|
  
- 积分
- 0
- 威望
- 0
- 包包
- 483
|
作者：John Arne Dahl, Philippe Collas作者单位：Institute of Basic Medical Sciences, Department of Biochemistry, Faculty of Medicine, University of Oslo, Oslo, Norway . H3 R, `1 G5 E8 E# |
& { ?* G; n# U( x, k( n8 a+ Y
$ p) r; V' k1 I. h
0 W6 m" F' o5 p: L% G 6 m9 Y1 r# a/ k
3 K+ s7 Z# H: b
$ K+ w! N3 P+ _2 o+ u8 d2 ~
+ f9 T/ d/ d& L0 c
, y: l G" j. }0 ?' n$ l 5 \ H& o- R& f/ ^) i
* R) A5 M3 b' a1 N
: n6 x+ F4 |3 \5 r; J8 I
6 G4 }, l O" n. g( G
【摘要】' e2 [ p/ D8 d+ \7 @
Chromatin immunoprecipitation (ChIP) is a key technique for studying protein-DNA interactions and mapping epigenetic histone modifications on DNA. Current ChIP protocols require extensive sample handling and large cell numbers. We developed a quick and quantitative (Q2)ChIP assay suitable for histone and transcription factor immunoprecipitation from chromatin amounts equivalent to as few as 100 cells. DNA-protein cross-linking in suspension in presence of butyrate, elimination of background chromatin through a tube shift after washes, and a combination of cross-link reversal, protein digestion, increased antibody-bead to chromatin ratio, and DNA elution into a single step considerably improve ChIP efficiency and shorten the procedure. We used Q2ChIP to monitor changes in histone H3 modifications on the 5' regulatory regions of the developmentally regulated genes OCT4, NANOG, LMNA, and PAX6 in the context of retinoic-acid-mediated human embryonal carcinoma cell differentiation. Quantitative polymerase chain reaction analysis of precipitated DNA unravels biphasic heterochromatin assembly on OCT4 and NANOG, involving H3 lysine (K)9 and K27 methylation followed by H3K9 deacetylation and additional H3K27 trimethylation. Di- and trimethylation of H3K4 remain relatively unaltered. In contrast, PAX6 displays histone modifications characteristic of repressed genes with potential for activation in undifferentiated cells. PAX6 undergoes H3K9 acetylation and enhanced H3K4 trimethylation upon transcriptional activation. Q2ChIP of the transcription factor Oct4 demonstrates its dissociation from the NANOG promoter upon differentiation. This study is, to our knowledge, the first to reveal histone modification changes on human OCT4 and NANOG regulatory sequences. The results demonstrate ordered chromatin rearrangement on developmentally regulated promoters upon differentiation.6 K& X Q( o! w, [: F! F9 Q2 b
3 g1 ]1 s+ ?, Q) |
Disclosure of potential conflicts of interest is found at the end of this article.
1 ?5 A6 I% n" i% ^% N" v# e ? 【关键词】 Chromatin immunoprecipitation Differentiation Embryonal carcinoma cell Histone acetylation Histone methylation; {) L6 @! z; h; U: f+ N
INTRODUCTION) }& j: B1 \6 b
/ g+ t3 _( c7 d5 E
Interactions between proteins and DNA are essential for cellular functions such as genomic stability, DNA replication and repair, chromosome segregation, transcription, and epigenetic silencing of gene expression. Chromatin immunoprecipitation (ChIP) . Limitations of current ChIP procedures, however, are a requirement for large numbers of cells (107 per immunoprecipitation), exhaustive sample handling, antibody cost, time, and significant loss of material. This makes current ChIP protocols unsuitable for analysis of small or precious samples and tedious for analysis of many samples in parallel.9 j9 l) D& b1 s1 c4 H4 _9 \
1 g) D, X5 c- B# a, V' X
Maintenance of a pluripotent state in embryos and embryonic stem (ES) cells is conferred by at least two developmentally regulated homeodomain transcription factors, Oct4 and Nanog . No report exists, however, on histone modifications on human OCT4 and NANOG regulatory elements.
! j+ \7 M1 u g# E4 ?4 s
9 ]: Y9 ]& A6 YUsing a quick and quantitative (Q2)ChIP assay suitable for chromatin from cell numbers up to 105-fold lower than those required for conventional ChIP, we monitored changes in six histone modifications on the promoters of developmentally regulated genes in differentiating human EC cells. Q2ChIP unravels an unexpected biphasic heterochromatinization of OCT4 and NANOG in response to RA stimulation, whereas PAX6, a gene repressed in undifferentiated cells, acquires histone modifications compatible with transcriptional activation. Q2ChIP also shows a dynamic association of the Oct4 transcription factor with the NANOG promoter during differentiation. This study is, to our knowledge, the first to reveal histone modification changes on human OCT4 and NANOG regulatory sequences.7 B+ X, }3 j2 Z- Y% U) G
3 V3 z4 ]. y# a H4 w6 Q' k
MATERIALS AND METHODS
( n# a- E+ C9 z/ I
: s+ e0 e# o6 H4 n5 F; ?$ s0 Y1 tAntibodies and Reagents
! E7 L! m% n/ Y; G; o
8 Y b' s6 P6 NAntibodies against H3K9ac (catalog number 06-942), H3K9m2 (catalog number 07-441), H3K9m3 (catalog number 07-442), and H3K27m3 (catalog number 05-851) were from Upstate (Charlottesville, VA, http://www.upstate.com). Antibodies against H3K4m2 (ab7766) and H3K4m3 (ab8580) were from Abcam (Cambridge, U.K., http://www.abcam.com). Rabbit anti-Oct4 antibodies (H-65) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com). Other reagents were from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com) unless otherwise stated.
