myc via an intron 1 x box in undiffer

Activation of protein kinase C induces nuclear translocation of RFX1 and down-regulates c-

myc via an intron 1 X box in undifferentiated leukemia HL-60 cells*

Lei Chen, Lucinda Smith, Martin R. Johnson, Kangsheng Wang, Robert B. Diasio, and Jeffrey

Bingham Smith‡

Department of Pharmacology & Toxicology and Comprehensive Cancer Center, Schools of

Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294

Running title: Role of RFX1 in the down-regulation of c-myc by PKC

‡To whom correspondence should be addressed: Jeffrey B. Smith, Ph.D., Department of

Pharmacology & Toxicology, Schools of Medicine and Dentistry, University of Alabama at

Birmingham, Birmingham, AL 35294-0019; Tel: (205) 934-7434; Fax: (205) 975-5841; E-mail:

[email protected]

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 28, 2000 as Manuscript M002645200 by guest on M

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ABSTRACT

Treatment of human promyelocytic leukemia cells (HL-60) with phorbol 12-myristate 13-

acetate (PMA) is known to decrease c-myc mRNA by blocking transcription elongation at sites

near the first exon/intron border. Treatment of HL-60 cells with either PMA or bryostatin 1

(Bryo), which acutely activates protein kinase C (PKC), decreased the levels of myc mRNA and

Myc protein. The inhibition of Myc synthesis accounted for the drop in Myc protein because PMA

treatment had no effect on Myc turnover. Treatment with PMA or Bryo increased nuclear protein

binding to MIE1, a c-myc intron 1 element that defines an RFX1-binding X box. RFX1 antiserum

supershifted MIE1-protein complexes. Increased MIE1 binding was independent of protein

synthesis and abolished by a selective PKC inhibitor, which also prevented the effect of PMA on

myc mRNA and protein levels and Myc synthesis. PMA treatment increased RFX1 in the nuclear

fraction and decreased it in the cytosol without affecting total RFX1. Transfection of HL-60 cells

with myc-reporter gene constructs showed that the RFX1-binding X box was required for the

down-regulation of reporter gene expression by PMA. These findings suggest that nuclear

translocation and binding of RFX1 to the X box cause the down-regulation of myc expression

which follows acute PKC activation in undifferentiated HL-60 cells.

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The c-myc protooncogene encodes nuclear proteins, which heterodimerize with Max and

regulate the expression of genes implicated in cell growth (size), metabolism, differentiation,

apoptosis, tumorigenesis, and genomic stability (1-4). Activation of the c-myc gene is a crucial

oncogenic determinant in a wide variety of human cancers (1, 3). Uncontrolled expression of the

normal Myc proteins is associated with a wide variety of animal and human tumors, with almost a

third of breast and colon carcinomas having elevated c-myc expression (1). Mammalian cells

express multiple Myc polypeptides: Myc1, Myc2, and MycS, which are produced by initiation of

translation at different codons (5). Normal cell function depends on tightly modulated Myc protein

levels. Myc proteins and myc mRNA turnover rapidly (t1/2 < 30 min) in eukaryotic cells, and

multiple redundant systems regulate myc transcription and translation (1-3). Myc and MycS

proteins are degraded by the tightly regulated ubiquitin-proteasome system (6, 7), and stabilization

of Myc has been suggested to be caused by certain cancer-associated mutations (6).

Overexpression of Myc in mammalian cells blocks differentiation, predisposes to malignant

transformation, and can initiate apoptosis (8-10).

Initiation of c-myc transcription at the two major promoters (P1 and P2) is under the

control of several protein factors and DNA elements (2). Furthermore, c-myc was the first

eukaryotic gene shown to be regulated at the level of transcription elongation (11, 12). Premature

transcription termination near the first exon/intron junction depends on initiation at the predominant

P2 promoter and explains the early phase of c-myc down-regulation following induction of

differentiation (2, 11-13). For example, in human promyelocytic leukemia HL-60 cells, induction

of differentiation along either the monocytic/macrophage pathway by phorbol 12-myristate 13-

acetate (PMA)1 and perhaps by 1,25-dihydroxyvitamin D3, or along the granulocytic pathway by

retinoic acid or DMSO, blocks c-myc transcription near the first exon/intron border (11-15).

Protein kinase C β plays a critical role in the differentiation response to PMA, retinoic acid, and

1,25-dihydroxyvitamin D3 (16-18). Cotransfection of Burkitt’s lymphoma cells with a c-myc-

reporter gene construct together with myc gene fragments suggested that the 5’ half of the first

intron contained sequences that competed for one or more putative negative regulatory factors

(19). Remarkably somatic mutations in a 20 bp c-myc intron 1 element (called MIE1 or MIF-1)

abolished nuclear protein binding to the element and were associated with c-myc activation in

Burkitt’s lymphoma cell lines (20). Burkitt’s lymphoma mutations also appeared to be clustered in

two additional protein-binding elements (MIE2 and MIE3) that were just downstream of MIE1


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(21). The functional significance of the intron 1 elements in myc expression remains to be

established. Deletion of MIE1 and MIE2 had no effect on c-myc-driven reporter gene expression,

and deletion of MIE3 modestly increased reporter gene activity in transfected cells (21). Recently

five tandem repeats of MIE1 were shown to suppress the activity of the SV40 promoter in

hepatocarcinoma cell lines (22, 23).

MIE1 essentially consists of a regulatory factor X (RFX) consensus binding site (5’-

GTNRCC(0-3N)RGYAAC), which is called an X box, EP element, or MDBP site (22-25). X

boxes are key positive elements in the promoters of MHC class II (22-24) and interleukin-5

receptor α chain (28). EP elements are enhancers of genes encoded by hepatitis B virus, polyoma

virus, cytomegalovirus, and Epstein-Barr virus (29, 30). MDBP sites occur in a wide variety of

mammalian genes and bind RFX when they are methylated at CpG dinucleotides or when they

contain TpG or TpA at the analogous positions of the methylated cytosine (24, 31).

