademicoupcomhmgarticle-abstract251430292525781by gueston 14 February 2018

(Mecp2thorn) female mice have a wide range in phenotype severitydue to random X-chromosome inactivation (2ndash5)

Mecp2 encodes a ubiquitously-expressed intrinsically disor-dered protein (IDP) that in murine neurons binds DNA at nearhistone-octomer levels (67) Characteristic of an IDP MeCP2 istargeted by a multitude of post-translational modificationswhich determine cofactor association and may account for itsseemingly endless roles in maintaining cellular homeostasis (8)To this effect the mechanistic contribution of mutant MeCP2 toRTT pathology is highly complex and remains elusive

As the culprit gene of a devastating neurological disorderMECP2 has been largely studied in relation to central nervoussystem (CNS) development maturation and signaling In neu-rons MeCP2 can repress gene transcription by binding the nu-clear receptor co-repressorsilencing mediator for retinoid orthyroid-hormone receptors (NCoRSMRT) complex at MeCP2amino acids 302ndash306 (910) MeCP2-NCoRSMRT suppresses tran-scription by delivering the histone deacetylase HDAC3 to targetgenes which removes acetyl marks left by histone acetyltrans-ferases resulting in a closed chromatin state (1112) A R306Cmissense mutation in mouse Mecp2 abolishes interaction withthe NCoRSMRT co-repressor complex and prevents MeCP2-mediated transcriptional repression Importantly this mutationis sufficient to cause severe RTT-like phenotypes including hin-dlimb clasping hypoactivity and reduced brain weight thushighlighting the importance of the NCoRSMRT-HDAC3 interac-tion with MeCP2 in RTT pathology Furthermore the amino acid302ndash306 region of human MECP2 contains a missense mutationhotspot causing classical RTT in patients (1013)

Mouse models have established the vital role of the NCoR co-repressor complex in energy metabolism NCoR-HDAC3 regu-lates the circadian metabolic cycle of the liver governing thefluctuation between lipid and glucose utilization in hepatocytesduring lightdark cycles (1415) Loss of Hdac3 from the liver ofembryonic or adult mice promotes a constitutively active tran-scriptional environment which increases expression of de novolipogenesis enzymes resulting in fatty liver disease (1617)Further Hdac3 liver deletion increases transcription of severalgenes in the cholesterol biosynthesis pathway elevating serumcholesterol One target of HDAC3 is squalene epoxidase (Sqle) arate-limiting enzyme of the committed cholesterol biosynthesispathway whose transcription is increased 170-fold in re-sponse to Hdac3 liver deletion (161819) Curiously a forwardgenetic suppressor screen found that a nonsense mutation inSqle improved RTT-like motor symptoms and overall health inMecp2 null mice This was intriguing as both RTT patients andMecp2 mutant mice can have increased serum cholesterol andtriglycerides (20ndash22) Furthermore treatment of Mecp2 hemizy-gous male and heterozygous female mice with HMG-CoA reduc-tase inhibitors (statins) phenocopies symptom improvement bySqle mutation (20) Notably RTT-like phenotypes are improvedfollowing treatment with fluvastatin a hydrophilic statin thatdoes not readily cross the blood-brain barrier suggesting thatMeCP2 plays a role in regulating systemic cholesterol metabo-lism (2324) As such we hypothesized that genetic loss of Mecp2would cause metabolic perturbations in mice Here we reportthat Mecp2 deletion causes fatty liver disease metabolic syn-drome insulin resistance and alters overall energy homeosta-sis We show that hepatic MeCP2 works in conjunction with theNCoR-HDAC3 corepressor complex to orchestrate transcriptionof enzymes in the triglyceride and cholesterol synthesis path-ways In support of this liver-specific loss of Mecp2 is suffi-ciently deleterious to increase lipogenic enzyme transcriptionleading to fatty liver however these mice are insulin sensitive

suggesting that MeCP2 plays a role in insulin signaling outsideof the liver

ResultsMecp2 deletion increases lipogenic enzyme expressionresulting in non-alcoholic fatty liver

Surprisingly we observed a consistent pale coloration of129Mecp2Y livers (Fig 1A) Oil Red O staining suggested an accu-mulation of lipid vesicles and subsequent quantification of lipidsin Mecp2Y null liver showed a 2-fold increase in liver triacylgly-cerol (TAG) over wild-type (Fig 1B) We examined hepatic expres-sion of genes in the fatty acid and TAG synthesis pathways atthree (pre-symptomatic) and eight (symptomatic) weeks of ageExpression of four enzymes involved in fatty acid synthesis (ace-tyl-CoA carboxylase A and B [Acca Accb] fatty acid synthase[Fasn] and stearoyl-CoA desaturase 1 [Scd-1]) was increased inpre- and post-symptomatic Mecp2Y mice liver Expression offatty acid translocase (Cd36) a transporter of fatty acids and per-ilipin 2 and 5 (Plin2 and Plin5) proteins that coat TAG-dense lipiddroplets was also increased However levels of sterol-regulatoryelement-binding protein 1c (Srebp-1c) the transcription factor re-sponsible for regulating the fatty acid synthesis pathway wasnot altered in Mecp2Y liver (Fig 1CndashE) (2526)

Transcription of HMG-CoA reductase (Hmgcr) squalene epoxi-dase (Sqle) and lanosterol synthase (Lss) enzymes of the choles-terol biosynthesis pathway were all increased 3-fold in129Mecp2Y liver by 8 weeks of age (Fig 1CndashE) Concurrently129Mecp2Y null mice have increased serum cholesterol by sixweeks of age (20) Therefore we analyzed the expression andmaturation of SREBP2 the transcription factor responsible for ex-pression of enzymes in the cholesterol biosynthesis pathway (27)However there were no differences in levels of Srebp2 transcript(Fig 1C and D) nor levels of unspliced or mature nuclear nSREBP2between 129Mecp2Y null and wild-type livers (Fig 1F and G)These data suggest that a SREBP-independent pathway is respon-sible for aberrant hepatic lipogenesis in Mecp2Y null miceNotably Mecp2thornheterozygous female mice also accumulate fatin their livers by 6 months of age indicating that mosaic loss ofMecp2 is sufficient to cause fatty liver (Supplementary MaterialFigure S1A and B)

Impaired glucose and insulin homeostasisin Mecp2 mice

Non-alcoholic fatty liver disease is closely linked to glucose intol-erance and insulin resistance Mecp2Y mice display hyperglyce-mia in the fed state at 8-weeks of age but glucose levels aresimilar to wild-type when fasted (Supplementary Material FigureS2A and B) Even so plasma insulin levels of fasted and non-fasted Mecp2Y mice are significantly greater than wild-type litter-mates at both pre- and post-symptomatic ages (4 and 8 weeks re-spectively) (Fig 2A and B) Concurrently 129Mecp2Y null mice areglucose intolerant at both four and eight-weeks of age and insulinresistance is apparent by 9-weeks (Supplementary Material FigureS2CndashE) Glucose intolerance also arises in 129Mecp2thornheterozy-gous females by 20-weeks of age and insulin resistance is evidentat 24-weeks of age (Supplementary Material Figure S3AndashC)

To critically assess global insulin sensitivity Mecp2Y nullmice were subjected to hyperinsulinemic-euglycemic clampstudies (Fig 2C) In this gold standard assay for assessing insulinresistance [3-3H]-glucose and insulin are infused in fastedunrestrained mice to maintain a euglycemic lsquoclampedrsquo state

3030 | Human Molecular Genetics 2016 Vol 25 No 14


(100ndash140 mgdl serum glucose) The radioisotope allows for mea-surement of basal and clamped hepatic glucose production (HGP)and glucose disappearance or disposal (Rd) These studies re-vealed that Mecp2Y null mice require a lower rate of exogenousglucose infusion (GIR) to maintain euglycemia (Fig 2D) Whenclamped insulin infusion did not decrease HGP in mutant mice(Fig 2E) Further Mecp2Y mice have a lower rate of whole-bodyinsulin-stimulated glucose disposal (Rd) (Fig 2F) Together thesedata reflect a state of profound insulin resistance in Mecp2 nullliver skeletal muscle and white adipose tissues (WATs) To thiseffect glucose uptake by Mecp2 null WAT and muscle is markedlysuppressed (Fig 2G) However glucose uptake is increased in the

cerebral cortex and hypothalamusthalamus regions of the brainpossibly due to the insulin-independent nature of the major glu-cose transporters (GLUTs) of the brain