; p9 o% G* G3 w% K4 V/ n- Z* a/ o/ I. Z2 g: N
Cells- k4 ^: T/ U! p6 N1 b6 ?4 o
7 j; H- D3 n r: L# I- H
Undifferentiated human teratocarcinoma NCCIT cells (American Type Culture Collection, Manassas, VA, http://www.atcc.org) were established from a mediastinal germ cell tumor and can differentiate into derivatives of the three germ layers .( O' q6 ~9 H+ ^4 O
1 @: g! I; [, ]; V
Q2ChIP) L* k- s, d3 B; w2 n0 Y% B
) Z5 o' [ d' [+ T: VAntibody-Bead Complexes. Paramagnetic beads (Dynabeads Protein A; Dynal Biotech, Oslo, Norway, http://www.dynalbiotech.com) were washed twice in RIPA buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl) and resuspended in 1 volume of RIPA buffer. Beads (10 µl) were added to 90 µl of RIPA buffer and 2.4 µg of primary antibody (specified in the text) in a 0.2-ml polymerase chain reaction (PCR) tube and incubated on a rotator (Stuart SB3; Barloworld Scientific, Staffordshire, U.K., http://www.barloworld-scientific.com; 40 rpm) for 2 hours at 4¡ãC. Note that, for this and subsequent procedures (except when noted), PCR tubes were in the form of 8-tube strips and handled in parallel in an aluminum rack fitted with a magnet and chilled on ice.) z& O" \7 i% q* O1 U
' O3 R5 t3 @ s8 o: @+ U9 @% W* a
DNA-Protein Cross-Linking. Immediately before cell harvest, 20 mM the histone deacetylase inhibitor sodium butyrate was added to the culture medium (Fig. 1). Butyrate was also added to all solutions thereafter unless otherwise indicated. Cells were harvested by trypsinization, washed, and resuspended to 1¨C2 x 106 cells per milliliter in phosphate-buffered saline (PBS). Formaldehyde was added to 1% (vol/vol) and, after 8 minutes of fixation, was stopped with 125 mM glycine for 5 minutes. All following steps were performed on ice or at 4¡ãC.
7 \! d* F5 x4 F* j+ U! C8 [3 O( M1 w! p
Figure 1. Development of Q2ChIP. (A): Cross-linking in presence of sodium butyrate. NCCIT cells were cross-linked in suspension in 1% formaldehyde without or with 20 mM sodium butyrate prior to sonication and immunoprecipitation of H3K9ac or H3K9m2. Precipitated DNA was analyzed by real-time polymerase chain reaction (PCR) in triplicates using GAPDH promoter-specific primers and data expressed as ChIP efficiency relative to the XL procedure (mean ¡À SD relative ChIP efficiency; duplicate experiment). (B): Tube shift: H3K9ac and H3K9m2 were precipitated under XL NaB conditions. ChIP material was washed and, while in TE buffer, transferred to a clean 0.2-ml tube (tube shift ) or kept in the same tube (tube shift ¨C) for further processing. Precipitated GAPDH promoter DNA was analyzed by real-time PCR (mean ¡À SD fold enrichment of precipitated DNA relative to no antibody control . Conventional ChIP was also applied to 2 A260 units chromatin in a 100 µl reaction volume (mean ¡À SD fold enrichment of precipitated DNA relative to no-antibody control; 2¨C4 experiments). Abbreviations: Ab, antibody; ChIP, chromatin immunoprecipitation; Q2ChIP, quick and quantitative ChIP; Rel., relative; XL, without 20 mM sodium butyrate; XL NaB, with 20 mM sodium butyrate.
2 n; g9 H- H: t0 A/ O) ?: t: S+ q G+ c. s
Lysis, Sonication. Cross-linked cells were washed twice by sedimentation and suspension in 0.5¨C1 ml of PBS/20 mM butyrate and lysed for 5 minutes by a sixfold dilution in lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, protease inhibitor cocktail 1 mM PMSF) containing 20 mM butyrate. Aliquots of 200 µl in 1.5-ml tubes were sonicated, each for 10 x 30 seconds on ice (Labsonic-M, 3-mm probe; cycle 0.5, 30% power; Sartorius AG, Goettingen, Germany, http://www.sartorius.com) with 30-second pauses on ice to produce chromatin fragments of 500 base pairs. Note that, when preparing chromatin from 100,000 cells, samples (120 µl) were sonicated in 0.5-ml tubes. The lysate was sedimented at 10,000g for 10 minutes, and the supernatant, except the upper lipid layer, was collected and DNA (chromatin) concentration determined by A260 from an aliquot diluted 100-fold.% d* D* B9 S" C1 `4 O# Z
6 Q% Q8 |/ P. YImmunoprecipitation. Chromatin was diluted to a concentration of 2 A260 units in RIPA buffer containing 20 mM butyrate. Chromatin (100 µl, 2 A260 units) was transferred to a 0.2-ml tube containing antibody-bead complexes in RIPA buffer held to the tube wall by a magnet. Beads were released into the chromatin suspension and rotated at 40 rpm for 2 hours at 4¡ãC. In dilution experiments, chromatin was further diluted in RIPA buffer to produce chromatin from 104, 103, and 102 cell equivalents (eq.) as specified in the text.
! W1 b7 P8 _9 F2 e" s- K& N; u) K: M
" m8 [8 U$ w: bWashes. Immune complexes (ChIP material) were washed three times by capturing and releasing the beads in fresh 100 µl of RIPA buffer and once in TE buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Each wash lasted for 4 minutes on a rotator (40 rpm) at 4¡ãC.- `+ V e7 l! \: d/ ?
/ I d0 q- n" Z4 q5 N. vDNA Elution, Cross-Link Reversal, Proteinase K Digestion. ChIP material suspended in TE, pH 8.0, was transferred to a new 0.2-ml tube. Beads were captured, TE was removed, and 150 µl of elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 20 mM sodium butyrate, 50 mM NaCl) containing 1% SDS and 50 µg/ml proteinase K were added. Samples were incubated for 2 hours at 68¡ãC on a Thermomixer (Eppendorf AG, Hamburg, Germany, http://www.eppendorf.de; 1,300 rpm) with constant vortexing. After capturing the beads, the supernatant was recovered and the ChIP material was reincubated in 150 µl of elution buffer/SDS/proteinase K for 5 minutes on the Thermomixer. Both supernatants were pooled. ~6 F6 P( j- o$ I2 L
# u% S9 y! s3 c* T2 I) ^DNA Isolation. Elution buffer (200 µl) was added to the eluted ChIP material (300 µl), and DNA was extracted once with phenol-chloroform isoamylalcohol and once with chloroform isoamylalcohol and was ethanol-precipitated. The Q2ChIP procedure is summarized in Figure 2 with indications on which step is compatible with ¨C20¡ãC freezing of the preparations.
