University of Southern California Dissertations and Theses

Collecting epigenetic information with DNA methylation footprinting

(USC Thesis Other)

Collecting epigenetic information with DNA methylation footprinting

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content C o l l e c t i n g e p i g e n e t i c i n f o r m a t i o n w i t h D N A m e t h y l a t i o n f o o t p r i n t i n g
B y
J i n g q i Y u
A T h e s i s P r e s e n t e d t o t h e
F A C U L T Y O F T H E U S C K E C K S C H O O L O F M E D I C I N E
U N I V E R S I T Y O F S O U T H E R N C A L I F O R N I A
I n P a r t i a l F u l f i l l m e n t o f t h e
R e q u i r e m e n t s f o r t h e D e g r e e
M A S T E R O F S C I E N C E
( B I O C H E M I S T R Y A N D M O L E C U L A R M E D I C I N E )
A u g u s t 2 0 2 2
C o p y r i g h t 2 0 2 2 J i n g q i Y u
i i
A c k n o w l e d g e m e n t s
I w oul d l i ke t o of f e r m y gr a t i t ude t o m y a dvi s or D r . Y i f a n L i u a nd c o- m e nt or D r . Y a l i D ou f or
gi vi ng m e t he oppor t uni t y t o c onduc t t hi s pr oj e c t . I a m gr a t e f ul f or t he i r m e nt or s hi p, f e e dba c k,
a nd s uppor t t hr oughout t hi s pr oc e s s . I w oul d a l s o t ha nk m y s upe r vi s or , D r . X ue qi ng W a ng f or hi s
gui da nc e a nd f or t e a c hi ng m e t he t e c hni que s r e qui r e d f or t hi s r e s e a r c h. I a m e xt r e m e l y gr a t e f ul
f or t he a s s i s t a nc e pr ovi de d by D r . W e nt a o Y a ng f or hi s bi oi nf or m a t i c a na l ys i s . I w oul d a l s o l i ke
t o t ha nk D r . L i a ng S ha a nd M a s t e r T i a nya ng B a i a nd Z i Y a ng f or t he i r gui da nc e a nd he l p. I
w oul d l i ke t o t hi nk D r . W ooj i n A n f or hi s
i ns i ght s .
N one of t hi s w oul d ha ve be e n pos s i bl e w i t hout t he s uppor t of m y pa r e nt s . I w a nt t o t ha nk
t he m f or t he i r e nc our a ge m e nt , l ove , a nd e s pe c i a l l y f or be l i e vi ng i n m e .
i i i
T a b l e o f C o n t e n t s
A c k n ow l e d ge m e n t s ........................................................................................................................ i i
L i s t of F i gu r e s ................................................................................................................................ i v
A b s t r ac t .......................................................................................................................................... vi
C h ap t e r O n e : I n t r od u c t i on ........................................................................................................... 1
E p i ge n e t i c s .................................................................................................................................. 1
C ol l e c t i n g e p i ge n e t i c i n f or m at i on w i t h s h or t - r e ad s e q u e n c i n g ............................................. 1
C ol l e c t i n g e p i ge n e t i c i n f or m at i on w i t h D N A m e t h yl at i on f oot p r i n t i n g ............................... 4
D N A N
6
- ad e n i n e m e t h yl at i on ( 6m A ) ........................................................................................ 5
C al l i n g 6m A at t h e s i n gl e m ol e c u l e l e ve l w i t h S M R T C C S .................................................... 6
R e s e ar c h S t r at e gy ...................................................................................................................... 8
C h ap t e r T w o: M e t h od s ................................................................................................................ 12
C e l l c u l t u r e ................................................................................................................................12
C on s t r u c t i on of 6m A - M T as e s ove r e xp r e s s i on ve c t or s ......................................................... 12
C on f i r m at i on of p l as m i d s ........................................................................................................ 13
M T as e p r od u c t i on .................................................................................................................... 13
I n vitro m e t h yl at i on of D N A .................................................................................................... 14
S at u r at e d m e t h yl at i on （ I n vitro m e t h yl at i on of c h r om at i n） ........................................... 15
I m m u n of l u or e s c e n t s t ai n i n g ....................................................................................................15
S M R T s e q u e n c i n g s am p l e p r e p ar at i on .................................................................................. 16
C h ap t e r T h r e e : R e s u l t s ............................................................................................................... 17
6m A M T as e p u r i f i c at i on ......................................................................................................... 17
I n vi t r o m e t h yl at i on ................................................................................................................. 29
I m m u n of l u or e s c e n t s t ai n i n g ....................................................................................................34
P r e p ar at i on an d an al ys i s of S M R T s e q u e n c i n g s am p l e ....................................................... 38
C h ap t e r F ou r : D i s c u s s i on ............................................................................................................ 43
R e f e r e n c e s ..................................................................................................................................... 46
i v
L i s t o f F i g u r e s
F i gu r e 1.................................................................................................................................... 3
F i gu r e 2.................................................................................................................................... 4
F i gu r e 3.................................................................................................................................... 7
F i gu r e 4.................................................................................................................................... 8
F i gu r e 5.................................................................................................................................. 17
F i gu r e 6.................................................................................................................................. 18
F i gu r e 7.................................................................................................................................. 19
F i gu r e 8.................................................................................................................................. 20
F i gu r e 9.................................................................................................................................. 21
F i gu r e 10................................................................................................................................ 22
F i gu r e 11................................................................................................................................ 23
F i gu r e 12................................................................................................................................ 24
F i gu r e 13................................................................................................................................ 25
F i gu r e 14................................................................................................................................ 26
F i gu r e 15................................................................................................................................ 27
F i gu r e 16................................................................................................................................ 28
F i gu r e 17................................................................................................................................ 29
F i gu r e 18................................................................................................................................ 31
F i gu r e 19................................................................................................................................ 32
F i gu r e 20................................................................................................................................ 33
F i gu r e 21................................................................................................................................ 34
F i gu r e 22................................................................................................................................ 36
F i gu r e 23................................................................................................................................ 37
F i gu r e 24................................................................................................................................ 38
v
F i gu r e 25................................................................................................................................ 39
F i gu r e 26................................................................................................................................ 40
F i gu r e 27................................................................................................................................ 42
F i gu r e 28................................................................................................................................ 43
vi
A b s t r a c t
A m a i n que s t i n e pi ge ne t i c s i s t o r e ve a l c hr om a t i n or ga ni z a t i on, e s pe c i a l l y di s t r i but i on of
c hr om a t i n- a s s oc i a t e d pr ot e i ns ( C A P s ) . A t pr e s e nt , t hi s i s us ua l l y a c hi e ve d by c ol l e c t i ng
c hr om a t i n f r a gm e nt s of i nt e r e s t s ( of t e n ge ne r a t e d by nuc l e a s e di ge s t i on) a nd s hor t - r e a d
s e que nc i ng. W e a r e de ve l opi ng a ne w a ppr oa c h i n w hi c h t he l i ne a r or ga ni z a t i on of m ul t i ki l oba s e
c hr om a t i n f i be r s i s t r a ns c ode d i nt o N
6
- a de ni ne m e t hyl a t i on ( 6m A ) s i t e s on t he i r c ons t i t ue nt
D N A m ol e c ul e s by a l t e r e d m e t hyl t r a ns f e r a s e ( M T a s e ) a c c e s s , a nd s ubs e que nt l y de c ode d by
l ong- r e a d s e que nc i ng. W e ha ve pur i f i e d a nd c ha r a c t e r i z e d 6m A M T a s e s , E c oG I I a nd H i a 5, bot h
of w hi c h c a n m e t hyl a t e a de ni ne i n m os t s e que nc e c ont e xt s . W e ha ve pe r f or m e d ge ne r a l 6m A
f oot pr i nt i ng, i n w hi c h s a t ur a t e m e t hyl a t i on by E c oG I I or H i a 5 r e ve a l s gl oba l di s t r i but i on of
C A P s . W e ha ve a l s o t e s t e d t a r ge t e d 6m A f oot pr i nt i ng, i n w hi c h E c oG I I or H i a 5 i s gui de d by a n
a nt i body t o de pos i t 6m A i n t he vi c i ni t y of a s pe c i f i c C A P . O ur a ppr oa c h c a n pot e nt i a l l y pr ovi de
