|
 
- 积分
- 368
- 威望
- 368
- 包包
- 2103
|
回复 xuyang0317 的帖子5 n0 f# g, L; ~- B/ y S
; `9 Z. I' I/ O5 r" K* b
INTRODUCTION
6 [, B1 n, H: J0 gMammalian DNA methylation at 5-cytosine plays critical roles in
' A4 @* j# K3 i. I4 l1 Emany biological processes, including genomic imprinting, cell-
' I! R3 {4 N3 o1 xfate determination, chromatin architecture organization, and% s9 G+ j8 n2 W8 `( J( ^& Q- i
regulation of gene expression (Bird, 2002; Jaenisch and Bird,
& n2 @6 c5 E5 R: k5 w2003; Smith and Meissner, 2013). Genetic studies have revealed
n; |! e) w( _* c( ], R, ^9 `that DNA methylation is essential for mammalian development
8 J' @% ]" V- o' |( i/ T: ^6 Iand adaptation to environmental signals (Jaenisch and Bird,
6 {- n: m# X' x3 c5 w# u$ g& Q2003). Abnormal DNA methylation has been observed in cancer
! a+ d- U6 d1 i! {2 Sand neurological disorders (Robertson, 2005). Owing to the; z1 G8 T2 Z+ Y" d
advancement in sequencing technologies, single-nucleotide! s/ u0 C3 B. s; h/ o% ^. K
resolution methylation maps for many types of human and
" i- C1 s5 R2 c# nmouse cells and tissues have been depicted (Lister et al.,1 g) V, _: N: s1 K& _
2009, 2013). Importantly, these maps have allowed for the iden-
7 q& X9 f# P9 K, l# Htification of differentially methylated regions (DMRs) at base pair
1 L: x* D9 b3 Nresolution during different stages of normal development as well
8 F$ m1 V" O+ \, E$ Das disease (De Jager et al., 2014; Landau et al., 2014). However,
0 h" _- P! n3 ~2 u& u$ f* \investigation of the functional significance of these DMRs re-' r* o/ s" W" C# H
mains a challenge due to lack of appropriate molecular tools
$ N: N0 t% k9 N8 N, Z5 W5 u8 S7 bthat enable efficient editing of DNA methylation in a targeted
- H$ z7 |2 ~- S4 ?% omanner." a4 j6 k' p8 c/ H+ f7 G
We set out to establish such a toolbox by hybridization of the
1 A& A. q/ S: x1 hkey enzymes in the DNA methylation pathway with reprogram-/ y. s U: L7 u: Q5 E) I6 f( K3 \
mable sequence-specific DNA-targeting molecular machinery.) F5 F6 K' M; U* w) o' D" \
DNA methylation is established by two de novo DNA methyl-
) F9 e' P! D# A4 W* Rtransferases (Dnmt3a/b) and is maintained by Dnmt1 (Smith8 O: W7 I) m7 B4 a/ I W. z
and Meissner, 2013). Gene activation during development is1 o/ Q5 Z9 V7 \- d- E4 m0 K5 L
associated with demethylation of promoter and enhancer se-
: a: p! g( m+ E" N. m2 S k( |/ Wquences with the best-understood mechanism being passive
2 O4 |; |# O0 I) }0 D, kdemethylation by inhibition of Dnmt1. In addition, demethyla-' l* c4 i: x1 g$ |, n9 _9 P
tion can be achieved through oxidation of the methyl group by
. ]8 [: d2 f F: V# Q; qTET (ten-eleven translocation) dioxygenases to form 5-hydroxy-
4 N+ X6 k' w6 c2 b# I0 wmethylcytosine (5-hmC), and then restoration into unmodified+ D" Y) e- d. u* D
cytosines by either DNA replication-dependent dilution or DNA
, j0 G9 Y; W- Q7 ]8 S( Aglycosylase-initiated base excision repair (BER), a process
- a' n3 p1 w& W3 i; _( M5 Y6 ltermed as active demethylation and proposed to operate during" {9 _! }) ^5 U! F1 \
specific developmental stages suchaspreimplantation embryos1 }/ a5 K* ~" H6 F
or in post-mitotic neurons (Wu and Zhang, 2014).& k. e. P, w5 K J
Clusteredregularlyinterspacedpalindromicrepeats(CRISPR),
0 [2 y0 t! w7 x+ m" e% l5 j; ia type II bacterial adaptive immune system, has been modified, a+ E z; l8 C, N" j
to target theCas9nuclease to the desiredgenomic lociwithseq-. Y m" ^1 _9 ~: i
uence-specific guide RNAs for genome editing (Cong et al.,+ j; L c H% p/ N
2013; Jinek et al., 2012; Mali et al., 2013). Importantly, a catalyt-
( Y3 s% ~7 y: \3 Dically inactive Cas9 (dCas9) was generated and engineered in+ L B$ t$ c7 {2 p! Q' R
severalsystems as a DNAtargeting moduleto bringeffector pro-
/ @: P) M' S( Y* v$ C$ _: c4 kteins such as transcriptional activator/suppressor, chromatin# K+ E. p( q% w8 L
modifier, and green fluorescence protein to regulate gene' t2 R* B+ v7 `! \1 G
expression, to modify chromatin, and to image genomic loci
7 V( J; J# x3 s& e8 t+ frespectively (Chen et al., 2013; Gilbert et al., 2013; Hilton et al.,
7 W9 Z3 n& P) ^0 u2015; Jinek et al., 2012; Konermann et al., 2015; Qi et al., 2013).1 z9 D! N. ?+ z2 r* M) W
In this study, we demonstrate that fusion of dCas9 with the
( T# x9 V. i3 g$ L1 u: Z% tTet1 enzymatic domain or Dnmt3a allows for targeted erasure
, y1 y0 `* J8 ~9 v. c% P$ gor establishment of DNA methylation, respectively. As a proof
3 W# l9 o6 w* g" S' I! nof principle, we first induced alterations to DNA methylation
& s6 e. r/ y! j0 c# Nin two synthetic methylation reporters integrated in mouse
/ p+ P3 e6 z/ }6 cembryonic stem cells (mESCs). We further show that targeted
1 s6 J2 Q$ P& s6 _& udemethylation of BDNF promoter IV is sufficient to activate4 z0 n5 j7 w# Q
its expression in mouse cortical neurons, and that targeted
- ?. s! k8 v' o0 F0 p. tdemethylation of a MyoD distal enhancer promotes reprogram-
2 v/ c# U p0 R8 Nming of fibroblasts into myoblasts and facilitates myotube for-
# O. p) m- K8 A1 G. M dmation. With dCas9-Dnmt3a, we demonstrate that targeted
& W# h" u& n- g% n6 Y& ]methylation at CTCF binding sites is able to block CTCF recruit-
' J9 L2 e p1 Cment and to alter the expression of genes in the neighborhood
: P$ |: R r! t5 Y, u* C5 Jloop by increasing their interaction frequencies with the super-
9 |+ A! M- l! n" ]0 E: o1 denhancers insulated in the targeted loops. Furthermore, lentiviral* q0 l! f3 y8 x) p
delivery of dCas9-Tet1 with target gRNAs into mice enabled: }( d$ X3 l( N7 T7 z2 Y& A
in vivo activation of a methylation reporter by demethylation of
$ `. z* O! a3 Z/ q: Kits promoter. Thus, dCas9-Tet1 and dCas9-Dnmt3a provide
7 G- K6 f* q$ P, Npowerful tools to investigate the functional significance of DNA0 E7 f1 Q% z5 i I" j
methylation in a locus-specific manner. |
|
发表于 2016-10-7 08:47