; y3 H7 U* k- p6 G" Y3 D! {7 j3 l
$ i# L1 Q+ r, y) y9 y0 `& T, k1 jFigure 2. Diagram summary of the quick and quantitative ChIP procedure. See Materials and Methods for details. Abbreviations: bp, base pairs; ChIP, chromatin immunoprecipitation; h, hours; min, minutes; PCI, phenol-chloroform isoamylalcohol; PCR, polymerase chain reaction; PF, paraformaldehyde; TE, Tris EDTA.5 g) X8 a1 {" \5 ?: _/ |
9 r: R- y' r% t' x6 a T
ChIP Data9 A$ [4 ~7 M6 j. a- g
) j# Y$ q- T% W+ f& [ChIP is assumed to precipitate less than 1% of nucleosomes, thus real-time PCR data for antibody-bound fractions were compared to a 1/100 dilution of input DNA in Figure 3. One-sample t tests were used to compare the level of a histone modification to that of a reference sample, paired t tests were used to compare two samples, and analysis of variance (ANOVA) was used to compare three or more samples.
/ D, @* O: b2 Q; B1 x8 q2 W1 A( c! m: p* V0 a) [; a: d
Figure 3. Quick and quantitative chromatin immunoprecipitation (Q2ChIP) analysis of histone H3 modifications in undifferentiated NCCIT cells on regulatory regions of developmentally regulated genes. (A): Regulatory regions examined by Q2ChIP on the OCT4 PP, PE, and DE and on the promoter of NANOG, LMNA, PAX6, and GAPDH. Five primer pairs were used for OCT4 (OCT4-A, B, C, D, E). Regions amplified by polymerase chain reaction are indicated by bars and nucleotide number relative to the ATG translation initiation site. Position of the TSS is indicated. (B¨CF): Q2ChIP analysis of (B) PP, PE, and DE of OCT4, (C) NANOG, (D) LMNA, (E) PAX6, and (F) GAPDH promoters. Data are expressed as fold enrichment of DNA associated with indicated immunoprecipitated histone modifications relative to a 1/100 dilution of input chromatin (mean ¡À SD; triplicate experiment). Abbreviations: Ab, antibody; DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; TSS, transcription start site.
, T O5 V2 o! O* i! I1 O% J
3 d" A, {( G6 X; \Polymerase Chain Reaction
( A. s z4 C: a* d! w
( ~" B7 \& S! d- k6 `2 c BImmunoprecipitated DNA was analyzed in triplicates by real-time PCR starting from 5 µl of template DNA (from a total of 150 µl) on a MyiQ Real-Time PCR Detection System using iQ SYBR Green (Bio-Rad, Hercules, CA, http://www.bio-rad.com). PCR conditions were 95¡ãC for 3 minutes and 40 cycles of 95¡ãC for 30 seconds, 60¡ãC (58¡ãC for PAX6) for 30 seconds, and 72¡ãC for 30 seconds. Reverse transcription (RT)-PCR analyses were performed in triplicate from 200¨C1,000 ng of total RNA using the Iscript cDNA synthesis kit (Bio-Rad) and PCR conditions mentioned above using GAPDH for normalization. Real-time ChIP and RT-PCR primers used are listed in Table 1.
9 ]0 A$ p( T% l- R Q$ o/ ^
% k# n( \- C' Q- y+ g; C: J# iTable 1. Real-time ChIP PCR and RT-PCR primers used in this study Y; i& k& F6 F9 h7 b/ y
% ~# l6 Z7 e5 o3 n1 M! ` P8 ZRESULTS
2 D/ C& @, W( ~) U
7 u" B/ p% w: Q" ~4 F+ U- WThe Q2ChIP Assay; g+ T6 }6 S" H. X6 H
) I7 O1 b+ P& h1 t7 dThe conventional ChIP protocol we used as a starting point for development of Q2ChIP was that of Spencer and colleagues . Quantitative PCR analysis of ChIP products using GAPDH promoter-specific primers showed that amounts of precipitated DNA associated with H3K9ac or H3K9m2 were similar after in-suspension or after in-flask cross-linking (data not shown). In-suspension fixation also minimized cell loss and considerably shortened sample handling. Thus, ChIP efficiency after in-suspension cross-linking was preserved relative to a conventional protocol.
& r; ~2 S3 F9 ~. l N. c( i' K0 {7 r9 K7 V+ y% Y' k
Histone lysine acetylation is a labile modification that is degraded unless histone deacetylase (HDAC) inhibitors are used in ChIP buffers. With the aim of preserving histone acetylation, we compared the amounts of GAPDH promoter DNA associated with immunoprecipitated H3K9ac or H3K9m2 from NCCIT cells collected and cross-linked without or with 20 mM the HDAC inhibitor sodium butyrate. Butyrate was also added to all buffers after either cross-linking treatment. Figure 1A shows that cross-linking in the presence of 20 mM butyrate enhanced H3K9ac ChIP efficiency by approximately threefold, leaving H3K9m2 ChIP efficiency unaffected.