RFX proteins are the chief component of nuclear complexes previously referred to as MDBP,

MIF-1, NF-X, EF-C, or EP protein (21, 24, 27, 31). RFX family members (RFX1-5) have a

highly conserved winged-helix DNA binding domain (32). RFX proteins homo- and

heterodimerize with one another and up- or down-regulate transcription of target genes in a DNA

context-dependent manner (27, 33-35). RFX1, which appears to be ubiquitously expressed in

mammalian cells, has an N-terminal activation domain and a C-terminal repression domain that

overlaps the dimerization domain (27, 28, 31). The functional regions can neutralize one another

resulting in a nearly inactive transcription factor (34). Association of RFX1with other family

members, with non-RFX proteins such as c-Abl, or with other DNA-bound proteins apparently

determines whether it has enhancer or silencer activity, although the determinants of the activity

are not understood (33-36).

In this study, we measured the rates of synthesis and degradation of Myc proteins

following the treatment of undifferentiated HL-60 cells with PMA or Bryo. Bryo, like PMA, binds

to the zinc finger C1 domain of conventional (α, β, γ) and novel (δ, ε, η, θ, µ) isoforms of PKC,

which turns on its kinase function and concomitantly predisposes it to ubiquitinylation and

degradation by the 26S proteasome (37, 38, 39). In contrast to PMA, Bryo fails to induce

differentiation of HL-60 cells and prevents the induction of HL-60 differentiation by PMA (40).

Bryo, which is in clinical trials as an anticancer agent, apparently down-regulates PKC more

rapidly and efficiently than PMA (41). Our results indicate that a brief activation of PKC in


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undifferentiated HL-60 cells decreased the Myc protein by blocking its synthesis without affecting

its turnover. Studies of HL-60 cells transfected with c-myc-luciferase reporter constructs suggest

that the RFX-binding X box of intron 1 is essential for the down-regulation of myc by PKC. We

also show, for the first time, that PMA treatment induced nuclear translocation of RFX1. Our

findings suggest that nuclear translocation and binding of RFX1 to the X box contributes to the

down-regulation of myc expression following acute activation of PKC.


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MATERIALS AND METHODS

Cell culture and protein concentration. HL-60 cells were grown in RPMI 1640

containing 15% FBS, 100 units/ml penicillin G and 0.1 mg/ml streptomycin. The medium was

diluted with fresh medium three-times per week and cell density was kept below a million per ml.

The cells were collected by centrifugation and incubated in RPMI 1640 containing the additions

indicated in the figure legends. Protein concentration was measured by the BCA method (Pierce

Chemical) with bovine serum albumin as a standard.

Plasmids. pMPCAT (42), which contains a 3.2 kb HindIII/SacI fragment (nt -2238 to

+936) of human c-myc upstream of the CAT gene, was used to produce the luciferase reporter

constructs. A 2 kb fragment of the c-myc (nt –1058 to +936 relative to P1) was excised from

pMPCAT with KpnI and EcoRV and inserted into the pGL3 control vector at the KpnI and HindIII

(blunt) sites in place of the SV40 promoter. The 2 kb c-myc promoter was upstream of the SV40

enhancer and the firefly luciferase gene. The same procedure was used to subclone the myc

promoter mutants from pMPCAT∆287 and pMPCAT∆220 (21). pMP-Luc∆14, which lacked the

14 bp intron 1 X box (nt 3004-3017) was produced by overlap extension polymerase chain

reaction mutagenesis with the following forward and reverse primers, respectively: 5’-TTT TCT

CAG ATG GGG CTG GGG TGG GGG GTA and 5’-CCC AGC CCC ATC TGA GAA AAG TGT

CAA TAG. Each of the constructions was validated by sequencing, carried out on double-

stranded DNA with dye-terminator chemistry, and the products were resolved using an ABI Prism

377 automated sequencer.

Electroporation and dual-luciferase assay. HL-60 cells were collected by centrifugation

and rinsed once with antibiotic-free RPMI 1640, and 20 million cells were suspended with 0.8 ml

of this medium. Electroporation was done at room temperature at 350 V and 960 µF with a Gene

Pulser (Bio-Rad) and 15 µg of the indicated myc-reporter vector and 15 µg of pRL-TK (Promega)

to control for transfection efficiency. After electroporation the cells were placed on ice for 30 min

prior to dilution with 20 ml of RPMI 1640 containing 10% FBS without antibiotics. To assay

luciferase the cells were harvested by centrifugation, rinsed once with room temperature PBS, and

suspended with passive lysis buffer (Promega). The cells were lysed by three freeze-thaw cycles

using liquid nitrogen and a room temperature water bath. The protein concentration of the

samples was measured, and each was diluted to 3 µg/µl with lysis buffer. Luciferase activity (60


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µg) was determined with the Dual-Luciferase Reporter Assay System (Promega) as recommended

by the manufacturer. Statistical analysis was done by two-tailed Student’s t test.

Western blot analyses. For Myc protein analysis HL-60 cells were lysed with hot

(>95oC) SDS lysis buffer which contained 1% (w/v) SDS, 2 mM EDTA, 2 mM EGTA, and 10

mM Tris-HCl, pH 7.4. Sample of containing 30 µg protein were fractionated by SDS-PAGE on a

10% gel (Myc proteins) or 7% gel (MIBP1 or RFX1). Proteins were electrophoretically

transferred to a PVDF membrane (Millipore Corp.), and the membrane was blocked for 1 h at

room temperature with 5% (w/v) nonfat dry milk in TBS. TBS contained (per liter): 8 g of NaCl,

0.2 g of KCl, 3 g of Tris base, and was adjusted to pH 7.4 at room temperature, and in the case of

TBST, 0.05% (w/v) Tween 20. Membranes were incubated overnight at 4oC with 2 µg/ml OP10

primary antibody in TBS containing 1% nonfat dry milk. For western analysis of MIBP1, RFX1

and NFκB, the membrane was incubated overnight at 4oC with a mouse monoclonal antibody to

NFκB (200 fold dilution, F-6, Santa Cruz Biotechnology, Inc.), rabbit antiserum to MIBP1 (250

fold dilution), rabbit antiserum to RFX1 (1000 fold dilution), or the respective preimmune serum

in TBS containing 1% nonfat dry milk. The preparation and specificity of anti-MIBP1 and anti-

RFX1 were described previously (22). Membranes were rinsed and processed with horseradish

peroxidase-conjugated goat anti-mouse IgG (Transduction Laboratories) or with horseradish

peroxidase-conjugated goat anti-rabbit IgG (BioSource) and a chemiluminescent substrate as

described (7). Films were scanned and analyzed with a model GS-670 Imaging Densitometer

using Molecular Analyst software (Bio-Rad).