Mecp2 deletion induces macrophage infiltration adiposeinflammation and increased lipolysis

Lipolysis is precisely regulated by hormones such as insulin Weasked whether Mecp2 loss and subsequent insulin resistancewould affect adipose lipolysis Basal levels of circulating glyceroland non-esterified fatty acids (NEFA) were significantly

Figure 1 Mecp2 null mice develop fatty liver and aberrant hepatic lipogenic enzyme expression (A) Representative livers from eight-week old mice Liver sections

were subjected to Oil Red O staining Scale bar represents 50 lM (B) Levels of TAGs and total cholesterol in liver of eight-week old male mice (n frac14 6ndash7 mice per geno-

type) (C) mRNA expression in liver dissected from three-week old male mice (n frac14 3ndash4 mice per genotype) quantified by real-time PCR (D) mRNA expression in liver dis-

sected from eight-week old male mice (n frac14 6ndash7 mice per genotype) quantified by real-time PCR (E) A simplified schematic of the enzymes and transcription factors in

the hepatic cholesterol and TAG synthesis pathways (F) Western blot of immature SREBP2 and mature nuclear SREBP2 (nSREBP2) in liver lysates from eight-week old

male Mecp2Y mice (n frac14 4ndash5 mice per genotype) (G) Quantification of nSREBP2SREBP2 western blot normalized to GAPDH BndashD G Values are mean 6 SEM ns no signif-

icance P lt 005 P 001 P lt 0001 versus wild-type littermates Statistical analyses were performed using Studentrsquos t-test

3031Human Molecular Genetics 2016 Vol 25 No 14 |


increased at eight-weeks in Mecp2Y null mice and at 24-weeksin Mecp2thornheterozygous female mice compared with wild-typelittermates (Fig 2H and Supplementary Material Figure S3D)suggesting an increased rate of lipolysis A selective b3-adrener-gic receptor agonist CL316243 (CL) was used to measure re-sponse to lipolysis induction Lipolysis stimulation resulted inan approximately 2-fold increase in circulating glycerol andfatty acids in both male and female Mecp2 mutant and wild-type mice (Fig 2H and Supplementary Material Figure S3D)

No increase in basal circulating NEFA or glycerol was observedwhen lipolysis was examined at earlier time points (4-weeks ofage in Mecp2Y males and 12-weeks of age in Mecp2thorn females)indicating that glucose intolerance precedes these events(Supplementary Material Figures S2F and S3E)

Adipose expansion and increased lipolysis are often accompa-nied by WAT inflammation As such we investigated inflamma-tory markers in serum and in epididymal (gonadal) WAT usinggene expression analysis Serum leptin a pro-inflammatory

Figure 2 Mecp2 null mice are insulin resistant resulting in perturbed glucose uptake (A) Serum insulin concentration in Mecp2Y and wild-type (thornY) littermates at

four-weeks of age in the fed state (n frac14 5ndash7 mice per genotype) or following a 6-h fast (n frac14 10ndash12 mice per genotype) (B) Serum insulin concentration at eight-weeks of

age in the fed state or following a 6-h fast (n frac14 10ndash12 mice per genotype) (C) A schematic showing the hyperinsulinemic-euglycemia clamps protocol (n frac14 4ndash5 mice per

genotype) performed at eight-weeks of age (D) The GIR needed to maintain euglycemia (100ndash140 mgdl serum glucose) in response to insulin clamps (E) HGP in the

basal and clamped state (F) Rate of insulin-stimulated glucose disposal (Rd) (G) The rate of glucose uptake in WAT muscle cerebral cortex hypothalamus and thala-

mus cerebellum and hippocampus (H) In vivo lipolysis assays in 4-h fasted male mice at eight-weeks of age Serum glycerol and NEFAs levels are shown at basal met-

abolic state (basal) and 15-min after lipolysis induction by injection with CL316243 (CL) a b3-adrenoceptor agonist (n frac14 7ndash9 mice per genotype) (I) mRNA expression of

inflammatory markers TNFa CD68 IL6 and IL10 in epididymal WAT from eight-week male mice (n frac14 6ndash8 mice per genotype) quantified by real-time PCR (J)

Representative epididymal WAT sections stained for F4F80thorn cells at eight-weeks Scale bar represents 100 lM (K) Number of crown-like structures in epididymal WAT

sections (n frac14 3 mice per genotype) at 9-weeks AB DndashI K Values are mean 6 SEM ns no significance P lt 005 P 001 P 0001 versus wild-type littermates

Statistical analyses were performed using Studentrsquos t-test



factor was highly elevated in Mecp2Y serum while there was noobservable difference in adiponectin (Supplementary MaterialsFigure S2G and H) Mecp2Y null WAT has increased expression ofmacrophage-derived interleukin-6 (IL-6) and TNFa cytokines im-plicated in dramatically increasing adipocyte lipolysis and CD68a macrophage marker but exhibit no change in the anti-inflammatory marker IL10 (28) (Fig 2I) These data indicate mac-rophage infiltration in Mecp2Y WAT which was confirmed byincreased F4F80 immunohistochemistcal staining in gonadalWAT (Fig 2J) Adipose macrophages in Mecp2Y null WAT formeda significantly larger number of crown-like structures whichenvelope necrotic adipocytes (Fig 2K)

Mecp2 deletion modifies whole body substratemetabolism

To further characterize metabolic changes in Mecp2Y null micewe assessed energy expenditure (EE) and substrate metabolismby indirect calorimetry over a 20-h period (11-h dark period cu-mulative 9-h day period) following a 4-h acclimation period129Mecp2Y null male mice weigh significantly more than wild-type (thornY) littermates by seven weeks of age due to an expan-sion of white and brown adipose tissue despite a decrease inlean mass (Fig 3A and B and Supplementary Material FigureS4A and B) Because Mecp2Y mice are heavier at eight weeks ofage we employed analysis of covariance (ANCOVA) to controlfor both the weight factor and genotype (29) (Fig 3C) Mecp2Ynull mice display a slight but significant decrease (Pfrac14 003) inEE during the day but have no difference in EE at night (Fig 3D)This is somewhat surprising considering severe hypoactivity isa hallmark of symptomatic (eight-week old) Mecp2Y null mice

(3) As expected both wild-type and Mecp2 null mice displayeddiurnal rhythms in their whole-body EE and respiratory ex-change ratio (RER) RER an indicator of energy derived from oxi-dizing glucose (RERfrac14 10) or fatty acids (RERfrac14 07) increases forboth genotypes at night reflective of food intake and carbohy-drate utilization (Fig 3E) However Mecp2 null mice have amarkedly decreased RER versus wild-type littermates duringboth day and night cycles consistent with preferential lipid oxi-dation over carbohydrate metabolism (Fig 3E and F)

MeCP2 regulates expression of liver lipogenicenzymes through interaction with the NCoRHDAC3corepressor complex

In neurons MeCP2 interacts with the nuclear receptor co-repressor 1 (NCoR1) complex which includes the proteins his-tone deactylase 3 (HDAC3) and transducin-(beta) like 1 X-linkedreceptor 1 (TBLR1) to facilitate gene repression (910) In hepato-cytes the NCoR-HDAC3-TBLR1 corepressor complex is a potentregulator of deactylase-dependent gene transcription As suchwe hypothesized that this corepressor complex contributes tothe metabolic perturbations in Mecp2 null mice To address thisquestion we first assessed native MeCP2 ability to bind theNCoR complex in vivo in the liver Co-immunoprecipitation ofHDAC3 indeed revealed association with MeCP2 as well as itscorepressor complex partners NCoR1 and TBLR1 (Fig 4A) Thisbinding partnership was validated using three different HDAC3immunoprecipitation (IP) antibodies all of which pulled downMeCP2 in complex (not shown)

Next we investigated the effect of MeCP2 loss on HDAC3binding and activity Specifically we performed ChIP-qPCR to