9 H7 {, B- x; `( O8 [ @( B# `: \3 b
Background DNA caused by nonspecific binding of chromatin to tube walls is expected to increase with reduced ChIP reaction volumes due to increased surface-to-volume ratio. To reduce background, while in TE buffer, H3K9ac and H3K9m2 ChIP complexes were transferred to a clean 0.2-ml tube before DNA elution ("tube shift") rather than being processed in the same tube. PCR analysis of precipitated GAPDH promoter DNA shows that tube shift resulted in a more than fivefold increase in ChIP specificity (Fig. 1B). We interpret enhanced specificity with tube shift as a result of elimination of contaminating chromatin bound to the tube wall.2 H" P. i8 b5 q( h3 S: P
- H0 b" n8 `4 u2 e* m1 h) {Conventional ChIP protocols include SDS elution steps of the precipitated DNA followed by a 6-hour cross-link reversal at 68¡ãC and a 2-hour proteinase K digestion at 55¡ãC . In order to shorten the procedure and minimize sample handling, we combined SDS elution, cross-link reversal, and proteinase K digestion into a single step. We first showed that shortening the 68¡ãC cross-link reversal step from 6 to 2 hours, and combining cross-link reversal and proteinase K digestion, did not affect ChIP efficiency or specificity (data not shown). Secondly, SDS (1%) elution, cross-link reversal, and proteinase K (50 µg/ml) digestion were combined into a single 2-hour step at 68¡ãC in 150 µl of elution buffer. We refer to this procedure as the "short protocol." Alternatively, ChIP samples were processed according to a standard "long protocol" as follows: 2 x 15-minute elution in 1% SDS, 6-hour cross-link reversal at 68¡ãC, and 2-hour proteinase K digestion at 55¡ãC (long protocol). Enrichment of GAPDH promoter DNA associated with H3K9ac or H3K9m2 achieved with the long protocol was similar with the short protocol (p > .1; t test; Fig. 1C). Furthermore, variation in the amount of recovered DNA was significantly reduced with the short protocol (p
+ R$ [! i! w. E3 I' T* U/ Z
+ H) C% r- t1 T6 N& e$ A5 t8 }0 ?As a first validation of the Q2ChIP assay, we directly compared the specificity of H3K9ac and H3K9m2 Q2ChIP of the GAPDH promoter with that achieved by conventional ChIP with in-suspension cross-linking . When comparing Q2ChIP with conventional ChIP in a 500-µl reaction volume, both protocols yielded similar specificities (p > .05, t test; Fig. 1D). Moreover, a direct comparison between Q2ChIP and conventional ChIP carried out in 100 µl (i.e., with the same amount of input chromatin), Q2ChIP specificity was enhanced fivefold (p 8 S0 {; ~+ i& G3 ? D: g! h
7 l0 v2 S. l3 j$ ]- N. d' Y0 yIntegration of all steps examined above followed by real-time PCR analysis of precipitated and purified DNA resulted in a quick and quantitative ChIP procedure we refer to as Q2ChIP. The Q2ChIP assay may be carried out over 1 day with PCR and data analysis taking place the day after, in contrast to conventional ChIP, which may take as long as 3¨C5 days. A flow diagram of Q2ChIP is provided in Figure 2, and the procedure is detailed under Materials and Methods. Additional validation of the Q2ChIP assay is provided in the following experiments.( A+ Q- n$ p+ z' r% x
' b1 Z2 r& e2 m8 BEpigenetic Histone Modifications on OCT4, NANOG, and Differentiation Marker Genes in Undifferentiated EC Cells: P& H* q0 K& {& G
% _' {& V {$ T5 O2 BTo further validate Q2ChIP, we first monitored six histone H3 modifications on the promoters of the developmentally regulated genes OCT4 and NANOG expressed in undifferentiated NCCIT cells . PAX6, in contrast, is upregulated during differentiation and is essentially not expressed in NCCIT cells (see below). Promoter regions examined are illustrated in Figure 3A. Q2ChIP analysis of five regions across the OCT4 PP, PE, and DE unraveled consistent histone marks associated with transcription (Fig. 3B). Acetylated H3K9 (H3K9ac) was enriched 8¨C22-fold over input chromatin levels, with a trend toward highest acetylation on the PP (detected with OCT4-E:PP primers; Fig. 3B). Di- and trimethylated forms of H3K4 (H3K4m2 and H3K4m3) were also strongly detected (p .2; Fig. 3C). This is in agreement with NANOG expression in undifferentiated NCCIT cells. Therefore, in undifferentiated NCCIT cells, regulatory regions of OCT4 and NANOG are enriched in euchromatic marks.' ]4 [' t! j. x/ N; x
( W! ~- g( H2 o" \5 {We next examined histone modifications on the promoter of the LMNA gene, which encodes A-type lamins only in lineage committed or differentiated cells . The LMNA promoter region examined (Fig. 3A) displayed nearly basal levels of H3K9 acetylation and significant di- and trimethylation of H3K4 (p
. V+ x. l4 z, Z2 d2 F6 w
5 o4 j$ F9 n$ ?& _1 B/ j6 j: AWe also determined histone changes on the promoter of PAX6 (Fig. 3A), a developmentally controlled gene upregulated during pancreas, eye, and brain development . Lastly, we showed that the GAPDH housekeeping gene promoter (Fig. 3A) was marked by H3K9ac, H3K4m2, H3K4m3, and by a lack of heterochromatin histone modifications, as anticipated from a constitutively expressed gene (Fig. 3F).5 h% t& I$ |/ n$ c$ x
# W& y! d, @. [: x8 _1 c
Collectively, these results indicate that Q2ChIP can be applied to identify histone modifications on regulatory sequences of pluripotency and differentiation marker genes. Undifferentiated NCCIT cells are characterized by euchromatic packaging of OCT4 and NANOG consistent with expression of these genes and assembly of a facultative repressive mark (H3K27m3) on LMNA and PAX6 with nevertheless a potential for expression upon differentiation.