Immunoprecipitations and pulse-labeling of Myc. For immunoprecipitation a sample of

the SDS lysate (usually 0.5 mg protein) was diluted ten fold with immunoprecipitation buffer

which contained (in mM): 70 NaCl, 50 NaF, 1 EDTA, 1 EGTA, 0.2 mM sodium orthovanadate,

0.2 phenylmethylsulfonyl fluoride, 1% Triton X-100, 0.5% NP-40, and 10 Tris-HCl, pH 7.4. Two

µg of the C-8 anti-Myc monoclonal (C-8, Santa Cruz Biotechnology), or 5 µl of anti-MIBP1, anti-

RFX1, or preimmune sera were added, and the solution was rotated overnight at 4oC, with 30 µl

of a 50% suspension of protein A agarose (Life Technologies) present during the last h.

Immunoprecipitates were collected by centrifugation and washed twice with each of two buffers,

which differed only NaCl concentration: the first buffer contained (in mM): 1 EDTA, 1 EGTA,

500 NaCl, 0.5% NP40, 1% Triton and 10 Tris HCl, pH 7.4; the second buffer lacked NaCl.

Finally immunoprecipitates were washed twice with 10 mM Tris-HCl, pH 7.4, extracted with 20


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µl 2X SDS sample loading buffer, and incubated for 5 min in a boiling water bath. Proteins were

fractionated by SDS-PAGE (10% gel), and the dried gel was fluorographed with Kodak PPB film

to visualize [35S] labeled proteins. For pulse-labeling experiments, the cells were labeled with

[35S]Met/Cys and subjected to the treatments indicated in the figure legends. The [35S] labeled c-

Myc2 band was cut out of the gel and [35S] was quantified by liquid scintillation counting. To

determine the effects of the cell treatments on the overall translation rate, some cells were labeled

with 5 µCi [35S]Met/Cys for 10 min prior to lysis with >95oC SDS buffer and treated with or

without 20 nM PMA or Bryo. Proteins (30 µg) were fractionated by SDS-PAGE (10% gel) and

fluorographically visualized.

Pulse-chase labeling of Myc. HL-60 cells (107 cells per condition) were rinsed twice

with PBS and once with Met/Cys free culture medium. Pulse-labeling was usually done for 60

min with medium containing 10% of the usual concentration of Cys and Met, and 0.15 mCi of

[35S]Met/Cys. Labeling was stopped by addition of 10 mM each of Met and Cys. After the

indicated chase interval the cells were lysed with hot SDS buffer as described above, and Myc

was immunoprecipitated from a sample containing 0.5 mg protein as described above with the

OP10 antibody. Half-lives were determined by nonlinear regression curve fitting to a single

exponential decay equation.

c-myc northern blot analysis. Total RNA was extracted by the acidified guanidinium

thiocyanate-phenol chloroform method with Trizol as recommended by the manufacturer (Life

Technologies) and quantified by absorbance at 260 nm. RNA samples (10 µg) were size

fractionated by electrophoresis on 1% agarose gel containing (in mM): 20 MOPS, pH 7.4, 1

EDTA, 5 sodium acetate, 0.2 M formaldehyde, and 0.5 µg/ml ethidium bromide. RNA samples

contained 50% formamide. The gel was illuminated with a UV lamp and photographed to compare

the quality and quantity of the rRNA. RNA was transferred to Duralon (UV) membranes

(Stratagene) by downward capillary transfer with the Turboblotter (Schleicher and Schuell) and

cross-linked to the membrane with a Stratalinker 1800 (Stratagene). Membranes were

prehybridized with 6 ml QuikHyb (Stratagene) for 10 min at 68oC in a roller-bottle oven. 32P

labeled cDNA probe (3-5 µCi/ml, >109 cpm/µg) was mixed with 0.1ml of denatured salmon

sperm DNA (10 mg/ml) and added to the roller-bottle. After 2 h at 68oC the membrane was

washed twice at room temperature for 15 min with twice concentrated sodium chloride sodium

citrate (SSC) containing 0.1% SDS and twice at 60oC for 15 min with SSC containing 0.1% SDS.


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SSC contained 8.8 g NaCl and 4.4 g sodium citrate per liter and was adjusted to pH 7.0 with HCl.

A 1.4 kbp Cla1-EcoRI fragment of pHSR-1 of human c-myc (ATCC 41010) was agarose gel

purified and labeled using [α-32P] dCTP and the Klenow fragment of DNA polymerase I (Life

Technologies). c-myc transcript was quantified by autoradiography with Konica PPB film and an

intensifying screen for <24 h at -70oC. Autoradiograms were scanned and analyzed with a model

GS-670 Imaging Densitometer using Molecular Analyst software (Bio-Rad).

Nuclear extracts. The cells (3 x 107 per condition) were rinsed and subjected to hypotonic

lysis without mechanical disruption in buffer A, which contained (in mM): 10 Hepes-Tris, pH 7.9,

10 KCl, 1.5 MgCl2, 5 sodium pyrophosphate, 1 sodium orthovanadate, 0.5 DTT, 0.5 PMSF, and 20

nM calyculin A. The cells were incubated on ice for 10 min and observed by phase-contrast

microscopy to determine that >95% lysis had occurred. A nuclear pellet was obtained by

centrifugation (16,000 x g for 10 s) and extracted with buffer B for 20 min on ice with intermittent

dispersal by pipetting. Buffer B contained (in mM): 20 Hepes-Tris, pH 7.9, 25% glycerol, 420

NaCl, 1.5 MgCl2, 0.2 EDTA, 5 sodium pyrophosphate, 1 sodium orthovanadate, 0.5 DTT, 0.5

PMSF, and 100 nM calyculin A. Particulate material was removed by centrifugation for 15 min at

16,000 x g at 4oC and the supernatant was used for EMSA. Nuclear extracts were diluted 3 fold

with buffer C which contained: 20 mM Hepes-Tris, pH 7.9, 20% glycerol, 100 mM KCl, 0.5 mM

DTT, 0.2 mM EDTA, and the following phosphatase inhibitors: 2 nM calyculin A, 1 mM Na3VO4,

and 5 mM sodium pyrophosphate. Nuclear extracts (~1 µg/µl protein) were used immediately for

EMSA and then frozen in liquid N2 and stored at -80oC.