ne w i nf or m a t i on on c hr om a t i n or ga ni z a t i on, e s pe c i a l l y t he c oor di na t e d bi ndi ng of ne i ghbor i ng
C A P s a t t he s i ngl e - m ol e c ul e l e ve l .
1
C h a p t e r O n e : I n t r o d u c t i o n
E p i g e n e t i c s
I n t he nuc l e us of e uka r yot i c c e l l s , D N A i s hi e r a r c hi c a l l y or ga ni z e d i nt o t he c hr om a t i n. A t i t s
ba s i c l e ve l , ~ 150 bp of D N A w r a ps a r ound c or e hi s t one s , f or m i ng t he nuc l e os om e ( K or nbe r g a nd
L or c h, 1999) . E nor m ous a m ount s of e pi ge ne t i c i nf or m a t i on i s s t or e d i n t he c hr om a t i n, e s pe c i a l l y
i n t he di s t r i but i ons of nuc l e os om e s ( w i t h a m yr i a d of hi s t one m odi f i c a t i ons ) a nd ot he r c hr om a t i n-
a s s oc i a t e d pr ot e i ns ( A l l i s a nd J e nuw e i n, 2016) . A m ong t he s e pr ot e i ns , t r a ns c r i pt i on f a c t or s ( T F s )
a nd ot he r nonhi s t one pr ot e i ns punc t ua t e nuc l e os om e a r r a ys i nt o r e gul a t or y r e gi ons .
C o l l e c t i n g e p i g e n e t i c i n f o r m a t i o n w i t h s h o r t - r e a d s e q u e n c i n g
T he m os t s i gni f i c a nt que s t i n e pi ge ne t i c s / c hr om a t i n bi ol ogy i s t o pr of i l e di s t r i but i on of
c hr om a t i n- a s s oc i a t e d pr ot e i ns ( C A P s ) . O ve r t he l a s t 10- 15 ye a r s , r e s e a r c he r s ha ve m a de gr e a t
pr ogr e s s i n m a ppi ng t he s e di s t r i but i ons w i t h s hor t - r e a d s e que nc i ng t e c hnol ogi e s . A t pr e s e nt , t hi s
i s us ua l l y a c hi e ve d by ge ne r a t i ng a nd c ol l e c t i ng c hr om a t i n f r a gm e nt s of i nt e r e s t , w hi c h a r e t he n
i de nt i f i e d by s hor t - r e a d s e que nc i ng t e c hnol ogi e s a nd m a ppe d ba c k t o r e f e r e nc e ge nom e ( F i g.1 )
( J ohns on; M i kke l s e n e t a l ., 2007; R obe r t s on e t a l ., 2007) . N uc l e a s e s ( e .g., D N a s e I , M N a s e , a nd
T n5 t r a ns pos a s e ) a r e r out i ne l y us e d f or c hr om a t i n f r a gm e nt a t i on, e i t he r a s a ge ne r a l pr obe of
c hr om a t i n s t r uc t ur e ( D N a s e - s e q, M N a s e - s e q, a nd A T A C - s e q) ( B ue nr os t r o e t a l ., 2013; S ong a nd
C r a w f or d, 2010; Z a r e t , 1999) or s pe c i f i c a l l y t a r ge t e d ( C U T & R U N a nd C U T & T a g) ( K a ya - O kur
e t a l ., 2019; S ke ne a nd H e ni kof f , 2017) . F or e xa m pl e , i n c hr om a t i n i m m unopr e c i pi t a t i on ( C hI P ) ,
s hor t f r a gm e nt s of c hr om a t i n w i t h your f a vor i t e pr ot e i n/ m odi f i c a t i on —i n t he r a nge of m ono- or
ol i go- nuc l e os om e s —a r e c ol l e c t e d, a nd s ubs e que nt l y s e que nc e d ( P a r k, 2009) . T F pr of i l i ng i s
2
m os t l y c a r r i e d out us i ng c hr om a t i n I m m unopr e c i pi t a t i on ( C hI P ) t ha t ha s be e n pe r f e c t e d s i nc e i t
w a s f i r s t i nt r oduc e d ove r 30 ye a r s a go ( A r dui e t a l ., 2018) . W i t h C hI P - s e q, ba s e - pa i r r e s ol ut i on
m a ppi ng of T F s ha s be c om e f e a s i bl e . A pa r a di gm i s e m e r gi ng f r om t hi s de l uge of s hor t - r e a d
s e que nc i ng da t a : a r r a ys of c hr om a t i n- a s s oc i a t e d pr ot e i ns , i n pa r t i c ul a r nuc l e os om e s , a r e
s t r a t e gi c a l l y pos i t i one d a t ke y ge nom i c l a ndm a r ks , i nc l udi ng t r a ns c r i pt i on e nha nc e r s a nd
pr om ot e r s ( J i a ng a nd P ugh, 2009) . E s s e nt i a l l y, t he pos i t i ona l i nf or m a t i on of C A P s i s t r a ns c ode d
i nt o c ut t i ng s i t e s on D N A m ol e c ul e s , be c a us e of a l t e r e d nuc l e a s e a c c e s s . I t i s w or t h not i ng t ha t
da t a f or nuc l e a s e f oot pr i nt i ng a r e i nt r i ns i c a l l y poor a nd s pa r s e a t t he s i ngl e m ol e c ul e l e ve l : a
c hr om a t i n f r a gm e nt , c or r e s pondi ng t o a C A P bi ndi ng e ve nt , i s de f i ne d onl y by i t s t w o e nds . D ue
t o s t oc ha s t i c f l uc t ua t i ons , t he s e da t a a r e noi s y, a nd t he r e f or e of t e n ne e d t o be a ggr e ga t e d t o
pr oduc e s t a t i s t i c a l l y s i gni f i c a nt s i gna l s a t t he e ns e m bl e or popul a t i on l e ve l . M or e i m por t a nt l y, t he
c onne c t i vi t y i nf or m a t i on be t w e e n C A P s c oe xi s t i ng on t he s a m e D N A m ol e c ul e i s l os t i n s hor t
f r a gm e nt s .
3
F i gu r e 1
C ol l e c t i n g e p i ge n e t i c i n f or m at i on w i t h s h or t - r e ad s e q u e n c i n g
D N A i s br oke n i nt o s hor t f r a gm e nt s t ha t a r e a m pl i f i e d ( c opi e d) a nd t he n s e que nc e d t o pr oduc e ‘ r e a ds ’ . T he f r a gm e nt s
a r e s e que nc e d by ne xt ge ne r a t i on s e que nc i ng, I l l um i na .
4
F i gu r e 2
C ol l e c t i n g e p i ge n e t i c i n f or m at i on w i t h D N A m e t h yl at i on f oot p r i n t i n g
T he 6m A - m e t hyl t r a ns f e r a s e E c oG I I t e t he r e d t o t he t a r ge t pr ot e i n, s t e nc i l s out t he f oot pr i nt of c hr om a t i n- a s s oc i a t e d
pr ot e i ns by de pos i t i ng 6m A i n unpr ot e c t e d ge nom i c D N A . 6m A i s s ubs e que nt l y i de nt i f i e d by S M R T C C S , r e ve a l i ng t he l oc a l
c hr om a t i n.
H ow e ve r , t hi s pa r a di gm i s a s ynt he s i s a t t he e ns e m bl e or popul a t i on l e ve l . T he c ur r e nt t r a di t i ona l
s hor t - r e a d s e que nc i ng r e l i e s on di ge s t i on or s he a r i ng D N A i nt o s hor t f r a gm e nt s f or e nr i c hm e nt ,
f ol l ow e d by N G S , w hi c h pr oduc e s s hor t s e que nc i ng r e a ds t ypi c a l l y unde r 250 ba s e pa i r s ( bp) i n
l e ngt h. S hor t f r a gm e nt l e ngt hs a r e of t e n ne c e s s a r y f or a c hi e vi ng a de qua t e bi ndi ng- s i t e r e s ol ut i on
w i t h t he s e t e c hni que s . A l t hough i t i s pos s i bl e t o m a p m ul t i pl e pr ot e i n- D N A i nt e r a c t i ons on s hor t
r e a ds , s he a r i ng t he D N A i nt o s hor t f r a gm e nt s c a n de s t r oy j oi nt l ong- r a nge bi ndi ng i nf or m a t i on,
a nd i t hi nde r s t he a bi l i t y t o pha s e r e a ds t o m e a s ur e ha pl ot ype - s pe c i f i c pr ot e i n- D N A i nt e r a c t i ons
( A l t e m os e e t a l ., 2022) . S hor t r e a d t e c hnol ogi e s ha ve i nhe r e nt l i m i t a t i ons s uc h a s G C bi a s ,
di f f i c ul t i e s m a ppi ng t o r e pe t i t i ve e l e m e nt s , t r oubl e di s c r i m i na t i ng pa r a l ogous s e que nc e s , a nd
di f f i c ul t i e s i n pha s i ng a l l e l e s . M os t i m por t a nt l y, a s l ong- r a nge i nf or m a t i on i s not pr e s e r ve d i n
s hor t f r a gm e nt s , di s t r i but i on pa t t e r ns a t t he s i ngl e m ol e c ul e l e ve l r e m a i n e l us i ve . I t hi nde r s t he
a bi l i t y t o pha s e r e a ds t o m e a s ur e ha pl ot ype - s pe c i f i c pr ot e i n- D N A i nt e r a c t i ons .
5
C o l l e c t i n g e p i g e n e t i c i n f o r m a t i o n w i t h D N A m e t h y l a t i o n f o o t p r i n t i n g
R e c e nt de ve l opm e nt i n l ong- r e a d s e que nc i ng t e c hnol ogi e s r e s ol ve s t he s e obs t a c l e s a nd ope ns a
door t o a c c e s s t he l ong- r a nge e pi ge ne t i c i nf or m a t i on. S i ngl e m ol e c ul e r e a l - t i m e ( S M R T )
s e que nc i ng r out i ne l y ge ne r a t e s r e a ds > 100kb i n l e ngt h. T he pos i t i on f or a C A P c a n be
a l t e r na t i ve l y r e gi s t e r e d by D N A m e t hyl a t i on, a s i n D a m I D a nd N O M e - s e q ( K e l l y e t a l ., 2012;
S t e e ns e l a nd H e ni kof f , 2000) . E s s e nt i a l l y, t he pos i t i ona l i nf or m a t i on of C A P s i s t r a ns c ode d i nt o
m e t hyl a t i on s i t e s on D N A m ol e c ul e s , be c a us e of a l t e r e d m e t hyl t r a ns f e r a s e ( M T a s e ) a c c e s s
( A bdul ha y e t a l ., 2020; A l t e m os e e t a l ., 2022; S hi pony e t a l ., 2020a ; S t e r ga c hi s e t a l .,
2020) ( F i g.2 ) . T hi s a ppr oa c h ha s t he a dva nt a ge of pr e s e r vi ng t he c onne c t i vi t y i nf or m a t i on
be t w e e n j oi nt bi ndi ng e ve nt s on i ndi vi dua l D N A m ol e c ul e s . I t pr ovi de s t he a bi l i t y t o m a p
p r o t e i n - D N A i n t e r a c t i o n s w i t h h i g h r e s o l u t i o n o n n a t i v e , l o n g , s i n g l e , s e q u e n c e d D N A m o l e c u l e s ,
w hi l e s i m ul t a ne ous l y m e a s ur i ng e ndoge nous D N A m odi f i c a t i ons a nd s e que nc e va r i a t i on.
F ur t he r m or e , t he da t a f or D N A m e t hyl a t i on f oot pr i nt i ng c a n be r i c h a nd de ns e on i ndi vi dua l
D N A m ol e c ul e s , of t e n de c or a t e d w i t h hundr e ds of m e t hyl a t i on s i t e s ove r m ul t i - kb. E a c h bi ndi ng
e ve nt i s de f i ne d by m ul t i pl e m e t hyl a t i on s i t e s , a l l ow i ng i t t o be c a l l e d w i t h hi gh c onf i de nc e e ve n
a t t he s i ngl e m ol e c ul e l e ve l . T he a bi l i t y t o r e s ol ve he t e r oge ne ous bi ndi ng e ve nt s a l s o l e a ds t o
m or e a c c ur a t e a s s e s s m e nt of C A P di s t r i but i on a t t he e ns e m bl e l e ve l .
D N A N
6
- a d e n i n e m e t h y l a t i o n ( 6 m A )
F i r s t i de nt i f i e d a nd c ha r a c t e r i z e d a s a D N A m odi f i c a t i on i n ba c t e r i a , 6m A w a s f or a l ong t i m e
onl y know n i n a f e w e uka r yot e s , i nc l udi ng t he c i l i a t e T e t r ahy m e na t he r m ophi l a ( A m m e r m a nn e t
a l ., 1981; C um m i ngs e t a l ., 1974; G or ovs ky e t a l ., 1973; R a e a nd S t e e l e , 1978) . D N A 6m A i n
T e t r ahy m e na , di s c ove r e d ove r 40 ye a r s a go, i s hi ghl y a bunda nt (∼ 1% of a de ni ne ) . I t i s e nr i c he d
6
i n l i nke r D N A of s t e r e ot ypi c a l nuc l e os om e a r r a ys dow ns t r e a m of t r a ns c r i pt i on s t a r t s i t e s , a s a n
i nt e gr a l pa r t of t he e pi ge ne t i c l a nds c a pe ( K a r r e r a nd V a nN ul a nd, 2002) . M or e r e c e nt l y, 6m A ha s
be e n i de nt i f i e d i n t he ge nom i c D N A of a w i de r a nge of e uka r yot i c or ga ni s m s , e s pe c i a l l y pr ot i s t s
a nd ba s a l f ungi ( F u e t a l ., 2015) . H ow e ve r , a de ni ne ba s e s i n D N A a r e a l m os t c om pl e t e l y de voi d
of e ndoge nous m e t hyl a t i on i n a ni m a l s a nd pl a nt s . W e t he r e f or e a i m t o e m pl oy a N
6
- a de ni ne
D N A m e t hyl t r a ns f e r a s e ( 6m A - M T a s e ) t ha t c a n a c c e s s pr ot e i n- D N A i nt e r f a c e s a t nuc l e ot i de
r e s ol ut i on. H e r e , t he 6m A - M T a s e s i s us e d a s a qua nt i t a t i ve pr obe of D N A a c c e s s i bi l i t y, t o obt a i n
l ong- r a nge e pi ge ne t i c i nf or m a t i on a t t he s i ngl e m ol e c ul e l e ve l .
C a l l i n g 6 m A a t t h e s i n g l e m o l e c u l e l e v e l w i t h S M R T C C S
A s a s e que nc i ng by s ynt he s i s t e c hnol ogy, S M R T s e que nc i ng de t e c t s 6m A by i t s pe r t ur ba t i on t o
D N A pol ym e r a s e ki ne t i c s , s pe c i f i c a l l y i nc r e a s e s i n t he t i m e be t w e e n nuc l e ot i de i nc or por a t i ons ,
or t he i nt e r - pul s e dur a t i on ( I P D ) ( F i g.3 ) . E r r or r a t e s f or s i ngl e pa s s r e a ds a r e hi gh, a t m or e t ha n