% [) J' \$ d' r- ~9 [2 b# N7 h6 E: h7 M6 D! H0 Y# p( X
Q2ChIP Unravels Heterochromatinization of OCT4 Regulatory Elements upon RA-Mediated Differentiation3 j. P* k9 w. h+ Q% n
1 f1 j& n- ]* T2 @5 g( ?& dSeven days of stimulation with 10 µM RA induced differentiation of NCCIT cells. The cells formed spheres in bacterial culture plates and acquired an early neurogenic phenotype upon subsequent replating on poly-L-lysine (Fig. 4A). Induction of differentiation was confirmed by downregulation of OCT4 and NANOG expression and upregulation of PAX6 expression (p + G" d- q- [7 D
9 ?! H5 j6 X/ k) RFigure 4. Quick and quantitative chromatin immunoprecipitation (Q2ChIP) analysis of histone H3 modifications on developmentally regulated promoters upon RA-mediated differentiation of NCCIT cells. (A): NCCIT cells were cultured with 10 µM RA ( RA) for 7 days on bacterial plates to induce sphere formation. Spheres were trypsinized, replated onto poly-L-lysine-coated glass, and cultured for 4 days ( RAwithdrawal). Bars, 25 µm. (B): Quantitative reverse transcription-polymerase chain reaction analysis of OCT4, NANOG, PAX6, and LMNA expression after 7 days of RA stimulation (yellow bars) and after replating cells (red bars). Data are expressed as mean ¡À SD fold change mRNA level relative to untreated cells (triplicate experiment). (C¨CG): Q2ChIP analysis of indicated histone modifications on the (C) OCT4, (D) NANOG, (E) LMNA, (F) PAX6, and (G) GAPDH promoters in unstimulated cells (¨CRA) after RA stimulation for 7 days ( RA) and after replating ( RAwithdrawal) (mean ¡À SD fold change relative to undifferentiated cells; duplicate experiment). Abbreviations: Ab, antibody; DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; RA, retinoic acid.
8 J" R% a% ?, ]/ @4 b: E" e2 m& i% q9 ~- A
Q2ChIP analysis revealed discrete changes in histone modifications on OCT4 triggered by RA. After 7 days of stimulation, active transcription marks were surprisingly stable. H3K9ac, H3K4m2, and H3K4m3 levels remained significant and unchanged on OCT4 PP, PE, and DE (p > .1; Fig. 5C, insets) despite transcriptional downregulation. However, RA increased di- and trimethylation of H3K9 on these regions (p
6 G8 V0 a+ S' q0 @+ ?' |* i4 a9 A
% o3 x) [( e6 p) M# ^9 @Figure 5. Quick and quantitative chromatin immunoprecipitation (Q2ChIP) is compatible with a reduction in amount of input chromatin. Indicated modified histones were immunoprecipitated by Q2ChIP from chromatin prepared from 105 NCCIT cells and diluted to 10,000, 1,000, and 100 cell eq. Precipitated DNA was analyzed by real-time polymerase chain reaction and data expressed as in Figure 3 (mean ¡À SD of two experiments). (A): OCT4-D:PE, (B): OCT4-E:PP, (C): NANOG, (D): PAX6, (E): GAPDH. Abbreviations: Ab, antibody; eq., equivalents; PE, proximal enhancer; PP, proximal promoter.! ?4 _. J5 P8 ~8 C
# M) j; g2 F0 M4 J- E# }: F5 Q
Removal of RA and replating cells on poly-L-lysine were determinant in promoting heterochromatinization on OCT4 on the basis of the following observations (Fig. 4C): (a) a 5¨C10-fold deacetylation of H3K9 on OCT4 PP, PE, and DE (p 0 g- W: X: i, U4 i5 j1 C
( [3 |* R2 A' p9 P/ xThe NANOG Promoter Is More Resistant to Assembly of Repressive Histone Marks
|& W8 c* C4 x1 L! S% W
" D n1 Y/ _/ W. vDespite downregulation of expression, the dynamics of histone H3 modifications on NANOG differed from that of OCT4. RA stimulation maintained H3K9ac, H3K4m2, and H3K4m3 levels, enhanced H3K9m2 level (p .05; Fig. 4D). RA removal and cell replating on poly-L-lysine, however, triggered 90% deacetylation of H3K9 (p 2 V. L3 p( u; `# V5 j( r( x
+ Q7 Z, z( e: q2 S, J7 w2 c) t6 DPAX6 and LMNA Promoters Undergo H3K9 Acetylation upon RA-Mediated Differentiation0 ]( e% ^+ a, ~& g
9 M1 ]- n: d- J2 f& i' v/ Z" G4 ]5 dLMNA mRNA levels were not significantly upregulated upon RA stimulation (Fig. 4B). Acetylation of H3K9 on the LMNA promoter was observed only after RA stimulation; however, this effect was not maintained after replating on poly-L-lysine (Fig. 4E). No significant changes in H3K9m2, H3K9m3, and H3K27m3 were observed.7 B7 L9 _9 O* ?- W" [, e7 A
( ~& |& g; h+ c2 E4 S
In contrast to LMNA, PAX6 was transcriptionally activated after RA stimulation (Fig. 4B). Accordingly, the promoter was strongly hyperacetylated on H3K9 (p
6 S& X9 A4 U$ I" ~4 p' f3 e4 Y; b& Q% ^& a* F
Determination of Amount of Chromatin Compatible with Q2ChIP
! K# \6 P/ |, w$ P% [! k6 X, f* c0 ~
We next determined the lower limit of input chromatin amount compatible with Q2ChIP. Sonicated chromatin was prepared from 105 NCCIT cells and diluted to 10¨C1,000-fold to produce chromatin amounts corresponding to that of 104, 103, or 102 cells (10,000, 1,000, or 100 cell eq.). Modified histones were immunoprecipitated by Q2ChIP and association with two regions of OCT4, NANOG, PAX6, and GAPDH was determined by real-time PCR as reported earlier (Fig. 5). Histone binding pattern to OCT4-D:PE detected in chromatin from 104 cell eq. was similar to that detected with 105 cell eq. (see Fig. 3B). Furthermore, the Q2ChIP pattern was preserved with dilutions to 103 and 102 cell eq. We also noted that enrichment of precipitated DNA was greater with increasing dilutions of input chromatin (Fig. 5A). This was most likely due to an increased antibody-bead complex/histone ratio in the assay as chromatin was diluted, because antibody concentration remained constant under all conditions. Similar observations were consistently made for Q2ChIP analysis of OCT4-E:PP (Fig. 5B) and NANOG (Fig. 5C) as well as for the inactive PAX6 promoter (Fig. 5D) and for the constitutively expressed GAPDH promoter (Fig. 5E). Moreover, in each example, standard deviations of the relative amounts of precipitated DNA tended to augment as chromatin dilution increased (in particular for 100 cell eq.; Fig. 5), reflecting more variable quantitative PCR efficiencies with lower amount of template. Enhanced variation was particularly evident in the analysis of PAX6 (Fig. 5D) due to intrinsic lower PCR efficiency with the PAX6 primers (not shown). Detection limit is probably reached with 100 cell eq. in our assay, as shown by enhanced variation and, in some instances, slightly enhanced background (no antibody control; Fig. 5A, 5C, 5D). Nevertheless, the results demonstrate that Q2ChIP can be used with up to 105-fold reduced amounts of input chromatin compared with standard ChIP protocols without affecting patterns of histone binding.5 h6 d0 }+ [4 ]! e/ ]& _, Y
