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Comp l ex d i sease , gender and ep i gene t i cs

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Ann als of Medic ine . 2006; 38: 530– 544

REV IEW AR TICLE

als of Medic ine . 2006; 38: 530– 544 REV IEW AR TICLE Complex disease, gender

Complex disease, gender and epigenetics

ZACHARY KAMINSKY, SUN-CHONG WANG & ARTURAS PETRONIS

The Krembil Family Epigenetics Laboratory, Centre for Addiction and Mental Health, Toronto, Canada

Abstract Gender differences in susceptibility to complex disease such as asthma, diabetes, lupus, autism and major depression, among numerous other disorders, represent one of the hallmarks of non-Mendelian biology. It has been generally accepted that endocrinological differences are involved in the sexual dimorphism of complex disease; however, specific molecular mechanisms of such hormonal effects have not been elucidated yet. This paper will review evidence that sex hormone action may be mediated via gene-specific epigenetic modifications of DNA and histones. The epigenetic modifications can explain sex effects at DNA sequence polymorphisms and haplotypes identified in gender-stratified genetic linkage and association studies. Hormone-induced DNA methylation and histone modification changes at specific gene regulatory regions may increase or reduce the risk of a disease. The epigenetic interpretation of sexual dimorphism fits well into the epigenetic theory of complex disease, which argues for the primary pathogenic role of inherited and/or acquired epigenetic misregulation rather than DNA sequence variation. The new experimental strategies, especially the high throughput microarray-based epigenetic profiling, can be used for testing the epigenetic hypothesis of gender effects in complex diseases.

Key words: Chromatin, DNA methylation, epigenetics, epigenomic profiling, histones, hormone, microarrays, sexual dimorphism

Si mple and comp lex gene tic disease s

In the past 20 years, there has been a major effort int o the developm ent of experi mental and comput a- tional strat egies for the ide ntification of molecul ar caus es of hum an compl ex diseas e. Unl ike simp le Me ndeli an disorde rs, for wh ich the cl oning of dis ease gen es has becom e a rou tine procedur e, ide ntification of the molecul ar g enetic basis of com plex disease repres ents a m ajor challe nge to hum an biologists . Although the genes for some rare early on set and familial case s o f complex diseas es (e.g. colon canc er, breast cancer and Alzheime r’s dis ease) have been ident ified, the overw helming pr oportion of non-Mend elian pathology r emains une xplained . Resea rch in compl ex no n-Mendeli an dis eases is reachin g an unprec edented level of so phisticati on and sc ale: from buildi ng m assive da tabases of DN A seque nce variants (single nucle o- tid e polymo rphism s (SNPs), hap lotype maps (H apMap)) to screening thousands of polymo rph- isms in thousan ds of affec ted individua ls and

contro ls. Why is it that compl ex diseas es are so resista nt to the discov ery of the etiologi cal factors ? I n an attemp t to addre ss this questi on, it is importan t t o note that compl ex diseas es, in compa rison to simple ones, exhibit numero us epi demiologic al, clini cal an d molecul ar differe nces. Com plex diseas es as a rule are commo n (mo re than 1 case per 1000 individua ls), with spora dic cases do minating ove r the familial ones, wh ile simp le on es are rare (less than 0.1%) an d predo minantly famili al. Phenotypi c differe nces (dis- cordance ) in ident ical twins have been one of the hallmark s of com plex non-Me ndelian diseas e. Conco rdance of mono zygotic twin s reaches only , 15% in breas t canc er, 20% in ulcer ative colitis, 25%–30% in multipl e sclero sis, 25%–45% in dia- betes, 50% in schizophre nia and 40%–70% for Alzheim er’s diseas e (1). Com plex dis eases usually exhibit a significant ly later age at onset, while the overw helming maj ority ( , 90%) of Me ndelian dis- eases have an onset before p uberty and only 1% after 40 years of age (2). Majo r psyc hosis, asthma, multipl e sclerosis, infla mmato ry bowe l disease, rheum atoid arthrit is an d numero us othe r compl ex

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dis eases exhi bit major fluctuations in dis ease sever- ity, and in some cases age-depe ndent decli ne of sym ptoms and eve n full recovery from a dis ease. E pidemiol ogical studies have reve aled that quite often the risk of developi ng complex disease in offsp ring depen ds on the sex of affec ted parent. For exam ple, asthma, bipolar disorde r and epilepsy are m ore often trans mitted from the affec ted mothe r, wh ile type 1 diabe tes se ems to b e more oft en trans mitted from the affec ted father. Fina lly, one of the most common and well kno wn no n-Mendeli an featu res of complex diseas e i s differenti al suscept - ibilit y t o a diseas e i n males and females , o r sexual dimo rphism (2). This article will revie w epide mio- logic al an d gen etic findings of gen der effects in com plex diseas e. The other objectiv es are: 1) to int roduce the conce pt of epigen etics an d dem on- strat e that gende r effec ts are consis tent with the epi genetic int erpret ation of compl ex diseas e; and 2) to desc ribe experi mental strategi es for the ident ifica- tion of the molecul ar substrat e o f gen der effects, wh ich may cont ribute to uncoveri ng the fund amen- tal molecul ar mechanis ms of compl ex diseas e.