Electrophoretic mobility shift assays (EMSA) and supershift assays. Binding reactions,

which contained nuclear extract (2 µg protein), 12 µl buffer C, 1 µl poly (dI-dC) (1 µg/µl), were

incubated for 10 min at 25oC in the absence or presence of the indicated competitor double

stranded oligonucleotide or 1 µl of rabbit antiserum to MIBP1, RFX1, or the respective

preimmune serum. [32P] labeled double stranded MIE1, (0.1 ng, ~20,000 cpm) was added and

incubation continued for 30 min. After the addition of gel loading buffer (2 µl), which contained

250 mM Tris-HCl, pH 7.4, 0.2% bromphenol blue, and 40% glycerol, the entire reaction was

loaded onto a 4% acrylamide gel which had been prerun for 1 h at 100 V at 4oC. Electrophoresis

was for 1 h at 100 V and 4oC, and the gel was dried and autoradiographed. The gel buffer

contained (in mM): 380 glycine, 50 Tris, 2 EDTA, and had a pH of 8.5.


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Complementary strands of the oligonucleotides (Life Technologies, Inc.), having the

sequences indicated in Figure 5B, were mixed in annealing buffer ( 20 mM Tris-HCl, pH 7.4, 50

mM NaCl, and 1 mM EDTA), incubated at 65oC for 10 min and allowed to cool slowly (>4 h) to

room temperature. The double stranded oligonucleotides were 5' end labeled with [32P] using T4

kinase (Life Technologies, Inc.) and PAGE purified on a 20% gel which had been prerun for 1 h at

150 V.

Materials. Three antibodies which recognize c-Myc were used: OP10 (Calbiochem)

which is a monoclonal to the Myc epitope tag (amino acids 410-419); a rabbit polyclonal to c-Myc

(cat. no. 06-340, Upstate Biotechnology) and C-8 (Santa Cruz Biotechnology, Inc.) which is a

monoclonal produced by immunization with full length human c-Myc. [α-32P] dCTP (3,000

Ci/mmol) and [35S]Met/Cys (>1000 Ci/mmol, EXPRE35S35S) was from Dupont NEN. Bryo, PMA,

and Bis were dissolved in dimethylsulfoxide and added to culture medium from thousand-fold

concentrated stock solutions.


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RESULTS

Activation of PKC decreases c-myc mRNA and protein levels and Myc2 synthesis.

Treatment of HL-60 cells with Bryo or PMA for 4 h markedly decreased the steady state level of

the 64 kDa Myc2 protein (Figure 1A). The decrease in Myc2 protein depended on the

concentration of Bryo or PMA. At 1 nM neither compound affected the Myc2 level, and at 10 nM

each compound almost maximally decreased Myc2 (Fig. 1A). PMA maximally decreased Myc2

by 82%, whereas Bryo maximally decreased Myc2 by 58% (Fig. 1B). Bis, a selective inhibitor of

PKC (43), completely prevented PMA or Bryo from decreasing Myc2 (Figure 2, top panel).

Treatment of the cells with Bis alone moderately increased Myc2, which may be caused by the

inhibition of residual PKC activity in cells not treated with PMA or Bryo (Fig. 2). In addition to

decreasing the Myc2 level, treatment with 20 nM PMA or Bryo for 4 h decreased the level of c-

myc mRNA (Fig. 2, middle panel). Bis prevented either PMA or Bryo from decreasing the c-myc

transcript in the cells (Fig. 2). These results indicate that the decreases in myc mRNA and protein

were produced by the activation of PKC. The 67 kDa Myc1 and 45-50 kDa MycS proteins are

much less abundant in HL-60 cells than Myc2.2 Although we quantified MycS protein levels and

synthesis rates, we present only Myc2 data because all of the treatments described had essentially

the same effects on MycS and Myc2.2

To determine whether the activation of PKC affected Myc translation, the cells were

treated with PMA or Bryo for 1 h and pulse-labeled with [35S]Met /Cys for 10 min. Pulse labeling

of Myc2 increased linearly between 10 and 30 min.2 Treatment with 20 or 200 nM PMA or Bryo

for 1 h markedly decreased Myc2 labeling (Fig. 3A). Treatment with 20 nM PMA decreased the

pulse-labeling of Myc2 to 45 ± 3% control (n = 3). Simultaneous treatment with Bis prevented

PMA from decreasing Myc2 labeling (Fig. 3B). Treatment with Bis alone slightly increased Myc2

labeling (Fig. 3B), in agreement with the effects of Bis on the Myc2 level (Fig. 2). Treatment

with PMA had no effect on general protein synthesis which was determined by the rate of

incorporation of [35S]Met /Cys into protein.2 These results show that a 1 h treatment with PMA or

Bryo decreased the rate of Myc2 synthesis in HL-60 cells. Treatment of the cells with 20 nM

PMA for 1 h decreased myc mRNA to 42 ± 3% control (n = 3) (Fig. 3C). These findings show

that a 1 h treatment with PMA produced similar decreases in the myc mRNA and Myc2 synthesis.

Treatment of the cells with 20 nM Bryo for 1 h decreased myc mRNA level and Myc2 synthesis

similarly to PMA.2


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Down-regulation of myc depends on the X box of MIE1. To determine whether the

previously identified myc intron 1 elements (MIEs) were involved in the down-regulation of myc

by PMA, we transfected HL-60 cells with a c-myc-luciferase reporter vector which contained a 2

kb c-myc cDNA upstream of the SV40 enhancer and luciferase gene (Fig. 4A). The 2 kb myc

cDNA consisted of 1057 bp upstream of exon 1, exon 1 (554 bp), and the first 387 bp of intron 1.

Two myc deletion mutants, pMP-Luc∆287, which lacked all three MIEs, and pMP-Luc∆220,

which lacked MIE2 and MIE3 were used to determine whether one or more of the MIEs affected

reporter gene expression (Fig. 4A). HL-60 cells were transfected with the pMP-Luc wild type or

mutant vectors and 18 h later half of the cells were treated with 20 nM PMA for 6 h. PMA

treatment significantly decreased myc-driven luciferase expression by 44 ± 2% (p < 0.005) in

cells transfected with pMP-Luc and by 40 ± 4% (p < 0.02) in cells transfected with the deletion

mutant which lacked MIE2 and MIE3 (Fig. 4B). In cells transfected with pMP-Luc deletion mutant

which lacked all three MIEs, PMA treatment had no significant effect on luciferase expression (p =

0.19) (Fig. 4B). These findings are consistent with the hypothesis that only MIE1 is required for

the down-regulation of myc-driven luciferase expression by PMA. Next we deleted only the 14 bp

X box to determine whether it was required for the down-regulation of myc. PMA had no

significant effect on reporter gene expression in cells transfected with pMP-Luc∆14 (p = 0.44)

(Fig. 4B). Luciferase expression in the untreated cells was essentially the same for each of the

pMP-Luc constructs. These findings indicate the down-regulation of reporter gene expression by

PMA required the myc intron 1 X box.

