Recently, we affinity purified several MLL complexes from human cells and demonstrated that MLL1 plays critical roles in his-tone H3 lysine-4 methylation and Hox gene regulation [21].. The
Trang 1upon exposure to a food contaminant mycotoxin,
deoxynivalenol
Khairul I Ansari1, Imran Hussain1, Hriday K Das2and Subhrangsu S Mandal1
1 Department of Chemistry and Biochemistry, The University of Texas at Arlington, TX, USA
2 Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA
Elucidating the regulatory network of proto-oncogenes
in normal healthy cells and under toxic stress is
impor-tant for understanding the mechanism of human
dis-eases [1–5] Mixed lineage leukemias (MLLs) are a set
of evolutionary conserved genes that are often
rear-ranged and misregulated in acute lymphoblastic and
myeloid leukemias [1,2,6] Humans encode several
MLL protein families, such as MLL1, MLL2, MLL3,
MLL4 and Set1 [1,7–14] In general, they are master
regulators of homeobox (Hox) genes which are critical
for cell differentiation and development [1,2,15,16]
Because of their importance in gene regulation and
dis-ease, researchers have purified MLL proteins from
human cells and have demonstrated that MLLs posses
histone H3 lysine-4-specific methyl-transferase activities
and play a critical role in gene activation [9,17–20]
MLLs exist as multiprotein complexes inside cells with
several common protein subunits such as Ash2, Wdr5,
Rbbp5 and CGBP [1,9,10,19,21] Recently, we affinity purified several MLL complexes from human cells and demonstrated that MLL1 plays critical roles in his-tone H3 lysine-4 methylation and Hox gene regulation [21] We also demonstrated that downregulation of MLL1 results in cell-cycle arrest in the G2⁄ M phase indicating its critical role in cell-cycle progression [22] Although recent discoveries of MLL-associated his-tone H3 lysine-4-specific methyl-transferase activities have shed significant light on the complex function of MLLs in gene regulation, little is known about their own regulation in normal cells or in cells under stress [1] However, it has been reported that certain chemo-therapeutic stresses result in MLL rearrangement and misregulation, leading to the development of secondary leukemias in humans [23,24] These observations indi-cated that MLL1 is stress-responsive gene Herein, we studied the effect of a potential carcinogenic
mycotox-Keywords
deoxynivalenol; mixed lineage leukemia;
MLL; mycotoxin; Sp1
Correspondence
S S Mandal, Department of Chemistry and
Biochemistry, The University of Texas at
Arlington, Arlington, TX 76019, USA
Fax: +1 817 272 3808
Tel: +1 817 272 3804
E-mail: smandal@uta.edu
(Received 2 February 2009, revised 29
March 2009, accepted 7 April 2009)
doi:10.1111/j.1742-4658.2009.07055.x
Mixed lineage leukemias (MLLs) are histone-methylating enzymes with critical roles in gene expression, epigenetics and cancer Although MLLs are important gene regulators little is known about their own regulation Herein, to understand the effects of toxic stress on MLL gene regulation,
we treated human cells with a common food contaminant mycotoxin, deoxynivalenol (DON) Our results demonstrate that MLLs and Hox genes are overexpressed upon exposure to DON Studies using specific inhibitors demonstrated that Src kinase families are involved in upstream events in DON-mediated upregulation of MLL1 Sequence analysis demonstrated that the MLL1 promoter contains multiple Sp1-binding sites and impor-tantly, the binding of Sp1 is enriched in the MLL1 promoter upon expo-sure to DON Moreover, antisense-mediated knockdown of Sp1 diminished DON-induced MLL1 upregulation These results demonstrated that MLL1 gene expression is sensitive to toxic stress and Sp1 plays crucial roles in the stress-induced upregulation of MLL1
Abbreviations
ChIP, chromatin immunoprecipitation; DON, deoxynivalenol; Hox, homeobox; MLL, mixed lineage leukemia.
Trang 2in, deoxynivalenol (DON) on the regulation of MLL1.