Figure 3 Mecp2 deletion shifts whole body substrate metabolism (A) Total weight of Mecp2Y and wild-type (thornY) littermates (n frac14 10 mice per genotype) (B) Lean and

fat mass (n frac14 7ndash8 mice per genotype) (C) Linear regression for EE as a function of genotype and body weight separated by day (thornY y frac14 00228 x ndash 01398 r2frac1404247

Mecp2Y y frac14 00102 x thorn 01159 r2 frac14 06417) and night (thornY y frac14 00209 x ndash 00245 r2frac1404716 Mecp2Y y frac14 00125 x thorn 01709 r2 frac14 07990) periods (D) EE adjusted for

weight (average weight frac14 2435g) using ANCOVA for wild-type control and Mecp2Y mice (n frac14 7ndash9 mice per genotype) at eight-weeks (E) Respiratory rate (VCO2VO2)

across a 20-h period (F) Average respiratory rate separated by day and night periods Values are mean 6 SEM ns no significance P lt 005 P 001 P 0001 ver-

sus WT EE averages were adjusted for weight using ANCOVA and statistical analyses were performed using Studentrsquos t-test



assess the binding of HDAC3 to regulatory regions surroundingthe transcription start sites (TSS) of several rate-limiting en-zymes that are upregulated in Mecp2 null liver We found thatloss of MeCP2 significantly hinders the capability of HDAC3 tobind regulatory regions surrounding the TSS of Sqle (Fig 4B)Fasn (Fig 4C) and Cd36 (Fig 4D) in the liver HDAC3 binding atthe promoter region of the attachment region binding protein(Arbp) gene was used as a negative control (Fig 4B) Deletion ofHDAC3 from the liver or genetic abolishment of its deacetylaseactivity results in increased local histone acetylation in liverspecifically of the transcriptional activating marker Histone 3 ly-sine 27 acetylation (H3K27ac) (151630) Therefore we hypothe-sized that a reduction in chromatin-bound HDAC3 in Mecp2 nullmice would result in increased H3K27ac at the same locationsRemarkably we saw increased accumulation of H3K27ac atall three genes in Mecp2 null liver lysates correlating with de-creased HDAC3 binding (Fig 4BndashD)

A conditional deletion of Mecp2 from mouse liverdissociates fatty liver and insulin resistance

In light of these findings we hypothesized that Mecp2 loss fromthe liver alone would be sufficient to cause fatty liver disease

through aberrant expression of genes such as Sqle To test thishypothesis we generated a B6Mecp2 liver-specific deletedmouse line We removed Mecp2 from Albumin-expressing cellsby breeding B6Mecp2tm1Bird mice (Mecp2floxthorn) in which exons 3and 4 are flanked by loxP sites to B6Alb-Cre21Mgn BAC transgenicmice Alb regulatory elements drive Cre expression in trans-genic mice during embryonic development (as early as E155)and after birth in hepatocytes with some restricted expressionin bile ducts Recombination of the floxed Mecp2 allele is robustbut not complete in the Mecp2floxYAlb-Cre liver resulting in a90 decrease in Mecp2 transcription as evidenced by qRT-PCR(Fig 5A) However recombination does not occur in the brain ofMecp2floxYAlb-Cre mice and they did not develop neurologicalsymptoms as expected (Supplementary Material Figure S5Aand B) It is important to note that the Mecp2flox allele(Mecp2tm1Bird) utilized in these studies is hypomorphic resultingin an approximate 20 decrease in Mecp2 mRNA in littermatecontrol males lacking Cre (Fig 5B) (31) Therefore we antici-pated that male mice carrying this allele would display mildphenotypes due to a decrease in MeCP2 dosage

Conditional deletion of Mecp2 from hepatocytes increasestranscription of genes in the lipogenesis pathways including therate-limiting enzymes of cholesterol biosynthesis Hmgcr and Sqle

Figure 4 MeCP2 interacts in murine hepatocytes with the NCoR-HDAC3 complex and loss of MeCP2 prevents lipogenic enzyme repression at the locus (A) IP of NCoR1

MeCP2 and TBLR1 from murine liver nuclear extracts using an antibody to HDAC3 i input (B) Anti-HDAC3 and anti-H3K27ac ChIP-qPCR of sites surrounding the Sqle

TSS (n frac14 3 mice per genotype) (C) Anti-HDAC3 and anti-H3K27ac ChIP-qPCR of sites surrounding the Fasn TSS (n frac14 3 mice per genotype) (D) Anti-HDAC3 and anti-

H3K27ac ChIP-qPCR of sites surrounding the Cd36 TSS (n frac14 3 mice per genotype) Values are mean 6 SEM ns no significance P lt 005 P 001 P 0001 versus

WT Statistical analyses were performed using Studentrsquos t-test



as well as rate-limiting enzymes of the triglyceride synthesispathway Scd1 and Fasn (Fig 5C) Similarly to a liver-specific dele-tion of Hdac3 B6Mecp2floxYAlb-Cre mice develop fatty liver asevidenced by Oil Red O staining and a quantitative assessment ofliver lipids (Fig 5D and E) Perturbation of these pathways trans-lates to an increase in serum cholesterol in B6Mecp2floxYAlb-Crebut not in wild-type Alb-Cre or B6Mecp2floxY littermate controls(Table 1) B6Mecp2floxYAlb-Cre mice gain weight through an

expansion of fat mass however B6Mecp2floxY littermates dis-play the same phenotype Therefore we cannot attribute weightgain to a direct MeCP2 mechanism in the liver

Fatty liver is closely associated with metabolic syndrometherefore we performed glucose tolerance and insulin resis-tance tests in tissue-specific deleted B6Mecp2 null animals andcontrol littermates Mecp2floxYAlb-Cre mice are not glucose in-tolerant nor insulin resistant (Fig 5F and G) Furthermore

Figure 5 Loss of Mecp2 from the liver dissociates fatty liver and insulin resistance (A) PCR amplification of the Mecp2 locus in mouse liver shows presence of a hemizy-

gous Mecp2flox allele in mice lacking the Alb-Cre transgene Offspring with the genotype Mecp2floxY Alb-Cre have recombination of the Mecp2 floxed locus to a null al-

lele This recombination does not occur in the brain of Mecp2floxY Alb-Cre mice (B) Mecp2 mRNA expression in liver (n frac14 5 per genotype) (C) mRNA expression in liver

dissected from 6-month old male mice (n frac14 5 per genotype) (D) Livers from 6-month-old mice subjected to Oil Red O staining Scale bar represents 100 lM (E) Levels of

liver lipids of from 6-month-old male mice (nfrac146ndash7 per genotype) (F) Glucose tolerance tests (GTT) Glucose was administered intraperitoneally after a 5-h fast and

blood glucose was measured at the indicated times Male mice were tested at 6 months of age (n frac14 6ndash8 mice per genotype) (G) Insulin tolerance tests (ITT) Insulin was

administered intraperitoneally after a 4-h fast and blood glucose was measured at the indicated times Male mice were tested at 6 months of age (n frac14 4ndash5 mice per ge-

notype) BndashC EndashG Values are mean 6 SEM ns no significance P lt 005 P 001 P 0001 Statistical analyses were performed using one-way analysis of variance

(ANOVA) followed by Tukeyrsquos post-hoc analysis



Mecp2floxYAlb-Cre display no significant increase in circulatingfatty acids or glycerol (Table 1) As such Mecp2 loss from theliver dissociates fatty liver and insulin sensitivity

DiscussionThought to be entirely a neurological disorder RTT research hasfocused on the role of Mecp2 in the CNS however the wide vari-ety of phenotypes in Mecp2 null mice and RTT patients points tovital roles for Mecp2 outside the nervous system (32ndash36) Herewe show that Mecp2 null male mice develop severe dyslipidemiadue to aberrant transcription of rate-limiting de novo lipogenesisand cholesterol synthesizing enzymes in the liver Strikinglyexpression of neither Srebp1c nor nSREBP2 the transcriptionalregulators of fatty acid and cholesterol synthesis respectivelychanged in Mecp2Y liver (2737) As such we hypothesized thatincreased transcription of cholesterol and de novo lipogenesisenzymes results from a direct loss of MeCP2 transcriptional re-pression at targeted loci rather than increased SREBP activitysimilar to a liver-specific deletion of Hdac3