10% f or c a l l i ng r e gul a r ba s e s a nd e ve n hi ghe r f or ba s e m odi f i c a t i ons . R e c e nt pr ogr e s s i n S M R T
s e que nc i ng gr e a t l y i nc r e a s e s t he pol ym e r a s e r e a d l e ngt h, e na bl i ng e f f e c t i ve i m pl e m e nt a t i on of
C i r c ul a r C ons e ns us S e que nc i ng ( C C S , by c om bi ni ng r e a ds f r om m ul t i pl e pa s s e s of t he s a m e
D N A t e m pl a t e ) f or a c c ur a t e c a l l i ng of 6m A s i t e s a t t he s i ngl e m ol e c ul e l e ve l .
7
F i gu r e 3
(
B as e M od i f i c at i on : D i s c ove r t h e E p i ge n om e
C a l l i ng 6m A by S M R T s e que nc i ng. D e t e c t s ba s e m odi f i c a t i ons us i ng t he ki ne t i c s of t he pol ym e r i z a t i on.
8
F i gu r e 4
T h e s c h e m at i c of S M R T an al ys i s
A bi oi nf or m a t i c pi pe l i ne f or e f f i c i e nt 6m A c a l l i ng a t t he s i ngl e m ol e c ul e l e ve l , w i t h S M R T C C S da t a a s t he i nput .
S t i l l , c a l l i ng 6m A on i ndi vi dua l D N A m ol e c ul e s r e pr e s e nt s a m a j or t e c hni c a l c ha l l e nge . T he
a va i l a bl e s of t w a r e ( i pdS um m a r y) i s opt i m i z e d f or c a l l i ng 6m A a t t he e ns e m bl e l e ve l , a nd
t he r e f or e c om put a t i ona l l y ve r y i ne f f i c i e nt i n pr oc e s s i ng m i l l i ons of r e a ds . N um e r ous noi s y or
a bnor m a l r e a ds a l s o ne e d t o be r e m ove d. W e ha ve s ys t e m a t i c a l l y a ddr e s s e d t he s e i s s ue s a nd
de ve l ope d a bi oi nf or m a t i c pi pe l i ne f or c a l l i ng 6m A us i ng S M R T C C S da t a . ( F i g.4 ) . W e ha ve
us e d i t t o s t udy T e t r a hym e na t he r m ophi l a , a uni c e l l ul a r e uka r yot e i n w hi c h t hi s D N A ba s e
m odi f i c a t i on i s a bunda nt a nd a s s oc i a t e d w i t h t r a ns c r i pt i on a c t i va t i on.
R e s e a r c h S t r a t e g y
W e w i l l s t a r t by c onve r t i ng t he f oot pr i nt of t he nuc l e os om e a nd ot he r c hr om a t i n- a s s oc i a t e d
pr ot e i ns t o D N A m e t hyl a t i on pa t t e r ns ( r e f e r r e d t o a s M E T & R U N ; F i g. 2) . T he c onve r s i on i s
pe r f or m e d by us i ng t he s e que nc e non- s pe c i f i c 6m A - M T a s e E c oG I I or H i a 5 t o m e t hyl a t e ge nom i c
D N A . T hi s pr oc e s s i s l i ke N O M e - s e q i n w hi c h nuc l e os om e s a r e r e ve a l e d by a t e t he r e d 5m C -
m e t hyl t r a ns f e r a s e ( K e l l y e t a l ., 2012) . I t i s s o di f f i c ul t y t o de t e c t 5m C by S M R T s e que nc i ng t ha t
i t s us e ha s be e n pr e c l ude d i n M E T & R U N . I n t he c onve r s i on pr oc e s s , t he t e t he r e d M T a s e c a n
9
m e t hyl a t e a ny of t he hundr e ds of A ’ s i n unpr ot e c t e d ge nom i c D N A a r ound a s i ngl e bi ndi ng s i t e
( F i g. 3) ( M ur r a y e t a l ., 2018; S t e r ga c hi s e t a l ., 2020) . A s t he r e i s ha r dl y a ny 6m A i n t he nuc l e a r
D N A of m a m m a l i a n c e l l s , M E T & R U N c a n ha ve ve r y hi gh s i gna l - t o- noi s e r a t i os , pot e nt i a l l y
e xc e e di ng e s t a bl i s he d a ppr oa c he s l i ke C hI P - s e q a nd C U T & R U N . F ur t he r m or e , M E T & R U N c a n
m a p a t ne a r - ba s e pa i r r e s ol ut i on ( w i t h s a t ur a t e m e t hyl a t i on of a c c e s s i bl e A ’ s ) , a nd e s t a bl i s h t he
l oc a l c hr om a t i n ove r kb- s i z e d r e gi ons . M E T & R U N c a n a l s o ut i l i z e i t s hi gh s e ns i t i vi t y t o de t e c t
di s t a l c ont a c t s , t hus r e ve a l i ng t he 3D c hr om a t i n or ga ni z a t i on a r ound bi ndi ng s i t e s . M E T & R U N
t he r e f or e c om pa r e s f a vor a bl y w i t h N O M e - s e q, w hi c h de t e c t s 5m C by e i t he r bi s ul f i t e s e que nc i ng
( M ur r a y e t a l ., 2018) or na nopor e ( O N T ) s e que nc i ng ( S hi pony e t a l ., 2020b; W a ng e t a l ., 2019) ,
a nd s uf f e r s f r om l ow r e s ol ut i on a nd hi gh e ndoge nous 5m C ba c kgr ound.
I n t hi s pr oj e c t , w e s ought t o c ha r a c t e r i z e t w o nons pe c i f i c N 6- a de ni ne D N A m e t hyl t r a ns f e r a s e
( m 6A - M T a s e ) w i t h hi gh e f f i c i e nc y, hi gh s t a bi l i t y, a nd a m ol e c ul a r w e i ght l i ke t ha t of nons pe c i f i c
nuc l e a s e s s uc h a s D N a s e I , f oc us i ng on t he i r a bi l i t y t o a c c e s s pr ot e i n- D N A i nt e r f a c e s a t
nuc l e ot i de r e s ol ut i on. W e s e l e c t e d E c oG I I a nd H i a 5, w hi c h c a n m e t hyl a t e a de ni ne s i t e s i n a ny
s e que nc e c ont e xt , ge ne r a t i ng hi gh 6m A de ns i t y ne e de d f or hi gh r e s ol ut i on m a ppi ng.
T he y a r e t a gge d by pr ot e i n A or pr ot e i n G . W e i s ol a t e d f our di s t i nc t nons pe c i f i c D N A
m 6A M T a s e s a nd de m ons t r a t e d t ha t t r e a t m e nt of na ke d a nd c hr om a t i ni z e d D N A t e m pl a t e s w i t h
i nc r e a s i ng vol um e of e a c h e nz ym e r e s ul t e d i n i nc r e a s i ng i n a de ni ne m e t hyl a t i on. F or na ke d
D N A , w e t e s t e d pU C 19 ve c t or , w hi c h i s a s m a l l , hi gh c opy num be r pl a s m i d w i t hout e ndoge nous
6m A s i t e s t o i nt e r f e r e w i t h our e xpe r i m e nt . T he r e a c t i on w a s m oni t or e d by D pnI di ge s t i on. D pnI
i s a m e t hyl a t i on- de pe nde nt r e s t r i c t i on e nz ym e c a n onl y c ut a G A T C s i t e i f i t s a de ni ne i s
m e t hyl a t e d. W e a l s o ve r i f i e d t he e nz ym a t i c a c t i vi t y of our hom e m a de m e t hyl t r a ns f e r a s e s i n
10
m e t hyl a t i ng m a m m a l i a n c hr om a t i n a nd m oni t or e d t he r e a c t i on by i m m uno- f l uor e s c e nc e s t a i ni ng
a nd D pnI di ge s t . A s D N A m e t hyl a t i on pr e f e r e nt i a l l y oc c ur s a t l i nke r D N A , D pnI a l s o
pr e f e r e nt i a l l y c ut s l i nke r D N A , ge ne r a t i ng a nuc l e os om e l a dde r i n a ga r os e ge l e l e c t r ophor e s i s .
W e e m pha s i z e t ha t S M R T s e que nc i ng, i n pa r t i c ul a r c i r c ul a r c ons e ns us s e que nc i ng ( C C S ; H i F i
s e que nc i ng) i s c r i t i c a l f or m a ki ng hi ghl y a c c ur a t e a nd s e ns i t i ve 6m A c a l l s a t t he s i ngl e m ol e c ul e
l e ve l ( W e nge r e t a l ., 2019) . S M R T s e que nc i ng de t e c t s 6m A by i t s pe r t ur ba t i on t o D N A
pol ym e r a s e ki ne t i c s , s pe c i f i c a l l y i nc r e a s i ng t i m e be t w e e n nuc l e ot i de i nc or por a t i ons , or t he
i nt e r pul s e dur a t i on ( I P D ) ( F i g.3 ) . C om pa r e d t o L ong- r e a d s e que nc i ng, t r a di t i ona l s hor t r e a ds
s e que nc i ng l a c ks t he i nf or m a t i on a bout j oi nt bi ndi ng e ve nt s ( F i g.3 ) . T hi s know l e dge ga p i s
r e a di l y f i l l e d by 6m A f oot pr i nt i ng a nd l ong- r e a d s e que nc i ng. T he da t a ge ne r a t e d by our a ppr oa c h
a r e hi ghl y r e l e va nt i n ba s i c a nd t r a ns l a t i ona l c a nc e r r e s e a r c h. C oope r a t i ve bi ndi ng unde r pi ns
m a ny ke y pr oc e s s e s i n t r a ns c r i pt i on r e gul a t i on. T r a ns c r i pt i ona l dys r e gul a t i on, i m pa c t i ng m a ny of
t he s e c oope r a t i ve pr oc e s s e s , i s a c r i t i c a l a s pe c t of c a nc e r bi ol ogy, w i de l y i m pl i c a t e d i n c a nc e r
di a gnos i s a nd pr ognos i s . T r a ns c r i pt i on a ddi c t i on, of t e n i nvol vi ng ke y t r a ns c r i pt i on
f a c t or s / c of a c t or s w i t h s i gm oi d dos e r e s pons e c ur ve s s ugge s t i ve of hi gh c oope r a t i vi t y, ha s l a t e l y
e m e r ge d a s a pr om i s i ng t a r ge t f or c a nc e r t r e a t m e nt ( B r a dne r e t a l ., 2017; Z a nc ona t o e t a l ., 2018) .
O ur pr opos e d a ppr oa c h w i l l s he d ne w l i ght on a l l t he s e c oope r a t i ve r e l a t i ons hi ps , pr ovi de de e p
m e c ha ni s t i c i ns i ght s , a nd i de nt i f y t he r a pe ut i c oppor t uni t i e s . R e c e nt pr ogr e s s i n S M R T
s e que nc i ng gr e a t l y i nc r e a s e s t he pol ym e r a s e r e a d l e ngt h, e na bl i ng e f f e c t i ve i m pl e m e nt a t i on of
C i r c ul a r C ons e ns us S e que nc i ng ( C C S , by c om bi ni ng r e a ds f r om m ul t i pl e pa s s e s of t he s a m e
D N A t e m pl a t e ) . T he m e t hyl a t e d s a m pl e s w e r e s ubj e c t e d t o S M R T s e que nc i ng. A s a pr oof - of -
c onc e pt , w e w a nt t o us e M E T & R U N t o pr of i l e R N A pol ym e r a s e I ( P ol I ) bi ndi ng t o t he
r i bos om a l R N A ( r R N A ) ge ne s , w hi c h a r e t he s t r uc t ur a l c om pone nt s of t he nuc l e ol us . W e
11
s e l e c t e d s om e of t he r e a ds m a ppe d t o t he 28S r i bos om a l R N A ge ne . T he r e s ul t r e ve a l s t hr e e
di s t i nc t c hr om a t i n s t a t e s a t 28S r R N A ge ne . I n f ut ur e s t udi e s , w e w i l l f ur t he r e xpl or e P ol I
dyna m i c s dur i ng c e l l c yc l e pr ogr e s s i on a nd unde r va r i ous pe r t ur ba t i ons .
12
C h a p t e r T w o : M e t h o d s
C e l l c u l t u r e
H E K 293T c e l l s ( A T C C C R L - 11268 ) w e r e m a i nt a i ne d i n D M E M ( hi gh gl uc os e , w i t h G l ut a M A X ,
w i t h phe nol r e d, w i t hout s odi um pyr uva t e ; G i bc o 10566016) s uppl e m e nt e d w i t h 10% F e t a l
bovi ne S e r um ( A 3382001) a nd 1% P e n S t r e p ( G i bc o 15070063) a t 37° C i n 5% C O 2 . O C I - A M L 1
c e l l s w e r e m a i nt a i ne d i n R P M I - 1640 w i t h L - gl ut a m i ne ( R 8758- 24) s uppl e m e nt e d w i t h 15%
F e t a l B ovi ne S e r um ( A 3382001) a nd 1% P e n S t r e p ( G i bc o2199839) a t 37° C i n 5% C O 2 .
C o n s t r u c t i o n o f 6 m A - M T a s e s o v e r e x p r e s s i o n v e c t o r s
F our pl a s m i d c ons t r uc t s w e r e ne e de d f or ove r - e xpr e s s i on of pA - E c oG I I , pG - E c oG I I , pA - H i a 5
a nd pG - H i a 5 i n E . c ol i . T he pE T 28a H i s - S U M O c ons t r uc t f or ove r - e xpr e s s i on of H i s -
S U M O t a gge d pr ot e i ns w a s a l a b s t oc k. C odon opt i m i z e d pA - E c oG I I ( G e nB a nk: A F S T 01000004)
w a s s ynt he s i z e d by I D T a nd c l one d i nt o t he pE T 28a H i s - S U M O ve c t or us i ng B a m H I a nd X hol
r e s t r i c t i on s i t e s t o ge ne r a t e t he pE T 28a H i s - S U M O - pA - E c oG I I ve c t or . T he pA G - H i a 5 c ons t r uc t
w a s obt a i ne d f r om A ddge ne . T he pG , pG - H i a 5 a nd H i a 5 f r a gm e nt s w e r e a m pl i f i e d by P C R . T he
pG f r a gm e nt w a s c l one d i nt o t he pE T 28a H i s - S U M O - pA - E c oG I I ve c t or a t B a m H I a nd S bf I s i t e s
t o r e pl a c e t he pA f r a gm e nt a nd ge ne r a t e t he pE T 28a H i s - S U M O - pG - E c oG I I ve c t or . T he pG H i a 5
f r a gm e nt w a s c l one d i nt o t he pE T 28a - H i s - S U M O ve c t or a t B a m H I a nd X hoI s i t e s t o ge ne r a t e t he
pE T 28a H i s - S U M O - pG - H i a 5 ve c t or . T he H i a 5 f r a gm e nt w a s c l one d i nt o t he pE T 28a H i s -
S U M O - pA - E c oG I I ve c t or a t S bf I a nd X hol s i t e s t o ge ne r a t e t he pE T 28a H i s S U M O - pA - H i a 5
ve c t or . C l oni ng w a s pe r f or m e d i n X L 1- B l ue C om pe t e nt E s c he r i c hi a c ol i C e l l s ( N E B C 2992) .