Notably, DON is a toxin produced by pathogenic
fungi during the infection of cereal crops and is often
linked with various acute and chronic human diseases,
including cancer [25–27] Herein, we report that MLL1
and its target Hox genes are upregulated upon
expo-sure to DON and transcription factor Sp1 plays
criti-cal roles in the DON-mediated upregulation of MLL1
Results
DON induces expression of MLL
To understand the effects of mycotoxic stress on MLL
expression, we treated cultured human cells
(H358 cells) with varying concentrations of DON (up
to 33 lm) for 7.5 h We isolated RNA from the treated
and untreated control cells and subjected it to
RT-PCR with primers specific to MLL1 and Set1 As seen
in Fig 1A,B, treatment with DON induced two- to five-fold overexpression of MLL1 and Set1 in a con-centration-dependent manner MLL1 overexpression
by DON was more dramatic (8.3-fold) at the protein level (lane 4, Fig 1C,D) The decrease in expression of MLL1 and Set1 at 10 h or longer (Fig 1C,D) is likely caused by cell death induced by DON Because MLL1
is upregulated upon exposure to DON, we analyzed the expression of several other proteins (such as Rbbp5, Wdr5 and Ash2) known to interact with MLL1 [9,21] We also analyzed the effect of DON on expression of some MLL1 target Hox genes (HoxA2, HoxA7, HoxB1, HoxB7, etc.) Importantly, similar to MLL1, Rbbp5 and Wdr5 were overexpressed upon treatment with DON, whereas Ash2 was not affected significantly (Fig 2) Similarly, HoxA7, HoxA2 and HoxB1were overexpressed, whereas HoxB7 was down-regulated upon exposure to DON (Fig 2 and data not shown) The upregulation of MLL1, its several
inter-Incubation time (h)
1 2 3 4 5 6
0 2.5 5 7.5 10 15
β -actin MLL1 Set1
0
2
4
6
8
10
0 h 2.5 h
5 h 7.5 h
10 h
15 h
1
2
3
4
5
6
0.33 µ M
3.3 µ M
33 µ M
β -actin
MLL1
Set1
1 2 3 4 5 6 7 8
0 0.33 3.3 33
DON (µ M )
B D
Fig 1 DON-induced expression of MLL 1 and Set1 (A) Human lung cancer cells (H358) were treated with varying concentra-tions (0–33 l M) of DON for 7.5 h Total RNA was subjected to RT-PCR analysis with primers specific to b-actin (control), MLL 1 and Set1 Each experiment was duplicated for accuracy (B) Quantification of MLL 1 and Set1 expression as seen in (A) Bars indi-cate SEM (C) Total protein extracts from DON (3.3 l M DON for various time points) treated H358 cells were analyzed by wes-tern blot using anti-actin (control), anti-MLL 1 and anti-Set1 Ig (D) Quantification of expressed proteins as seen in (C) relative
to actin.
Water DON
1 2 3 4 5 6 7 8
β -actin
Rbbp5
Ash2
Wdr5
HoxA7
HoxB7
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
A B
Fig 2 DON-induced expression of MLL 1- int-eracting and target genes (A) Human lung cancer cells (H358) were treated with 3.3 l M DON for 7.5 h Total RNA was subjected to RT-PCR analysis with primers specific for b-actin (control), Rbbp5, WDR5, Ash2, HoxA7 and HoxB7 Lanes 1–4, untreated control; lanes 5–8, treated with DON (B) Quantification of gene expression level as seen in (A) Bars indicate SEM.