MeCP2 represses gene transcription through associationwith chromatin-remodeling complexes containing Type I his-tone deactylases (3839) which cannot readily bind promoter re-gions in the absence of MeCP2 resulting in aberranttranscription of targets (40) In neurons NCoR1-HDAC3 associ-ates with MeCP2 to regulate gene transcription in an activity-dependent manner (910) The NCoR1-HDAC3 complex regulatesa wide range of vital cellular processes its disruption is impli-cated in metabolism inflammatory pathways and perturbedCNS development (reviewed in 41) Whole-body deletion ofHdac3 results in embryonic death limiting its mechanistic studyto individual tissues and organ systems (42) Hepatic HDAC3regulates the diurnal balance between gluconeogenesis andlipid synthesis thereby balancing energy oxidation versus stor-age during the fasted and fed states respectively Liver-specificdeletion of Hdac3 increases activating acetylation marks at thepromoters of enzymes in the cholesterol and triglyceride syn-thesizing pathways including Sqle This results in aberranttranscription of the de novo lipogenesis pathway in a Srebp-inde-pendent manner subsequently leading to fatty liver diseaseand increased serum cholesterol (15ndash17) We hypothesized thatMeCP2 interacts with the NCoR-HDAC3-TBLR1 complex in theliver to repress enzyme transcription (20) Indeed hepaticMeCP2 interacts in vivo with endogenous HDAC3 NCoR1 andTBLR1 in the liver Loss of MeCP2 decreases HDAC3 binding

resulting in an open chromatin state as defined by increasedH3K27ac marks at the Sqle Fasn and Cd36 loci (Fig 6) This his-tone modification profile in the liver is highly consistent withloss of enzymatically active HDAC3 (30)

In addition to fatty liver Mecp2 null mice develop glucose in-tolerance and hyperinsulinemia as early as four weeks of agebefore onset of neurological phenotypes Insulin resistance inthese animals is global as evidenced by poor glucose uptake inmuscle and WAT when under a euglycemic insulin clampedstate Lipolysis is significantly increased in Mecp2 null mice ateight-weeks of age but not as early as four-weeks This timingsuggests that increased lipolysis and subsequent WAT inflam-mation is a downstream effect of hyperinsulinemia and glucoseintolerance Consistent with increased lipolysis a decreasedRER in Mecp2Y mice during both day and night cycles indicatesa preferential shift in energy utilization from carbohydrate tolipids This may be due to the inability of skeletal muscle to uti-lize glucose properly in its insulin resistant state in which casethe tissue will switch to fatty acid oxidation for energy

Neither transport nor usage of glucose in the CNS is effectedby insulin action (4344) Glucose is transported into the brainthrough a gradient sink such that a faster rate of glucose me-tabolism (glycolysis) in the brain allows for faster uptake of glu-cose from the blood via brain-specific GLUTs As such wehypothesize that increased glucose uptake by the Mecp2Y nullbrain may be due to an upregulation in insulin-insensitiveGLUT or abnormal neuronal glycolysis in response to synaptichyper-excitability reported in Mecp2 mutant neurons (45ndash48)Further investigation of this phenotype will shed light on thismechanism and may inform on a root cause of poor neuronalcommunication in the Mecp2 mutant brain

Here we show for the first time that MeCP2 directly controlsexpression of rate-limiting metabolic enzymes such as Sqle inthe liver As such loss of MeCP2 from the liver causes aberrantexpression of lipogenesis pathways leading to dyslipidemia andnon-alcoholic fatty liver disease Although the brain regulatesenergy metabolism through neuroendocrine feedback we rea-soned that MeCP2 deletion specifically from the liver would alsoresult in fatty liver disease Surprisingly this is only the secondtime a conditional deletion of Mecp2 outside of the CNS hasbeen published (49) Consistent with our hypothesis liver-specific deletion of Mecp2 recapitulates most of the phenotypesof a whole-body deletion the mice have increased de novo lipo-genesis fatty liver and increased serum cholesterol This ge-netic experiment further supports a hepatocyte-autonomous

Table 1 Metabolic and serum parameters in liver-specific Mecp2 null mice

Parameter 6 months

B6thornY B6Alb-Cre B6Mecp2floxY B6Mecp2floxY Alb-Cre

Weight (g) 338 6 08 356 6 08ns 417 6 19 418 66 20Fat mass (g) 64 6 06 78 6 06ns 132 6 15 121 6 13Lean mass (g) 222 6 03 227 6 04ns 231 6 06ns 232 6 08ns

Cholesterol (mgdl) 1135 6 75 1029 6 144ns 1352 6 106ns 1665 6 142Triglycerides (mgdl) 469 6 68 487 6 41ns 490 6 59ns 421 6 24ns

NEFA (mEl) 06 6 01 08 6 01ns 07 6 01ns 07 6 01ns

Glycerol (mgdl) 242 6 17 332 6 38ns 305 6 25ns 329 6 12ns

Leptin (ngml) 152 6 29 159 6 31ns 426 6 55 422 6 49

nfrac146ndash7 mice per genotype Values represent mean 6 SEM Statistics were performed using one-way ANOVA followed by Tukeyrsquos post-hoc analysis Ns no significance

P lt 005

P 001

P 0001 compared with B6thornY Male (WT)



role for Mecp2 in repression of enzymes of the cholesterol andtriglyceride biosynthesis pathways Dissociation of fatty liverand insulin resistance is reminiscent of an Alb-Cre induced liverdeletion of Hdac3 (17) Mecp2 deleted from Sim-1 or Pomc hypo-thalamic cells results in obesity reduced Pomc expression andsubsequent leptin resistance (5051) It is unknown if thesehypothalamic-specific Mecp2 deletion models develop insulinresistance However we would predict that dysfunctional sys-temic feedback between the hypothalamus adipose and liverin the whole-body Mecp2 null animals account for insulin resis-tance and excessive lipolysis which are absent in a liver-specific deletion of Mecp2

Importantly we found that Mecp2 heterozygous (Mecp2thorn) fe-male mice develop similar metabolic phenotypes to the malenull mice indicating that a mosaic dose of MeC2 is not sufficientto maintain metabolic homeostasis in mice Fatty liver diseasehas not been reported in RTT patients in the literatureHowever we and others have reported abnormal cholesteroland triglycerides levels in RTT patients (212252) Furthermoreglucose intolerance has been reported in pediatric RTT patients(53) Certainly our data open the possibility that RTT should bereclassified as a neurological disorder with systemic metaboliccomponents and suggest that RTT patients should be examinedfurther for metabolic anomalies Before labelled as a monogenicneurological disorder RTT was popularly thought to be a meta-bolic disease of mitochondria Abnormal carbohydrate metabo-lism increased reactive oxygen species load and poormitochondrial function have all been reported in RTT patientsor Mecp2 mouse models (54ndash57) These and other metabolicanomalies can be utilized as biomarkers to alert physicians ofimpending complications associated with Rett syndrome

In addition to a severe systemic dyslipidemia we have previ-ously reported perturbed cholesterol homeostasis in the brainsof Mecp2Y null mice (20) Cholesterol is a vital molecule forbrain development and health as it is heavily incorporated inmyelin sheaths and lipid rafts and is required for axonal guid-ance synaptogenesis and dendrite formation (58) Genetic per-turbations in brain cholesterol homeostasis cause a host ofsevere life-threatening neurological disorders (reviewed in 59)We showed that cholesterol-lowering statins successfully im-proved motor phenotypes overall health and increased lifespanin Mecp2 mutant mice (20) Our data here suggest that statintreatment primarily influenced improvement of phenotypesthrough altered brain lipids Strikingly statins account for onlyone family of numerous metabolic modulators many of whichare being developed to treat lipid accumulation in Type II diabe-tes and many of which cross the blood brain barrier Furtherstudy will illuminate new therapeutic options in highly target-able metabolic pathways that may prevent the progressing mor-bidities of Rett syndrome