Activation of PKC increases protein binding to MIE1 DNA. Treatment of HL-60 cells

with 20 nM PMA or Bryo for 1 h increased MIE1-binding activity in the nuclear fraction (Fig.

5A). Two MIE1-protein complexes with slightly different electrophoretic mobilities were

observed by EMSA (Fig. 5). Treatment with Bis eliminated the increases in both MIE1-protein

complexes produced by PMA or Bryo (Fig. 5A), as expected if the increases depended on the

activation of PKC. The specificity of the binding to MIE1 was determined by addition of

competitor oligonucleotides with the indicated sequences (Fig. 5B). A 10 fold excess of

unlabeled MIE1 was sufficient to completely block the MIE1-protein binding (Fig. 5C). A 100

fold excess of duplex MIE2 and MIE3 oligonucleotides relative to the 32P labeled MIE1 probe had

no effect on binding (Fig. 5C). Therefore, the MIE1-protein complexes were specific for MIE1.

A 10 fold excess of the duplex BL1+2 (Burkitt’s lymphoma mutation 1 + 2) oligonucleotide, a


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mutant with two substitutions in the 3’ half of the MIE1 X box (Fig. 5B), had no effect on

[32P]MIE1 binding, and a 100 fold excess of the X box mutant only partially reduced binding (Fig.

5C). This finding confirms the role of the X box in nuclear protein binding to MIE1 as reported

previously (20, 24). These results show that a brief treatment of undifferentiated HL-60 cells with

PMA or Bryo is sufficient to increase specific MIE1-binding activity in the nuclear fraction.

Treatment of the cells with PMA for 0.5, 1, 2, 3, and 4 h increased MIE1-binding activity.2

However, a 48 h treatment of the cells with 0.1 µM PMA, which induced differentiation, as

indicated by the attachment and elongation of cells on the culture surface, had no effect on MIE1

binding.2 A 48 h treatment with 0.1 µM Bryo, which failed to induce attachment and

differentiation, also had no effect on MIE1 binding.2 These findings indicate that MIE1-binding

activity returned to the basal level between 4 and 48 h of PMA treatment , and they suggest that

there is no difference in MIE1 binding activity between undifferentiated and differentiated HL-60

cells, as observed by Erhlich and coworkers (24).

Presence of RFX1 and MIBP1 in MIE1 DNA-protein complexes. Supershift analysis of

MIE1-protein complexes was carried out with antisera to RFX1 and MIBP1 (Fig. 6). MIBP1 is a

160 kDa protein which is present in MIE1 complexes and apparently associates with RFX1 (22).

The formation of the both MIE1-protein complexes depended on the addition of the nuclear extract

as expected (Fig. 6A). A 10 fold excess of unlabeled duplex MIE1, but not the BL1+2 MIE1

mutant, abolished the supershifted complexes indicating that 32P MIE1 binding was specific (Fig.

6A). Antiserum to RFX1 supershifted both of the complexes, but the MIBP1 antiserum

supershifted only the slower mobility (“Complex 1”) (Fig. 6A). This finding suggests that both of

the MIE1-protein complexes contained RFX1, but only the slower mobility complex contained

MIBP1, as reported MIE1-protein complex from HeLa cells (22). Western blot analysis of

nuclear extracts from undifferentiated HL-60 cells confirmed that the antisera specifically

recognized proteins with the expected electrophoretic mobility of RFX1 and MIBP1, respectively

(Fig. 6B). The preimmune sera were not reactive with MIBP1 or RFX1 (Fig. 6B).

PMA treatment increases nuclear accumulation of RFX1, but not MIBP1. Treatment of

HL-60 cells with PMA for 1 h increased RFX1 protein in the nuclear extract as determined by

western blot (Fig. 6B and 7A). PMA increased nuclear fraction RFX1 2.5 ± 0.2 fold (n = 4, p =

0.001). NFκB was also increased in the nuclear fraction by PMA treatment (Fig. 7A), which is

known to induce NFκB translocation from the cytoplasm to the nucleus (41). PMA treatment


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decreased RFX1 in the cytosol and had no effect on total cell RFX1 (Fig. 7A). PMA treatment had

no effect on the level of MIBP1 in the nuclear fraction (Fig. 7B). The increase in nuclear RFX1

was independent of protein synthesis. Blockade of protein synthesis with cycloheximide had no

effect on the accumulation of RFX1 in the nuclear fraction (Fig. 7B). Blockade of protein or RNA

synthesis with cycloheximide or actinomycin D, respectively, had no effect on increased MIE1-

binding activity produced by PMA (Fig. 7C). These findings indicate that the increases in nuclear

extract RFX1 and MIE1-protein binding were independent of protein synthesis.

Lack of effect of PMA treatment on Myc protein turnover. Although decreased synthesis

appeared to cause the fall in Myc protein level, pulse-chase labeling experiments were done to

determine whether activation of PKC also affected Myc turnover. HL-60 cells were labeled with

[35S]Met /Cys for 1 h in the presence or absence of 20 nM PMA. Excess unlabeled Cys and Met

were added to terminate the labeling, and after the indicated chase interval, the cells were lysed

and [35S] labeled Myc was immunoprecipitated and fractionated by SDS-PAGE. The gel was

fluorographed and [35S] labeled Myc1 plus Myc2 was quantified by scintillation counting. These

experiments indicated that [35S] labeled Myc had a half-life of 23 ± 2 min in the cells (Fig. 8).

Following the PMA treatment, [35S] labeled Myc disappeared with a half life of 22 ± 2 min

(Figure 8).


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DISCUSSION

It has been known for some time that premature termination of transcription plays a

critically important role in the down-regulation of c-myc in the early phase of the response to

differentiation inducing compounds in undifferentiated HL-60 cells (11-15). During the later

phase of differentiation of HL-60 cells, however, a loss of transcriptional initiation occurs (45).