3 T! `2 |# E' c0 w7 |- MQ2ChIP Reveals Dynamic Oct4 Binding to the NANOG Promoter. @# D8 Z! Y6 ~+ Q K
+ E& E4 z' y, R" W
We examined whether Q2ChIP was also applicable to assess transcription factor binding to DNA. The developmentally regulated transcription factor Oct4 would be expected to bind the NANOG promoter on the Sox/Oct binding site in undifferentiated, but not in differentiated, cells. Indeed, Q2ChIP analysis of 105 cells showed that Oct4 bound to the NANOG promoter in undifferentiated NCCIT cells but was dissociated from the promoter in RA-differentiated cells (Fig. 6A). In contrast, Oct4 was not significantly associated with the control GAPDH promoter, as anticipated (Fig. 6A). In addition, we showed that Oct4 binding to NANOG was also clearly detected by Q2ChIP analysis of 10,000 and 1,000 cell eq. (Fig. 6B). We concluded that Q2ChIP is suitable for analysis of both histone modifications and transcription factor binding from greatly reduced amounts of chromatin relative to conventional ChIP.
, D: f6 @# b( w4 X1 h
) Z) d8 p4 F* ~0 o1 RFigure 6. Quick and quantitative (Q2)ChIP unravels a dynamic association of Oct4 with the NANOG promoter under RA-mediated differentiation. (A): Oct4 was immunoprecipitated by Q2ChIP from chromatin of 105 unstimulated NCCIT cells (NCCIT) or RA-stimulated/replated cells (NCCIT RA). Oct4 binding to the NANOG and GAPDH promoters was examined by real-time polymerase chain reaction. Control ChIPs were done with beads only (mean ¡À SD from duplicate experiment). (B): Oct4 Q2ChIP with reduced chromatin amount. Chromatin from 105 NCCIT cells was diluted to 10,000 and 1,000 cell eq. and Oct4 binding to NANOG and GAPDH was assessed by Q2ChIP as in (A) (mean ¡À SD from two experiments). Abbreviations: Ab, antibody; ChIP, chromatin immunoprecipitation; eq., equivalents; RA, retinoic acid.
1 m8 |( q+ B6 c# C
5 E! K3 L- ?$ z% e, aDISCUSSION
( A! }& u+ |. z5 S! l( X# u' `- A0 `6 w# u+ n. Q
Q2ChIP in the Context of Other Current ChIP Protocols! s* l% m, Z% L. n
, C2 |( w7 X0 F4 n. o6 hAlteration of several steps in a conventional ChIP assay globally increases the efficiency of Q2ChIP over that of conventional ChIP. Cells are cross-linked in suspension rather than in flasks, making the protocol identical for cells growing in suspension or adherent and enhancing cell recovery. A HDAC inhibitor is included in the cell harvest and cross-linking steps, as opposed to after cross-linking, to maximize the amount of precipitated acetylated histones. Nevertheless, ChIP specificity is conserved. A tube shift step is introduced after the last washing step, eliminating background material that remains on the tube wall and enhancing specificity of the recovered precipitated material. We also combined cross-link reversal, proteinase K digestion, and DNA elution into a single 2-hour step, which substitutes for the 9-hour process of current protocols. ChIP efficiency is maintained, and we observe a marked reduction in the variation of the amount of H3K9ac recovered compared with conventional ChIP. Chromatin can easily be prepared from as low as 105 cells and diluted 1,000-fold to produce reliable templates for Q2ChIP. Indeed, histone binding patterns are maintained with chromatin from as low as 100 cell equivalents. Furthermore, efficiency of Q2ChIP is enhanced with diluted chromatin due to the increased ratio of precipitating antibody-bead complex to target histone (Fig. 5E). Lastly, we demonstrate that Q2ChIP can be used for analysis of transcription factor binding to DNA.8 w( I# R8 O( m, K5 L% ]
, ?8 i' T% ^ v* t+ d$ G8 jWhile this paper was in preparation, a carrier ChIP (CChIP) procedure designed for small cell numbers was reported . CChIP may be applicable to cross-linked chromatin, but this remains to be shown. The Q2ChIP procedure reported here is fast and includes a cross-linking step and thus, unlike NChIP, is applicable to the analysis of histone and nonhistone proteins. A combination of CChIP with Q2ChIP should create possibilities to unravel epigenetic modifications unexplored to date in embryos and ES cells.
* d7 ^# @' W* n* }3 C7 @, k; m7 f$ C4 ?) d. s& z, \3 Q) V
Remodeling of the NANOG Promoter During EC Cell Differentiation
2 w1 _) f8 \/ y* l+ C# X. v( X1 o" l
5 p" b6 w6 t+ r" rCooperative interaction between Nanog, Oct4, and Sox2 on their respective promoters is essential for transcriptional activation of these genes .3 D- e* q0 U' B: ~7 G k
8 N& i9 ~( d( G4 v# pHistone Modifications on OCT4 During EC Cell Differentiation
0 Z4 x; ^2 \$ n l/ H" @# v8 M3 v, A
' f- ?2 `8 Q8 p0 s; cAs with NANOG, the OCT4 regulatory locus contains epigenetic marks indicative of transcriptional activation in undifferentiated NCCIT cells. Likewise, in mouse ES and EC cells, Oct4 displays acetylated and unmethylated H3K9 and di- and trimethylated H3K4 .
