High-level expression of the globin genes requires a locus control region LCR located far upstream of the embryonic e-globin gene and composed of five DNaseI hypersensitive HS sites that
Trang 1locus by TFII-I and upstream stimulatory factor
Valerie J Crusselle-Davis, Zhuo Zhou, Archana Anantharaman, Babak Moghimi, Tihomir Dodev, Suming Huang and Jo¨rg Bungert
Department of Biochemistry and Molecular Biology, Center for Mammalian Genetics, Shands Cancer Center, Powell Gene Therapy Center, Genetics Institute, University of Florida, College of Medicine, Gainesville, FL, USA
Gene expression is regulated at multiple steps
involv-ing the relocation of genes in the nucleus, the
modifi-cation of chromatin structure and, ultimately, the
recruitment of transcription complexes [1–3] Each step
is regulated by transcription factors that interact in a
sequence-specific manner with DNA control elements
located in promoters or enhancers [4] These
sequence-specific binding proteins recruit coregulators that
modify histones, mobilize nucleosomes or recruit
components of the basal transcription machinery [5]
Tissue-specific genes are often regulated by
tissue-specific activators and repressors that act in concert
with ubiquitously expressed transcription factors
The sequential stage-specific expression of the five
b-like globin genes is regulated by gene proximal
regulatory elements that recruit transcription factors either activating or repressing gene expression [6] The regulation of globin gene transcription involves the recruitment of chromatin modifying activities that regulate accessibility to subregions of the globin gene locus in a developmental stage-specific manner High-level expression of the globin genes requires a locus control region (LCR) located far upstream of the embryonic e-globin gene and composed of five DNaseI hypersensitive (HS) sites that are 200–400 bp in size and separated form each other by 2–4 kbp [7–9] The LCR HS sites function together in a synergistic or additive manner to stimulate globin gene expression [10–12] There is increasing evidence that transcription
of at least some, perhaps highly expressed, genes takes
Keywords
coregulator; globin genes; locus control
region; transcription
Correspondence
J Bungert, Department of Biochemistry and
Molecular Biology, College of Medicine,
University of Florida, 1600 SW Archer Road,
PO Box 100245, Gainesville, FL 32610, USA
Fax: +1 352 392 2853
Tel: +1 352 273 8098
E-mail: jbungert@ufl.edu
(Received 16 May 2007, revised 3 October
2007, accepted 5 October 2007)
doi:10.1111/j.1742-4658.2007.06128.x
Upstream stimulatory factor and TFII-I are ubiquitously expressed helix-loop-helix transcription factors that interact with E-box sequences and or initiator elements We previously demonstrated that upstream stimulatory factor is an activator of b-globin gene expression whereas TFII-I is a repressor In the present study, we demonstrate that upstream stimulatory factor interacts with the coactivator p300 and that this interaction is restricted to erythroid cells expressing the adult b-globin gene Further-more, we demonstrate that Suz12, a component of the polycomb repressor complex 2, is recruited to the b-globin gene Reducing expression of Suz12 significantly activates b-globin gene expression in an erythroid cell line with
an embryonic phenotype Suz12 also interacts with the adult b-globin gene during early stages of erythroid differentiation of mouse embryonic stem cells Our data suggest that TFII-I contributes to the recruitment of the polycomb repressor complex 2 complex to the b-globin gene Together, these data demonstrate that the antagonistic activities of upstream stimula-tory factor and TFII-I on b-globin gene expression are mediated at least in part by protein complexes that render the promoter associated chromatin accessible or inaccessible for the transcription complex
Abbreviations
ChIP, chromatin immunoprecipitation; EPO, erythropoietin; ES, embryonic stem; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HDAC, histone deacetylase; HS, hypersensitive; LCR, locus control region; MEL, murine erythroleukemia; PRC, polycomb repressor complex; siRNA, short interfering RNA; USF, upstream stimulatory factor.