Ge nder and comp lex disease : epidem iolog ical and mo lecular gene tic findings

Sexua l dimorph ism in compl ex diseases presen ts with an unev en diseas e frequ ency in m e n and wome n: althoug h both gen ders can be affec ted, on e i s m ore sus ceptible. Multip le sclerosis , rheuma - toid arthrit is, Crohn ’s diseas e, panic disorde r, stru ctural heart diseas e and hyp erthyro idism are m ore common in females , while males are more often affected with autism, Hi rschspr ung’s dis ease, ulcer ative coli tis, Parkin son’s dis ease, alcohol ism, alle rgies and asthma (especi ally at young age) (3) (Fig ure 1). In psyc hiatri c dis eases such as Al zheimer ’s dis ease, schizophre nia, alcoho lism, and m ood and anxiety disorde rs, psyc hopatho logy exhi- bits a numbe r o f differenc es betw een the sexes in rates of illne ss as well as the course of illne ss (4). It is impo rtant to note that se x effe cts in com plex dis eases shoul d be differe ntiated from those in sin gle-gen e disorde rs where sex chromos ome- linked gen es are know n t o be the caus e o f sexual dimorph - ism. In simple Me ndelian disor ders sexual dimorph - ism is clear-c ut and usually affec ts only one of two se xes. For exampl e, on ly m ales (excludi ng so me rare case s o f e.g. skewed X inactivati on) are affecte d with hemo philia and Du chenne’s m uscular dystr ophy, but only female s can have Rett ’s syndrome (a gain, with some rare exception s). Se x horm ones have been the usual ‘culprit ’ i n the expla nation of gende r effec ts in various

Complex diseas e, gender an d epige netics

531

Ke y m e ssages

N

Sexua l dimorph ism in compl ex diseas es presen ts with an uneven disease frequ ency in m e n and wome n: althoug h both gen ders can be affected, one is m ore susce ptible.

N

It can be hyp othesized that differenti al susce ptibility to compl ex diseas e i n m ales and females is mediate d b y sex hor mone- ind uced diffe rences in the epigenet ic regu lation of genes .

N

The epidem iological and molecula r eviden ce of gender effe cts in variou s com plex diseas es warrant s dedica ted molec ular studies that would unco ver underl ying epigenet ic mech anisms and genomic sites that are the prima ry ta rgets of sex horm one actio n.

morpho logica l and phy siologic al differenc es betwee n males and female s (5). Difference s in endo crine mediate d developme nt and maint enance thro ughout the lives of m ales and female s are the se cond factor next to the sex chromos omes that def ine the sexes and are lik ely to drive a d y namic dive rgence of epigenet ic patterns betw een the sexes . From bra in studies it is known that differ ential effec ts of androge ns and estroge ns contribu te to neu ral deve l- opment , affecting progra mmed cell deat h, cellular migratio n, syna ptogene sis and axon al migrati on, an d formatio n of sexuall y distinct neuro nal ci rcuits (6,7). Other studies have show n that an drogens are direc tly related to neurite arb orizati on, while estrogens are responsi ble for synapse format ion and initiat ion of cellular commun icatio n (8). Difference s i n the circulati ng hormone conce ntrations between sexes orchest rate the dive rgence of behav ior and cogniti ve developme nt in males through two related pro cesses known as m asculiz ation and defemi nizatio n mediate d by the es trogen receptor a and b , respecti vely (9). Additiona lly, gona dal hor mones are impl icated in sex differe nces obse rved in neura l protect ion to trauma or ische mic brain injury , with clinical report s sho wing a highe r reco very rate in females than in m ales, an d p rogestero ne and estroge n treatme nts dem onstratin g a neu roprote c- tive effect (5). Nu merous other diseas e-asso ciated traits are differenti ally affected by the sex horm ones. For exampl e, in obes ity, wh ich is a risk factor for coronary arter y disease, diabetes, osteo arthritis , a n d some m etabolic dis orders (10,11) , hor mones have been impl icated in the contro l o f a d ipose tissu e prolifera tion and estroge n and testos terone r eplace- ment thera py in olde r wome n and m e n resultin g i n a reduction of obesity (11). Estrogen replacem ent also

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532 Z. Kamins ky et al.

i a] A t : 20:41 3 March 2007 532 Z. Kamins ky et al. Figure

Figure 1. Complex non-Mendelian diseases displaying differential susceptibility to complex diseases (based on epidemiological data obtained from Harrison’s ‘Principles of Internal Medicine’ 15th edition, 2001) (111).

at tenuates Alzheime r’s diseas e (AD) sym ptoms in pos tmenopa usal wome n (12). Ad ditionall y, AD- affec ted men have sign ificant ly low er levels of te stostero ne in the brain compare d t o contro ls (1 3). Animal models of disease offer a valua ble res ource f o r stu dying the hormon e-associ ated as pects of suscept ibility to com plex diseas e. Fem ale no nobese diabeti c (NO D) mic e are m ore oft en affec ted by type I diabe tes than the m ale m ice of the sa me strain (14). Neo natal sex hormone m anipula tion demon strated that disease phenoty pe in cast rated mal es and ovarie ctomize d females inc reased and decrea sed, respectiv ely (15). It is interestin g t o note that gende r effec ts have also been detect ed in genet ic lin kage and associati on stu dies. There is now an incre asing list of molecul ar gen etic find ings indicatin g that genetic mark ers on au tosome s also can be gen der dependen t (Table I). For exampl e, gen etic linkage studies in autism reve aled that 17q 11 at markers D17S12 94 and D1 7S798 reac hed genome-w ide sign ificance with the linkage strat ificati on on the basis of gen der ( P 50.008) (16). Anothe r group performe d a search for suscep tibility loci stratifi ed for the sex of the affec ted pro band using a numbe r o f markers on chrom osomes 2q, 7q , 9 p , 15q and 16p (17 ). Ma le- fema le and female-fe male pairs show ed evid ence for