+ Z4 Y- }+ y+ q0 p- B# j' \, C& B/ G( Z o* j, Z
Our results unexpectedly unravel a biphasic epigenetic modulation of the OCT4 regulatory region upon RA stimulation. RA causes hypermethylation of H3K9 and H3K27 yet maintains H3K9 acetylation, and only subsequent removal of RA and replating on poly-L-lysine results in H3K9 deacetylation throughout the OCT4 PP, PE, and DE and enhances assembly of heterochromatin marks. We found consistent deacetylation of H3K9 and demethylation of H3K4m2 throughout the OCT4 promoter, reflecting heterochromatin assembly. Marked increases in H3K9m3 were also prominent in the PP (detected by OCT4-E:PP), the 3' region of the PE (detected by OCT4-D:PE), and the 5' region of the DE (detected by OCT4-A:DE). The OCT4-C:PE and OCT4:B:DE regions displayed a moderate increase in H3K9m3, whereas H3K9m2 was more prominent. These results suggest a highly localized assembly of heterochromatin marks. The OCT4 promoter harbors RA-responsive elements in the PP and PE, which in murine EC and ES cells are responsible for RA-mediated inactivation of Oct4 . No RA-responsive element has been identified to date in the DE of OCT4; however, we observed a clear methylation response on lysine 9 and 27 of H3 in this region after RA stimulation. Yet, the absence of clear H3K9m3 in the distal PE (OCT4-C:PE) and in the proximal DE (OCT4-B:DE) argues against a uniform spreading of heterochromatin on OCT4. Rather, H3K9m3 in the distal DE (OCT4A:DE) may be brought about by the deposition of histone methyltransferases or by the action of enzymes assembled in the PP/PE region through DNA looping.
, B7 o* B, b+ L7 T7 ^& X' M* y7 h8 J
In accordance with our findings, the early stage of RA differentiation of mouse ES cells is associated with H3K9 methylation to a greater extent than H3K9 deacetylation or H3K4 demethylation and our observations may reflect the analysis of a mixed cell population or differential marking of OCT4 alleles. Alternatively, the data suggest that specific lysine (K9) residues in a nonacetylated state on nucleosomes are "reserved" for immediate methylation upon transcriptional inactivation, whereas acetylated lysines must undergo deacetylation prior to methylation. Either putative mechanism would result in epigenetic mosaicism between cells in response to RA.& p! X, m1 N9 e0 _- w4 m+ W( F& k
+ n" M8 N, V- W a
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST4 j: q7 K! `( C1 |) F
+ {" X. O" P- |$ f& yThe authors indicate no potential conflicts of interest.- C3 u( H/ A: |2 k" o- O
. S: I/ v( ?3 r4 E& j* z/ P" XACKNOWLEDGMENTS
( \0 O4 n; w/ |& i2 Q. d3 z
4 a$ h3 F0 X/ j# `We thank K. Gaustad for assistance with RT-PCR and A. Noer and Dr. A.-M. Håkelien for critical reading of the manuscript. This work was supported by the Research Council of Norway (FUGE, YFF, and STORFORSK programs), the Norwegian Cancer Society, and the Norwegian Stem Cell Network.7 O+ u: e0 F& p: J) s
【参考文献】0 ^1 C% S5 d1 q9 G
% O$ E' \. C3 @2 Q5 Y* s' ^$ j
' ]* U& ?, C/ u KO'Neill LP, Turner BM. Immunoprecipitation of chromatin. Methods Enzymol 1996;274:189¨C197.
$ c! l2 v' p ]1 o0 E6 B" P6 I7 e k# L& T' { S
Strahl BD, Allis CD. The language of covalent histone modifications. Nature 2000;403:41¨C45.
* R' x) f3 S$ T2 ?) D, \# e J) }
( Z, F4 i9 U& b. ?Orlando V. Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem Sci 2000;25:99¨C104.
" B+ g/ I& q: j w- J& |
' K4 I; D8 Z8 L+ P- e0 g6 j; G1 tRodriguez BA, Huang TH. Tilling the chromatin landscape: Emerging methods for the discovery and profiling of protein-DNA interactions. Biochem Cell Biol 2005;83:525¨C534./ R8 P, p4 }( i. m9 m1 e- N0 K2 ^
* q; `8 D8 f3 Y; {* E4 f# TLachner M, Jenuwein T. The many faces of histone lysine methylation. Curr Opin Cell Biol 2002;14:286¨C298.
4 E$ I# z: y; `- Y
8 [9 d) C; v, b+ Y; O' z& lLaird PW. Cancer epigenetics. Hum Mol Genet 2005;14:R65¨CR76.
0 N1 f! H+ V" J! |& g# o) h$ p) K/ r
$ v+ S9 [) w* @" x# [Morgan HD, Santos F, Green K et al. Epigenetic reprogramming in mammals. Hum Mol Genet 2005;14:R47¨CR58.: B e3 v ~( F5 w
( h; G/ G" n- Z2 h' t( L
Lee JH, Hart SR, Skalnik DG. Histone deacetylase activity is required for embryonic stem cell differentiation. Genesis 2004;38:32¨C38.
, |2 n# d5 G: i. g; y; _7 ?5 x0 \3 K e2 L+ q2 m% y
Feldman N, Gerson A, Fang J et al. G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis. Nat Cell Biol 2006;8:188¨C194.0 Y: X* ^4 @ P8 C' I
7 C. {. q, `3 T6 O% K1 d& e
Scholer HR, Ruppert S, Suzuki N et al. New type of POU domain in germ line-specific protein Oct-4. Nature 1990;344:435¨C439.
9 G0 }: b* Q1 T) p6 t2 I5 P3 T4 P |
Nichols J, Zevnik B, Anastassiadis K et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998;95:379¨C391.
