M a trix /S c a ffo ld A tta c h m e n t Regions
The e u k a ry o tic genom e is organised in a lo o p w is e m a n ne r w ith c h ro m a tin lo o p s c o rre s p o n d in g to fu n c tio n a l d o m a in s ; M a tr ix a n d S c a ffo ld A tta c h m e n t R egions (M A R s /S A R s ) c o n s titu te the base o f these loo p s. M A R s /S A R s are several h u n d re d b p in le n g th and are A T -ric h sequences w h ic h a n ch o r c h ro m a tin lo o p s to the c h ro m o so m e n u c le a r m a tr ix o r s c a ffo ld th e re b y , e s ta b lis h in g a to p o lo g ic a lly in d e p e n d e n t d o m a in (M irk o v itc h et al., 1984; C o c k e rill and G arrard, 1986; L a e m m li et al., 1992). It has been s h o w n th a t these A T -ric h sequences c o n ta in to p o iso m e ra se I I cleavage sites and possess stro n g n u c le o tid e -u n p a irin g p ro p e rtie s (Bode et a l., 1992), w h ic h fa c ilita te u n z ip p in g o f th e d o u b le h e lix d u r in g
tra n s c rip tio n in itia tio n o r D N A re p lic a tio n (H om b e rg er, 1989; O p s te lte n et al., 1989; D ijk w e l and H a m lin , 1995). The in te ra c tio n o f M A R s /S A R s w it h the n u cle a r sca ffo ld is n o t d e te rm in e d b y precise m o tifs , b u t b y s tru c tu ra l features o f A T -ric h regions, such as a n a rro w m in o r groove w h ic h have to be o f a certain len g th to e x h ib it a specific in te ra c tio n (A d a ch i et al., 1989; Kas et al., 1989).
Multiple faces of M A R s / S A R s . I t has been sh o w n th a t the M A R s /S A R s are a sso cia te d w it h s e v e ra l typ e s o f gene r e g u la to r y e le m e n ts. M a n y M A R s /S A R s w ere fo u n d in the v ic in ity o f p ro m o te rs and enhancers and some M A R s /S A R s w ere sh o w n to act as b o u n d a ry in s u la tin g elem ents. (H y rie n et al., 1997; D avie, 1995; D ijk w e l and H a m lin , 1995). For exam ple, it has been fo u n d th a t an A T -ric h d o m a in in the h u m a n th y ro tro p in -p gene p ro m o te r has a sile n cin g effect on its p ro m o te r, th u s a ctin g as a silencer e le m e n t (K im et al., 1996). O th e r M A R s /S A R s w e re fo u n d in close association w ith enhancer elem ents (Casser and L a e m m li, 1986; C o c k e rill and C a rra rd , 1986) w it h a recent re p o rt suggesting th a t some M A R s /S A R s are able to m a in ta in e xp re ssio n even w h e n th e enha n ce r is d e le te d (W ie rsm a et al., 1999). A T -ric h regions w ere also fo u n d w it h in the LCRs, e.g. o f the h u m a n C D 2 (Lake et al., 1990) and p -g lo b in genes (B o u lik a s , 1993). Some SARs w h e n placed in fla n k in g p o s itio n s o f the re p o rte r gene w e re able to s tim u la te e x p re s s io n o f th e v a rio u s h o m o lo g o u s a n d h e te ro lo g o u s re p o rte rs in te g ra te d in the genom e, b u t n o t in tra n s ie n t tra n s fe c tio n assays, and so act as b o u n d a ry elem ents (L a e m m li, 1992). A T - ric h regions w ere also fo u n d w ith in the g yp sy in s u la to r (Spana and Corces, 1990; N a b iro c h k in et al., 1998), as w e ll as w it h in in s u la to rs o f the h u m a n
a p o lip o p ro te in B and alpha 1 - a n titry p s in gene lo c i (K alos a n d F o u rn ie r, 1995; N a m c iu et al., 1998).
T hus, it appears th a t M A R s /S A R s are associated w it h v a rio u s re g u la to ry ele m e n ts w h ic h act v ia c h ro m a tin lo o p in g an d th e e s ta b lis h m e n t o f s tr u c tu r a l/fu n c tio n a l d o m a in . In a d d itio n , evid e n ce has e m e rg e d th a t a tta ch m e n t to the n uclear m a trix is re q u ire d fo r D N A re p lic a tio n (D ijk w e l a n d H a m lin , 1995). W h ile recent data show s th a t M A R s /S A R s p la y an im p o rta n t ro le in m ito tic chrom osom e co n de n sa tio n and m a in ten a n ce o f ch ro m o so m a l stru c tu re (H a rt and L a e m m li, 1998).
CpG Isla n d s and M e th y la te d D N A
C pG isla n d s have been fo u n d at the 5'-ends (in c lu d in g fir s t exon) o f a ll housekeeping genes and a large n u m b e r o f tissue specific genes (Cross and B ird ; 1995). The C pG isla n d s in h o u se ke e p in g genes are c o n s titu tiv e ly u n m e th y la te d , w hereas in m o st tissu e -spe cific genes th e y are g e n e ra lly m o d ifie d in every cell typ e except those th a t a c tu a lly express the gene. The m é th y la tio n p a tte rn o f the genes is established de n o vo w it h each ce ll cycle (Y e iv in and R azin, 1993). The n o n m e th y la te d C pG isla n d s c o rre s p o n d to n u c le o s o m e - fr e e p r o m o t e r r e g io n s , w h e r e th e c h r o m a t in is unde ra ce tylate d and lacks histone H I (Cross and B ird ; 1995). M é th y la tio n o f the C pG islands causes the tra n s c rip tio n a l repression o f the loca l gene and such a m ech an ism o f gene in a c tiv a tio n w as fo u n d in several tu m o u rs w h e re the tu m o u rs occured due to h y p e rm e th y la tio n o f p l6 , p l5 , V H L and E-cad tu m o u r suppressor genes (G onzalgo and Jones, 1997; B a y lin et al..