13
C o n f i r m a t i o n o f p l a s m i d s
T o c onf i r m t he i r s uc c e s s f ul c ons t r uc t i on, t he pl a s m i ds w e r e a m pl i f i e d i n ka na m yc i n s e l e c t i on
m e di um , pur i f i e d by m i ni pr e p ( Q i a ge n) , a nd di ge s t e d by di a gnos t i c r e s t r i c t i on e nz ym e s . E a c h of
t he f our pl a s m i ds s houl d ge ne r a t e t w o f r a gm e nt s of s pe c i f i c s i z e s . T he s a m pl e ha vi ng t he r i ght
s i z e d f r a gm e nt s w a s f ur t he r c onf i r m e d by S a nge r s e que nc i ng.
M T a s e p r o d u c t i o n
pE T 28a - pA - H i a 5, pE T 28a - pG - H i a 5, pE T 28a - pA - E c oG I I a nd pE T 28a - pG - E c oG I I ve c t or s w e r e
t r a ns f or m e d i nt o T 7 E xpr e s s l ys a t e c om pe t e nt E s c he r i c hi a c ol i c e l l s ( N E B C 3010I ) f or
r e c om bi na nt pr ot e i n e xpr e s s i on. O ve r ni ght c ul t ur e s w e r e a dde d t o 50m l c ul t ur e s of L B m e di um
s uppl e m e nt e d w i t h 50 μ g/ m l ka na m yc i n t o s c a l e up ove r ni ght c ul t ur e a ga i n. O ve r ni ght c ul t ur e s
w e r e a dde d t o t w o 1.5L c ul t ur e s of L B m e di um s uppl e m e nt e d w i t h 50 μ g/ m l ka na m yc i n a nd
gr ow n w i t h s ha ki ng a t 37° C t o a n O D 600 of 0.5- 0.6. I s opr opyl β - D - 1- t hi oga l a c t opyr a nos i de
( I P T G ) w a s a dde d a t a f i na l c onc e nt r a t i on of 1m M a nd gr ow n f or a n a ddi t i ona l ove r ni ght a t 16° C
w i t h s ha ki ng. C e l l s w e r e pe l l e t e d a t 5000 x g f or 30 m i n a t 4° C ( a l l s ubs e que nt s t e ps a t 4° C ) a nd
r e s u s p e n d e d i n 1 0 0 m l l y s i s b u f f e r ( 1 0 m M H E P E S , p H 7 . 5 ; 1 M N a C l ; 1 0 % g l y c e r o l ; 0 . 5 % N P 4 0 ;
10m M i m i da z ol e ) s uppl e m e nt e d w i t h 2X C om pl e t e , E D T A - f r e e P r ot e a s e I nhi bi t or C oc kt a i l
( S I G M A P 8340- 5M L ) a nd P M S F ( S I G M A P 7626- 25G ) . C e l l s w e r e l ys e d by pr obe s oni c a t i on
( Q s oni c a Q 125) f or 30 m i n on t he m i xt ur e of i c e a nd w a t e r a t 30% a m pl i t ude , 15 s e c onds on, 30
s e c onds of f t he n c e nt r i f uge d f or 30m i ns a t 33000 x g. N i c ke l N T A A ga r os e B e a ds ( 100
m L ) ( G ol d B i o H - 350- 100) w a s pr e pa r e d by w a s hi ng 1.5 m L s of s l ur r y w i t h 15 m L s w a s h buf f e r
( 10 m M T r i s , pH 7.5; 250 m M N a C l ; 10% G l yc e r ol ; 0.5% N P - 40; 20 m M i m i da z ol e ) a nd
14
c e nt r i f uge d a t 600 x g f or 5 m i n, r e pe a t i ng t hr e e t i m e s . T he c l a r i f i e d c e l l l ys a t e a nd N i c ke l N T A
A ga r os e B e a ds w e r e c om bi ne d a nd r ot a t e d a t 4° C f or 1 hr be f or e pour i ng t he c ol um n. T he
c ol um n w i t h l ys a t e a nd N i c ke l N T A A ga r os e B e a ds w a s w a s he d w i t h 20 m L s of di s t i l l e d w a t e r
a nd 15 m L s of w a s h buf f e r be f or e a ddi ng 5 m L s of e l ut i on buf f e r ( 10 m M T r i s , pH 7.5; 250
m M N a C l ; 10% G l yc e r ol ; 0.5% N P - 40; 250 m M i m i da z ol e ) . T he e l ua t e w a s a dde d t o di a l ys i s
C a s s e t t e s ( S l i de - A - L yz e r ™ D i a l ys i s C a s s e t t e s , 3.5K M W C O , 3 m L ) a nd di a l yz e d a ga i ns t 3L of
di a l ys i s buf f e r ( 10 m M T r i s , pH 7.5; 250 m M N a C l ; 10% G l yc e r ol ) ove r ni ght a t 4 ° C . T he f i na l
s a m pl e w a s s uppl e m e nt e d w i t h 60% gl yc e r ol a nd s t or e d a t - 20 ° C f or da i l y us e . T he m a j or pa r t
w i t h 1m M D T T a nd 0.1m M E D T A w a s s t or e d a t - 80 ° C f or s t or a ge . 10 μ l of t he f i na l s a m pl e
w a s m i xe d w i t h 10μ l 1.25x L D S S a m pl e B uf f e r ( N uP A G E ) , i nc uba t e d f or 10 m i nut e s a t 98° C
a nd r un on a 10% B i s - T r i s ge l us i ng M O P S S D S r unni ng buf f e r ( N uP A G E ) a l ongs i de a
P r e c i s i on
P l us P r ot e i n K a l e i dos c ope P r ot e i n l a dde r ( B I O - R A D ) . T he pr ot e i n ge l w a s s t a i ne d us i ng
S i m pl yB l ue S a f e S t a i n ( I nvi t r oge n) a nd i m a ge d us i ng a n A z ur e B i os ys t e m s 300.
I n v i t r o m e t h y l a t i o n o f D N A
pU C 19 pl a s m i d ge ne r a t e d f r om da m - / dc m - c om pe t e nt E .c ol i ( N E B ) w a s l i ne a r i z e d by E c oR I . 1ug
of pur i f i e d l i ne a r i z e d pl a s m i d w a s m e t hyl a t e d w i t h E c oG I I or H i a 5 i n a 20 μ L r e a c t i on
( 20m M T r i s - H C l pH 8.0 or T r i s - H C l pH 8.5 or 1x C ut S m a r t ( N E B 7204S ) , 160μ M / 800 μ M S A M ,
2m M E D T A , 0.5m M E G T A ) . R e a c t i ons w e r e pe r f or m e d f or 1h a t 37° C or ove r ni ght a t 30° C a nd
que nc he d w i t h a ddi t i on of 1% S D S a nd pr ot e i na s e K i nc uba t i on a t 50° C f or 1h. M e t hyl a t e d
pl a s m i d w a s pur i f i e d w i t h Z ym o R e s e a r c h D N A C l e a n & C onc e nt r a t or - 5 a nd di ge s t e d w i t h D pnI .
C ont r ol pU C 19 di ge s t i ons a nd m e t hyl a t e d s a m pl e s w e r e r e s ol ve d on 1% a ga r os e ge l .
15
S a t u r a t e d m e t h y l a t i o n （ I n v i t r o m e t h y l a t i o n o f c h r o m a t i n）
F i x c e l l s : 10 m i l l i on c e l l s w e r e pe l l e t e d a t 100 g f or 5 m i nut e s a nd r e s us pe nde d i n 500 μ L of
c om pl e t e m e di a . C e l l s w e r e f i xe d by a ddi ng 9.5 m L of m e t ha nol dr opw i s e ove r a ge nt l e vor t e x
a nd i nc uba t e d on i c e f or 15 m i nut e s . F i xe d c e l l s w e r e w a s he d t w i c e w i t h c ol d P B S a nd
c e nt r i f uge d a t 500 g f or 5 m i nut e s i n a s w i ngi ng buc ke t r ot or . C e l l s w e r e t he n f l a s h f r oz e n on dr y
i c e or us e d di r e c t l y f or m e t hyl a t i on.
F i xe d c e l l s w e r e i nc uba t e d w i t h 500 μ L of buf f e r A ( 20 m M H E P E S pH 7.9, 10 m M K C l , 20%
gl yc e r ol , 0.5m M S pe r m i di ne , 1x P r ot e a s e i nhi bi t or c oc kt a i l ) s uppl e m e nt e d w i t h 120 μ g of
R N a s e A f or 6 m i nut e s on i c e . T he y w e r e w a s he d onc e w i t h a m e t hyl t r a ns f e r a s e buf f e r ( 50 m M
T r i s - H C l pH 8.0, 2 m M E D T A pH 8.0, 0.5 m M E G T A pH 8.0, 1x pr ot e a s e i nhi bi t or c oc kt a i l ) a t
500 g f or 5 m i nut e s . C e l l s w e r e r e s us pe nde d i n m e t hyl t r a ns f e r a s e buf f e r w i t h 2 μ L of e i t he r
pA / pG - E c oG I I a nd 160 μ M S - A de nos yl m e t hi oni ne ( S A M ) or pA - / pG - H i a 5 a nd 800 μ M S A M t o
a r e a c t i on vol um e of 100 μ L . M e t hyl a t i on r e a c t i on w a s pe r f or m e d a t 37° C f or 1 hour . G e nom i c
D N A w a s e xt r a c t e d w i t h N E B M ona r c h H M W gD N A e xt r a c t i on ki t a c c or di ng t o m a nuf a c t ur e r
i ns t r uc t i ons . P ur i f i e d ge nom i c D N A w a s di ge s t e d w i t h D pnI ( N E B - R 0176L ) ove r ni ght a t 37° C
a nd r un on 1% a ga r os e ge l .
I m m u n o f l u o r e s c e n t s t a i n i n g
T he f i xe d c e l l s w e r e pe r m e a bi l i z e d i n P B S c ont a i ni ng 0.5% T r i t on X - 100 f or 15 m i nut e s . A f t e r
pe r m e a bi l i z a t i on, s a m pl e s w e r e t r e a t e d w i t h 2N H C l f or 30 m i n a nd s ubs e que nt l y ne ut r a l i z e d f or
10 m i nut e s w i t h 0.1 M s odi um bor a t e buf f e r pH 8.5. T he n, t he s a m pl e s w e r e bl oc ke d f or 2h i n
bl oc ki ng s ol ut i on ( 5% donke y s e r um i n P B S , R N a s e A w a s a dde d t o a f i na l c onc e nt r a t i on of 100
16
m g/ m l ) , f ol l ow e d by i nc uba t i on w i t h N 6 - m A a nt i bodi e s ( 1: 200, S yna pt i c S ys t e m s , 202- 003) .
P r i m a r y a nt i bodi e s w e r e i nc uba t e d ove r ni ght a t 4° C , f ol l ow e d by s pe c i e s a ppr opr i a t e s e c onda r y
a nt i bodi e s ( 1: 1000, A l e xa 488; I nvi t r oge n M ol e c ul a r P r obe s , E uge ne , O R ) w i t h i nc uba t i on f or 1
hour . N uc l e i w e r e s t a i ne d w i t h D A P I , a nd s l i de s w e r e t he n m ount e d us i ng F l uor om ount
( C a l bi oc he m , S a n D i e go, C A ) . I m a ge s w e r e t a ke n us i ng a L e i c a D M 4000 U pr i ght m i c r os c opy.
S M R T s e q u e n c i n g s a m p l e p r e p a r a t i o n
G e nom i c D N A w a s e xt r a c t e d f r om t he m e t hyl a t e d c e l l s us i ng W i z a r d G e nom i c D N A P ur i f i c a t i on
K i t ( P r om e ga , A 1120) , s he a r e d t o 3- 5kb i n l e ngt h w i t h M e ga r upt or ( D i a ge node D i a gnos t i c s ) , a nd
us e d t o ge ne r a t e s e que nc i ng l i br a r i e s f or t he S e que l I I S ys t e m .
17
C h a p t e r T h r e e : R e s u l t s
6 m A M T a s e p u r i f i c a t i o n
D N A 6m A i s ge ne r a t e d by t he a ddi t i on of a m e t hyl gr oup f r om S - a de nos yl - L - m e t hi oni ne
( A doM e t / S A M ) t o e nc yc l i c N H 2 a t t he s i xt h pos i t i on of t he pur i ne r i ng i n a de ni ne by s pe c i f i c
m e t hyl t r a ns f e r a s e e nz ym e s . ( L uo a nd H e , 2017) ( F i g.5 ) .
F i gu r e 5
S c h e m at i c f r om ad e n i n e t o N 6- m e t h yl ad e n i n e .
T he a de ni ne i s m e t hyl a t e d i nt o N 6- m e t hyl a de ni ne by e xoge nous m e t hyl t r a ns f e r a s e , E c oG I I or H i a 5. T he a ddi t i on of S - a de nos yl L -
m e t hi oni ne w i l l c ont r ol t he i ni t i a t i on of t he r e a c t i on.
T o obt a i n a l a r ge a m ount of m e t hyl t r a ns f e r a s e w i t h hi gh pur i t y a nd hi gh a c t i vi t y, w e i s ol a t e d a nd
c l one d m 6A - M T a s e s a nd pur i f i e d t w o ki nds of m e t hyl t r a ns f e r a s e s , E c oG I I a nd H i a 5, w hi c h a r e
t a gge d by pr ot e i n A a nd pr ot e i n G . T he pr oj e c t i s ba s e d on pr e vi ous w or k w i t h N 6- a de ni ne
m e t hyl a t i on( 6m A ) i n e uka r yot e s . T he f i r s t s t e p I t ook ove r i s t he opt i m i z a t i on of pA - E c oG I I
pur i f i c a t i on. T he w hol e e xpe r i m e nt w a s r e pe a t e d a c c or di ng t o t he pr e vi ous pr oc e dur e ( F i g.6 ) .
18
F i gu r e 6
P r ot e i n ove r e xp r e s s i on an d p u r i f i c at i on f r om b ac t e r i a
W e f ound t ha t a l a r ge a m ount of t a r ge t pr ot e i n e xi s t e d i n t he pe l l e t a nd di d not e xpos e i nt o l ys a t e
a f t e r s oni c a t i on ( F i g.7) . T he yi e l d of pA - E c oG I I pr oduc e d by t he pr e vi ous pr ot oc ol i s t oo l ow t o
us e i n t a r ge t e d m e t hyl a t i on ( F i g.8 ) . W e s e t up s e ve r a l c ont r ol s t o t r a c k t he s t e p i n w hi c h m os t of
t he pr ot e i n w a s l os t . C om pa r e d t o L B c ul t ur e , T B m e di a doe s not c ha nge t he yi e l d t oo m uc h. I n
t he I P T G c onc e nt r a t i on t i t r a t i on e xpe r i m e nt , w e ve r i f y t ha t t he e xpr e s s i on of pr ot e i n i nduc e d by
0.4m M I P T G i s t he hi ghe s t ( F i g. 9, 10) a s e t dom a i n ( W D R 5) pur i f i e d by a l a b m e m be r a s a
pos i t i ve c ont r ol a nd ge t r i d of s om e pos s i bi l i t i e s , l i ke t he e f f i c i e nc y of N i c ke l be a ds , m a nua l
ope r a t i on, a nd di f f e r e nt bi ndi ng t i m e ( F i g. 11) . A c c or di ng t o t he S D S ge l r e s ul t s , m os t of t he
s ol ubl e pr ot e i n i s f r e e i n t he f l ow t hr ough.
19
F i gu r e 7