linkage to chrom osome 15q markers ( D15S11 7D15S12 5, P 5 0.0011), while male pairs only dem on- strated sign ificance on 16p ( D16S40 7 D16S497 , P 5 0.026) with a trend toward s signifi cance obser ved on 7q ( D7S 480 D7S530 , P 5 0.075) (17). In major dep ression studies, a genome -wide lin kage study in a sample of 110 Utah ped igrees with a history of major depressi on ident ified sign ificant logarit hm of odd scores (LOD ) signifyin g linka ge at 12q22 -q23.2 mark ers speci fic only to males ( D12S1300 , LOD 5 4.6, P 50.000 03; D12S 1706 LOD 56.1, P 50.000 0007) (18 ). Anothe r genome scan reve aled a numbe r o f putat ive loci of linkage to major dep ressi on females on ly, with the m ost promine nt lin kage on 2q 33-q35 display ing LOD scores of 6.3 and 6.9 at D2 S2321 and D2S2208 , respecti vely (1 9). A h aplotype analysis was per- formed on the TRA X/DISC1 region previou sly associa ted with schizophre nia in a Scottish popula- tion (20). A case-c ontrol assoc iation study ident ified signifi cant as sociation of DISC 1 with bipola r dis- order in wome n ( P 5 0.0002 6) (20). Anothe r study invest igating 28 SNPs inclu ding TRAX , DISC 1, a n d DISC 2 in a F i nnish coho rt of 458 individu als determ ined a signifi cant undert ransmi ssion of the HEP3 hap lotype to affec ted females ( P 5 0.00024), sugges ting tha t this hap lotype may confe r a

Complex diseas e, gender an d epige netics

533

Table I. Some loci and markers exhibiting gender-specific evidence for association (A) and linkage (B) in complex diseases and traits. Not all significant findings from each reference are reported. A. Association studies:

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Disease or trait Gene

Polymorphism (allele, genotype, haplotype)

Sex

Statistic

Reference

Age-related maculopathy

DRD2

141C Ins/Del

Female

P v0.05

(112)

DRD3

MscI Gly/Gly

Female

P v0.05

(112)

Alzheimer’s disease TFAM COMT Asthma

rs1937 G/G RS4680, RS4680, RS737865

Female

P v0.05

(113)

Female

P v0.05

(114)

Cox-2

765 C/C

Female

P50.01

(115)

Atopy Angiotensinogen gene Angiotensinogen gene Autism

235 Met/Thr

Female

P50.04

(116)

-6 A/G

Female

P50.016

(116)

SLC6A4

rs140700

Male

P v0.05

(117)

Behcet’s disease (uveitis)

CCL2

22518 GA/AA -403 AA 22076 AA/AA or AA/AT

Female

P50.02

(118)

CCR5

Male

P50.005

(118)

CCL2

Male

P50.02

(118)

Bipolar disorder

GPR78

rs1282

Female

P50.038

(119)

MTHFR Hepatic Lipase (HL) CHD metabolic syndrome LRPAP PRCP LDLR Multiple sclerosis IFNG IFNG Obsessive compulsive disorder DRD2 with COMT Osteoporosis MTHFR APOE APOE APOE Parkinson’s disease

677 CT and TT 250 G/A

Female

P50.023

(120)

Male

P50.025

(121)

LRPAP1_1b

Female

P50.0003

(122)

PRCP_1

Male

P50.039

(122)

LDLR_3b

Male

P50.046

(122)

325 A

M ale

P50.044

(123)

761C/A

Male

P50.050

(123)

A2A2 with NlaIII

Male

P50.035

(124)

677 TT

Female

P v0.05

(125)

Haplotype CGTC

Male

P50.001

(126)

Haplotype GGTT

Male

P50.002

(126)

Haplotype GATC

Male

P50.006

(126)

DJ1

rs161799

Female

P50.03

(127)

DJ1

rs226253

Female

P50.002

(127)

Schizophrenia

DISC1

Hep3 Haplotype

Female

P50.00024

(21)

GPR78

rs1282

Female

P50.015

(119)

PLP1

rs475827

Male

P50.0294

(128)

Type I diabetes AR Longevity APOE APOC-I ACE Myocardial infarction FXIII-A ERa

CAG Repeat

Female

P50.03

(129)

Allele E2, E3, E4 Hpa I RFLP Homozygous ALU insertion

Female

P v0.03

(130)

Female

P v0.05

(130)

Female

P v0.05

(130)

Leu34

Male

P v0.001

(131)

397 C/C

Male

P50.003

(132)

CCR2

64I Val/Ile

Female

P v0.01

(133)

Whole blood serotonin levels Chromosome 10 Chromosome 17

D10S1677

Female

P50.0005

(134)

ITGB3_33

Male

P50.0003

(134)

534

Z. Kamins ky et al.

Table I. continued

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B. Linkage studies:

Disease or trait Genomic Location

Marker

Sex

Statistic

Reference

Autism

15q

D15S117-D15S125

Female

MLS52.62

(17)

17q11

D17S1294-D17S798

Male

P50.008

(16)

7q

D7S480-D7S530

Male

MLS52.55

(17)

16p

D16S407-D16S497

Male

MLS52.48

(17)

Major depression

3centr

D3S2323

Female

LOD52.9

(135)

18q

D18S1270

Female

LOD53.5

(135)

2q35

D2S2208

Female

LOD56.87

(19)

2q33

D2S2321

Female

LOD56.3

(19)

1p36

D1S450

Female

LOD53.03

(136)