Materials and MethodsAnimals

All animal experiments were conducted under protocols ap-proved by local Animal Care and Use Committees in the AALACor CCAC-accredited animal facilities at Baylor College ofMedicine or the Toronto Center for Phenogenomics respec-tively Congenic 129Mecp2tm11BirdY (Mecp2Y null) feature a de-letion of the last two exons (exons 3ndash4) of the Mecp2 transcriptMecp2Y and wild-type male offspring were obtained by back-crossing Mecp2tm11Birdthorn females to males of the 129SvEvS6Tacstrain Mecp2Y liver-specific deletion lines (Mecp2floxY Alb-Cre)conditional-ready control (Mecp2floxY) lines (exons 3 and 4flanked by loxP sites) and wild-type littermates were obtainedby crossing B6JMecp2tm1Birdthorn (Mecp2floxthorn) heterozygous femalemice to male mice heterozygous for the Albumin-Cre transgeneon a C57BL6J background Mice were fed a standard diet(Harlan Teklad 2918) ad libitum consisting of 18 protein 6fat and 44 carbohydrates Mice were housed in a 12-h lightdark cycle facility and were euthanized between the hours of 3PM and 5 PM (ZT 8ndash10) to control for circadian rhythm fluctua-tions Lean mass and fat mass was determined in live mice us-ing a Bruker Minispec NMR body composition analyzer

Histology

A small section of liver (5 mm3) was extracted from Mecp2Y(eight-weeks of age) and age-matched wild-type littermatesLiver was placed immediately in 10 neutral buffered formalinand was fixed for 48 h at room temperature The liver waswashed with exchanges of PBS and was incubated in increasingsucrose gradients (15 then 30) in PBS overnight at 4C Tissuewas frozen in OCT and 7 lm slices were prepared Sections werethen stained in 03 Oil Red O in isopropanol and then counter-stained with hematoxylin for 5 s

Quantitative PCR

Tissue (liver WAT) was snap-frozen and stored at 80 until pro-cessed for RNA extraction Details are described inSupplementary Materials Primers are listed in SupplementaryMaterial Table S1

Figure 6 A schematic model of metabolic phenotypes in Mecp2 null mice

MeCP2 co-ordinates hepatic lipid metabolism in vivo through binding HDAC3

which deactylates histone tails leading to reduced transcription at lipogenic

loci In the absence of MeCP2 HDAC3 fails to bind promoter regions which pre-

vents the removal of histone aceylation marks This leads to downstream aber-

rant transcription of enzymes such as Sqle which subsequently increases

hepatic triglyceride and cholesterol production Mecp2 null mice then develop

severe metabolic syndrome including reduced insulin signaling and glucose up-

take (indicated by red lines) resulting in increased lipolysis and shifting the bal-

ance between energy storage and expenditure



Hyperinsulinemic-euglycemic clamps

Mice were anesthetized and a midline neck incision was made toexpose the jugular vein A microcannula was inserted into thejugular vein threaded into the right atrium and anchored at thevenotomy site Mice were allowed to recover for 4 days with adlibitum access to water and food Following an overnight fast theconscious mice received a primary infusion (10 uCi) and thena constant rate intravenous infusion (01 uCimin) ofchromatography-purified [3-3H]-glucose using a syringe infusionpump For determination of basal glucose production blood sam-ples were collected from the tail vein after 50 55 and 60 min oflabeled glucose infusion After 60 min mice received a primingbolus of insulin (40 mUkg body weight) followed by a 2-h insulininfusion (4 mUkgmin) Simultaneously 10 glucose was in-fused using another infusion pump at a rate adjusted to maintainthe blood glucose level at 100ndash140 mgdl (euglycemia) Blood glu-cose concentration was measured every 10 min by a glucometerAt 100 110 and 120 min blood was collected to measure HGP un-der clamped conditions To estimate insulin-stimulated GLUT ac-tivity in individual tissues 2-[14C]deoxyglucose was administeredas a bolus (10 uCi) at 45 min before the end of the clamps After120 min mice were euthanized and tissues were extracted Brainregions were rapidly dissected from both hemispheres of thebrain Thalamic and hypothalamic tissue was pooled Glucoseuptake in different tissues was calculated from plasma by tissueenrichment of 2-[14C]deoxyglucose by gas chromatography-massspectrometry

Tolerance tests and lipolysis analysis

In vivo lipolysis analyses were performed by the Diabetes andEndocrinology Research Center at BCM Details are described inSupplementary Materials

Nuclear extraction

Freshly dissected liver (50 mg) was washed with 1X volume ofice-cold PBS and centrifuged at 500 g for 5 min at 4C Tissuewas then transferred cleanly to a dounce homogenizer on icecontaining 500 ll of cold NP40 lysis buffer (10 mM Hepes pH793 mM MgCl2 10 mM KCl 05 NP40 05mM DTT and CompleteEDTA-free protease inhibitor cocktail (Roche CA USA)) Tissuewas incubated in buffer on ice for 45 min and then tissue wasdisrupted with 10 strokes of the tight pestle in the dounce tissuehomogenizer Homogenized tissue was transferred into a newcold microfuge tube and centrifuged at 2000 g for 10 min at 4CThe supernatant was removed and the pellet was resuspendedin 250 ll of Benzonase extraction buffer (10 mM Hepes pH79 3mM MgCl2 280 mM NaCl 02 mM EDTA pH80 05 NP40 05mM DTT and Complete EDTA-free protease inhibitor cocktail)with 250 U of Benzonase Nuclei were incubated in extractionbuffer for 1 h on at rotator at 4C Following incubation lysateswere centrifuged at 17 000g for 20 min at 4C Lysates were snapfrozen and stored at 80

Co-immunoprecipitation

IP was carried out using the Millipore Catch and Release kit(Millipore MA USA) Following the instructions 500 lg of nu-clear lysates was incubated with 12 lg of anti-HDAC3 (ab7030)10 lg of anti-HDAC3 (Upstate 06-890) or 10 lg of anti-HDAC3(SantaCruz H-303) at 4C for 3 h Following elution half of theimmunoprecipitated sample was loaded onto an SDS-PAGE gel

for downstream western blotting Input samples represent 25 lgof total nuclear extracted protein

Western blotting

The following antibodies were used for western blotting anti-MeCP2 (Millipore 07-013) at a concentration of 1700 in 3 Milk-PBS anti-SREBP2 (ab30682) at a concentration of 1400 in 5Milk-TBST anti-GAPDH (Cell Signaling 5174) at a concentrationof 18000 in 5 Milk-TBST anti-NCoR1 (sc-1609) at a concentra-tion of 1450 in 1 casein-TBST anti-TBLR1 at a concentrationof 11200 in 1 casein-TBST For co-IP a light-chain specific sec-ondary antibody (Jackson ImmunoResearch) was used to avoidheavy chain detection at 50kDa Quantification of band intensitywas achieved using ImageJ (NIH) and normalized to GAPDH

Chromatin-IP

ChIP was performed as described in (60) Details are provided inSupplementary Materials

Indirect calorimetry

Respiratory metabolic function was assessed using the OxymaxDeluxe System indirect calorimeter equal flow eight chambersystem (Columbus Instruments Columbus Ohio) and analyzedusing Oxymax Windows V332 Software Details are describedin the Supplementary Materials

Subjective health assessments

Mice were assayed for general health once per week from 5weeks of age to 24-weeks of age Scoring was blinded by geno-type Mice were scored using the assessment published in (61)where a score of 0 is not different from wildtype 1 is more se-vere and 2 is very severe Mice were assessed for limbclaspingtremors home-cage activity and grooming for a combined pos-sible score of 0ndash8

Rotarod

At 5-months of age male mice were placed on a grooved rotat-ing rod at a speed of 4 rpm Over the course of 5 min the revo-lution rate increased steadily to a maximum of 40 rpm Thetime each mouse stayed on the rod was recorded for four trialsspread across two consecutive days totaling eight trials foreach mouse with a 30-min break between trials A trial endedwhen the mouse fell from the rotating rod spun with the rodfor two consecutive revolutions or successfully stayed on therod for 5 min

Statistics

Values are expressed at mean 6 SEM The significance of the dif-ferences in mean values among different groups was evaluatedby two-tailed Student t-tests or for comparing means of morethan two groups one-way ANOVA followed by Tukeyrsquos post-hocanalysis P-values lt 005 were considered statistically signifi-cant (Plt 005frac14 P 001 frac14 P 0001frac14 ) Linear regressionStudent t-test and ANOVA was analyzed using GraphPad Prism6