" }: o! J% i& A4 `0 p. L
?+ ?) F8 J- QNiwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372¨C376.
8 f0 Q: G4 m; X, h: w4 o. k, f( y2 T/ C U- \1 K8 O4 V
Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;113:631¨C642.
2 Y, {3 Z P& _- q3 F* E3 p. G5 D) o' n# V- ]
Yeom YI, Fuhrmann G, Ovitt CE et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 1996;122:881¨C894.; R; a3 @5 W; P( m2 L' v
* v% s/ w5 p! }: a- ^. \/ B& v& p
Schoorlemmer J, van PA, van Den EM et al. Characterization of a negative retinoic acid response element in the murine Oct4 promoter. Mol Cell Biol 1994;14:1122¨C1136.
2 n' O' g K3 P. d6 s& R( G" A3 Z4 \) c: j/ ?! G
Chew JL, Loh YH, Zhang W et al. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol 2005;25:6031¨C6046.3 X. |" F+ P6 E; |+ L6 l f. k& G! ~
5 {, C/ {' H- i' G: w1 P" xRodda DJ, Chew JL, Lim LH et al. Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 2005;280:24731¨C24737.
' J4 \, E. I. _% f! Z' w, b ^# ~ [
0 ]1 Y1 {% j5 N' ]! MBoyer LA, Lee TI, Cole MF et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005;122:947¨C956." B; ~3 Q3 h6 y, R# q
; n; X g$ C! v3 b" F1 Z2 }Loh YH, Wu Q, Chew JL et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 2006;38:431¨C440.1 T$ \" q9 ]: u: T
5 Z& Y; O D& u
Minucci S, Botquin V, Yeom YI et al. Retinoic acid-mediated down-regulation of Oct3/4 coincides with the loss of promoter occupancy in vivo. EMBO J 1996;15:888¨C899.
/ E# @+ u+ \9 l1 `' p6 T
4 e e% U2 |- A) \4 ^. lO'Neill LP, Vermilyea MD, Turner BM. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat Genet 2006;38:835¨C841.
/ F* v$ G3 s. X& Y* V. x8 h# {, ~3 K8 M* ]
Damjanov I, Horvat B, Gibas Z. Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, NCCIT. Lab Invest 1993;68:220¨C232.
# N0 i- _, U+ l+ k( n; f. T6 k- x5 D( ~, V D
Taranger CK, Noer A, Sorensen AL et al. Induction of dedifferentiation, genome-wide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells. Mol Biol Cell 2005;16:5719¨C5735.
4 j$ p0 S2 E$ v9 I- C7 p& q) ~4 p8 g8 w* \* |8 k( i" u" D' t0 G
Bledi Y, Domb AJ, Linial M. Culturing neuronal cells on surfaces coated by a novel polyethyleneimine-based polymer. Brain Res Protoc 2000;5:282¨C289.: E& q' _" ]! a6 E3 Y" F$ p
( w* ?. t( s% `; v) ]" H/ nNoer A, Sørensen AL, Boquest AC et al. Stable CpG hypomethylation of adipogenic promoters in freshly isolated, cultured and differentiated mesenchymal stem cells from adipose tissue. Mol Biol Cell 2006;17:3543¨C3556.
& V, `: E# N' U( @( j, \- E; D, k: e- m/ i! `8 H' B" e
Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.
/ \# j! X' {9 \" ^) [1 { _& q! t- d; M/ U+ W6 A$ T$ \& y
Spencer VA, Sun JM, Li L et al. Chromatin immunoprecipitation: A tool for studying histone acetylation and transcription factor binding. Methods 2003;31:67¨C75.: {3 L# [1 ]$ `
% G5 r+ H b8 G& O
Hutchison CJ, Worman HJ. A-type lamins: Guardians of the soma? Nat Cell Biol 2004;6:1062¨C1067. @7 u y& g7 l: C, G5 U
8 l7 s# N9 q- X. M" u) p# s9 G
Kleinjan DA, Seawright A, Mella S et al. Long-range downstream enhancers are essential for Pax6 expression. Dev Biol 2006;299:563¨C581.4 g" R, X: l9 u
7 M+ r3 p/ Q9 T) W
Simpson TI, Price DJ. Pax6; a pleiotropic player in development. Bioessays 2002;24:1041¨C1051.0 f7 b9 `* X1 h, t+ P
; v: Y4 h' ?1 X8 ^& w& b2 @( o' x$ d! |Azuara V, Perry P, Sauer S et al. Chromatin signatures of pluripotent cell lines. Nat Cell Biol 2006;8:532¨C538.
q+ q/ z) G) V% x/ l: F" D8 ^, O
0 y% x0 M" y; x1 rKubicek S, Jenuwein T. A crack in histone lysine methylation. Cell 2004;119:903¨C906.' x. a8 l$ ?6 J Q
+ }6 \- n8 e! z2 Z" r# \Orlando V. Polycomb, epigenomes, and control of cell identity. Cell 2003;112:599¨C606.( }; t1 j" r0 w
, d' g( u' ]: J# D
Nordhoff V, Hubner K, Bauer A et al. Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences. Mamm Genome 2001;12:309¨C317.
& W' l* N+ \, t/ [+ E7 Y
0 U% T. L* { U! a pBen-Shushan E, Sharir H, Pikarsky E et al. A dynamic balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and retinoic acid receptor:retinoid X receptor heterodimers regulates Oct-3/4 expression in embryonal carcinoma cells. Mol Cell Biol 1995;15:1034¨C1048./ T+ ~" b( E3 \0 X, f
2 r1 n; ~" q) } C
Fuhrmann G, Chung AC, Jackson KJ et al. Mouse germline restriction of Oct4 expression by germ cell nuclear factor. Dev Cell 2001;1:377¨C387.
+ w+ F9 \2 A- N j& @; P @4 b9 [4 |
Martin C, Zhang Y. The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol 2005;6:838¨C849. |
|
发表于 2009-3-5 00:55
发表于 2015-6-1 10:35