T h e am ou n t of t ar ge t e d p r ot e i n , p A - E c oG I I , af t e r s on i c at i on .
G r oup A . A 1. T he a m ount of pr ot e i n i n t he T 7 e xpr e s s e d ba c t e r i a pe l l e t i n a 10ul s a m pl e . T he s i z e of t a r ge t e d pr ot e i n i s a r ound
70kD a . A 2. T he a m ount of pr ot e i n i n t he l ys a t e .
G r oup B . B S A t i t r a t i on. B 1. 0.025ug/ ul , B 2. 0.05ug/ ul , B 3. 0.075ug/ ul , B 4. 0.1ug/ ul . B 5. 0.125ug/ ul , B 6. 0.2ug/ ul , B 7. 0.25ug/ ul .
20
F i gu r e 8
P r od u c t i on of s t oc k p A - E c oG I I
G r oup A . B S A t i t r a t i on. A 1. 0.25ug/ ul , A 2. 0.5ug/ ul , A 3.1.0ug/ ul , A 4.2.0ug/ ul .
G r oup B . S t oc k pr ot e i n. B 1. 2ul s t oc k. B 2. 1ul s t oc k.
21
F i gu r e 9
E xp r e s s i on am ou n t of T 7 e xp r e s s e d c on s t r u c t i on
A 1. T he e xpr e s s e d pr ot e i n i n l ys a t e , i nduc e d by 0.01m M I P T G i n L B m e di a . A 2. T he e xpr e s s e d pr ot e i n i n l ys a t e , i nduc e d by
0.05m M I P T G i n L B m e di a . A 3. T he e xpr e s s e d pr ot e i n i n l ys a t e , i nduc e d by 0.1m M I P T G . A 4. T he e xpr e s s e d pr ot e i n i n
l ys a t e , i nduc e d by 0.4m M I P T G i n L B m e di a . A 5. T he e xpr e s s e d pr ot e i n i n t he pe l l e t , i nduc e d by 0.1m M I P T G i n L B m e di a .
B 1. T he e xpr e s s e d pr ot e i n i n l ys a t e , i nduc e d by 0.1m M I P T G i n T B m e di a . B 2. T he e xpr e s s e d pr ot e i n i n t he pe l l e t , i nduc e d
by 0.1m M I P T G i n T B m e di a .
22
F i gu r e 10
V e r i f i c at i on of t h e e f f i c i e n c y of b e ad s b i n d i n g
G r oup A . P r ot e i n i n t he f l ow t hr ough. A 1. 0.01m M I P T G i nduc t i on i n L B m e di a . A 2. 0.05m M I P T G i nduc t i on i n L B m e di a .
A 3. 0.1m M I P T G i nduc t i on i n L B m e di a . A 4. 0.4m M I P T G i nduc t i on i n L B m e di a . A 5. 0.1m M I P T G i nduc t i on i n T B
m e di a . G r oup B . L e f t pr ot e i n a f t e r be a ds bi ndi ng. B 1. 0.01m M I P T G i nduc t i on i n L B m e di a . B 2. 0.05m M I P T G i nduc t i on i n
L B m e di a . B 3. 0.1m M I P T G i nduc t i on i n L B m e di a . B 4. 0.4m M I P T G i nduc t i on i n L B m e di a . B 5. 0.1m M I P T G i nduc t i on
i n T B m e di a .
I n a ddi t i on, t he s oni c a t i on t i m e a nd m e t hod ha ve be e n opt i m i z e d but t he e f f e c t of i m pr ovi ng t he
yi e l d i s not obvi ous . T he l a s t t r y i s t o i nc r e a s e t he s a l t c onc e nt r a t i on f r om 350m M t o 1M i n t he
l ys i s buf f e r . F or t una t e l y, t he f r e e pr ot e i n i n t he f l ow t hr ough r e duc e d a nd t he pe r c e nt of pr ot e i n
bi ndi ng on t he be a d i nc r e a s e d f r om 6.25% t o 16% . T he yi e l d i n t he opt i m i z e d pr ot oc ol i s 15m g,
w hi c h i s f i ve t i m e s w ha t w a s be f or e . A f t e r pr ot e i n pur i f i c a t i on, w e obt a i ne d f our hom e m a de
m e t hyl t r a ns f e r a s e s a nd t e s t e d t he i r pur i t y on 10% S D S ge l ( F i g.12 ) . T he s e c onda r y ba nd w a s not
obvi ous , a nd t he pur i t y of pr ot e i n c a n s a t i s f y t he r e qui r e m e nt s f or t he ne xt s t e p. T o pr e l i m i na r i l y
ve r i f y t he a c t i vi t y of t he e nz ym e , w e s e t up f i ve t i m e s t i t r a t i on of e nz ym e vol um e on pU C 19
w hi c h ha s no e ndoge nous 6m A s i t e s . M e t hyl t r a ns f e r a s e s w i l l f unc t i on on t he pl a s m i d. T he
m e t hyl a t e d s a m pl e s w e r e di ge s t e d w i t h D pnI a nd D pnI I , w hi c h c a n onl y c ut a
G A T C s i t e i f i t s a de ni ne i s m e t hyl a t e d a nd onl y c ut a G A T C s i t e w i t hout m e t hyl a t i on
( F i g.13,14 ) .
23
F i gu r e 11
WD R 5 c om p ar i s on an d b i n d i n g t i m e t i t r at i on .
G r oup A . W D R 5 ve r i f i c a t i on. A 1. W D R 5 i n t he f l ow t hr ough. A 2. W D R 5 bi ndi ng on t he be a ds .
G r oup B . B i ndi ng t i m e t i t r a t i on of pA - E c oG I I . B 1. pA - E c oG I I i n t he f l ow t hr ough. B 2. pA - E c oG I I bi ndi ng on t he be a ds f or 2
hour s . B 3. pA - E c oG I I bi ndi ng on t he be a ds f or 5 hour s . B 4. pA - E c oG I I bi ndi ng on t he be a ds ove r ni ght .
24
F i gu r e 12
P ol yac r yl am i d e ge l c on t ai n i n g p u r i f i e d p r ot e i n A an d G t agge d E c oG I I an d H i a5
G r o u p A . p A - E c o G I I A 1 . 0 . 5 u l . A 2 . 1 u l . A 3 . 2 u l . G r o u p B . p G - E c o G I I B 1 . 0 . 5 u l . B 2 . 1 u l . B 3 . 2 u l . G r o u p C . p A - H i a 5 C 1 . 2 u l .
C 2.1ul . G r oup D . D 1. 2ul . D 2.1ul .
25
F i gu r e 13
S c h e m at i c of e n z ym at i c ve r i f i c at i on on n ak e d D N A .
M T a s e s w i l l m e t hyl a t e a ny a de ni ne on t he na ke d D N A . D pnI onl y c ut s t he m e t hyl a t e d a de ni ne w i t hi n G A T C .
26
F i gu r e 14
D p n I an d D p n I I f u n c t i on p r i n c i p l e
D pnI r e qui r e s m e t hyl a t i on of a de ni ne i n G A T C r e c ogni t i on s e que nc e . D pnI I w i l l onl y c l e a ve f ul l y- a de nom e t hyl a t e d da m
s i t e s a nd he m i - a de nom e t hyl a t e d da m s i t e s 60X m or e s l ow l y.
27
F i gu r e 15
D i f f e r e n t e f f e c t of u s i n g d i f f e r e n t vol u m e n e w m ad e p A - E c oG I I .
G r oupA . N e w m a de pA - E c oG I I m e t hyl a t i on. A 1. N on- di ge s t i on i nput . A 2. N o m e t hyl a t i on s a m pl e di ge s t e d by D pnI I . A 3.
0.25ul e nz ym e m e t hyl a t e d s a m pl e di ge s t e d by D pnI . A 4. 0.25ul e nz ym e m e t hyl a t e d s a m pl e di ge s t e d by D pnI I . A 5.0.5ul
e nz ym e m e t hyl a t e d s a m pl e di ge s t e d by D pnI . A 6. 0.5ul e nz ym e m e t hyl a t e d s a m pl e di ge s t e d by D pnI I . A 7. 1.0ul e nz ym e
m e t hyl a t e d s a m pl e di ge s t e d by D pnI . A 8. 1.0ul e nz ym e m e t hyl a t e d s a m pl e di ge s t e d by D pnI I .
G r oup B . S t oc k m e t hyl a t i on. B 1.1.0ul e nz ym e m e t hyl a t e d s a m pl e di ge s t e d by D pnI . B 2. 1.0ul e nz ym e m e t hyl a t e d s a m pl e
di ge s t e d by D pnI I .
28
F i gu r e 16
T r ou b l e s h oot i n g of m i s s e d f r agm e n t s .
A 0. N o m e t hyl a t i on s a m pl e di ge s t e d by D pnI I .
G r oup A . E f f e c t ve r i f i c a t i on of s e c onda r y pr ot e i n. A 1. pA - E c oG I I m e t hyl a t i on s a m pl e w i t hout di ge s t i on. A 2. S t oc k
m e t hyl a t i on s a m pl e w i t hout di ge s t i on.
G r oup B . E f f e c t ve r i f i c a t i on of di l ut i ng D pnI 1 i n 10. B 1. pA - E c oG I I m e t hyl a t i on s a m pl e di ge s t e d by D pnI . B 2. pA - E c oG I I
m e t hyl a t i on s a m pl e di ge s t e d by D pnI I . B 3. S t oc k e nz ym e m e t hyl a t i on s a m pl e di ge s t e d by D pnI I . B 4. S t oc k e nz ym e
m e t hyl a t i on s a m pl e di ge s t e d by D pnI .
29
F i gu r e 17
F ou r h om e m ad e m e t h yl t r an s f e r as e s f i ve - t i m e t i t r at i on on p U C 19.
T he vol um e r a nge s f r om 0.025ul t o 0.4ul .
G r oup A . pA - H i a 5 m e t hyl a t i on. G r oup B . pG - H i a 5 m e t hyl a t i on. G r oup C . pA - E c oG I I m e t hyl a t i on. G r oup D . pG - E c oG I I
m e t hyl a t i on.
W e c om pa r e d t he e nz ym a t i c a c t i vi t y of ne w m a de pA - E c oG I I a nd s t oc k ( F i g.15 ) a nd f ound t ha t
t he f r a gm e nt s pa t t e r n m e t hyl a t e d by ne w pA - E c oG I I m i s s e d s om e f r a gm e nt s . W e t r i e d t o ge t r i d
of t he e f f e c t of s e c onda r y pr ot e i n i n t he pur i f i e d e nz ym e by s e t t i ng up a c ont r ol gr oup i n w hi c h
t he m e t hyl a t e d s a m pl e s r un di r e c t l y on S D S ge l w i t hout D pnI di ge s t i on. A nd i n t he ne xt s t e p, w e
t r i e d 1i n 10 di l ut i ng t he D pnI i n t he 10ul r e a c t i on s ys t e m ( F i g.16 ) a nd t he di s a ppe a r e d f r a gm e nt s
r e c ove r e d a ga i n. W he n H i a 5 m e t hyl t r a ns f e r a s e s t a gge d by pr ot e i n A a nd pr ot e i n G a r e a va i l a bl e ,
w e ve r i f y t he e nz ym a t i c a c t i vi t y vi a t he s a m e r out e . A f t e r t he opt i m i z a t i on of pr ot oc ol , t he
s pe c i f i c c ondi t i ons i n t he r e a c t i on s ys t e m ha ve be e n s e t t l e d a nd w e di d a 5 t i m e s t i t r a t i on of
e nz ym e vol um e . A c c or di ng t o t he w a t e r s he d, w e c a n di s t i ngui s h t ha t E c oG I I i s m or e a c t i ve t ha n
H i a 5( F i g. 17) .
I n v i t r o m e t h y l a t i o n
W e ha ve r e c ons t i t ut e d e nz ym a t i c a l l y a c t i ve M T a s e s ( pA - E c oG I I , pG - E c oG I I , pA - H i a 5 a nd pG -
H i a 5) a nd pe r f or m e d i n vi t r o m e t hyl a t i on of t he c hr om a t i n f r om a hum a n l e uke m i a c e l l l i ne ( O C I -
A M L 3) a nd m ous e c e l l l i ne ( H M M ) ( F i g.18) . M a m m a l i a n ge nom i c D N A l a c ks 6m A , s o
30
e s s e nt i a l l y a l l 6m A s i t e s r e ve a l e d by S M R T C C S a r e a t t r i but a bl e t o t he a dde d M T a s e s ( F i g.19) .
W e e xpe c t t ha t t he m a m m a l i a n c hr om a t i n w i l l be m e t hyl a t e d a f t e r i nc uba t i on w i t h t he
i nvol ve m e nt of S A M .
W e ha ve t e s t e d t he m e t hyl a t e d e f f e c t of i nc uba t i ng 0.5 hour s , 1 hour a nd 2 hour s ( F i g.20 ) .
C ons i de r i ng t he e f f i c i e nc y a nd di f f e r e nt e f f e c t s unde r di f f e r e nt i nc uba t i on t i m e , w e f i nd t ha t t he
be s t m e t hyl a t e d t i m e i s 1 hour . A f t e r i nc uba t i on, t he m e t hyl a t e d c hr om a t i n i s e xt r a c t e d by
M ona r c h ki t ( N E B T 3015- 21) . D pnI di ge s t i on i s a ki nd of m e a s ur e m e nt of t he qua l i t y of
m e t hyl a t e d s a m pl e s . A s D N A m e t hyl a t i on pr e f e r e nt i a l l y oc c ur s a t l i nke r D N A , D pnI a l s o
pr e f e r e nt i a l l y c ut s l i nke r D N A , ge ne r a t i ng a nuc l e os om e l a dde r you s e e i n t he a ga r os e ge l
( F i g.20 ) .
31
F i gu r e 18
E n z ym at i c ve r i f i c at i on on c h r om at i n of O C I - A M L 3 an d m ou s e H M M
G r oup A . M e t hyl a t i on on M ous e H M M . A 1.A 2. S t oc k e nz ym e m e t hyl a t i on s a m pl e s ha ve no di ge s t i on i nput a nd a r e di ge s t e d
by D pnI . A 3.A 4. pA - E c oG I I m e t hyl a t i on s a m pl e s ha ve no di ge s t i on i nput a nd a r e di ge s t e d by D pnI .
G r oup B . M e t hyl a t i on on O C I - A M L 3. B 1.B 2. S t oc k e nz ym e m e t hyl a t i on s a m pl e s ha ve no di ge s t i on i nput a nd a r e di ge s t e d
by D pnI . B 3.B 4. pA - E c oG I I m e t hyl a t i on s a m pl e s ha ve no di ge s t i on i nput a nd a r e di ge s t e d by D pnI .
32
F i gu r e 19
N u c l e os om e l ad d e r oc c u r r e n c e .
T he m e t hyl a t e d s a m pl e s a r e di ge s t e d i nt o f r a gm e nt s w hi c h s how a nuc l e os om e l a dde r on t he 1% a ga r os e ge l .
33
F i gu r e 20
I n c u b at i on t i m e e xp l or at i on on O C I - A M L 3
A 1.A 3.A 5.A 7. T he i nput s a m pl e c or r e s ponds t o t he i r ow n i nc uba t i on t i m e . A 2.0.5 hour s m e t hyl a t e d s a m pl e . A 4. 1- hour
m e t hyl a t e d s a m pl e . A 6. S t oc k 1- hour m e t hyl a t e d s a m pl e . A 8.2 hour s m e t hyl a t e d s a m pl e .
34
F i gu r e 21
E f f e c t of f i xat i on on O C I - A M L 3 s at u r at i on m e t h yl at i on .