In this report we observed that acute activators of conventional and novel isoforms of PKC,

namely Bryo and PMA, rapidly and markedly decreased the steady state level of Myc protein and

mRNA in undifferentiated HL-60 cells (Fig. 1 and 2). Treatment with PMA or Bryo for 1 h

strongly inhibited the rate of Myc synthesis (Fig. 3). Because the half-life of the Myc protein was

unaffected by PMA (Fig. 8), the inhibition of Myc synthesis explained the decrease in the Myc

level. For this inference to be correct the Myc protein must have a relatively short half-life in

untreated HL-60 cells, which it does (~20 min) (Fig. 8).

Our findings indicate that PMA or Bryo markedly increased nuclear protein binding to

MIE1 as determined by EMSA (Fig. 5). Protein binding depended on the MIE1 X box because

substitution of two nucleotides in the 3’ half of the X box strongly decreased the ability of the

BL1+2 oligonucleotide to compete with MIE1 (Fig. 5C). Supershift analysis with antiserum to

RFX1 showed that it was present in both of the MIE1-protein complexes that were resolved by

EMSA (Fig. 6). Only one of the complexes contained MIBP1, which is a 160 kDa uncharacterized

MIE1 binding protein from HeLa cells (22). Increased MIE1-protein binding depended on PKC

activation, but was independent of protein synthesis (Fig. 5 and 7). Bis, a selective inhibitor of

PKC (41), which prevented the down-regulation of myc mRNA and protein by PMA or Bryo, also

abolished their effects on MIE1-binding activity (Fig. 5A).

The mechanism by which acute activation of PKC increased protein binding to MIE1

appears to be indirect and due at least in part to the nuclear translocation of RFX1 (Fig. 7). Thus

we have been unable to detect the 32P labeled RFX1 following immunoprecipitation from 32P

labeled cells.2 Although 32P labeling of MIBP1 was readily detected, PMA treatment had no effect

on the labeling.2 Nuclear translocation of RFX1 could explain the PMA- or Bryo-evoked increase

in the MIE1 complex that contained MIBP1 because this complex also contained RFX1 (Fig. 6A,

“Complex 1”). In agreement with this idea, MIBP1 and RFX1 coimmunoprecipitated from HeLa

cell nuclear extracts in the absence of MIE1 (22). No RFX nuclear localization signals have been

reported, and there appear to be no reports of a dynamic change in the subcellular localization of


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an RFX family protein. One possible explanation for nuclear translocation of RFX1 is the

phosphorylation of an RFX1 associated and cotranslocated protein in response to PKC activation.

Although RFX proteins are most well known as essential transactivators of MHC class II

genes and as a cellular transactivators of pathogenic viruses such as hepatitis B virus (27, 29, 30),

RFX1 appears to be ubiquitously expressed in mammalian cells (25, 27, 28), including

undifferentiated HL-60 cells as determined by western blot analysis (Fig. 6 and 7) and northern

blot analysis (28). The present results suggest that RFX1 binding to the X box of intron 1 has

silencer activity towards myc expression and are consistent with the apparent silencer function of

tandem MIE1 repeats towards SV40 promoter activity in hepatocarcinoma cell lines (22, 23).

Apparently the interaction of RFX proteins with other DNA bound proteins determines whether it

has enhancer or silencer activity , although the determinants of the activity are unknown (36).

A recent report by Pan and Simpson (18) of the suppression of c-myc following a 2 day

treatment of HL-60 cells with 1,25-dihydroxyvitamin D3 implicated MIE1 and activation of PKC,

in agreement with our study using acute PKC activators. However, they suggested that the

homeobox HOXB4 protein was a major MIE1-binding protein and that the 1,25-dihydroxyvitamin

D3 treatment down-regulated c-myc by increasing the level of HOXB4 (18). It is not known if

HOXB4 complexes with RFX1 or other MIE1 binding proteins. The following evidence supports

the view that RFX1 plays a role in the rapid down-regulation of c-myc following acute activation

of PKC: 1) down-regulation of myc-driven reporter gene expression by PMA was abolished by

deletion of the RFX-binding X box (Fig. 4); 2) acute treatment with a PKC activator, PMA or

Bryo, increased protein binding to the X box of MIE1 (Fig. 5); 3) RFX1 was present in the MIE1-

protein complexes (Fig. 6); 4) a selective inhibitor of PKC prevented PMA or Bryo from

increasing MIE1-binding activity and decreasing myc expression (Fig. 2, 3, and 5); and 5) PMA

treatment increased RFX1 in the nuclear fraction and decreased it in the cytosol (Fig. 7). It is

noteworthy that PMA and Bryo only transiently increased MIE1-protein binding.2 We observed no

difference in MIE1-binding activity between undifferentiated and differentiated (2 day PMA

treatment) HL-60 cells as reported previously (24).2 The lack of increased MIE1-binding activity

in differentiated cells is consistent with the role of transcription termination near the first

exon/intron junction in the early response to a stimulus of differentiation (14, 45). Hence, acute

induction of nuclear translocation and binding of RFX1 to the intron 1 X box produced by PMA

correlates with the rapid onset of the blockade of transcription elongation following the addition of


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PMA or other stimuli of differentiation, in contrast to the later phases of myc down-regulation

which involve a loss of transcriptional initiation (14, 45). Bryo treatment downregulated

endogenous c-myc similarly to PMA (this report), however Bryo fails to induce a differentiation

response and antagonizes differentiation produced by PMA (40). PKC activation by Bryo is

shorter in duration than that produced by PMA because Bryo more rapidly and efficiently

downregulates PKC (41). Additional studies are needed to identify critical differences in the

cellular responses to PMA and Bryo which are subsequent to the rapid downregulation of c-myc.

In conclusion, the present findings implicate nuclear translocation of RFX1 and an intron 1

X box in the early phase of the down-regulation of c-myc produced by acute PKC activators in

undifferentiated HL-60 cells. Considering the pivotal role of myc overexpression in malignant

tumors (1, 2), biochemical understanding of myc regulation by RFX1 should help to devise novel

strategies for silencing myc.

ACKNOWLEDGMENTS

We thank Drs. Maria Zajac-Kaye for the generous gift of the pMPCAT constructs and

antisera to RFX1 and MIBP1; G. R. Pettit for the bryostatin 1; and Svetlana A. Shestopal for

helpful discussions.