Trang 2Regulation of the globin genes involves the action of
many transcription factors, some of which have been
characterized in detail GATA-1, EKLF and NF-E2
are hematopoietic transcription factors that have all
been shown to participate in LCR function and
b-glo-bin gene expression [6] In addition to these tissue
restricted transcription factors, ubiquitously expressed
transcription regulatory proteins such as Sp1, upstream
stimulatory factor (USF) and TFII-I have also been
demonstrated to regulate globin gene expression
[18,19] Previous studies have shown that the
helix-loop-helix protein USF activates b-globin gene
expres-sion and interacts with E-box elements located in LCR
element HS2 and in the b-globin downstream
moter region [20] TFII-I, another helix-loop-helix
pro-tein, interacts with the b-globin initiator sequence and
represses b-globin gene expression TFII-I exerts part
of its function by recruiting histone deacetylase
(HDAC) to the b-globin gene promoter and rendering
the chromatin inaccessible for transcription complexes
[20]
The polycomb repressor complex (PRC) has
origi-nally been identified in Drosophila, in which it plays an
important role in regulating the expression of segment
polarity genes, including the Hox gene cluster [21]
Homologous proteins have also been identified in
mammalian cells There are two main PRC complexes,
PRC1 and PRC2 PRC2 contains the histone
methyl-transferase Ezh2, which methylates lysine 27 on the
histone H3 N-terminal tail [21] This modification is
absent or reduced in promoters of transcribed genes
PRC1 interacts with PRC2 and contains subunits that
recruit DNA methyltransferases Current models
pro-pose that the PRC2 complex initially represses gene
activity by H3K27 methylation Subsequent
interac-tions with the PRC1 complex appear to stabilize the
repressed chromatin structure by recruitment of DNA
methyl-transferases [22]
In the present study, we demonstrate that USF
interacts with coactivator p300 in mouse
erythroleuke-mia cells that express the b-globin gene No
inter-b-globin gene promoter to establish an inaccessible chromatin configuration
Results
CBP⁄ p300 interacts with USF and the b-globin promoter in MEL but not in K562 cells
We have shown previously that USF is required for high-level b-globin gene expression in MEL cells [20] USF functions as a classical transcription factor that is able to stimulate transcription in in vitro transcription systems [23] Recent data from the Felsenfeld labora-tory have shown that USF is also a critical part of a chromatin boundary in the chicken b-globin gene locus [24] It was shown that USF interacts with CBP⁄ p300, suggesting that it can function at least in part by recruiting chromatin modifying activities MEL cells expressing a dominant negative mutant of USF exhib-ited a reduction in Pol II loading to LCR element HS2 and to the b-globin gene promoter [20] At the same time, we observed a reduction in the recruitment of CBP and p300 to these sites
To examine in more detail whether USF recruits co-activators to the b-globin gene locus, we first examined the recruitment of different coactivators to regions in the globin gene locus (Fig 1A) The data show that CBP and p300 are efficiently crosslinked at the tran-scribed b-globin gene promoter and at LCR element HS2 in MEL cells (Fig 1B) No interactions were detected at the repressed embryonic ec-globin gene promoter (Fig 1B) In K562 cells, CBP and p300 interact efficiently with HS2 but not with the repressed b-globin gene promoter (Fig 1C) There is some inter-action between CBP and the expressed e- and c-globin genes in K562 cells but the interaction appears to be less efficient compared to LCR HS2
We next analyzed interactions between USF and p300