Trang 3acting proteins or selected target Hox genes upon
exposure to DON indicated that expression of these
proteins is sensitive to toxic stress
Notably, we analyzed the effects of DON on cell
growth and determined the cytotoxicity (IC50) towards
H358 cells using a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromideassay, as described
previ-ously [28] Upon treatment with 3.3 lm DON, up to 5,
47 and 68% of H358 cells were killed at 7.5, 24 and
72 h post treatment, respectively The IC50 value is
determined to be 1 lm These results demonstrated
that DON is significantly cytotoxic towards human
cells
Src kinase inhibitor suppressed the DON-induced
upregulation of MLL1
To understand potential mechanism of DON-mediated
upregulation of MLL1 and Hox genes, we examined
the involvement of different DON-responsive signaling
pathways Because DON is known to induce ribotoxic
stress that instigates various signaling cascades,
includ-ing MAP⁄ Src kinases [29–33], we initially examined
whether inhibition of MAP⁄ Src kinase activation had
any effect on DON-induced upregulation of MLL1
We treated cells with a Src kianse inhibitor (PP2) or a
MAP kinase inhibitor (PD98059) and then exposed the
cells to DON As expected, MLL1 was upregulated
upon treatment with DON (lanes 1 and 4–7, Fig 3)
However, upon treatment with PP2, DON-induced
expression of MLL1 and HoxA7 was suppressed in a
concentration-dependent manner at both the mRNA
and protein levels (compare lanes 4–7 with lane 1,
Fig 3A,B) These results indicated that Src kinases
play a critical role in regulating upstream events that
lead to MLL1 and HoxA7 upregulation by DON
Notably, PP2 has no significant effect on
DON-induced expression of Set1, Rbbp5, Ash2 and Wdr5
(data not shown) suggesting the involvement of
alter-nate pathways Because MLL1 induction was
sup-pressed by Src kinase inhibitor (PP2), we examined
whether MAP kinases are also involved in
DON-medi-ated MLL1 upregulation However, application of
PD98059 did not have any significant effect on
DON-induced upregulation of MLL1, indicating no
involve-ment of MAP kianses in this process (Fig 3C)
Sp1 plays a critical role in DON-induced MLL1
upregulation
To understand the mechanism of MLL1 upregulation
by DON, we analyzed the MLL1 promoter for the
presence of various cis-elements recognized by specific
transcription factors (such as Sp1, AP2), particularly those known to be activated by mycotoxins [27,29–36] Interestingly, we found the presence of multiple Sp1-binding sites in the MLL1 promoter ()3000 to +500 nucleotide region; Fig 4) To investigate possi-ble role of Sp1 in MLL1 gene regulation, we knocked down Sp1 in H358 cells by using Sp1-specific antisense and then analyzed the expression of MLL1 in the absence and presence of DON (3.3 lm) As seen in
Fig 5A,B, treatment with Sp1 antisense effectively knocked down Sp1 expression at both the mRNA and the protein level (compare lanes 1 with 3) Upon knockdown of Sp1, the basal level of MLL1 expression was not significantly affected at the mRNA or the
PP2 (µ M )
– –
1 2 3 4 5 6 7
β -actin MLL1
HoxA7 Ash2
10 0.1 1 10 25
A
+ + + +
PP2 (µ M ) 10 0.1 1 10 25
β -actin MLL1 Ash2
DON (3.3 µ M )
1 2 3 4 5 6 7
– – – + –
B
PD89059 (µ M )
1 2 3 4 5 6 7
β -actin MLL1
– – – –
25 1 5 25 50
C
Fig 3 Effect of DON on expression of MLL 1 and HoxA7 genes in presence of MAP ⁄ Src kinase inhibitor PP2 and PD98059 H358 cells were treated 0.1, 1, 10 and 25 l M PP2 and 1, 5, 25 and
50 l M PD98059 for 1 h prior to treatment with 3.3 l M DON for an additional 7.5 h (A) RT-PCR analysis of RNA extract from cells treated with PP2 with primers specific to b-actin (control), MLL1, Ash2 and HoxA7 (B) Western blot of the total proteins extract of the cells treated with PP2 with antibodies specific for b-actin, MLL 1 and Ash2 (C) RT-PCR analysis of RNA extract from cells treated with PD98059 with primers specific to b-actin (control) and MLL1 Each experiment was performed in duplicate.