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FOOTNOTES*This work was supported by grant HL44408 from the National Institutes of Health.

‡To whom correspondence should be addressed: Dr. Jeffrey B. Smith, Department of

Pharmacology & Toxicology, Volker Hall G133E, 1670 University Blvd., UAB, Birmingham, AL

35294-0019. Tel.: 205-934-7434; Fax: 205-975-5841; E-mail: [email protected]

1The abbreviations used are: AD, actinomycin D; Bis, bisindoylmaleimide; Bryo, bryostatin 1;

CAT, choramphenicol acetyltransferase; CHX, cycloheximide; DMSO, dimethylsulfoxide; DTT,

dithiothreitol; EMSA, electrophoretic gel mobility shift assay; FBS, fetal bovine serum; MIBP1,

myc intron binding protein 1; MIE, myc intron 1 element; PAGE, polyacrylamide gel

electrophoresis; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RFX, regulatory

factor X; SDS, sodium dodecylsulfate; TBS, Tris buffered saline.

2Chen, L., Smith, L., and Smith, J. B., unpublished data


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FIGURE LEGENDS

Figure 1. Down-regulation of Myc2 protein following the treatment of HL-60 cells with Bryo

or PMA. A. HL-60 cells (107 per condition) were incubated with the indicated concentration of

Bryo or PMA for 4 h in RPMI 1640 containing 2% FBS. The cells were lysed with >95oC SDS

buffer, and proteins (30 µg) were fractionated by SDS-PAGE for western blot analysis with the

OP10 monoclonal antibody. B. Graph of Myc2 protein level determined as indicated for A.

Values are mean ± S.E. (n = 3 experiments).

Figure 2. Effects of Bryo, PMA, and Bis on myc mRNA and Myc2 protein. HL-60 cells (107

per condition) were incubated for 30 min in RPMI 1640 containing 2% FBS and 4 µM Bis as

indicated. Then Bryo or PMA (20 nM) was added, and 4 h later the cells were lysed with >95oC

SDS buffer and proteins (30 µg) were subjected to western blot analysis (top panel). For

determination of myc mRNA, the cells (3 x107 each) were treated with Bis, Bryo, and PMA as

described for western analysis, and total RNA was extracted, size fractionated, and subjected to

northern blot analysis (middle panel). 28S rRNA was visualized by ethidium bromide staining

(bottom panel).

Figure 3. Effects of PMA, Bryo, or Bis on Myc2 synthesis in HL-60 cells. A. HL-60 cells (107

each) were incubated in RPMI 1640 containing one-tenth the normal Met and Cys and treated with

the indicated concentration of PMA or Bryo for 1 h. Then the cells were labeled with 0.15 mCi

[35S]Met/Cys for 10 min and lysed with >95oC SDS buffer. c-Myc was immunoprecipitated from

lysate proteins (0.2 mg), fractionated by SDS-PAGE (10% gel), and visualized by fluorography.

B. The cells were incubated in RPMI 1640 containing one-tenth the normal Met and Cys for 10

min with or without 4 µM Bis as indicated. PMA or Bryo (20 nM) was added for 1 h. Then the

cells were pulse-labeled for 10 min and processed to quantify [35S] labeled Myc2 as indicated for

part A. C. Northern blot analysis of myc mRNA following a 1 h treatment with PMA. HL-60 cells

(3 x 107 each) were incubated in the presence or absence of 20 nM PMA as indicated prior to

extraction of total RNA, which was size fractionated, and subjected to northern analysis. 28S

rRNA was visualized by ethidium bromide staining (bottom panel).


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Figure 4. Effects of c-myc intron 1 elements on the down-regulation of reporter gene

expression by PMA treatment of transfected HL-60 cells. A. Diagram of human c-myc-firefly

luciferase construct (pMP-Luc) and deletion mutants lacking all three MIEs (pMP-Luc∆287) or

lacking MIE2 and MIE3 (pMP-Luc∆220). pMP-Luc∆14 lacked the 14 bp X box of MIE1.

Nucleotide positions are relative to the P1 transcription start site. B. Change in firefly luciferase

expression produced by a 6 h treatment of transfected HL-60 cells with 20 nM PMA. After

electroporation with the indicated vector, the cells were incubated for 18 h in 20 ml of RPMI 1640

containing 10% FBS before adding PMA to half the cells from each electroporation. The cells

were cotransfected with pRL-TK vector and the Dual-Luciferase Assay System (Promega) was

used to measure myc-driven firefly luciferase and Ranilla luciferase activity as a control for

transfection efficiency. Values are mean ± S.E. for 5-7 experiments. Percentage change in

luciferase activity produced by PMA was significantly different from pMP-Luc to pMP-Luc∆287

(p = 0.003) and to pMP-Luc∆14 (p = 0.004), but not to pMP-Luc∆220 (p = 0.419).

Figure 5. Effects of PMA, Bryo, and Bis on protein binding to 32P labeled MIE1 duplex DNA.

A. EMSA autoradiogram obtained with nuclear extracts from HL-60 cells following treatment

with 20 nM PMA or Bryo for 1 h as indicated. Some cells were treated with 4 µM Bis for 30 min

before the addition of PMA and Bryo. Nuclear extracts were prepared and 2 µg protein was

incubated with 1 µg poly dI-dC for 10 min at 25oC as described in “Methods”. Then 0.1 ng 32P

labeled duplex MIE1 was added to each reaction and the incubation continued for 30 min. Binding

reactions were size-fractionated by Tris-glycine PAGE (4% gel). The positions of the two

protein-32P-MIE1 complexes are indicated. B. Diagram of c-myc intron 1 elements (MIEs) and

nucleotide sequences of the MIEs and the BL1+2 mutant. C. Specificity of the 32P-MIE1-protein

complexes was determined by addition of 1 or 10 ng (“10X” or “100X”) of the unlabeled duplex

MIE1, MIE2, MIE3, or the BL1+2 mutant of MIE1 to the binding reaction described in A.

Unlabeled competitor was present during the 10 min incubation of the nuclear extract with poly dI-

dC before the addition of 32P labeled MIE1.

Figure 6. Supershift analysis of 32P labeled MIE1 DNA-protein complexes. A. Antiserum to

RFX1 or MIBP1 or the corresponding preimmune serum (1 µl each) was added to the binding


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reaction which contained 0.1 ng 32P labeled MIE1, 2 µg nuclear extract from cells treated with 20

nM PMA for 1 h, 1 ng duplex MIE1 or the BL1+2 mutant as indicated. The indicated antiserum or

preimmune serum and the indicated duplex DNA competitor was present during the 10 min

incubation of the nuclear extract with poly dI-dC before the addition of 32P labeled MIE1. B.