in MEL and K562 cells by co-immunoprecipitation (Fig 1D) The results demonstrate that USF interacts with p300 in MEL cells but not in K562 cells This
Trang 3interaction is specific because no interactions between
HDAC3 and p300 are observed Taken together, the
data demonstrate that USF interacts with the
coactiva-tor p300 in erythroid cells and suggest that it recruits
p300 to specific regions in the b-globin gene locus
Interaction of Suz12 with the b-globin gene
promoter in K562 but not in MEL cells
TFII-I interacts with the b-globin initiator and
represses b-globin gene expression in embryonic
erythroid cells [20] We have shown previously that
TFII-I interacts with HDAC3 in K562 cells Polycomb
group proteins were originally identified as repressors
of gene expression during development in Drosophila
Recently, it was shown that the PRC2 is located at
and represses developmentally regulated genes in
undifferentiated, embryonic stem (ES) cells [22] To
examine whether PRCs are located at the b-globin
gene promoter in embryonic cells, we carried out
chro-matin immunoprecipitation (ChIP) experiments using
antibodies against Suz12, a component of PRC2, in
K562 and MEL cells (Fig 2) The data demonstrate
that Suz12 can be crosslinked to the repressed b-globin gene promoter in K562 cells (Fig 2A) but not to the transcribed bmaj-globin gene promoter in MEL cells (Fig 2B) We did not detect any interactions of Suz12 with the embryonic e-globin gene promoter The Suz12 antibody used in these experiments specifically recog-nizes both mouse and human proteins and Suz12 is expressed in both K562 and MEL cells as determined
in western blotting experiments (data not shown)
Interaction of TFII-I with HDAC3 and Suz12
We previously demonstrated that TFII-I interacts with HDAC3 and recruits this protein to the b-globin gene promoter in K562 cells In the present study, we ana-lyzed the interaction between TFII-I and HDAC3 in both MEL and K562 cells and show that this inter-action is restricted to K562 cells We next wished to examine whether the PRC2 complex, which interacts with the b-globin gene in K562 cells, could be recruited
to the gene by TFII-I Co-immunoprecipitation experi-ments demonstrate that TFII-I interacts with HDAC3
in K562 cells, consistent with our previous data, but
Fig 1 CBP ⁄ p300 interact with the b-globin gene locus and with USF1 in MEL cells but not in K562 cells (A) Schematic of the organization
of the human and mouse b-globin gene loci The human b-globin locus depicted on top consists of five genes which are expressed in a developmental stage-specific manner in erythroid cells as outlined The expression of the genes is regulated by a LCR composed of five DNaseI HS and located approximately 15–27 kbp upstream of the embryonic e-globin gene The murine b-globin gene locus, which is depicted on the bottom, consists of four genes which are expressed either in erythroid cells of the embryonic yolk sac (EY or bH1) or in definitive erythroid cells derived from fetal liver or bone marrow hematopoiesis (bmajand bmin) The murine LCR also contains multiple HS required for high-level globin gene expression K562 (B) and MEL cells (C) were subjected to ChIP analysis with antibodies against p300, CBP or with a nonspecific antibody (IgG) Purified DNA was analyzed by real-time quantitative PCR with primers specific for LCR HS2 or the e-, c-, b-, b-major or ec-globin gene promoters (D) K562 or MEL cell extracts were precleared with anti-(rabbit IgG) beads and precipitated with a-USF1, a-p300, a-CBP or a-HDAC3 (as negative control), and complexes were captured by incubation with anti-(rabbit IgG) beads Complexes were eluted off the beads with Laemmli buffer and incubation at 95 C for 10 min and loaded onto a 5% Ready gel (Bio-Rad) The membrane was probed with a-p300.