4q

D4S2631

Male

LOD52.6 ( P50.00027) LOD52.88 (P50.0001)

(135)

15q

D15S1515

M

ale

(135)

Early onset major depressive disease

12q22-q23.2

D12S1300

M

ale

P50.00003

(18)

Osteoporosis

18p11

D18S53-D18S478

Female

LOD52.83

(137)

20q13

D20S196-D20S173

Female

LOD53.2

(137)

2p

194 cM

Male

LOD53.97

(138)

4q

90 cM

Male

LOD54.16

(138)

10q21

D10S196-D10S537

Male

LOD54.42

(137)

Bone mineral density

3p22

76 cM

Female

LOD52.72

(139)

13q14

D13S788

Male

LOD53.46

(140)

Cortisol

10q

97 cM

Female

P50.024

(22)

Eosinophilia

10q

77 cM

Female

P50.0041

(22)

Serotonin

17q

70 cM

Female

P50.0049

(22)

Forced expiratory volume at 1 s/forced vital capacity

 

5q

154 cM

Male

P50.022

(22)

7p

55 cM

Male

P50.0025

(22)

High-density lipoprotein cholesterol

9q

117 cM

Male

P50.0084

(22)

Systolic blood pressure

8q

109cM

Male

P50.0007

(22)

Lymphocyte count

16q

129 cM

Male

P50.029

(22)

% Lymphocyte count per white blood cell count

 

19p

43 cM

Male

P50.027

(22)

Triglycerides

21q

30 cM

Male

P50.048

(22)

Whole blood serotonin levels

6q

183 cM

Female

Pv0.002

(134)

16p

56 cM

Male

Pv0.002

(134)

pr otective effect against the diseas e in wome n (21). Fi nally, a recent quant itative tra it loci [QTL] an alysis of variou s othe r compl ex dis eases found tha t o f the 17 QTLs inve stigate d, 11 were signifi - cant ly sexuall y dimorph ic (P v 0.05) (22). Among the sexuall y dimorph ic traits were trig lycerides , high- den sity lipopr otei n choleste rol, force d expira tory vol ume , all anthropom etric traits an d cortisol levels (2 2). While sex hormones have been the usual ‘culpr it’ in explanations of g ender effects in com plex d iseases

based on the myriad of data associating hormonal dif ferences with disease states and their critical involve- ment in human b iology (5), there are no underlying mech an isms proposed as to how such hormones pr edispose to or protect fr om a d isease relating to the specific molecular m echanisms of horm one action. The g ender-specif ic effects in genetic linkage an d association studies suggest that ch romosomes an d individual genes can be the targ et of sex hor mones. Wh ile such hormones cannot change DNA sequence, they can b e potent m od ifiers of epig enetic status,

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which controls gene ex pr ession and various other genomic activities. It is known that hormones, includ- in g sex hormones, can contr ol gene ex pr ession via ep igenetic m odifications ( 2). Theref or e, it can be hypo- thesi zed that differential susceptibility to complex d isease in ma les and females is media ted by sex ho rmone- induced differences in epig enetic regula tion of genes. In the next section, a brief intr oduction to epig enetics is pr ovid ed .

Ep igeneti cs and its relevance to comple x dise ase

E pigeneti cs refers to regulati on of vari ous genomic func tions that are brought abou t by herita ble, but pote ntially reve rsible chang es in DNA modificati on an d chroma tin structure (23 ). The epigenet ic inform ation is enco ded in two types of syne rgisticall y acti ng covalent modifi catio ns: DN A met hylation , m ore specific ally methy lated cytos ines ( met C), and chrom atin pro teins (m odificatio ns of chroma tin pro teins such as histone acetylatio n, met hylation , pho sphorylat ion) (2 4). In mammal s, DNA methy la- tion occ urs mos t comm only where cytosine is dire ctly followe d b y guanine, form ing wh at is known as a CpG dinucle otide. Cl usters of CpG din ucleo- tid es are referred to as CpG isla nds (25). The epi genetic compl exity confe rred is through DNA an d chromati n mod ification s that can direc tly affect the regulati on of gene activ ity (26–32 ), and othe r impo rtant genomic func tions (33 ,34) includin g gen etic reco mbinatio n (35 ) and DNA mutabi lity (36 ). Ma intenan ce of exist ing DNA methy lation and de nov o DNA m ethylati on is catalyzed by several types of enzymes know n a s DNA -methyl transferas es (37 ). The impo rtance of prop er maint enan ce of m ethylati on through out the g enome is highlight ed by the observat ion that a compl ete loss of main- te nance DNA -met hyltranf erase func tion results in deat h o f mice in early embryoge nesis (38). A large numbe r o f gen es exhibit an inverse correlati on bet ween the degree of DNA methy lation an d the m agnitude of gen e expres sion (39,40) . Trans - cri ption factor binding affini ty may be limited by the presence of methy lation at the binding sites (28 ,31). In addit ion to the ‘critic al site’ effec ts of met C, the den sity of met C in a gene regu latory region also cont ribute s t o gen e acti vity. DNA modifi cation acts in concer t with hist one modifi cations, anothe r epi genetic mecha nism. Alte rations in chroma tin stru cture occ ur through acetylati on, m ethylati on an d pho sphorylat ion of various histone ami no acid res idues includin g lysine, arg inine and serine (41– 45) . The informa tion within the patterns of such m odificati ons is sometime s referred to as the ‘hist one code’ (2 4). Epige netic modifi cations