Supplementary MaterialSupplementary Material is available at HMG online

Acknowledgements

We thank Misty Hill Jennifer Borkey Julie Ruston StephenMcDonald and Natalie Tully of the Justice lab for technical as-sistance We thank Dr Michael Wilson and Lina Antounians forvaluable guidance in ChIP experiments We also thank theCentre for Modeling Human Disease (CMHD) for assistance inIHC sample processing

Conflict of Interest statement None declared

FundingThis work was supported by the Rett Syndrome Research Trust(RSRT) [MJJ] and the National Institutes of Health [T32 GM008307to SMK P30 DK079638 to LCC] Funding to pay the Open Accesspublication charges for this article was provided by The Hospitalfor Sick Children Operating Funds [MJJ]

References1 Amir RE Van den Veyver IB Wan M Tran CQ Francke

U and Zoghbi HY (1999) Rett syndrome is caused by muta-tions in X-linked MECP2 encoding methyl-CpG-binding pro-tein 2 Nat Genet 23 185ndash188

2 Braunschweig D Simcox T Samaco RC and LaSalle JM(2004) X-Chromosome inactivation ratios affect wild-typeMeCP2 expression within mosaic Rett syndrome andMecp2thornmouse brain Hum Mol Genet 13 1275ndash1286

3 Guy J Hendrich B Holmes M Martin JE and Bird A (2001)A mouse Mecp2-null mutation causes neurological symp-toms that mimic Rett syndrome Nat Genet 27 322ndash326

4 Katz DM Berger-Sweeney JE Eubanks JH Justice MJNeul JL Pozzo-Miller L Blue ME Christian D CrawleyJN Giustetto M et al (2012) Preclinical research in Rett syn-drome setting the foundation for translational success DisModel Mech 5 733ndash745

5 Young JI and Zoghbi HY (2004) X-ChromosomeInactivation Patterns Are Unbalanced and Affect thePhenotypic Outcome in a Mouse Model of Rett SyndromeAm J Hum Genet 74 511ndash520

6 Adams VH McBryant SJ Wade PA Woodcock CL andHansen JC (2007) Intrinsic Disorder and AutonomousDomain Function in the Multifunctional Nuclear ProteinMeCP2 J Biol Chem 282 15057ndash15064

7 Skene PJ Illingworth RS Webb S Kerr ARW JamesKD Turner DJ Andrews R and Bird AP (2010) NeuronalMeCP2 is expressed at near histone-octamer levels and glob-ally alters the chromatin state Mol Cell 37 457ndash468

8 Ausio J Paz AM de and Esteller M (2014) MeCP2 the longtrip from a chromatin protein to neurological disordersTrends Mol Med 20 487ndash498

9 Ebert DH Gabel HW Robinson ND Kastan NR Hu LSCohen S Navarro AJ Lyst MJ Ekiert R Bird AP et al(2013) Activity-dependent phosphorylation of MeCP2 threo-nine 308 regulates interaction with NCoR Nature 499 341ndash345

10 Lyst MJ Ekiert R Ebert DH Merusi C Nowak JSelfridge J Guy J Kastan NR Robinson ND de LimaAlves F et al (2013) Rett syndrome mutations abolish the

interaction of MeCP2 with the NCoRSMRT co-repressor NatNeurosci 16 898ndash902

11 Guenther MG Barak O and Lazar MA (2001) The SMRTand N-CoR Corepressors Are Activating Cofactors forHistone Deacetylase 3 Mol Cell Biol 21 6091ndash6101

12 Li J Wang J Wang J Nawaz Z Liu JM Qin J and Wong J(2000) Both corepressor proteins SMRT and N-CoR exist in largeprotein complexes containing HDAC3 Embo J 19 4342ndash4350

13 Heckman LD Chahrour MH and Zoghbi HY (2014) Rett-causing mutations reveal two domains critical for MeCP2function and for toxicity in MECP2 duplication syndromemice eLife 3

14 Alenghat T Meyers K Mullican SE Leitner K Adeniji-Adele A Avila J Bucan M Ahima RS Kaestner KH andLazar MA (2008) Nuclear receptor corepressor and histonedeacetylase 3 govern circadian metabolic physiology Nature456 997ndash1000

15 Feng D Liu T Sun Z Bugge A Mullican SE AlenghatT Liu XS and Lazar MA (2011) A Circadian RhythmOrchestrated by Histone Deacetylase 3 Controls HepaticLipid Metabolism Science 331 1315ndash1319

16 Knutson SK Chyla BJ Amann JM Bhaskara SHuppert SS and Hiebert SW (2008) Liver-specific deletionof histone deacetylase 3 disrupts metabolic transcriptionalnetworks Embo J 27 1017ndash1028

17 Sun Z Miller RA Patel RT Chen J Dhir R Wang HZhang D Graham MJ Unterman TG Shulman GI et al(2012) Hepatic Hdac3 promotes gluconeogenesis by repress-ing lipid synthesis and sequestration Nat Med 18 934ndash942

18 Gill S Stevenson J Kristiana I and Brown AJ (2011)Cholesterol-dependent degradation of squalene monooxy-genase a control point in cholesterol synthesis beyondHMG-CoA reductase Cell Metab 13 260ndash273

19 Hidaka Y Satoh T and Kamei T (1990) Regulation of squa-lene epoxidase in HepG2 cells J Lipid Res 31 2087ndash2094

20 Buchovecky CM Turley SD Brown HM Kyle SMMcDonald JG Liu B Pieper AA Huang W Katz DMRussell DW et al (2013) A suppressor screen in Mecp2 mu-tant mice implicates cholesterol metabolism in Rett syn-drome Nat Genet 45 1013ndash1020

21 Justice MJ Buchovecky CM Kyle SM and Djukic A(2013) A role for metabolism in Rett syndrome pathogenesisNew clinical findings and potential treatment targets RareDis Austin Tex 1 e27265

22 Segatto M Trapani L Di Tunno I Sticozzi C Valacchi GHayek J and Pallottini V (2014) Cholesterol metabolism is al-tered in rett syndrome a study on plasma and primary cul-tured fibroblasts derived from patients PLoS One 9 e104834

23 Guillot F Misslin P and Lemaire M (1993) Comparison of flu-vastatin and lovastatin blood-brain barrier transfer using in vi-tro and in vivo methods J Cardiovasc Pharmacol 21 339ndash346

24 Saheki A Terasaki T Tamai I and Tsuji A (1994) In vivoand in vitro blood-brain barrier transport of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibi-tors Pharm Res 11 305ndash311

25 Matsumoto M Ogawa W Akimoto K Inoue H MiyakeK Furukawa K Hayashi Y Iguchi H Matsuki YHiramatsu R et al (2003) PKClambda in liver mediatesinsulin-induced SREBP-1c expression and determines bothhepatic lipid content and overall insulin sensitivity J ClinInvest 112 935ndash944

26 Shimomura I Matsuda M Hammer RE Bashmakov YBrown MS and Goldstein JL (2000) Decreased IRS-2 andincreased SREBP-1c lead to mixed insulin resistance and



sensitivity in livers of lipodystrophic and obob mice MolCell 6 77ndash86

27 Horton JD Goldstein JL and Brown MS (2002) SREBPsactivators of the complete program of cholesterol and fattyacid synthesis in the liver J Clin Invest 109 1125ndash1131

28 Guilherme A Virbasius JV Puri V and Czech MP (2008)Adipocyte dysfunctions linking obesity to insulin resistanceand type 2 diabetes Nat Rev Mol Cell Biol 9 367ndash377

29 Tschop MH Speakman JR Arch JRS Auwerx JBruning JC Chan L Eckel RH Farese RV Galgani JEHambly C et al (2012) A guide to analysis of mouse energymetabolism Nat Methods 9 57ndash63

30 You SH Lim HW Sun Z Broache M Won KJ andLazar MA (2013) Nuclear receptor co-repressors are re-quired for the histone-deacetylase activity of HDAC3 in vivoNat Struct Mol Biol 20 182ndash187