Trang 4protein level (Fig 5A,B, lanes 1 and 3) Interestingly,
however, DON-induced upregulation of MLL1
(mRNA and protein level) was suppressed to almost
normal levels under an Sp1 knocked down
environ-ment (Fig 5A,B, lanes 2 and 4) These results
indi-cated that Sp1 is critical for MLL1 regulation,
especially in presence of DON
Because the MLL1 promoter contains multiple
Sp1-binding sites and our results demonstrated that Sp1 is
critical in regulating MLL1 on exposure to DON, we
hypothesized that DON modulates binding of Sp1 to
the MLL1 promoter To confirm our hypothesis, we
treated H358 cells with 3.3 lm DON and subjected
them to chromatin immunoprecipitation (ChIP) using
anti-Sp1 Ig (Fig 5C,D) In parallel, we also performed
ChIP with an unrelated antibody (actin antiserum)
The immunoprecipitated DNA fragments were PCR
amplified using primers specific for MLL1 promoter
regions R1 ()497 to )593), R2 ()5 to )105),
MLL1-ORF (as control) and b-actin-MLL1-ORF (a second
unre-lated control) Our results demonstrated that no Sp1
was bound to the ORF region of actin in either the
absence or presence of DON (Fig 5C, upper, lanes
5 and 6) Similarly, Sp1 binding was not enriched in
the MLL1-ORF region in the presence of DON
(MLL1-ORF; Fig 5C, lanes 5 and 6) Interestingly,
however, the binding of Sp1 was significantly enriched
in the Sp1-binding sites of the MLL1 promoter regions
R1 and R2 in the presence of DON, although more
enrichment was observed in the R2 region (closer to
the transcription start site) (Fig 5C,D, lanes 5 and 6)
ChIP analysis showed no binding of b-actin to the
MLL1 promoters and ORF region, irrespective of the presence or absence of DON (Fig 5C, lanes 3 and 4) These results demonstrated that binding and enrich-ment of Sp1 to the MLL1 promoter regions (R1 and R2) in the presence of DON is specific and this dem-onstrates that Sp1 is crucial for transcriptional activa-tion of MLL1 under DON treatment
Furthermore, because phosphorylation of Sp1 is well known to be associated with toxic stress, we analyzed the state of Sp1 phosphorylation upon DON exposure [33,37] We performed immunoprecipitation of Sp1 using anti-Sp1 Ig (nonphosphorylated) from DON-treated and unDON-treated cells We analyzed the immuno-precipitates by western blot using both anti-Sp1 Ig (nonphosphorylated) and anti-phosphotyrosine Ig that recognize tyrosine-phosphorylated proteins Interest-ingly, upon DON treatment, the protein level of Sp1 was not significantly affected, although, the level of tyrosine-phosphorylated Sp1 was increased (Fig 5E) These results indicated that DON induces phosphory-lation of Sp1 and this might be linked with MLL1 upregulation
Discussion Because MLLs are proto-oncogenes and are known to
be rearranged or misregulated under chemotherapeutic stress, leading to secondary leukemias [23,24], elucidat-ing the stress responsive regulatory mechanism of MLL is important Herein, our studies showed that exposure to mycotoxin DON-induced expression of MLL1, several MLL interacting proteins and MLL target Hox genes Notably, MLL1 exists as a multipro-tein complex inside the cell with subunits like Ash2, Wdr5 and Rbbp5, and MLL1 executes its histone methyl-transferase activity and regulates target Hox genes in the context of the multiprotein complex [1,9,10] Therefore, because MLL1 and several Hox genes (HoxA7, HoxA2, etc.) were overexpressed upon exposure to DON, we anticipated that MLL-interact-ing proteins might be upregulated in a similar fashion However, our results demonstrated that although several MLL1-interacting proteins such as Wdr5 and Rbb5 were upregulated upon exposure to DON, Ash2 expression was not significantly affected These observations suggest that Ash2 is a unique component
of MLLs and may have other distinct functions that are yet to be revealed It is also possible that Ash2 is normally distributed in different protein complexes which may be redistributed (without being induced) under stress to compensate for the higher expression of MLL1 This aspect needs further investigation for complete understanding Similarly, although the MLL1
Fig 4 Sp 1- binding sites in the MLL 1 promoter MLL 1 gene
pro-moter (from )3000 to +500 bp) sequence was analyzed for
pres-ence of Sp 1 -binding sites (GGGCGG, GGCGGG, CCCGCC and
CCGCCC) using http://www.ifti.org/ promoter screening tool
Puta-tive Sp 1 Binding sites are underlined Transcription start site (ATG)
is shown as +1.