Trang 4not in MEL cells, in which the b-globin gene is
tran-scribed (Fig 3A) We also detected interactions
between TFII-I and Suz12 in K562 cells (Fig 3B) The
interaction between TFII-I and Suz12 is not as efficient
as that involving HDAC3 and it is not restricted to K562 cells, because interactions are also detectable in MEL cells (data not shown)
Reduction of Suz12 expression in K562 cells increases b-globin gene expression
We previously used SMART-pool short interfering RNA (siRNA) reagent from Dharmacon and effi-ciently reduced expression of TFII-I and HDAC3 [20] Reductions in both TFII-I and HDAC3 expression by more than 80% led to an approximately three-fold increase in b-globin gene expression To examine whether Suz12 and the PRC2 complex participate in the repression of the adult b-globin gene in K562 cells,
we reduced expression of Suz12 by RNA interference The western blot results demonstrate that siRNA transfected cells reveal a drastic reduction in Suz12 protein levels compared to mock transfected cells or cells transfected with nonspecific siRNA (Fig 4A) Expression of the adult b-globin gene was increased by three- to five-fold in cells transfected with Suz12 siRNA compared to mock or negative control siRNA transfected cells (Fig 4B) These results demonstrate that the PRC2 complex, or components thereof, partic-ipate in the repression of the adult b-globin gene in embryonic erythroid cells We also detected an increase
in the expression of the embryonic e-globin gene However, this increase was not as pronounced as the one seen for b-globin gene expression
The PRC2 complex contains the histone H3K27 specific histone methyltransferase Ezh2 [21] Ezh2 catalyzes the di- and tri-methylation of H3K27 [25] Using ChIP, we did not detect high levels of tri-methylated H3K27 at the globin gene locus in K562 cells, consistent with studies from the Blobel
labora-Fig 2 Suz12 interacts with the adult b-globin gene in K562 but not in MEL cells Antibodies against Suz12 and nonspecific IgG were used
in ChIP assays using K562 (A) and MEL (B) cells Quantitative PCR was performed with primers that amplified the promoters of the genes
as indicated.
A
B
Fig 3 Interaction of Suz12 and HDAC3 with TFII-I (A) Interaction
of HDAC3 with TFII-I in K562 but not MEL cells K562 or MEL cell
extract was precleared with anti-(a)-(rabbit IgG) beads and
precipi-tated with a-HDAC3 or IgG, and complexes were captured by
incu-bation with anti-(rabbit IgG) beads Complexes were eluted off the
beads with Laemmli buffer and incubation at 95 C for 10 min and
loaded onto a 10% Ready gel (Bio-Rad) The membrane was
probed with a-TFII-I and then stripped and probed with a-HDAC3
as a positive control (B) Interaction of Suz12 with TFII-I in K562
cells K562 extracts were precleared with anti-(rabbit IgG) beads
and precipitated with 2.5 lg of a-Suz12 or 2.5 lg of a-IgG (as
nega-tive control) antibodies The complexes were captured by
incuba-tion with anti-(rabbit IgG) beads Complexes were eluted off the
beads with Laemmli buffer and incubation at 95 C for 10 min and
loaded onto a 10% Ready gel (Bio-Rad) The membrane was
probed with a-TFII-I The lane labeled control represents a regular
western blot for TFII-I with protein extract from K562 cells.
Trang 5tory [26] In addition, the association of trimethylated
H3K27 was not significantly altered in cells that
express, or do not express, the adult b-globin gene
(data not shown)
Interaction of Suz12 with the bmaj-globin gene
promoter decreases during activation of the
bmaj-globin gene in differentiating murine ES cells
We next examined the association of Suz12 with the
bmaj-globin gene promoter during differentiation of
mouse ES cells We previously demonstrated that the
adult b-globin gene is expressed at low levels in ES
cell cultures incubated for 5 days with erythropoietin
(EPO), which mediates the differentiation and
prolif-eration of erythroid cells [17] High-level expression
of the adult b-globin gene was observed at day 12 in
the ES cell differentiation system Figure 5A,B
dem-onstrates that bmaj-globin gene expression is
up-regu-lated by more than 30-fold between days 5 and 12
We observed that the association of Suz12, TFII-I
and trimethylated H3K27 (H3K27me3) with the