31 Samaco RC Fryer JD Ren J Fyffe S Chao HT Sun YGreer JJ Zoghbi HY and Neul JL (2008) A partial loss offunction allele of Methyl-CpG-binding protein 2 predicts ahuman neurodevelopmental syndrome Hum Mol Genet 171718ndash1727

32 Chahrour M and Zoghbi HY (2007) The story of Rett syn-drome from clinic to neurobiology Neuron 56 422ndash437

33 Gong M Liu J Sakurai R Corre A Anthony S andRehan VK (2015) Perinatal nicotine exposure suppressesPPARc epigenetically in lung alveolar interstitial fibroblastsMol Genet Metab 114 604ndash612

34 Hara M Takahashi T Mitsumasu C Igata S Takano MMinami T Yasukawa H Okayama S Nakamura KOkabe Y et al (2015) Disturbance of cardiac gene expressionand cardiomyocyte structure predisposes Mecp2-null miceto arrhythmias Sci Rep 5

35 Hu B Gharaee-Kermani M Wu Z and Phan SH (2011)Essential role of MeCP2 in the regulation of myofibroblastdifferentiation during pulmonary fibrosis Am J Pathol 1781500ndash1508

36 Li C Jiang S Liu SQ Lykken E Zhao L Sevilla J Zhu Band Li QJ (2014) MeCP2 enforces Foxp3 expression to pro-mote regulatory T cellsrsquo resilience to inflammation ProcNatl Acad Sci USA 111 E2807ndashE2816

37 Murphy C Ledmyr H Ehrenborg E and Gafvels M (2006)Promoter analysis of the murine squalene epoxidase geneIdentification of a 205 bp homing region regulated by bothSREBPrsquoS and NF-Y Biochim Biophys Acta 1761 1213ndash1227

38 Bienvenu T and Chelly J (2006) Molecular genetics of Rettsyndrome when DNA methylation goes unrecognized NatRev Genet 7 415ndash426

39 Stancheva I Collins AL Van den Veyver IB Zoghbi Hand Meehan RR (2003) A mutant form of MeCP2 protein as-sociated with human Rett syndrome cannot be displacedfrom methylated DNA by notch in Xenopus embryos MolCell 12 425ndash435

40 Martinowich K Hattori D Wu H Fouse S He F Hu YFan G and Sun YE (2003) DNA Methylation-RelatedChromatin Remodeling in Activity-Dependent Bdnf GeneRegulation Science 302 890ndash893

41 Karagianni P and Wong J (2007) HDAC3 taking the SMRT-N-CoRrect road to repression Oncogene 26 5439ndash5449

42 Bhaskara S Chyla BJ Amann JM Knutson SK CortezD Sun ZW and Hiebert SW (2008) Deletion of HistoneDeacetylase 3 Reveals Critical Roles in S Phase Progressionand DNA Damage Control Mol Cell 30 61ndash72

43 Mergenthaler P Lindauer U Dienel GA and Meisel A(2013) Sugar for the brain the role of glucose in physiological

and pathological brain function Trends Neurosci 36587ndash597

44 Banks WA Owen JB and Erickson MA (2012) Insulin inthe brain there and back again Pharmacol Ther 136 82ndash93

45 Calfa G Hablitz JJ and Pozzo-Miller L (2011) Networkhyperexcitability in hippocampal slices from Mecp2 mutantmice revealed by voltage-sensitive dye imaging JNeurophysiol 105 1768ndash1784

46 Zhang W Peterson M Beyer B Frankel WN and ZhangZ (2014) Loss of MeCP2 from forebrain excitatory neuronsleads to cortical hyperexcitation and seizures J Neurosci OffJ Soc Neurosci 34 2754ndash2763

47 Kron M Howell CJ Adams IT Ransbottom MChristian D Ogier M and Katz DM (2012) Brain ActivityMapping in Mecp2 Mutant Mice Reveals Functional Deficitsin Forebrain Circuits Including Key Nodes in the DefaultMode Network that are Reversed with Ketamine TreatmentJ Neurosci Off J Soc Neurosci 32 13860ndash13872

48 Lundgaard I Li B Xie L Kang H Sanggaard S HaswellJDR Sun W Goldman S Blekot S Nielsen M et al (2015)Direct neuronal glucose uptake heralds activity-dependentincreases in cerebral metabolism Nat Commun 6 6807

49 Conti V Gandaglia A Galli F Tirone M Bellini ECampana L Kilstrup-Nielsen C Rovere-Querini PBrunelli S and Landsberger N (2015) MeCP2 affects skeletalmuscle growth and morphology through non cell-autonomous mechanisms PLoS One 10

50 Fyffe SL Neul JL Samaco RC Chao HT Ben-ShacharS Moretti P McGill BE Goulding EH Sullivan E TecottLH et al (2008) Deletion of Mecp2 in Sim1-expressing neu-rons reveals a critical role for MeCP2 in feeding behavior ag-gression and the response to stress Neuron 59 947ndash958

51 Wang X Lacza Z Sun YE and Han W (2014) Leptinresistance and obesity in mice with deletion of methyl-CpG-binding protein 2 (MeCP2) in hypothalamic pro-opiomelanocortin (POMC) neurons Diabetologia 57236ndash245

52 Sticozzi C Belmonte G Pecorelli A Cervellati FLeoncini S Signorini C Ciccoli L De Felice C Hayek Jand Valacchi G (2013) Scavenger receptor B1 post-translational modifications in Rett syndrome FEBS Lett101016jfebslet201305042

53 Cooke DW Naidu S Plotnick L and Berkovitz GD (1995)Abnormalities of thyroid function and glucose control insubjects with Rett syndrome Horm Res 43 273ndash278

54 Cronk JC Derecki NC Ji E Xu Y Lampano AESmirnov I Baker W Norris GT Marin I Coddington Net al (2015) Methyl-CpG binding protein 2 regulates microgliaand macrophage gene expression in response to inflamma-tory stimuli Immunity 42 679ndash691

55 De Felice C Della Ragione F Signorini C Leoncini SPecorelli A Ciccoli L Scalabrı F Marracino F MadonnaM Belmonte G et al (2014) Oxidative brain damage inMecp2-mutant murine models of Rett syndrome NeurobiolDis 68 66ndash77

56 Matsuishi T Urabe F Percy AK Komori H YamashitaY Schultz RS Ohtani Y Kuriya N and Kato H (1994)Abnormal carbohydrate metabolism in cerebrospinal fluidin rett syndrome J Child Neurol 9 26ndash30

57 Muller M and Can K (2014) Aberrant redox homoeostasisand mitochondrial dysfunction in Rett syndrome BiochemSoc Trans 42 959ndash964

58 Pfrieger FW and Ungerer N (2011) Cholesterol metabolismin neurons and astrocytes Prog Lipid Res 50 357ndash371



59 Kanungo S Soares N He M and Steiner RD (2013) Sterolmetabolism disorders and neurodevelopmentmdashan updateDev Disabil Res Rev 17 197ndash210

60 Schmidt D Wilson MD Spyrou C Brown GD HadfieldJ and Odom DT (2009) ChIP-seq using high-throughput

sequencing to discover protein-DNA interactions MethodsSan Diego Calif 48 240ndash248

61 Guy J Gan J Selfridge J Cobb S and Bird A (2007)Reversal of neurological defects in a mouse model of Rettsyndrome Science 315 1143ndash1147



ddw156-TF1

ddw156-TF2

ddw156-TF3

ddw156-TF4

(Mecp2thorn) female mice have a wide range in phenotype severitydue to random X-chromosome inactivation (2ndash5)

Mecp2 encodes a ubiquitously-expressed intrinsically disor-dered protein (IDP) that in murine neurons binds DNA at nearhistone-octomer levels (67) Characteristic of an IDP MeCP2 istargeted by a multitude of post-translational modificationswhich determine cofactor association and may account for itsseemingly endless roles in maintaining cellular homeostasis (8)To this effect the mechanistic contribution of mutant MeCP2 toRTT pathology is highly complex and remains elusive