O C I - A M L 3 c e l l s a r e f i xe d w i t h e t ha nol a nd m e t hyl a t e d w i t h A 1. pA - E c oG I I , A 2. pG - E c oG I I , A 3. pA - H i a 5, A 4. pG - H i a 5 or
m e t ha nol f i xa t i on a nd m e t hyl a t e d w i t h B 1. pA - E c oG I I , B 2. pG - E c oG I I , B 3. pA - H i a 5, B 4. pG - H i a 5. U ndi ge s t e d ge nom i c
D N A f i xe d by A 0. e t ha nol , B 0. m e t ha nol .
T o m oni t or t he s t a bl e s t a t us of c hr om a t i n, w e de t e r m i ne d t o f i x c e l l s . T w o s ol ve nt s a r e i nvol ve d
i n t he c e l l f i xa t i on, 100% m e t ha nol a nd 70% e t ha nol . T he D pnI di ge s t i on s how s t ha t t he
ge nom i c D N A s a m pl e s f i xe d by m e t ha nol a r e be t t e r ( F i g.21 ) .
I m m u n o f l u o r e s c e n t s t a i n i n g
W e t ook c ha r ge of a n i m m unof l uor e s c e nc e e xpe r i m e nt t o ge t t he vi s ua l r e s ul t s , w hi c h i s t he t hi r d
w a y t o ve r i f y t he e nz ym a t i c a c t i vi t y. F i r s t , w e w a nt t o ge t f a m i l i a r w i t h t he pr ot oc ol of
i m m unof l uor e s c e nc e . W e r e vi s e d s om e s t e ps of t he I F pr ot oc ol f or our m e t hyl a t i on a nd t e s t e d i t
us i ng pA - E c oG I I . T he r e s ul t f i gur e s how s s e ve r a l br i ght dot s , nuc l e ol i i ns i de t he nuc l e us , w hi c h
m e a ns t he r e vi s e d pr ot oc ol w or ks w e l l . A nd t hus , w e s e t up a gr oup e xpe r i m e nt f or di f f e r e nt
e nz ym e c om pa r i s on. A f t e r i n v i t r o m e t hyl a t i on by E c oG I I , H i a 5, or no M T a s e c ont r ol , w e
35
s t a i ne d t he m a m m a l i a n nuc l e i w i t h a 6m A - s pe c i f i c a nt i body. T he nuc l e i w e r e c ount e r s t a i ne d by
t he D N A dye D A P I . W e c a n s e e t ha t 6m A s i gna l s a r e m uc h s t r onge r i n t he M T a s e - t r e a t e d
s a m pl e s t ha n t he c ont r ol ( F i g.22 ) .
36
F i gu r e 22
M e t h yl at e d D N A b y M T as e s on O C I - A M L 3 s at u r at i on m e t h yl at i on .
A f t e r i n vi t r o m e t hyl a t i on by E c oG I I , H i a 5, or no M T a s e c ont r ol , t he O C I - A M L 3 nuc l e i a r e s t a i ne d w i t h a 6m A - s pe c i f i c
a nt i body. T he nuc l e i w e r e c ount e r s t a i ne d by t he D N A dye D A P I .
37
F i gu r e 23
E f f e c t of P ol 1 an t i b od y on H E K 293t c e l l l i n e .
H E K 293t c e l l s a r e s t a i ne d w i t h a P ol 1 a nt i body. T he nuc l e i w e r e c ount e r s t a i ne d by t he D N A dye D A P I .
38
F i gu r e 24
E f f e c t of P ol 1 an t i b od y on O C I - A M L 3 c e l l l i n e .
O C I - A M L 3 s t a i ne d w i t h a P ol 1 a nt i body. T he nuc l e i w e r e c ount e r s t a i ne d by t he D N A dye D A P I . F or t he
f ur t he r pl a n of m y pr oj e c t , w e pl a n t o e xpl or e t he c hr om a t i n s t a t us of P ol 1 a nd P ol 2 i n di f f e r e nt
c e l l a c t i vi t i e s . A s a s i m pl e e xpe r i m e nt , w e de c i de d t o us e a dhe s i on c e l l l i ne ( H E K 297t ) a nd
s us pe ns i on c e l l s ( O C I - A M L 3) t o t e s t t he e f f e c t of pol ym e r a s e I a nt i body ( F i g.23,24 ) .
P r e p a r a t i o n a n d a n a l y s i s o f S M R T s e q u e n c i n g s a m p l e
T he s e que nc i ng s a m pl e s c a n be obt a i ne d i n t w o w a ys . T he f i r s t w a y i s t o i nvol ve t he m a gne t i c
be a ds . W e t r i e d t o c a pt ur e f r a gm e nt s us i ng m a gne t i c be a ds a nd t he f r a gm e nt s be t w e e n 1kb t o
5kb a r e s e l e c t e d f or s e que nc i ng ( F i g.25 ) . T he ot he r w a y i s t o e xt r a c t t he t a r ge t e d f r a gm e nt s by
t he Q I A qui c k G e l E xt r a c t i on K i t . F or t hi s be nc h s a m pl e , t he f r a gm e nt s be t w e e n 3kb- 5kb a r e
s e l e c t e d ( F i g.26) .
39
F i gu r e 25
F r agm e n t s b e t w e e n 1- 5k b s e l e c t i on w i t h m agn e t i c b e ad s .
A 1. S a m pl e di ge s t i on pa t t e r n by D pnI . A 2. T he l e f t f r a gm e nt s a f t e r s e l e c t i on. A 3. T he D N A f r a gm e nt s 1- 5kb i n s i z e s e l e c t e d
by m a gne t i c be a ds .
40
F i gu r e 26
F r agm e n t s b e t w e e n 3- 5k b s e l e c t i on w i t h ge l e xt r ac t i on .
A . F r a gm e nt s 3- 5kb i n s i z e s e l e c t i on m e t hyl a t e d by pA - E c oG I I .
B . F r a gm e nt s 3- 5kb i n s i z e s e l e c t i on m e t hyl a t e d by pA - H i a 5.
F or t he f i r s t be nc h s e que nc i ng r e s ul t , w e s e l e c t e d s om e of t he r e a ds m a ppe d t o t he 28S r i bos om a l
R N A ge ne , w hi c h i s s t udi e d he r e a s a pr oof - of - c onc e pt . R i bos om a l r i bonuc l e i c a c i d ( r R N A ) i s a
t ype of non- c odi ng R N A w hi c h i s t he pr i m a r y c om pone nt of r i bos om e s , e s s e nt i a l t o a l l c e l l s .
C a l l i ng 6m A on i ndi vi dua l D N A m ol e c ul e s r e pr e s e nt s a m a j or t e c hni c a l c ha l l e nge . T he a va i l a bl e
s of t w a r e ( i pdS um m a r y) i s opt i m i z e d f or c a l l i ng 6m A a t t he e ns e m bl e l e ve l , a nd t he r e f or e
c om put a t i ona l l y ve r y i ne f f i c i e nt i n pr oc e s s i ng m i l l i ons of r e a ds . N um e r ous noi s y or a bnor m a l
r e a ds a l s o ne e d t o be r e m ove d. W e ha ve s ys t e m a t i c a l l y a ddr e s s e d t he s e i s s ue s a nd de ve l ope d a
bi oi nf or m a t i c pi pe l i ne f or c a l l i ng 6m A us i ng S M R T C C S da t a . D ue t o l a c k of e ndoge nous 6m A
i n hum a n ge nom i c D N A , e s s e nt i a l l y a l l 6m A s i t e s r e ve a l e d by S M R T s e que nc i ng w e r e
a t t r i but a bl e t o t he a dde d M T a s e s . A c c or di ng t o t he bi oi nf or m a t i c s r e s ul t of r a w da t a ( F i g.27) , w e
c a n s e e t ha t t he y ha ve di f f e r e nt D N A m e t hyl a t i on pa t t e r ns . F ur t he r a na l ys i s of i nt e r - 6m A
di s t a nc e s i n t he s e D N A m ol e c ul e s r e ve a l s t hr e e c l a s s e s , c or r e s pondi ng t o t hr e e di s t i nc t c hr om a t i n
s t a t e s a t 28 r R N A ge ne : a c t i ve r R N A ge ne w i t h di s pl a c e d nuc l e os om e s , i na c t i ve r R N A ge ne w i t h
41
i r r e gul a r l y s pa c e d nuc l e os om e s , a nd s i l e nt r R N A ge ne w i t h r e gul a r l y s pa c e d nuc l e os om e s
( F i g.28) .
42
F i gu r e 27
R aw d at a of 28s r i b os om al R N A f r om 1- 5k b f r agm e n t s .
R e s ul t s m a ppe d t o ge nom i c s . R e d dot i s a 6m A s i t e on t he c hr om a t i n.
43
F i gu r e 28
S our c e： K r e s oj a - R a ki c J , S a nt or o R . N uc l e ol us a nd r R N A G e ne C hr om a t i n i n E a r l y E m br yo D e ve l opm e nt . T r e nds G e ne t .
2019; 35( 11) : 868- 879. doi : 10.1016/ j .t i g.2019.06.005
P h as e b l ot .
T hr e e c l a s s e s of D N A m e t hyl a t i on pa t t e r ns a na l yz e d by i nt e r - 6m A di s t a nc e s i n t he s e D N A m ol e c ul e s . C or r e s pondi ng t o t hr e e
di s t i nc t c hr om a t i n s t a t e s a t 28S r R N A ge ne : A c t i ve r R N A ge ne w i t h di s pl a c e d nuc l e os om e s , i na c t i ve r R N A ge ne w i t h i r r e gul a r l y
s pa c e d nuc l e os om e s , a nd s i l e nt r R N A ge ne w i t h r e gul a r l y s pa c e d nuc l e os om e s .
C h a p t e r F o u r : D i s c u s s i o n
T he r e c e nt di s c ove r y of 6m A i n e uka r yot i c ge nom e s ope ns a ne w a nd pr om i s i ng di m e ns i on of
e pi ge ne t i c r e s e a r c h; how e ve r , m e t hods f or hi gh- r e s ol ut i on, c om pl e t e m a ppi ng of 6m A e ve nt s a r e
s t i l l l a c ki ng. H e r e w e pr ovi de a nove l a ppr oa c h a nd a n a na l yt i c a l f r a m e w or k f or 6m A
c ha r a c t e r i z a t i on i n e uka r yot i c ge nom e s us i ng S M R T s e que nc i ng.
M T a s e s p u r i f i c a t i o n
H e r e w e pur i f i e d i n l a r ge qua nt i t i e s t w o m e t hyl t r a ns f e r a s e s , E c oG I I a nd H i a 5, t a gge d by pr ot e i n
A a nd pr ot e i n G . W e dr a m a t i c a l l y i nc r e a s e d t he yi e l d of pA - E c oG I I by us i ng t he hi gh s a l t l ys i s
buf f e r . T he f i na l pr ot e i n c onc e nt r a t i on a f t e r pr ot oc ol opt i m i z a t i on w a s i nde e d m or e t ha n t hi r t y
t i m e hi ghe r t ha n t he or i gi na l s t oc k. D ur i ng t he t r oubl e s hoot i ng, w e ove r c a m e s om e m a j or
44
obs t a c l e s , a nd opt i m i z e d f a c t or s i nc l udi ng e xpr e s s e d t e m pe r a t ur e , I P T G c onc e nt r a t i on, c ul t ur e
m e di a , a nd t he s a l t c onc e nt r a t i on of t he l ys i s buf f e r . S t i l l , onl y 16% of t he t a r ge t pr ot e i n w a s
bound t o N i
2 +
be a ds . W e ha d t o i nc r e a s e t he i nput vol um e of t he ba c t e r i a l l ys a t e t o a c hi e ve t he
yi e l d ne e de d ( 4L f or pA - E c oG I I , c om pa r e d t o 1.5L f or W D R 5, a w e l l - be ha ve d pr ot e i n of s i m i l a r
s i z e ) . T he r e a s on f or l ow bi ndi ng e f f i c i e nc y of pA - E c oG I I r e m a i ns e l us i ve . W e s pe c ul a t e t ha t
pA - E c oG I I m a y i nt e r a c t w i t h t he H i s - S U M O m oi e t y of t he f us i on pr ot e i n a nd i nt e r f e r e w i t h
m e t a l c he l a t i on.
W he n t he ne w - m a de M T a s e s w e r e l oa de d on t he S D S ge l , t he ba nd of t a r ge t e d pr ot e i n w a s t he
m os t pr om i ne nt . H ow e ve r , t he r e w e r e a l s o s om e s e c onda r y ba nds , w hi c h m a y be t he de gr a da t i on
pr oduc t s of t he t a r ge t pr ot e i n or c ont a m i na t i ng ba c t e r i a l pr ot e i ns . F or t una t e l y, t he qua l i t y of our
pur i f i e d M T a s e s i s va l i da t e d i n our c ont r ol e xpe r i m e nt s , w hi c h c onf i r m e d t he hi gh M T a s e
a c t i vi t y, a nd r ul e d out nuc l e a s e a c t i vi t y a nd ot he r ne ga t i ve e f f e c t s .
I n v i t r o m e t h y l a t i o n ( D N A a n d c h r o m a t i n )
W e t e s t e d t he M T a s e a c t i vi t y of our hom e m a de e nz ym e s by i n v i t r o m e t hyl a t i on of pU C 19 a nd
m a m m a l i a n c hr om a t i n. A f t e r ve r i f yi ng t he e nz ym a t i c a c t i vi t y on pU C 19, w e us e d t he s a m e
nuc l e a r e xt r a c t i on buf f e r on m ous e H M M a nd l e uke m i a c e l l s . T he buf f e r w or ke d w e l l on m ous e
H M M a nd pr oduc e d t he pe r f e c t nuc l e os om e l a dde r pa t t e r n a f t e r D pnI di ge s t i on. H ow e ve r , t he
nuc l e os om e l a dde r pa t t e r n f or l e uke m i a c e l l s w e r e t oo w e a k. W e t r i e d us i ng N P - 40 i ns t e a d of
di gi t oni n. S i m ul t a ne ous l y, w e t r i e d us i ng l ow c onc e nt r a t i on of di gi t oni n. W e obt a i ne d t he
e xpe c t e d r e s ul t w i t h l ow de t e r ge nt c onc e nt r a t i on ( 0.01% di gi t oni n or N P - 40) . W e c hos e
di gi t oni n f or f ut ur e e xpe r i m e nt s , m a i nl y be c a us e of a l a c k of l i t e r a t ur e s uppor t f or us e of N P - 40
i n M T a s e a s s a ys .
45
I m m u n o f l u o r e s c e n t s t a i n i n g
T he pi l ot e xpe r i m e nt w a s pe r f or m e d on a t t a c he d c e l l s ( H E K 293t ) , w hi c h w e r e gr ow n on t he gl a s s
s l i de a nd c onve ni e nt f or I F pr oc e dur e s . H ow e ve r , t he c e l l l i ne s i nvol ve d i n our pr oj e c t ( l e uke m i a
or m ous e H M M ) w e r e s us pe ns i on c e l l s . T he r e f or e , t he pr ot oc ol w a s m odi f i e d a f t e r i ni t i a l