Trang 5target genes HoxA7, HoxA2 and HoxB1, along with
MLL1, were upregulated upon exposure to DON, we
observed that HoxB7expression decreased These
results suggested that the mechanism of regulation,
especially in presence of DON, is different for HoxB7
(as well as HoxA2 and HoxB1) and HoxA7, although
they are all targets of MLL1 in normal circumstances
(without DON) Nevertheless, our results showing the
DON-induced upregulation of MLL1 and related
pro-teins indicated that MLL1 and its associated genes are
sensitive to toxic stress
The effect of DON is very well studied in plants
[38,39] In mammalian cells, DON induces oxidative
stress, activates MAP⁄ Src kinases and induces
inflam-mation and oxidative stress-responsive genes such as
interleukins and cyclooxygenase [32,36,40–42] Using
RT-PCR analysis, we also observed that
interleukin-8 and cyclooxygenase are overexpressed in H35interleukin-8 cells upon exposure to DON, indicating the induction of oxidative stress in human cells, as reported earlier (data not shown) [36,41] Furthermore, using Src kinase inhibitor (PP2), we demonstrated that DON-induced MLL1 and HoxA7 gene upregulation were alleviated in the presence of PP2 These observations demonstrated that Src kinases are involved in upstream events in DON-mediated upregulation of MLL1 and HoxA7 Notably, our results demonstrated that application of PP2 has no significant effect on the DON-induced upregulation of other proteins such as Set1, Wdr5 and Rbbp5 (data not shown), suggesting the involvement of alternate pathways in the regulation
of these genes
Our sequence analysis demonstrated that the MLL1 promoter contains multiple binding sites for Sp1, a
Water DON Water DON Water DON Input Actin Sp1
Anti-serum
MLL1 ( ORF ) MLL1 ( R1 ) (R2)
β -actin
1 2 3 4 Water DON Water DON
Scramble antisense
28S rRNA Sp1 MLL1
Sp1 antisense
1 2 3 4 Water DON Water DON
Scramble antisense
β -actin Sp1 MLL1
Sp1 antisense
Sp1
Sp1-p
0 0.33 3.3 33 DON
(µ M )
0.0 0.2 0.4 0.6 0.8 1.0
Actin Sp1 Actin Sp1 Actin Sp1 Actin Sp1 Actin (ORF) MLL (ORF) MLL1 (R1) MLL1 (R2)
Water DON
ChIP anti-sera Target DNA region
A B
C E
D
Fig 5 Effect of knockdown of Sp 1 on
DON-induced upregulation of MLL1.
H358 cells were treated with Sp 1- specific
phosphorothioate antisense for 48 h
fol-lowed by treatment with 3.3 l M DON for
7.5 h (A) RT-PCR analysis of Sp 1 and MLL 1
using specific primers 28S rRNA was used
a quantitative control (B) Total protein was
analyzed by western blot using anti-actin
(control), anti-Sp 1 and anti-MLL 1 Ig (C)
DON-induced recruitment of Sp 1 in the
MLL 1 promoter H358 cells treated with
3.3 l M DON for 7.5 h were subjected to
ChIP assay using Sp 1 and actin antibodies.
Actin ChIP was used as a nonspecific
anti-body control ChIP DNA fragments were
PCR amplified using primer specific to
dif-ferent Sp 1- binding sites in the MLL 1
promot-ers b-actin (ORF): PCR-amplified ‘+712 to
+1011’ of b-actin (unrelated control); MLL 1
(ORF): PCR-amplified ‘+3190 to +3380’ of
MLL 1 gene (control); MLL 1 (R1 and R2)
PCR-amplified ‘ )497 to )593’ and ‘)5 to
)105’ of the MLL 1 promoter (D)
Quantifica-tion of Sp 1 recruitment as seen in (C) (E)
Western blot analysis of phosphorylated Sp 1
upon DON treatment H358 cells were
trea-ted 0–33 l M DON for 7.5 h The whole-cell
extracts were immunoprecipitated with
anti-Sp 1 Ig The Sp1 immunoprecipitate was
ana-lyzed by western blot using both anti-Sp 1
and anti-phosphotyrosine Ig.