As the culprit gene of a devastating neurological disorderMECP2 has been largely studied in relation to central nervoussystem (CNS) development maturation and signaling In neu-rons MeCP2 can repress gene transcription by binding the nu-clear receptor co-repressorsilencing mediator for retinoid orthyroid-hormone receptors (NCoRSMRT) complex at MeCP2amino acids 302ndash306 (910) MeCP2-NCoRSMRT suppresses tran-scription by delivering the histone deacetylase HDAC3 to targetgenes which removes acetyl marks left by histone acetyltrans-ferases resulting in a closed chromatin state (1112) A R306Cmissense mutation in mouse Mecp2 abolishes interaction withthe NCoRSMRT co-repressor complex and prevents MeCP2-mediated transcriptional repression Importantly this mutationis sufficient to cause severe RTT-like phenotypes including hin-dlimb clasping hypoactivity and reduced brain weight thushighlighting the importance of the NCoRSMRT-HDAC3 interac-tion with MeCP2 in RTT pathology Furthermore the amino acid302ndash306 region of human MECP2 contains a missense mutationhotspot causing classical RTT in patients (1013)

Mouse models have established the vital role of the NCoR co-repressor complex in energy metabolism NCoR-HDAC3 regu-lates the circadian metabolic cycle of the liver governing thefluctuation between lipid and glucose utilization in hepatocytesduring lightdark cycles (1415) Loss of Hdac3 from the liver ofembryonic or adult mice promotes a constitutively active tran-scriptional environment which increases expression of de novolipogenesis enzymes resulting in fatty liver disease (1617)Further Hdac3 liver deletion increases transcription of severalgenes in the cholesterol biosynthesis pathway elevating serumcholesterol One target of HDAC3 is squalene epoxidase (Sqle) arate-limiting enzyme of the committed cholesterol biosynthesispathway whose transcription is increased 170-fold in re-sponse to Hdac3 liver deletion (161819) Curiously a forwardgenetic suppressor screen found that a nonsense mutation inSqle improved RTT-like motor symptoms and overall health inMecp2 null mice This was intriguing as both RTT patients andMecp2 mutant mice can have increased serum cholesterol andtriglycerides (20ndash22) Furthermore treatment of Mecp2 hemizy-gous male and heterozygous female mice with HMG-CoA reduc-tase inhibitors (statins) phenocopies symptom improvement bySqle mutation (20) Notably RTT-like phenotypes are improvedfollowing treatment with fluvastatin a hydrophilic statin thatdoes not readily cross the blood-brain barrier suggesting thatMeCP2 plays a role in regulating systemic cholesterol metabo-lism (2324) As such we hypothesized that genetic loss of Mecp2would cause metabolic perturbations in mice Here we reportthat Mecp2 deletion causes fatty liver disease metabolic syn-drome insulin resistance and alters overall energy homeosta-sis We show that hepatic MeCP2 works in conjunction with theNCoR-HDAC3 corepressor complex to orchestrate transcriptionof enzymes in the triglyceride and cholesterol synthesis path-ways In support of this liver-specific loss of Mecp2 is suffi-ciently deleterious to increase lipogenic enzyme transcriptionleading to fatty liver however these mice are insulin sensitive

suggesting that MeCP2 plays a role in insulin signaling outsideof the liver

ResultsMecp2 deletion increases lipogenic enzyme expressionresulting in non-alcoholic fatty liver

Surprisingly we observed a consistent pale coloration of129Mecp2Y livers (Fig 1A) Oil Red O staining suggested an accu-mulation of lipid vesicles and subsequent quantification of lipidsin Mecp2Y null liver showed a 2-fold increase in liver triacylgly-cerol (TAG) over wild-type (Fig 1B) We examined hepatic expres-sion of genes in the fatty acid and TAG synthesis pathways atthree (pre-symptomatic) and eight (symptomatic) weeks of ageExpression of four enzymes involved in fatty acid synthesis (ace-tyl-CoA carboxylase A and B [Acca Accb] fatty acid synthase[Fasn] and stearoyl-CoA desaturase 1 [Scd-1]) was increased inpre- and post-symptomatic Mecp2Y mice liver Expression offatty acid translocase (Cd36) a transporter of fatty acids and per-ilipin 2 and 5 (Plin2 and Plin5) proteins that coat TAG-dense lipiddroplets was also increased However levels of sterol-regulatoryelement-binding protein 1c (Srebp-1c) the transcription factor re-sponsible for regulating the fatty acid synthesis pathway wasnot altered in Mecp2Y liver (Fig 1CndashE) (2526)

Transcription of HMG-CoA reductase (Hmgcr) squalene epoxi-dase (Sqle) and lanosterol synthase (Lss) enzymes of the choles-terol biosynthesis pathway were all increased 3-fold in129Mecp2Y liver by 8 weeks of age (Fig 1CndashE) Concurrently129Mecp2Y null mice have increased serum cholesterol by sixweeks of age (20) Therefore we analyzed the expression andmaturation of SREBP2 the transcription factor responsible for ex-pression of enzymes in the cholesterol biosynthesis pathway (27)However there were no differences in levels of Srebp2 transcript(Fig 1C and D) nor levels of unspliced or mature nuclear nSREBP2between 129Mecp2Y null and wild-type livers (Fig 1F and G)These data suggest that a SREBP-independent pathway is respon-sible for aberrant hepatic lipogenesis in Mecp2Y null miceNotably Mecp2thornheterozygous female mice also accumulate fatin their livers by 6 months of age indicating that mosaic loss ofMecp2 is sufficient to cause fatty liver (Supplementary MaterialFigure S1A and B)

Impaired glucose and insulin homeostasisin Mecp2 mice

Non-alcoholic fatty liver disease is closely linked to glucose intol-erance and insulin resistance Mecp2Y mice display hyperglyce-mia in the fed state at 8-weeks of age but glucose levels aresimilar to wild-type when fasted (Supplementary Material FigureS2A and B) Even so plasma insulin levels of fasted and non-fasted Mecp2Y mice are significantly greater than wild-type litter-mates at both pre- and post-symptomatic ages (4 and 8 weeks re-spectively) (Fig 2A and B) Concurrently 129Mecp2Y null mice areglucose intolerant at both four and eight-weeks of age and insulinresistance is apparent by 9-weeks (Supplementary Material FigureS2CndashE) Glucose intolerance also arises in 129Mecp2thornheterozy-gous females by 20-weeks of age and insulin resistance is evidentat 24-weeks of age (Supplementary Material Figure S3AndashC)

To critically assess global insulin sensitivity Mecp2Y nullmice were subjected to hyperinsulinemic-euglycemic clampstudies (Fig 2C) In this gold standard assay for assessing insulinresistance [3-3H]-glucose and insulin are infused in fastedunrestrained mice to maintain a euglycemic lsquoclampedrsquo state

















































































Parameter 6 months




NEFA (mEl) 06 6 01 08 6 01ns 07 6 01ns 07 6 01ns


Leptin (ngml) 152 6 29 159 6 31ns 426 6 55 422 6 49


P lt 005

P 001









Histology


Quantitative PCR


















Nuclear extraction





Western blotting


Chromatin-IP






Rotarod


Statistics





Acknowledgements












































































ddw156-TF1

ddw156-TF2

ddw156-TF3

ddw156-TF4















































































Parameter 6 months




NEFA (mEl) 06 6 01 08 6 01ns 07 6 01ns 07 6 01ns


Leptin (ngml) 152 6 29 159 6 31ns 426 6 55 422 6 49


P lt 005

P 001









Histology


Quantitative PCR


















Nuclear extraction





Western blotting


Chromatin-IP






Rotarod


Statistics





Acknowledgements












































































ddw156-TF1

ddw156-TF2

ddw156-TF3

ddw156-TF4






Histology


Quantitative PCR


















Nuclear extraction





Western blotting


Chromatin-IP






Rotarod


Statistics





Acknowledgements












































































ddw156-TF1

ddw156-TF2

ddw156-TF3

ddw156-TF4





Nuclear extraction





Western blotting


Chromatin-IP






Rotarod


Statistics





Acknowledgements












































































ddw156-TF1

ddw156-TF2

ddw156-TF3

ddw156-TF4


Acknowledgements












































































ddw156-TF1

ddw156-TF2

ddw156-TF3

ddw156-TF4











































ddw156-TF1

ddw156-TF2

ddw156-TF3

ddw156-TF4

mecp2 co-ordinates liver lipid metabolism with the ncor1/hdac3

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