Complex diseas e, gender an d epige netics

535

regulate genomi c functi oning not on ly in te rms of gene expr ession but al so in the supp ression of repetiti ve DNA sequenc es (46) and the formatio n of archite cturall y function al chroma tin structure s such as centrome ric regio ns (47). There has bee n significant pro gress in under standin g the compl e- mentary functi oning of epigenetic mecha nisms, i. e. how chang es in DNA modifi cation affec t chromati n conforma tion and vice versa (48–50). It is impo rtant to note that some studies have dem onstrated that genetic differe nces suc h a s single nucleo tide poly- morphis ms (SNPs ) can be pred ictors of diffe rentially methy lated alleles (51–53 ) al lowing for a reinte rpre- tation of the pote ntial importan ce of som e seemingl y function ally irre levant SN Ps associated with variou s compl ex dis eases. Sex effec ts lin ked to or associate d with speci fic SNPs or hap lotypes can be explaine d by hor mone-i nduced m odificati ons such that a specific allele or hap lotype only beco mes a risk factor after som e endocri nologicall y mediate d epi- genetic mod ification . There is incre asing evid ence that putativ e epi ge- netic misr egulati on of genes m a y explain numero us epidem iological, clinical, and m olecular compl ex- ities of compl ex dis ease (2). It has been argued that the heu ristic value of the epi genetic model of compl ex diseas e lies in the possibil ity of integr ating a variety of unrelated data int o a new the oretical framewo rk, providin g the basis for new experi men tal appro aches (54–58 ). Shifti ng the emphas is from DNA seque nce variat ion and a haz ardous envi ron- ment as the main et iological f actors to putat ive epigenet ic misr egulati on pro vides a new perspe ctive on the myria d of the above liste d non-Me ndelian features (preval ence of spo radic case s, discord ance of identic al tw ins, parenta l origin effe cts, la te age of diseas e onse t a n d fluctuatin g course, among othe rs) that cann ot be explaine d by m ore tradi tional mechanis ms (59). In the epig enetic m odel of compl ex non-Me ndelian dis ease, pathology is seen to result from a chain of unfavora ble epi genetic events tha t begi n w i t h a primary epi genetic defect, or preepi mutatio n, occ urring in the germl ine duri ng the error-pron e epi genetic- reprogr amming process (60,61) . Pre epimutat ion increases the risk of deve l- oping dis ease, but unlike the det erministi c DNA mutat ions in Me ndeli an disorde rs, a preepi mutatio n does no t nece ssarily ind icate that the disease is inevitabl e. Such preepi mutat ions may not caus e any immediat e clini cal problem s, and thus the age of onset may be dela yed for a rel atively long time. It may take decade s until the epigen etic misregu lation reaches a critica l threshol d beyond whic h the cell is no longer able to func tion norma lly. The phen otypic outcom e dep ends on the overall effe ct of a series of

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536 Z. Kamins ky et al.

pr e- and postn atal impac ts on suc h a preepi muta-

tion. Preepi mutatio ns are subjec t t o furth er chang es

by the mult idirectio nal effects of tissu e differenti a-

tion, stoc hastic events, some ext ernal enviro nmental factors (nutrition, med ications, etc.) (62–64), and

also the hormon al milieu. It is known that various

hor mones , inclu ding se x hormon es, have a signifi -

cant impac t o n gen e expres sion, and this is achiev ed

by chang ing chroma tin conformat ion (65 –68) an d/

or lo cal pattern of gene m ethylati on (6 9,70). Som e exam ples of how se x hormon es can infl uence

epi genetic regu lation of genes are provided below.

E pigeneti cs and sex hormone s

A numbe r o f earlier studies have demon strated the

dire ct effects of sex hormon e admini stration on

epi genetic stat es. An exampl e i s the effect of

es tradiol adm inistration on the methy lation stat us

of vari ous CpG dinucle otides locat ed in an estradi ol-

m

ediated regu latory regio n for the avian vitell ogenin

II

gene (6 9,71–73) . Two of these methy latable

cytos ines lo cated withi n the estradi ol-rece ptor bind -

ing site are activ ely demet hylated in response to

es tradiol tre atment in h ormone respo nsive tissues in

a stran d-spec ific manne r (69). This DNA demet hy-

lati on persists after transcri ption has ended (71,73)

an d has been sugg ested to res ult in a ‘mem ory effe ct’

res ulting in a quicke r ind uction of vite llogeni n I I

m RNA synth esis in response to subseq uent estradi ol

sti mulations (69). This finding highlight s the

fact that endocri nologica l influenc e can med iate

lo ng-lastin g epi genetic chang e t o l e vels of gene

tra nscription .

One of the m echanism s of sex hormon e actio n i s thro ugh the m olecular epi genetic sign atures of

par

ticular chromos omal regio ns that mod ulate the

acc

ess of transcripti on factors to the transcri bed

se quenc es. If DNA is tigh tly packed arou nd the

hist one prote ins, the acces s o f trans cription factors

to the ir respecti ve transcripti on factor binding sites is

res tricted (24,74– 76) . Such compac ted chrom atin is

supp orted by high den sity of met C i n the DN A

se quenc e and vari ous types of supp ressi ng mod ifica-

tions of ami no-term inal residues of histo ne proteins ,

e.g. deac etylation (75). Conve rsely, low den sity of

m e t C in the DNA seque nce and high levels of histo ne

ace

tylatio n unrav els the chroma tin, allowin g for

acc

ess of DN A binding el ements and tra nscription al

acti vation (24,74– 76).