Trang 6transcription factor that is well known to be activated
and phosphorylated under stress [29,33,34,36,43] The
literature relating to mycotoxin-mediated activation of
Sp1 and our results showing the presence of multiple
Sp1-binding sites in the MLL1 promoter, prompted us
to hypothesize that Sp1 plays a critical role in the
reg-ulation of MLL1, especially under mycotoxic stress
[33,37,43] Our studies demonstrated that
antisense-mediated knockdown of Sp1 suppressed the effects of
DON on upregulation of MLL1 In addition, the level
of Sp1 is enriched in the Sp1-binding regions of the
MLL1 promoter upon exposure to DON These results
demonstrated that Sp1 acts a mediator in translating
the effects of DON on MLL1 gene upregulation
Notably, cells respond to stress by activating signaling
pathways that regulate defense responsive genes
[36,38,39] An early step in the stress response includes
phosphorylation of the MAP⁄ Src kinases leading to
their activation [36] Sp1 and other Sp1 family
mem-bers are differentially acetylated, phosphorylated
and⁄ or glycosylated, and bind variants of a GC-rich
box in promoter of target genes Because the MLL1
promoter contains multiple Sp1-binding sites and is
regulated by Sp1, as well as the Src family of kinases
on DON treatment, we hypothesized that Sp1 is likely
phosphorylated and recruited to the MLL1 promoter,
resulting in its upregulation Our studies demonstrated
that Sp1 is phosphorylated upon exposure to DON
Although, at this point we could not directly analyze
recruitment of the phosphorylated Sp1 into the MLL1
promoter because of the unavailability of the
phospho-Sp1-specific antibody, the increased recruitment of the
Sp1 in the MLL1 promoter may be linked with
phos-phorylation of Sp1
In conclusion, we demonstrated that MLL1, several
MLL-associated proteins and Hox genes are
upregulat-ed upon exposure to mycotoxin DON via involvement
of Src kinase activation The transcription factor Sp1
plays critical role in upregulating MLL1 gene
expres-sion under mycotoxic stress Although further analysis
is needed to understand the detailed mechanism of
MLL gene (and other DON-responsive genes)
regula-tion in normal cell or under stress, our studies
estab-lished a novel link between MLL gene regulation, the
stress response and DON, and revealed critical
stress-responsive MLL1 gene regulatory pathways Although,
the mechanism is not clear, MLL is well known to be
rearranged and misregulated in various cancers and it
is likely that different types of stresses cause MLL
mis-regulation and rearrangement As exposure to DON
induces upregulation of MLL1, we hypothesize that
this may be one of the possible mechanism by which
DON exerts is carcinogenic action in human cells
Experimental procedures
Cell culture and treatments with DON Human cells (H358, a lung cancer, ATCC) were grown on RPMI media supplemented with 10% fetal bovine serum,
l-glutamine (1%) and penicillin⁄ streptomycin (0.1%) (Sigma, St Louis, MO, USA) For the toxin treatment, cells were grown to 80% confluence and treated with varying concentrations of DON (Sigma) for different times, as needed Total RNA and proteins were isolated from the treated and untreated cells and subjected to RT-PCR and western blot analysis For the RT-PCR analysis, each experiment was performed in two to four replicates in par-allel For the western blot analysis, proteins from replicate experiments were pulled together prior to SDS⁄ PAGE
Preparation of RNA, nuclear proteins and whole-cell extract
DON-treated and untreated cells were harvested by centrifu-gation at 500 g, resuspended in diethyl pyrocarbonate-treated buffer A (20 mm Tris⁄ HCl, pH 7.9, 1.5 mm MgCl2,