opt i m i z a t i on. S us pe ns i on c e l l s w e r e f i r s t f i xe d. P a r a f or m a l de hyde ( P F A ) ha s be e n w i de l y us e d a s
a c r os s - l i nki ng f i xa t i on a ge nt . H ow e ve r , P F A c a n da m a ge D N A , t hus not c om pa t i bl e w i t h
dow ns t r e a m a ppl i c a t i ons , i nc l udi ng S M R T s e que nc i ng. W e c om pa r e d m e t ha nol f i xa t i on a nd
e t ha nol f i xa t i on a nd c hos e t he f or m e r f or i t s m i ni m um pe r t ur ba t i on t o c hr om a t i n or ga ni z a t i on.
S us pe ns i on c e l l s w e r e a t t a c he d t o gl a s s s l i de s by c yt os pi n. W e opt i m i z e d t he c e nt r i f uga t i on s pe e d
t o e ns ur e c e l l a t t a c hm e nt w hi l e pr e s e r vi ng nuc l e a r m or phol ogy.
F r a g m e n t s e l e c t i o n
F or a c c ur a t e 6m A c a l l i ng f r om S M R T s e que nc i ng r e s ul t s , w e r e qui r e a t l e a s t 20x pa s s e s f or a
D N A m ol e c ul e . A s t he m e di a n r e a d l e ngt h f or S M R T s e que nc i ng i s ~ 100kb, our D N A f r a gm e nt
s i z e s houl d not e xc e e d 5kb. W e e m pl oye d t w o m e t hods f or f r a gm e nt s i z e s e l e c t i on. W e f i r s t us e d
S P R I be a ds t o s e l e c t D N A f r a gm e nt s 1- 5kb i n s i z e f or S M R T s e que nc i ng ( a f t e r D pnI di ge s t i on) .
N ot e t ha t 6m A i s r e qui r e d f or D pnI di ge s t i ons , t he r e f or e ge nom i c r e gi ons w i t h l ow 6m A de ns i t y
w e r e de pl e t e d i n t he s e f r a gm e nt s . L a t e r , w e s e l e c t e d f r a gm e nt s 3- 5kb i n s i z e by a ga r os e ge l
pur i f i c a t i on ( a f t e r P s t I or H i ndI I I di ge s t i on) . N ot e t ha t ne i t he r r e s t r i c t i on e nz ym e i s
6m A de pe nde nt , e ve n t hough t he y m a y be bl oc ke d by i nt e r na l 6m A . S M R T s e que nc i ng da t a
a na l ys i s s how e d t ha t P s t I or H i ndI I I di ge s t i on, c om bi ne d w i t h s i z e s e l e c t i on by ge l pur i f i c a t i on,
a c hi e ve d r e duc e d r e pr e s e nt a t i on of t he m a m m a l i a n ge nom e , a nd i nc r e a s e d c ove r a ge f or t a r ge t
r e gi ons . T hi s l a i d dow n t he gr oundw or k f or f ut ur e s t udi e s of c hr om a t i n or ga ni z a t i on of
m a m m a l i a n c e l l s us i ng 6m A f oot pr i nt i ng.
46
R e f e r e n c e s
A bdul ha y, N .J ., M c N a l l y, C .P ., H s i e h, L .J ., K a s i na t ha n, S ., K e i t h, A ., E s t e s , L .S ., K a r i m z a de h,
M ., U nde r w ood, J .G ., G ooda r z i , H ., a nd N a r l i ka r , G .J . ( 2020) . M a s s i ve l y m ul t i pl e x
s i ngl e m ol e c ul e ol i gonuc l e os om e f oot pr i nt i ng. E l i f e 9, e 59404.
A l l i s , C .D ., a nd J e nuw e i n, T . ( 2016) . T he m ol e c ul a r ha l l m a r ks of e pi ge ne t i c c ont r ol . N a t ur e
r e vi e w s G e ne t i c s 17, 487- 500.
A l t e m os e , N ., M a s l a n, A ., S m i t h, O .K ., S unda r a r a j a n, K ., B r ow n, R .R ., M i s hr a , R ., D e t w e i l e r ,
A .M ., N e f f , N ., M i ga , K .H ., a nd S t r a i ght , A .F . ( 2022) . D i M e L o- s e q: a l ong- r e a d, s i ngl e m ol e c ul e
m e t hod f or m a ppi ng pr ot e i n –D N A i nt e r a c t i ons ge nom e w i de . N a t ur e M e t hods , 1- 13.
A m m e r m a nn, D ., S t e i nbr uc k, G ., B a ur , R ., a nd W ohl e r t , H . ( 1981) . M e t hyl a t e d ba s e s i n t he
D N A of t he c i l i a t e S t yl onyc hi a m yt i l us . E ur J C e l l B i ol 24, 154- 156.
A r dui , S ., A m e ur , A ., V e r m e e s c h, J .R ., a nd H e s t a nd, M .S . ( 2018) . S i ngl e m ol e c ul e r e a l - t i m e
( S M R T ) s e que nc i ng c om e s of a ge : a ppl i c a t i ons a nd ut i l i t i e s f or m e di c a l di a gnos t i c s . N uc l e i c
A c i ds R e s 46, 2159- 2168.
B r a dne r , J .E ., H ni s z , D ., a nd Y oung, R .A . ( 2017) . T r a ns c r i pt i ona l a ddi c t i on i n c a nc e r . C e l l 168 ,
629- 643.
B ue nr os t r o, J .D ., G i r e s i , P .G ., Z a ba , L .C ., C ha ng, H .Y ., a nd G r e e nl e a f , W .J . ( 2013) .
T r a ns pos i t i on of na t i ve c hr om a t i n f or f a s t a nd s e ns i t i ve e pi ge nom i c pr of i l i ng of ope n c hr om a t i n,
D N A - bi ndi ng pr ot e i ns a nd nuc l e os om e pos i t i on. N a t ur e m e t hods 10, 1213- 1218. C um m i ngs ,
D .J ., T a i t , A ., a nd G odda r d, J .M . ( 1974) . M e t hyl a t e d ba s e s i n D N A f r om P a r a m e c i um a ur e l i a .
B i oc hi m B i ophys A c t a 374, 1- 11.
F u, Y ., L uo, G .Z ., C he n, K ., D e ng, X ., Y u, M ., H a n, D ., H a o, Z ., L i u, J ., L u, X ., D or e , L .C . , e t al .
( 2015) . N 6- m e t hyl de oxya de nos i ne m a r ks a c t i ve t r a ns c r i pt i on s t a r t s i t e s i n C hl a m ydom ona s . C e l l
161, 879- 892.
G or ovs ky, M .A ., H a t t m a n, S ., a nd P l e ge r , G .L . ( 1973) . ( 6 N ) m e t hyl a de ni ne i n t he nuc l e a r D N A
of a e uc a r yot e , T e t r a hym e na pyr i f or m i s . J C e l l B i ol 56, 697- 701.
J i a ng, C ., a nd P ugh, B .F . ( 2009) . N uc l e os om e pos i t i oni ng a nd ge ne r e gul a t i on: a dva nc e s t hr ough
ge nom i c s . N a t R e v G e ne t 10, 161- 172.
J ohns on, D . M or t a z a vi a , M ye r s R M , w ol d B ( 2007) G e nom e w i de m a ppi ng of i n vi vo
pr ot e i nD N a i nt e r a c t i ons . S c i e nc e 316, 1497- 1502.
K a r r e r , K .M ., a nd V a nN ul a nd, T .A . ( 2002) . M e t hyl a t i on of a de ni ne i n t he nuc l e a r D N A of
T e t r a hym e na i s i nt e r nuc l e os om a l a nd i nde pe nde nt of hi s t one H 1. N uc l e i c A c i ds R e s 30,
13641370.
47
K a ya - O kur , H .S ., W u, S .J ., C odom o, C .A ., P l e dge r , E .S ., B r ys on, T .D ., H e ni kof f , J .G ., A hm a d,
K ., a nd H e ni kof f , S . ( 2019) . C U T & T a g f or e f f i c i e nt e pi ge nom i c pr of i l i ng of s m a l l s a m pl e s a nd
s i ngl e c e l l s . N a t ur e c om m uni c a t i ons 10, 1- 10.
K e l l y, T .K ., L i u, Y ., L a y, F .D ., L i a ng, G ., B e r m a n, B .P ., a nd J one s , P .A . ( 2012) . G e nom e - w i de
m a ppi ng of nuc l e os om e pos i t i oni ng a nd D N A m e t hyl a t i on w i t hi n i ndi vi dua l D N A m ol e c ul e s .
G e nom e R e s 22, 2497- 2506.
K or nbe r g, R .D ., a nd L or c h, Y . ( 1999) . T w e nt y- f i ve ye a r s of t he nuc l e os om e , f unda m e nt a l
pa r t i c l e of t he e uka r yot e c hr om os om e . C e l l 98, 285- 294.
L uo, G .- Z ., a nd H e , C . ( 2017) . D N A N 6- m e t hyl a de ni ne i n m e t a z oa ns : f unc t i ona l e pi ge ne t i c
m a r k or bys t a nde r ? N a t ur e s t r uc t ur a l & m ol e c ul a r bi ol ogy 24, 503- 506.
M i kke l s e n, T ., K u, M ., J a f f e , D ., I s s a c , B ., L i e be r m a n, E ., G i a nnoukos , G ., A l va r e z , P .,
B r oc km a n, W ., K i m , T ., a nd K oc he , R . ( 2007) . O / ’ D onova n A , P r e s s e r A , R us s C , X i e X ,
M e i s s ne r A , W e r ni g M , J a e ni s c h R , N us ba um C , L a nde r E S , B e r ns t e i n B E : G e nom e - w i de m a ps
of c hr om a t i n s t a t e i n pl ur i pot e nt a nd l i ne a ge - c om m i t t e d c e l l s . N a t ur e 448, 553- 560. M ur r a y, I .A .,
M or ga n, R .D ., L uyt e n, Y ., F om e nkov, A ., C or r e a , I .R ., J r ., D a i , N ., A l l a w , M .B ., Z ha ng, X .,
C h e n g , X . , a n d R o b e r t s , R . J . ( 2 0 1 8 ) . T h e n o n - s p e c i f i c a d e n i n e D N A m e t h y l t r a n s f e r a s e M . E c o G I I .
N uc l e i c A c i ds R e s 46, 840- 848.
P a r k, P .J . ( 2009) . C hI P - s e q: a dva nt a ge s a nd c ha l l e nge s of a m a t ur i ng t e c hnol ogy. N a t R e v G e ne t
10, 669- 680.
R a e , P .M ., a nd S t e e l e , R .E . ( 1978) . M odi f i e d ba s e s i n t he D N A s of uni c e l l ul a r e uka r yot e s : a n
e xa m i na t i on of di s t r i but i ons a nd pos s i bl e r ol e s , w i t h e m pha s i s on hydr oxym e t hyl ur a c i l i n
di nof l a ge l l a t e s . B i os ys t e m s 10, 37- 53.
R obe r t s on, G ., H i r s t , M ., B a i nbr i dge , M ., B i l e nky, M ., Z ha o, Y ., Z e ng, T ., E us ki r c he n, G .,
B e r ni e r , B ., V a r hol , R ., a nd D e l a ne y, A . ( 2007) . G e nom e - w i de pr of i l e s of S T A T 1 D N A
a s s oc i a t i on us i ng c hr om a t i n i m m unopr e c i pi t a t i on a nd m a s s i ve l y pa r a l l e l s e que nc i ng. N a t ur e
m e t hods 4, 651- 657.
S hi pony, Z ., M a r i nov, G .K ., S w a f f e r , M .P ., S i nnot t - A r m s t r ong, N .A ., S kot he i m , J .M ., K unda j e ,
A ., a nd G r e e nl e a f , W .J . ( 2020a ) . L ong- r a nge s i ngl e - m ol e c ul e m a ppi ng of c hr om a t i n a c c e s s i bi l i t y
i n e uka r yot e s . N a t ur e m e t hods 17, 319- 327.
S hi pony, Z ., M a r i nov, G .K ., S w a f f e r , M .P ., S i nnot t - A r m s t r ong, N .A ., S kot he i m , J .M ., K unda j e ,
A ., a nd G r e e nl e a f , W .J . ( 2020b) . L ong- r a nge s i ngl e - m ol e c ul e m a ppi ng of c hr om a t i n a c c e s s i bi l i t y
i n e uka r yot e s . N a t M e t hods 17, 319- 327.
S ke ne , P .J ., a nd H e ni kof f , S . ( 2017) . A n e f f i c i e nt t a r ge t e d nuc l e a s e s t r a t e gy f or hi gh- r e s ol ut i on
m a ppi ng of D N A bi ndi ng s i t e s . e l i f e 6, e 21856.
48
S ong, L ., a nd C r a w f or d, G .E . ( 2010) . D N a s e - s e q: a hi gh- r e s ol ut i on t e c hni que f or m a ppi ng a c t i ve
ge ne r e gul a t or y e l e m e nt s a c r os s t he ge nom e f r om m a m m a l i a n c e l l s . C ol d S pr i ng H a r bor
P r ot oc ol s 2010, pdb. pr ot 5384.
S t e e ns e l , B .v., a nd H e ni kof f , S . ( 2000) . I de nt i f i c a t i on of i n vi vo D N A t a r ge t s of c hr om a t i n
pr ot e i ns us i ng t e t he r e d da m m e t hyl t r a ns f e r a s e . N a t ur e bi ot e c hnol ogy 18, 424- 428.
S t e r ga c hi s , A .B ., D e bo, B .M ., H a uge n, E ., C hur c hm a n, L .S ., a nd S t a m a t oya nnopoul os , J .A .
( 2020) . S i ngl e - m ol e c ul e r e gul a t or y a r c hi t e c t ur e s c a pt ur e d by c hr om a t i n f i be r s e que nc i ng.
S c i e nc e 368 , 1449- 1454.
W a ng, Y ., W a ng, A ., L i u, Z ., T hur m a n, A .L ., P ow e r s , L .S ., Z ou, M ., Z ha o, Y ., H e f e l , A ., L i , Y .,
Z a bne r , J . , e t al . ( 2019) . S i ngl e - m ol e c ul e l ong- r e a d s e que nc i ng r e ve a l s t he c hr om a t i n ba s i s of
ge ne e xpr e s s i on. G e nom e R e s 29, 1329- 1342.
W e nge r , A .M ., P e l us o, P ., R ow e l l , W .J ., C ha ng, P .- C ., H a l l , R .J ., C onc e pc i on, G .T ., E bl e r , J .,
F ungt a m m a s a n, A ., K ol e s ni kov, A ., a nd O l s on, N .D . ( 2019) . A c c ur a t e c i r c ul a r c ons e ns us
l ongr e a d s e que nc i ng i m pr ove s va r i a nt de t e c t i on a nd a s s e m bl y of a hum a n ge nom e . N a t ur e
bi ot e c hnol ogy 37, 1155- 1162.
Z a nc ona t o, F ., B a t t i l a na , G ., F or c a t o, M ., F i l i ppi , L ., A z z ol i n, L ., M a nf r i n, A ., Q ua r a nt a , E ., D i
B i a gi o, D ., S i gi s m ondo, G ., a nd G uz z a r do, V . ( 2018) . T r a ns c r i pt i ona l a ddi c t i on i n c a nc e r c e l l s i s
m e di a t e d by Y A P / T A Z t hr ough B R D 4. N a t ur e m e di c i ne 24 , 1599- 1610.
Z a r e t , K . ( 1999) . M i c r oc oc c a l nuc l e a s e a na l ys i s of c hr om a t i n s t r uc t ur e . C ur r e nt pr ot oc ol s i n
m ol e c ul a r bi ol ogy 45, 21.21. 21- 21.21. 17.