Chromat in mod ification can be direc tly affec ted by members of the nuc lear horm one recep tor (NHR ) superfa mily, and the steroid recep tor (SR) subs et of NHR is of par ticular interest as it contai ns

the androge n recep tor (AR) and es trogen recep tor (E R) (77). The se sex hormon e recep tors can have

activat ing or repres sing trans criptiona l activ ity depen dent on the presence or absence of ligand, respecti vely (78,79) . Steroi d hormone mediate d transcri ptional activ ation or repres sion result s from the SR recrui tment of coactiva tor and corepre ssor compl exes— protein compl exes wh ich associa te with variou s epigen etic modifi ers suc h a s histone deac e- tylases (HDA C), histone acetylt ransfera ses (HAT ) and histone methy ltransfe rases (HMT) (78,80) . I t i s these ‘co regulatory compl exes’ that achieve the epigen etic modifi catio n nece ssary for chrom atin remodeli ng, allowin g o r res tricting acc ess of tran- script ion factors an d RNA poly meras e I I a n d thus media ting the epigen etic effects of the sex hor mones (Figure 2). There are two primary types of core gulat ory compl exes, the first being the adenos ine trip ho- sphate (ATP)-d epend ant chromati n remodeli ng compl exes (Fig ure 2), such as the swit ch/sucros e nonfe rmenting (SWI/S NF) compl ex (77). The ATP-de pende nt chromati n remode ling compl exes may be p rimarily responsi ble for openi ng of chro- matin (81 ,82), form ation of nucleo some arrays , homol ogous stran d pairing and DNA trans cription (83). Only recentl y, the actio ns of SWI /SNF have been linked to changes in DNA met hylation thro ugh a mechanis m med iated by int eraction s with methy l CpG binding p rotein 2 (MeC P2) (84). SWI/SNF interac ts with both the E R and AR, and mutat ions in a core subu nit have been impl icated in the develop- ment of ovarian canc er (85). Histon e-modif ying compl exes cons titute the sec- ond type of coregul atory com plex that int eract with the sex hormon e recep tors. Coact ivators inclu de the cyclic aden osine mono phosphat e (cAM P) response element -bind ing pro tein (CBP)/p3 00, while the most widely studied corepre ssor compl exes are histone -modifyin g machi nes such as the nuc lear recep tor corepre ssor compl ex (NCoR ) and the silenci ng med iator of ret inoic acid an d thyro id hormon e receptor (SMR T) (77) (Figure 2). Histon e mod ification s m ediated by histone -modify- ing machi nes unrav el and compac t chromati n through ace tylatio n and deacety lation of lysine residu es on histone H4, respectiv ely, and alternati vely through met hylation of histone lysine and arginine res idues (74,86) . I n addit ion to recep tor agon ist bind ing, an tagonists to the proges- terone recep tor (PR) an d androge n receptor (AR) elicit an interac tion with NCoR and SM RT, demon strating that antago nistic actio ns are media ted through corepre ssor recrui tment (77). There are nume rous molecul ar m echanism s through which AR an d ER coactiva tor/core pressor- binding modulat e chrom atin struct ure and influe nce

Complex diseas e, gender an d epige netics

537

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Figure 2. In the absence of ligand or in the presence of antagonist, steroid receptors (SR) recruit transcriptional corepressor complexes to the DNA, producing an epigenetically silenced state through histone deacetylation or methylation. In the presence of hormone, S R recruit coactivator complexes, histone-modifying machines or ATP-dependent chromatin remodeling machines, t o acetylate histones and produce a euchromatic chromatin formation suitable for transcriptional activation.

epi genetic marks; however, the dis tributio n and expr ession of the g enes enco ding the core gulatory factors have been shown to vary between the sexes . Ge nder differe nces in the expression of steroid recep tor co-a ctivator- 1 (SCR-1 ), CBP/p300 , NCo R, an d S M R T were obser ved in vari ous tissu es in male and female rats (87). Furthe rmore, regu la- tion of trans cript levels can be influe nced by the

differenc es in circu lating hormon es between males and females , a s estradi ol differenti ally influe nced SCR-1 and SM RT trans cript le vels in the anterior pituitar y of m ale and female rats (87). The refore, despite bei ng ubiq uitousl y expresse d, the gen der- and tissu e-spec ific coregul ator trans cript differe nces have been sugg este d t o result in vari ability in the level of respo nse to horm one (87), a finding that

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538 Z. Kamins ky et al.