10 mm KCl, 0.5 mm dithiothreitol and 0.2 mm phenyl-methanesulfonyl fluoride), incubated on ice for 10 min and then centrifuged at 3500 g for 5 min The supernatant containing the cytoplasmic extracts was subjected to phenol– chloroform extraction followed by LiCl precipitation of cytoplasmic mRNA by incubating overnight at)80 C The mRNA was washed with diethyl pyrocarbonate treated 70% EtOH, air dried and resuspended in diethyl pyrocarbonate-treated water Nuclear proteins extracts were prepared from the nuclear pellets, as descried previously [21,22] For prepa-ration of whole-cell protein extracts cells were incubated in whole cells extract buffer (50 mm Tris⁄ HCl pH 8.0, 150 mm NaCl, 5 mm EDTA, NP-40, 0.2 mm phenylmethanesulfonyl fluoride, 1· protease inhibitors) for 20 min on ice The whole cell extract was separated from histone protein by centrifugation at 12 000 g for 10 min
RT-PCR and western blots Reverse transcription reactions were performed in a total volume of 25 lL containing 1 lg of total RNA, 2.4 lm of oligo-dT, 100 U of MMLV reverse transcriptase (Promega, Madison, WI, USA), 1· first strand buffer (Promega),
100 lm dNTPs, 1 mm dithiothreitol and 20 U of RNaseOut (Invitrogen, Carlsbad, CA, USA) This cDNA (1 lL) was used for PCR with primer pairs listed inTable 1 Each of the experiments was performed in two replicates for three times The normality of the data was analyzed by using t-test and analyses of the variants (ANOVA) were performed at 5% level of significance
Equivalent amount of proteins were analyzed in SDS⁄ PAGE and subjected to western blot analysis with
Trang 7specific antibodies MLL1, MLL2, Set1, Ash2 and Rbbp5,
antibodies were purchased from Bethyl laboratory
(Mont-gomery, TX, USA)
Immunoprecipitation and western blotting of Sp1
and phosphorylated Sp-1
For western blot analysis of the Sp1 expression, equivalent
amounts of whole-cell extract (DON-treated and untreated)
were separated in 8% SDS⁄ PAGE and subjected to western
blot analysis using anti-Sp1 Ig (Upstate, Waltham, MA,
USA) For the analysis of DON-induced phosphorylation of
Sp1, we performed immunoprecipitation of Sp1 from the
whole-cell protein extract using anti-Sp1 Ig, as described
ear-lier [21] The Sp1 immunoprecipitates were electrophoresed
in 8% SDS⁄ PAGE and subjected to western blot using both
anti-Sp1 (nonphosphorylated) and anti-phosphotyrosine Ig
(Upstate) that recognize tyrosine phosphorylated proteins
Antisense-mediated knockdown of Sp1
The Sp1 antisense (5¢-CTGAATATTAGGCATCACTCC
AGG-3¢) was transfected into H358 cells using Maxfect
transfection reagent (MoleculA) In brief, H358 cells were
grown to 60% confluence, washed twice with fetal bovine
serum-free RPMI media and then incubated with
transfec-tion reagent–antisense complex for 5 h in serum-free RPMI
prior to the addition of complete growth medium (with 10%
serum) Cells were then incubated for 48 h followed by
treat-ment with 3.3 lm DON for 7.5 h Cells were then harvested
for RNA, nuclear protein extraction or ChIP analysis A
scramble antisense without any sequence homology with Sp1
(5¢-CGTTTGTCCCTCCAGCATCT-3¢) was used as control
ChIP experiment
The ChIP assay was performed using H358 cells and
anti-Sp1 mAb (Bethyl lab) using EZ Chip chromatin
immuno-precipitation kit (Upstate) as described previously [21,22]
Immunoprecipitated DNA obtained from the ChIP was PCR amplified with different primers (specific to Sp1 rich sites in MLL1 promoter, Table 1)
Acknowledgements This work was supported by grants from Texas Advanced Research Program (00365-0009-2006) and American Heart Association (SM 0765160Y) We also thank Saoni Mandal and other Mandal lab members for critical discussions
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