1998). The reasons fo r such an aberrant h y p e rm e th y la tio n o f C pG islands is unclear and, in some cases, c o u ld be accounted fo r b y the increased a c tiv ity o f the D N A -m e th y ltra n s fe ra s e (D N A -M T A s e ) in tu m o u r cells.
H o w e v e r, m é th y la tio n p e r se m a y n o t be s u ffic ie n t fo r repression. Thus, b o th m e th y la te d and u n m e th y la te d tem plates are in itia lly e q u a lly active in X enopus oocyte n u c le i, b u t a fte r lo n g e r in c u b a tio n the m e th y la te d D N A converted to an in a ctive fo rm (Kass et al., 1997), suggesting a fo rm a tio n o f in a ctive c h ro m a tin stru c tu re in d u ce d b y the D N A m é th y la tio n .
This h ypothesis was fu rth e r su p p o rte d b y id e n tific a tio n o f the M e C P l and M eCP2 repressor p ro te in s capable o f b in d in g to m e th y la te d C pG D N A (M eehan et al., 1989; N a n et al., 1997). I t has been sh o w n th a t M eCP2 is able to re c ru it Sin3 and h is to n e acetylases ( H D A C l, H D A C 2 ) to c h ro m a tin c o n ta in in g m e th y la te d D N A to w h ic h it is b o u n d . These and o th e r data ind ica te th a t the D N A m é th y la tio n trig g e rs a sequence o f events le a d in g to the fo rm a tio n o f repressive c h ro m a tin s tru c tu re (N a n et al., 1998a; N a n et al., 1998b; Jones et al., 1998).
F in a lly , i t is m é th y la tio n w h ic h is re sp o n s ib le fo r th e p h e n o m e n o n o f im p r in tin g , re s u ltin g in differences in expression b e h a v io u r o f tw o alleles in h e rite d fro m each p a re n t. The im p o rta n c e o f m é th y la tio n in th e re g u la tio n o f an o v e ra ll gene expression m echanism w as v e rifie d in the D N A M T A s e k n o c k o u t e x p e rim e n t w h e re the m u ta n t m ice are le th a l at the e m b ry o n ic stage w hereas im p rin te d p a te rn a l m é th y la tio n o f several genes is absent in the m u ta n t m ice (L i et al., 1993).
Regions o f DNAse I Hypersensitivity
R e g u lato ry elem ents o f active genes and o rig in s o f re p lic a tio n s are ty p ic a lly associated w it h h y p e rs e n s itiv ity to d ig e s tio n b y D N A s e I (and o th e r nucleases) w h ic h is one to tw o o rd e rs o f m a g n itu d e h ig h e r th a n th e s e n s itiv ity o f b u lk c h ro m a tin (E lgin, 1981; Gross and G a rra rd , 1988; W u et a l, 1989). The regions o f the h y p e rs e n s itiv ity correspond to areas o f naked D N A , w he re the nucleosom es are e ith e r absent o r p a r tia lly d is ru p te d th u s re n d e rin g the re g io n s m o re accessible to D N A s e I. The m a jo rity o f the g e nom ic D N A is w o u n d a ro u n d nucleosom es and packaged in to h ig h e r o rd e r c h ro m a tin stru c tu re s and is th e re fo re , n o t re a d ily a v a ila b le to the D N A se I digest (McGhee et al., 1981). A ty p ic a l h y p e rs e n s itiv ity site is a bout 100-600 b p lo n g and contains b in d in g m o tifs o f the p ro te in s w h ic h b in d to the re g io n and d is to rt D N A w ith in the im m e d ia te v ic in ity (S talder et al., 1980). In such re g io n s histones are ty p ic a lly h y p e ra c e ty la te d an d C pG d in u c le o tid e s u n m e th y la te d (Pasin and Kadonaga, 1997; E den et al., 1998). Sites o f c o n s titu tiv e h y p e rs e n s ity u s u a lly co rre s p o n d to the o rig in s o f re p lic a tio n s and the re g u la to ry elem ents o f house ke e p in g genes, w hereas tis s u e - o r sta g e -sp e cific h y p e rs e n s itiv e sites o fte n c o in c id e w it h th e re g u la to ry elem ents o f genes active in th a t p a rtic u la r tissue o r stage o f d e v e lo p m e n t (S talder et al., 1980; Gross and G a rra rd , 1988; B erberich and Leffak, 1993; Boyes and Felsenfeld, 1996).
F in a lly , it is w o r th to n o tin g th a t a lth o u g h the tra n s c rip tio n a lly a ctive genes u s u a lly ha ve h y p e rs e n s itiv e re g u la to ry elem e n ts, the D N A s e I h y p e rs e n s itiv ity o f the re g u la to ry elem ents p e r se is n o t n e cessa rily an in d ic a to r o f the o n -g o in g tra n s c rip tio n a l a c tiv ity . Thus, th e fo rm a tio n o f
h y p e rs e n s itiv ity sites in the p -g lo b in LC R w as e sta b lish e d b e fo re the