coul d expl ain obs ervations of tissu e-specific differ- ence s in E R-medi ated response to ligand. AR and E R differ entially interact with core gula- to ry compl exes through binding to different types of a -h elix L XXLL motifs on the com plexes (88,89) . Thro ugh these interac tions, recent stu dies have sugg este d that differe nt typ es of ligand binding to the sex hormon e recep tors or conformat ional chang es over time may infl uence the ability to rec ruit vari ous cofact ors, as in the case of AR and E R wh ere chang es in coa ctivator- binding have been lin ked to prostat e and breas t canc er developm ent, res pectiv ely (90,91) . In a similar way, differenc es in AR and ER int eraction s with coactiva tor L XXLL m otifs (88) m ay result in the recruitme nt of differe nt epi genetic modifyin g coregul atory comp lexes bet ween the se xes. Finally, variou s studies have demon strated that tis sue-spec ific distributi on of the sex h ormone rec eptors vari es betw een the sexes (92) as do the ta rget gen es for trans cripti onal regu lation, and thus the epigenetic m odificati ons m ediated through the se x hormone recepto rs will lik ely vary by gen e and tis sue between the sexes. A study investigati ng the tra nscription al regulati on of the pro lactin gene te sted the effec ts of estradi ol administ ration in GH 4 cells (93). Estrad iol was found to have a sti mulating effec t on hist one H4 ace tylatio n i n the pr omoter regio n o f the pro lactin gene (93 ). In agr eemen t with othe r studie s, such histone modifi - cati ons are impl icated as necess ary factors in gen e tra nscription via the formation of a euchro matic stat e o f the pro moter regio n (94). Various form s o f hist one m ethylati on are assoc iated with estradi ol bind ing to the estroge n recep tor (ER) , affecting the tra nscription at the targe t prom oter in vitr o (77,95) an d furth er highlight ing that sex horm one-regul ated gen e transcripti on is associate d with the form ation of an epigen etic mark. In rat, es trogen treatme nt was sho wn to inc rease DNA methy lation on the prolact in gen e i n pituit ary an d live r tissues wi th subs equent red uction in mRNA amounts , sugges ting a direct effe ct on trans cription in a tissue-s pecific man ner (9 6). Prolac tin administ ration caused dem ethylati on of DNA in rat live r and kid ney in both mature and imm ature rat s (97). Pre natal expos ure to diethyl - sti lbestrol, a synthetic form of estrogen that has been impl icated in develo pment of cancer s i n huma ns and m ice in later life, perman ently alters the DNA m ethylati on pattern of the oncoge ne, c-fo s, i n mice, impl icating an epigen etic mecha nism for hor mone- ind uced carc inogene sis (98 ). Testo sterone and es tradiol administ ration have been shown to affect amyl oid precurso r protein (AP P) levels in AD m odel m ice (99 ). DNA methylati on levels at the APP

promot er exhibit a sex differ ence in mice an d are general ly highe r i n females (99). Increas ed le vels of estroge ns in transgen ic breas t cancer m odel m ice lead to epigen etic modifi cations in cancer rel evant genes involve d in cell cycle, cell pro liferati on an d apopto sis (100 ). Fina lly, inve stigation of the effe cts of estro gen on Japane se med aka fish demon strated that DNA met hylation varied bet ween tissues in a gende r-specific man ner (101).

Molecul ar strate gies for iden tificatio n of gende r specifi c epigen etic diffe rences

T h e e p i d e m i o l o g i c a l a n d m o l e c u l a r e v i d e n c e o f

g e n d e r e f f e c t s i n v a r i o u s c o m p l e x d i s e a s e s w a r r a n t

d

e d i c a t e d m o l e c u l a r s t u d i e s t h a t w o u l d u n c o v e r

u

n d e r l y i n g e p i g e n e t i c m e c h a n i s m s a n d g e n o m i c

s i t e s t h a t a r e t h e p r i m a r y t a r g e t s o f s e x h o r m o n e a c t i o n . A s m e n t i o n e d a b o v e , e v i d e n c e f o r l i n k a g e o r a s s o c i a t i o n o f a p a r t i c u l a r g e n o m i c r e g i o n c o u l d b e

a m a r k e r f o r d i f f e r e n t i a l e p i g e n e t i c m o d i f i c a t i o n o f t h a t r e g i o n , t h e r e f o r e t r a n s f o r m i n g t h o s e l o c i i n

T a b l e I i n t o c a n d i d a t e t a r g e t s f o r g e n d e r - s p e c i f i c

e p i g e n e t i c e v a l u a t i o n . S i t e - s p e c i f i c m e t h o d s f o r t h e

e p i g e n e t i c i n v e s t i g a t i o n o f c a n d i d a t e l o c i e x i s t , s u c h a s i n s o d i u m b i s u l f i t e m o d i f i c a t i o n - b a s e d m a p p i n g o f m e t h y l a t e d c y t o s i n e s , o r c h r o m a t i n i m m u n o p r e - c i p i t a t i o n a s s a y s f o r h i s t o n e m o d i f i c a t i o n s t u d i e s .

S o d i u m b i s u l f i t e m o d i f i c a t i o n i s c o n s i d e r e d t h e

g o l d s t a n d a r d i n D N A m e t h y l a t i o n m e a s u r e m e n t a n d f u n c t i o n s t h r o u g h s e l e c t i v e l y d e a m i n a t i n g

u n m e t h y l a t e d c y t o s i n e s t o u r a c i l w h i l e m e t h y l a t e d

c y t o s i n e s r e m a i n u n c h a n g e d . A f t e r p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) a m p l i f i c a t i o n , t h e r e l a t i v e

p r o p o r t i o n s o f D N A m e t h y l a t i o n c a n b e d e t e r -

m i n e d a t e a c h C p G d i n u c l e o t i d e t h r o u g h a v a r i e t y

o f m e t h o d s i n c l u d i n g s e q u e n c i n g o f c l o n e s ( 1 0 2 , 1 0 3 ) , p y r o s e q u e n c i n g ( 8 0 ) , M A L D I m a s s s p e c t r o m e t r y ( 1 0 4 , 1 0 5 ) , a n d S N a P s h o t ( 1 0 6 ) , a m o n g s t o t h e r s . C h r o m a t i n i m m u n o p r e c i p i t a t i o n

i s p e r f o r m e d w i t h a n t i b o d i e s f o r m o d i f i e d h i s t o n e r e s i d u e s , s u c h a s H 4 - K 9 m e t h y l a t i o n f o r e x a m p l e , f o l l o w e d b y r e a l t i m e P C R a m p l i f i c a t i o n o f a c a n d i d a t e r e g i o n i n o r d e r t o q u a n t i f y l e v e l s o f

h i s t o n e p r o t e i n m o d i f i c a t i o n ( 1 0 7 ) . U s e o f t h e s e

