|
 
- 积分
- 368
- 威望
- 368
- 包包
- 2103
|
回复 xuyang0317 的帖子
6 {9 M U1 |! E
' ]7 }4 m, S+ @3 V2 ~& DINTRODUCTION$ L0 I# l! L% }! A# V) P, B2 e
Mammalian DNA methylation at 5-cytosine plays critical roles in T8 z& A3 Q7 ^; U8 L% z, W
many biological processes, including genomic imprinting, cell-9 {4 N/ q* u. u: f3 g
fate determination, chromatin architecture organization, and
1 `% b K( g( t& m" C' T9 o5 kregulation of gene expression (Bird, 2002; Jaenisch and Bird,! K! v/ ]6 a5 s1 }1 Y
2003; Smith and Meissner, 2013). Genetic studies have revealed/ [/ Q4 z' T6 |
that DNA methylation is essential for mammalian development
# Y( f, c) Y3 @) h' f# X fand adaptation to environmental signals (Jaenisch and Bird,
" F7 [, S# I* H5 i4 U7 P2003). Abnormal DNA methylation has been observed in cancer: q- Q: a2 V) r7 u) N6 U2 T+ }5 j
and neurological disorders (Robertson, 2005). Owing to the% E9 s p. Z. q& P7 ~
advancement in sequencing technologies, single-nucleotide
: J, T2 L7 v2 S0 tresolution methylation maps for many types of human and
3 r/ Y0 @ O( x/ J" a8 Qmouse cells and tissues have been depicted (Lister et al.,
6 m7 F& d8 ]. P, c9 K9 ^1 k# U2009, 2013). Importantly, these maps have allowed for the iden-/ k( P) y0 K; F, \( Y
tification of differentially methylated regions (DMRs) at base pair
- t' P: B" Z: n& T% l/ J2 i/ Mresolution during different stages of normal development as well: t- z" i& I6 Y1 y
as disease (De Jager et al., 2014; Landau et al., 2014). However,, l' A* K2 C( i3 n" ?+ P2 [! v+ P
investigation of the functional significance of these DMRs re-
: c! ^% h% B4 O, `* {9 `mains a challenge due to lack of appropriate molecular tools9 Y7 Q$ f! c( A" |
that enable efficient editing of DNA methylation in a targeted- _1 u ^- N3 ^9 ]+ _
manner.
& w: M+ `8 V3 f! D% QWe set out to establish such a toolbox by hybridization of the
/ c0 j( t1 s) m6 _+ Gkey enzymes in the DNA methylation pathway with reprogram-9 F0 [' t" B9 G
mable sequence-specific DNA-targeting molecular machinery., y# T% ~+ n8 ]' \
DNA methylation is established by two de novo DNA methyl-/ t) V5 }3 T: D4 p# A8 M
transferases (Dnmt3a/b) and is maintained by Dnmt1 (Smith2 \% s! z3 c! j4 m
and Meissner, 2013). Gene activation during development is
! q- W* q6 b3 ^6 s5 ~ kassociated with demethylation of promoter and enhancer se-. d k" D/ P3 [" n0 Z |3 {# H
quences with the best-understood mechanism being passive1 p! h3 s) N# z# H' i
demethylation by inhibition of Dnmt1. In addition, demethyla-' ~. A# ^+ Z& d
tion can be achieved through oxidation of the methyl group by k8 m# H; p; H5 }
TET (ten-eleven translocation) dioxygenases to form 5-hydroxy-5 A8 t# ?5 Y4 {% d, n( u
methylcytosine (5-hmC), and then restoration into unmodified
- M* d/ N- g F4 \cytosines by either DNA replication-dependent dilution or DNA3 R7 D! _+ e/ ^' z J0 O
glycosylase-initiated base excision repair (BER), a process
8 Z2 s* C5 o5 T3 w) s/ stermed as active demethylation and proposed to operate during5 q4 D- p& N/ s/ a
specific developmental stages suchaspreimplantation embryos
/ z) v( b: J, P* _7 X7 Zor in post-mitotic neurons (Wu and Zhang, 2014).2 q; V, y( ~- {9 D, D3 p
Clusteredregularlyinterspacedpalindromicrepeats(CRISPR),
& B; r3 ] r1 E' ^2 sa type II bacterial adaptive immune system, has been modified6 J1 L* o$ b$ L: W7 d
to target theCas9nuclease to the desiredgenomic lociwithseq-
], K7 S+ k3 O' auence-specific guide RNAs for genome editing (Cong et al.,
d1 w, w1 O0 s/ ?# `4 e, t3 Y2013; Jinek et al., 2012; Mali et al., 2013). Importantly, a catalyt-
$ w# T# O" p( gically inactive Cas9 (dCas9) was generated and engineered in0 J ?' L1 y! I! i4 P9 o3 G+ D
severalsystems as a DNAtargeting moduleto bringeffector pro-
- C4 p; ]7 V/ ^ Nteins such as transcriptional activator/suppressor, chromatin3 Y) `3 `2 l5 F1 {1 s' _0 p
modifier, and green fluorescence protein to regulate gene
& T- Y: \/ Y) w! eexpression, to modify chromatin, and to image genomic loci
6 e. f1 d" j3 L) w* E3 m' ?8 d8 Vrespectively (Chen et al., 2013; Gilbert et al., 2013; Hilton et al.,
1 K( A. ?& f2 H& N) l! T: n. v2015; Jinek et al., 2012; Konermann et al., 2015; Qi et al., 2013).
3 p7 V% B/ p6 H/ T4 ?In this study, we demonstrate that fusion of dCas9 with the
7 I2 t$ P; } U# Q/ k) KTet1 enzymatic domain or Dnmt3a allows for targeted erasure
7 J. w g. I4 x: M& `or establishment of DNA methylation, respectively. As a proof
; x1 Q: P t/ Y, X; Oof principle, we first induced alterations to DNA methylation
" h9 g& D8 r0 d. k/ iin two synthetic methylation reporters integrated in mouse0 k. L8 `! t3 C5 R
embryonic stem cells (mESCs). We further show that targeted9 I- [9 [1 r7 g, H7 Q
demethylation of BDNF promoter IV is sufficient to activate G: Z- j8 s/ G1 _- {
its expression in mouse cortical neurons, and that targeted
% e) {/ c3 W' A3 v; y& Vdemethylation of a MyoD distal enhancer promotes reprogram-3 K" Q/ ~% k- C
ming of fibroblasts into myoblasts and facilitates myotube for-
4 k- m9 V$ I( u+ M; \ \mation. With dCas9-Dnmt3a, we demonstrate that targeted p5 p7 G* P0 Z5 M4 c4 y& b
methylation at CTCF binding sites is able to block CTCF recruit-' k9 g$ s! p! e. Q6 s2 l! i
ment and to alter the expression of genes in the neighborhood
( Y) K, i; s7 S+ R+ a6 Wloop by increasing their interaction frequencies with the super-
# M. g7 Q% F8 O) [" ~) Penhancers insulated in the targeted loops. Furthermore, lentiviral
7 [" H' ^# d4 H. y7 q3 b% }$ qdelivery of dCas9-Tet1 with target gRNAs into mice enabled* j( D: e; t0 |0 t T7 [
in vivo activation of a methylation reporter by demethylation of
5 S, ]+ j3 t4 O: y3 fits promoter. Thus, dCas9-Tet1 and dCas9-Dnmt3a provide
8 @: Q3 \' r* r; Fpowerful tools to investigate the functional significance of DNA! \3 J$ i( H$ _& J0 F
methylation in a locus-specific manner. |
|
发表于 2016-10-7 08:47