bmaj-globin gene promoter is high at day 5 but
undetectable at day 12 (Fig 5C) Quantitation of the
Suz12 levels at the bmaj-globin gene promoter
dem-onstrate that the changes between days 5 and 12 are
significant The control experiment demonstrates that
interaction of LCR HS2 associated dimethylated
H3K4 (H3K4me2) does not change during the
course of differentiation Neither Suz12, nor TFII-I
were found to associate with a control region located
between LCR elements HS2 and HS3 (data not
shown)
Discussion
We provide evidence that USF and TFII-I regulate b-globin gene expression through the recruitment of coactivator complexes that render the b-globin pro-moter accessible or inaccessible to the transcription complex USF recruits the histone acetyltransferase p300 to the b-globin promoter and this activity increases the accessibility for transcription factors TFII-I recruits HDAC3 and the PRC2 complex, which render the chromatin structure inaccessible to the tran-scription complex
Previous data from the Felsenfeld laboratory have shown that USF interacts with the coregulators p300, CBP, SET7⁄ 9 and PCAF and perhaps recruits these activities to a chromosomal boundary element [24] In the present study, we show that p300 and CBP are located at the promoter of the active adult b-globin gene and also at LCR element HS2 USF1 is observed
to interact with p300 exclusively in erythroid cells with
an adult phenotype These data suggest that USF1 recruits p300 to the promoter of the active b-globin gene to aid in transcriptional activation
CBP⁄ p300 is located at LCR HS2 in K562 cells, in which we did not detect interactions between USF and p300 This suggests that the recruitment of CBP⁄ p300
to the LCR, at least in K562 cells, is mediated by pro-teins other than USF, and potential candidates are GATA-1 and NF-E2, which have been shown to inter-act with CBP [27,28] Both of these proteins were shown to be required for histone acetylation of spe-cific regions in the globin gene locus However, the recruitment of CBP⁄ p300 to the adult b-globin gene
Fig 4 Suz12 represses b-globin gene expression in K562 cells K562 cells were nucleofected with Suz12 siRNA, nontargeting siRNA (neg),
or mock transfected (A) Protein was collected after 2 days and electrophoresed on gels for western blotting Blots were probed with a-Suz12 in the upper panel and then stripped and reprobed with a-GAPDH for a loading control (B) Relative b-, c- and e-globin expression in Suz12 knockdown cells RNA was collected, reverse transcribed, and analyzed by quantitative PCR Expression is set relative to either non-targeting siRNA (neg) samples or mock transfected cells, with GAPDH as the internal reference.
Trang 6promoter is mediated by USF We previously
demon-strated that the expression of a dominant negative
mutant of USF in MEL cells resulted in decreased
interactions between CBP⁄ p300 with both b-globin
gene promoter and LCR HS2, suggesting that, in adult
erythroid cells, USF is required for the recruitment of
CBP⁄ p300 to both the b-globin gene and the LCR
Expression of the dominant negative mutant also
reduced interactions of RNA polymerase II with LCR
HS2 and the b-globin gene promoter [20] E-box
motifs are present downstream of the transcription
start sites of both the b-globin promoter and HS2,
sug-gesting that USF is important for transcription in both
of these regions in adult erythroid cells [29]
Components of PRC2 are expressed at high levels
in embryonic tissues and are essential for the earliest
stages of vertebrate development They also have
been found to occupy a special set of developmental
genes in ES cells that must be repressed to maintain
pluripotency and that are poised for activation during
ES cell differentiation [30,31] As the adult b-globin
gene is repressed during the early stages of
develop-ment and is poised for activation, we reasoned that PCR2 could be involved in repressing b-globin expression at the embryonic and fetal stage of devel-opment
Reducing the activity of Suz12 in K562 cells led to
an increase in b-globin gene expression and also a modest increase in e-globin gene expression (Fig 4) It should be noted that the K562 cells were not induced