m

e t h o d s w o u l d a l l o w f o r t h e l o c u s - s p e c i f i c d e t e r -

m

i n a t i o n o f e p i g e n e t i c d i f f e r e n c e s i n p o p u l a t i o n s

s t r a t i f i e d f o r s e x ; h o w e v e r , w h i l e t h e s e c a n d i d a t e l o c i m a y r e p r e s e n t t a r g e t s o f e p i g e n e t i c m o d i f i c a - t i o n t h r o u g h t h e a c t i o n s o f s e x h o r m o n e s , t h e o v e r w h e l m i n g m a j o r i t y o f s e x h o r m o n e t a r g e t s i n t h e g e n o m e r e m a i n u n k n o w n . F o r t h i s r e a s o n , a

c o m p r e h e n s i v e e p i g e n e t i c i n t e r r o g a t i o n o f t h e e n t i r e g e n o m e w o u l d b e o p t i m a l f o r i d e n t i f y i n g t h e y e t u n k n o w n t a r g e t s o f s e x h o r m o n e - m e d i a t e d

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e p i g e n e t i c m o d i f i c a t i o n t h a t m a y b e a s s o c i a t e d w i t h s e x e f f e c t s i n d i s e a s e . The advent of microarr ay te chnolo gies that inter- rog ate a large numbe r o f DNA/R NA frag ments in a highl y paralle l fashion has also opened new oppor - tun ities for epi genetic studies (55,57,58, 108). The m icroarray- based large-sc ale DNA modificati on an alysis is base d o n interroga tion of the unme thy- late d (or hyperme thylated) fraction of genomic DN A o n the micro array cont aining oligonucl eotides that repres ent the gen omic regio ns of interest . E nrichme nt of the unme thylated fract ion of genomi c DN A is performe d through a numbe r of steps that include digest ion with m ethylati on-sensi tive

Complex diseas e, gender an d epige netics

539

restrict ion enz ymes. Such enzymes cut the unmethy- lated fract ion of gen omic DNA into short fragmen ts, wherea s the methy lated DNA se quences remain undige sted. In the next step, the restrict ion enzyme- treated DNA is ligat ed to DNA ad aptors an d subjecte d t o polymeras e chain reac tion (PC R). PCR primers compl ement ary to the adaptor sequenc es prefere ntially amp lify the sho rter DNA fragmen ts, namel y the unme thylated DN A fraction. Fluores cently labele d amp licons are the n hyb ridized to the microarr ays. The lower the deg ree of DNA methy lation at som e speci fic site of the gene of interest , the stron ger the hybridiza tion sign al seen on the array. Additiona lly, antib odies spe cific to

seen on the array. Additiona lly, antib odies spe cific to Figure 3. Loci identified as

Figure 3. Loci identified as significantly different in DNA methylation between male and female postmortem brain samples. DNA methylation profiles were measured by human CpG island microarrays in a common reference design. Each microarray interrogates 12,912 loci whose locations o n the chromosomes are shown in white in the plot. Data were logarithmically transformed and then normalized, by the print-tip loess method, to remove nonbiological effects. Vertically: human chromosomes 1 to 22. The t-statistic of the male group versus female group was calculated for each locus and its P -value was also obtained. Loci with P -values below 0.05 are painted black in the plot.

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540 Z. Kamins ky et al.

m ethylate d cytosine s have been develope d and

app lied in acc ordance with mic roarray technolo gy to interroga te DNA met hylation withi n the g enome (1 09). In addit ion to DNA m ethylati on, histo ne res idue modifi cation can be invest igated using chrom atin immunop recipitati on on micro array chips (Ch IP-on-c hip) (108 ).

As an example of the microarray-based approaches,

a recent analysis adds weight to the hypothesis that

some molecular causes of gender differences may manifest in different profiles o f epigenetic markers between the sexes. As a part of our ongoing study o n epigenomics of human brain w e performed a pilot analysis o f e pigenomic profiles of postmortem brain samples stratified for gender. The unmethylated fraction o f DNA extracted from the prefrontal cortex of postmortem brains was interrogated o n the 12K CpG island microarray (58,61). The group o f males (n520) was compared to the group o f females (n57). DNA methylation differences in males and females (P v0.05) identified by the microarray between the sexes are shown i n Figure 3. While this pilot data i s not corrected for multiple testing, the identified regions might serve as a starting point f or the in- depth epigenetic studies of sex differences, mapping of gender-dependent epigenetic e ffects across the entire genome, and analysis of the relevance of such sites t o various complex diseases.

Experime ntal techn iques applied to invest igate

lo ngitudinal chang es in gender- specific epigen etic

pat terns will be of particul ar int erest. Also, studies

on epigen etic effects of horm ones may allow for the ide ntification of critica l precli nical epi genetic stat es tha t coul d b e use d i n early diagnos tics as well as in des igning new epigen etic thera pies (110). It is our hop e that through an accelerat ion of molecul ar bio logy technolo gies of epigenet ic investiga tion and

a combi nation of an imal and human studies, the r ole of epigen etic factors in compl ex dis ease—bot h

m ediated through sex horm ones and othe r m echan- isms —will soon be elucidate d.

Ac know ledgeme nts

This res earch has been supp orted by the Ontari o

Me ntal Hea lth Foun dation, Canadia n Inst itutes for Hea lth an d Research , Nationa l Inst itute of Mental Hea lth (R01 MH074 127-01) , as well as NARSA D

an d the Stanley Foun dation. Zacha ry K aminsky is a

CIHR Doc toral Fellow. Special thanks to Sigr id Z iegler for her critical reading of the m anuscri pt.

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