Western blot analysis of MIBP1 and RFX1 in nuclear extracts from PMA-treated and untreated

HL-60 cells. The cells were treated with 20 nM PMA for 1 h and nuclear extracts were prepared

as described in “Methods”. Nuclear extract proteins (30 µg) were size fractionated by SDS-

PAGE (7% gel) and subjected to western blot analysis with the indicated serum.

Figure 7. Effect of PMA on the level of RFX1 in nuclear extracts, cytosol, and total cell

lysate. A. Western blot analysis of nuclear extract, cytosol, and total cell lysate from untreated

and PMA-treated cells. After the PMA treatment (20 nM for 1 h in RPMI 1640 containing 2%

FBS) a sample of the treated and untreated cells was extracted with >95oC SDS lysis buffer to

obtain the total cell lysate. Another sample of the cells was subjected to hypotonic lysis and the

cytosol was collected after centrifugation to remove nuclei. Nuclear extracts were prepared as

described in “Methods”. Cytosol was concentrated by with Centricon 10 concentrators, and

proteins (30 µg nuclear extract, 60 µg cytosol, or 100 µg total lysate) were size fractionated by

SDS-PAGE (7% gel) and subjected to western blot analysis with the indicated antiserum. B.

Western blot analysis of nuclear extracts (30 µg protein) from cells that were incubated with 10

µg/ml cycloheximide (“CHX”) or 2.5 µg/ml actinomycin D (“AD”) for 10 min prior to the

addition of 20 nM PMA as indicated. One h later nuclear extracts were prepared. C. EMSA was

carried out on nuclear extracts that were prepared from cells that were treated as described in B.

Figure 8. Lack of effect of PMA on the disappearance of [35S]labeled Myc in HL-60 cells.

A. HL-60 cells (107 each) were incubated with or without 20 nM PMA in medium containing a

tenth of the Met and Cys concentrations of RPMI 1640, 2% dialyzed FBS, and 0.15 mCi

[35S]Met/Cys for 1 h. Labeling was stopped by the addition of 10 mM each of Met and Cys, and

after the indicated interval, the cells were lysed with >95oC SDS buffer, and Myc was

immunoprecipitated, fractionated by SDS-PAGE, and visualized by fluorography. B. The

percentage of [35S]Myc remaining after the indicated interval was determined by liquid

scintillation counting of [35S]Myc1 plus Myc2 containing gel slices.


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Figure 1, Chen et al.

100

80

60

40

20

0

Myc

2 (%

con

trol

)

B

Con

trol Bryo PMA

10-3

10-2

100

10-1

10-3

10-2

100

10-1A

Myc2

µM


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PMA+

Bis

Con

trol

PMA

Bryo

+Bis

Bis

Bry

oMyc2

myc mRNA

28S rRNA


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Contro

l200 20 200 20

PMA BryonM

S Myc235

S Myc235

Contro

l

PMA

Bis PMA+B

is

A

B

PMA - +myc mRNA

28S rRNA

C


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% C

hang

e in

luci

fera

se e

xpre

ssio

n by

PM

A

B

- 50

- 40

- 30

- 20

- 10

0

10

20

30

pMP-Luc

pMP-Luc∆287

pMP-Luc∆220

pMP-Luc∆14

EXON 1

P1P2

HinP1Sau3A SstIpMP-Luc

pMP-Luc∆220

EXON 1

P1P2

HinP1 SstI

EXON 1

P1P2

HinP1Sau3A SstI

:deleted segment

KpnI

MIE1 2 3

KpnI

KpnI

Luc

Luc

Luc

MIE1: nt 3001-3020MIE2: nt 3139-3158MIE3: nt 3161-3194

(HinP1-SstI: ∆ nt 2981-3268)

(Sau3A-SstI: ∆ nt 3048-3268)

A

MIE1 2 3

MIE1 2 3

pMP-Luc∆287

pMP-Luc∆14EXON 1

P1P2

KpnILuc

MIE1 2 3

AGA GTAGTTATGGTAAC TGG:deleted segment


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PMA+

Bis

Con

trol

PMA

Bryo

+Bis

Bis

Bryo

P-MIE132

A C

Competitor MIE

1M

IE2

MIE

3B

L1+2

10x

100x

P-MIE132

Complex21 Complex

21

Complex21

EXON1 EXON2

P1P2

1 2 3

MIE1: 5’-AGAGTAGTTATG GTA ACTGGBL1+2: 5’-AGAGTAGTTATG ATT ACTGGMIE2: 5’-CCTTATGAATATATTCACGCMIE3: 5’-CTCCCGGCCGGTCGGACATTCCTGCTTTATTGT

MIEB


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Competitor

Nuclear extract

MIE1BL1+2

+ + + + + + + + + + + + + + + + + + + +

+ + + + +

antiserum none MIBP1 preimmuneMIBP1

RFX1 preimmuneRFX1

AComplex

21

B PMA - + - + - + - +

antiserum: MIBP1 pre-immuneMIBP1

RFX1 pre-immuneRFX1

217

126

73

MIBP1RFX1

kDa by guest on March 17, 2018

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Contro

l

PMA

PMA+

CHX

CHX

RFX1

MIBP1

B

PMA - +Nuclear extract

Cytosol

Total lysateNuclear extract

RFX

NFκΒ

A

Contro

lPMACH

X

PMA+

AD

PMA+

CHX

AD

C

P-MIE132

Complex21


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S Myc35

Control PMA

0 20 40 60 0 20 40 60Chase(min)

A

75

-PMA

+PMA

50

25

0

100

0 20 40 60

S M

yc (

% r

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ning

)35

Chase (min)

B


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Jeffrey B. SmithLei Chen, Lucinda Smith, Martin R. Johnson, Kangsheng Wang, Robert B. Diasio and

down-regulates c-myc via an intron 1 X box in undifferentiated leukemia HL-60 cellsActivation of protein kinase C induces nuclear translocation of RFX1 and

published online July 28, 2000J. Biol. Chem.

10.1074/jbc.M002645200Access the most updated version of this article at doi:

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myc via an intron 1 x box in undiffer

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