by hemin, which has been shown to increase the expression of e- and c-globin gene expression in these cells However, the effect of hemin on globin gene expression in these cells is relatively low [32] Neverthe-less, it is possible that the PRC complex represses globin gene expression in a differentiation and devel-opmental stage-specific manner This is consistent with previous findings demonstrating that the PRC2 complex localizes to developmentally regulated genes
in undifferentiated ES cells [30], and with our own data demonstrating that Suz12 and TFII-I interact with the bmaj-globin promoter during early but not late stages of EPO-induced ES cell differentiation [20] (Fig 5)
Fig 5 The interaction of Suz12 with the b-globin gene promoter decreases with increased b-globin gene expression during erythroid differ-entiation of mouse ES cells (A) RT-PCR analysis of e- and b-globin gene expression during erythroid differdiffer-entiation of mouse ES cells RNA was isolated from ES cells incubated in the presence of EPO for 5 or 12 days, as indicated The RNA was reverse transcribed and subjected
to RT-PCR using primers specific for the control genes Rex1 and b-actin as well as the embryonic e- and adult bmaj-globin genes (B) Quanti-tative RT-PCR analysis of bmaj-globin gene expression at days 5 and 12 of erythroid differentiation of mouse ES cells RNA was isolated from the cells at the indicated time points after addition of EPO and subjected to quantitative RT-PCR analysis using primers specific for the b maj -globin gene (C) Analysis of modified histones, Suz12 and TFII-I interactions with the bmaj-globin gene promoter at days 5 and 12 of erythroid differentiation Cells were collected at the indicated time points and subjected to ChIP analysis using antibodies specific for histone H3 dime-thylated at K4 (H3K4me2), histone H3 trimedime-thylated at K27 (H3K27me3), Suz12, TFII-I and the control IgG (D) Quantitative analysis of Suz12 interactions with the bmaj-globin gene promoter at days 5 and 12 of erythroid differentiation Cells were taken at the indicated time points and subjected to ChIP with the indicated antibodies The precipitated DNA was subjected to quantitative PCR using primers specific for the
b maj -globin gene In the left panel, chromatin was precipitated with antibodies specific for histone H3 dimethylated at K4 (H3K4me2) and analysed by quantitative PCR using primers specific for LCR element HS2.
Trang 7Suz12 has previously been shown to be required for
the di- and tri-methylation of H3K27, which are marks
for inactive chromatin [33] We did not observe high
levels of trimethylated H3K27 associated with the
b-globin gene promoter in K562 cells However,
trime-thylated H3K27 was present at the bmaj-globin gene
promoter in early stage EPO-induced ES cell cultures
(day 5) when the bmaj-globin gene is expressed at low
levels This modification was absent in day 12 cultures
that express high levels of the bmaj-globin gene
(Fig 5)
The interactions between TFII-I and Suz12 in K562
cells are weak This interaction may be unstable or
transient in nature In this context, it is interesting to
note that another initiator binding protein, YY1, has
also been shown to directly interact with both HDAC3
and the PRC complex [34,35] Furthermore, YY1 has
been implicated in the silencing of e-globin gene
expression in adult erythroid cells [36]
Suz12 could also be recruited to the b-globin gene
promoter through the interaction with other DNA
binding proteins involved in repressing the adult
glo-bin gene One possible candidate is BP1, a
homeo-domain-containing protein, which binds to a negative
regulatory element in the upstream b-globin gene
region and reduces expression [37,38] It is not
known how this protein represses b-globin gene
expression
Another corepressor recruited by TFII-I to the
glo-bin gene locus is HDAC3 TFII-I and HDAC3 interact
with each other exclusively in K562 cells and not in
MEL cells It has been observed that TFII-I and
HDAC3 have very similar expression patterns in the
developing mouse embryo [39] Therefore, the results
presented in the present study, along with the previous
studies, suggest that the transcription activity of
TFII-I may be controlled by HDAC3 during early
develop-ment Our data demonstrate that TFII-I functions as a
repressor of b-globin gene expression by recruiting
HDAC activity to the promoter This is consistent
with observations of the acetylation of the human
b-globin promoter region being reduced in erythroid
cells with an embryonic phenotype [16,40]
EKLF is present and active in both primitive and
adult erythroid cells but somehow is unable to activate
b-globin gene expression in embryonic erythroid cells
[41] Perhaps EKLF functions in conjunction with
other factors to activate b-globin gene expression and
these factors are not present or active in embryonic
cells Alternatively, EKLF requires an accessible
chro-matin structure to recruit nucleosome mobilizing
activ-ities to the promoter Initial accessibility may be
provided by proteins that recruit histone acetyl or
methyl transferases to the adult b-globin gene pro-moter, thereby counteracting the repressive activity of TFII-I
Experimental procedures
Cell culture
Human erythroleukemia (K562) cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and 5% penicillin-streptomycin Murine erythroleu-kemia (MEL) cells were grown in RPMI containing 10% fetal bovine serum and 5% antibiotic-antimycotic Cells were grown at 37C and maintained at a cell density of approximately 1–5· 105
cellsÆmL)1 Mouse ES cells were cultured and induced to differentiate with EPO as described
by Levings et al [17]
ChIP, co-immunoprecipitation
Chromatin immunoprecipitation was carried out as described by Leach et al [19] Briefly, cells were washed in NaCl⁄ Pi, crosslinked using 1% formaldehyde, and quenched with glycine After isolation of nuclei and lysis, the crosslinked chromatin was fragmented by sonication to average size fragments of 500 bp Chromatin fragments were precipitated with specific or nonspecific control (IgG) antibodies Antibody⁄ chromatin fragments were subjected
to several rounds of stringent washing The DNA was puri-fied from the chromatin fragments and analyzed by real-time quantitative PCR using pairs of primers specific for the murine or human b-globin gene locus as described
by Crusselle-Davis et al [20] in addition to mouse
CAT-3¢, downstream: 5¢-TCTTTGAGCCATTGGTCAGC-3¢); human HS2 (upstream: 5¢-CGCCTTCTGGTTCTGTG
AC-3¢); human c-globin promoter (upstream: 5¢-CCTTCA GCAGTTCCACACAC-3¢, downstream: 5¢-CTCCTCTGT GAAATGACCCA-3¢); human HS3 ⁄ 2 (upstream: 5¢-GTG ACCTCAGTGCCTCAGAA-3¢, downstream: 5¢-ACCTAT
glyceralde-hyde 3-phosphate dehydrogenase (GAPDH) promoter (upstream: 5¢-ACGTAGCTCAGGCCTCAAGACCTTG-3¢,
GA-3¢)
Immunoprecipitation was carried out essentially as described by Crusselle-Davis et al [20] The antibodies used
in the experiments comprised: monomethyl-histone H3 Lys27 07-448, trimethyl-H3 Lys27, Suz12 07-379 (Upstate, Charlotteville, VA, USA); USF1 (H-86) sc-8983, p300 (N-15) sc-584, CBP (A-22) sc-369, GAPDH (FL-335) sc-25778 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA); Suz12 ab12073, TFII-I ab10464 (Abcam, Cambridge, MA,
Trang 8transfected K562 cells after 48, 72 and 96 h, as described
previously [20] Relative expression was determined using
real-time RT-PCR and primers as described by
Crusselle-Davis et al [20] In addition, human c-globin primers
(upstream: 5¢-TGAATGTCCAAGATGCTGGA-3¢,
down-stream: 5¢-CATGATGGCAGAGGCAGAG-3¢) were used
Protein isolation and western blotting
Protein isolation and western blotting was performed as
described by Leach et al [19] A total of 20 lg of protein
was loaded onto 7.5% or 5% Ready gels (Bio-Rad,
Hercu-les, CA, USA), electrophoresed, and transferred to nylon
membranes The detection of proteins on the membranes
was performed using the ECL Plus system as described in
the manufacturer’s instructions (Amersham Pharmacia
Bio-tech, Piscataway, NJ, USA) The primary antibodies used
were the same as those used in the ChIP and
immunopre-cipitation assays in addition to HDAC3 (H-99) sc-11417
(Santa Cruz) The secondary antibodies used were as
described by Crusselle-Davis et al [20] The antibodies were
used at concentrations recommended in the manufacturer’s
guidelines
Acknowledgements
We are grateful to our colleagues I-Ju Lin, Shermi
Liang, Kunjal Gandhi and JoAnne Andersen for
assistance and stimulating discussions This work was
supported by grants from the NIH (DK 52356) and
American Heart Association
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