Abstract (if available)

Abstract A main quest in epigenetics is to reveal chromatin organization, especially distribution of chromatin-associated proteins (CAPs). At present, this is usually achieved by collecting chromatin fragments of interests (often generated by nuclease digestion) and short-read sequencing. We are developing a new approach in which the linear organization of multikilobase chromatin fibers is transcoded into N6-adenine methylation (6mA) sites on their constituent DNA molecules by altered methyltransferase (MTase) access, and subsequently decoded by long-read sequencing. We have purified and characterized 6mA MTases, EcoGII and Hia5, both of which can methylate adenine in most sequence contexts. We have performed general 6mA footprinting, in which saturate methylation by EcoGII or Hia5 reveals global distribution of CAPs. We have also tested targeted 6mA footprinting, in which EcoGII or Hia5 is guided by an antibody to deposit 6mA in the vicinity of a specific CAP. Our approach can potentially provide new information on chromatin organization, especially the coordinated binding of neighboring CAPs at the single-molecule level.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Epigenetic dysregulation in acute myeloid leukemia (AML) with MLL1 aberrations

PDF

Limit of detection analysis for cell-free DNA methylation using targeted bisulfite sequencing

PDF

DNA methylation inhibitors and epigenetic regulation of microRNA expression

PDF

Understanding DNA methylation and nucleosome organization in cancer cells using single molecule sequencing

PDF

DNA methylation as a biomarker in human reproductive health and disease

PDF

Transcriptional regulation by epigenetic mechanisms

PDF

The kinetic study of engineered MBD domain interactions with methylated DNA: insight into binding of methylated DNA by MBD2b

PDF

Epigenetic mechanisms driving bladder cancer

PDF

Cancer-recurrent missense mutations in SET domain alter KMT2A methyltransferase activity

PDF

Developing MLL1 degraders as therapeutics for leukemia

PDF

The relationship between DNA methylation and transcription factor binding in colon cancer cells

PDF

Exploring stem cell pluripotency through long range chromosome interactions

PDF

Characterization of zebrafish phf6 CRISPR mutant phenotypes

PDF

Evaluating the therapeutic potential of targeting CBX8 in MLLr leukemia

PDF

Identification and characterization of PR-Set7 and histone H4 lysine 20 methylation-associated proteins

PDF

A novel screening approach to identify regulators of polycomb-dependent gene silencing

PDF

The role of nuclear GRP78 in regulation of EGFR expression in lung cancer

PDF

Functional DNA methylation changes in normal and cancer cells

PDF

Determining the epigenetic contribution of basal cell identity in cystic fibrosis

PDF

DNA methylation review and application

Asset Metadata

Creator Yu, Jingqi (author)

Core Title Collecting epigenetic information with DNA methylation footprinting

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2022-08

Publication Date 07/22/2022

Defense Date 05/06/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag epigenetics,long-read sequencing,methyltransferase；chromatin-associated proteins,N6-adenine methylation,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Dou, Yali (committee chair), An, Woojin (committee member), Liu, Yifan (committee member)

Creator Email jingqiyes@163.com,jingqiyu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111373923

Unique identifier UC111373923

Legacy Identifier etd-YuJingqi-10925

Document Type Thesis

Format application/pdf (imt)

Rights Yu, Jingqi

Type texts

Source 20220722-usctheses-batch-961 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu