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Biochimica et Biophysica Ac

Blockade of murine erythroleukemia cell differentiation by

hypomethylating agents causes accumulation of discrete small poly(A)�

RNAs hybridized to 3V-end flanking sequences of hmajor globin gene

Ioannis S. Vizirianakis*, Asterios S. Tsiftsoglou

Laboratory of Pharmacology, Department of Pharmaceutical Sciences, School of Health Sciences, Aristotle University of Thessaloniki,

Thessaloniki GR-54124, Greece

Received 15 July 2004; received in revised form 2 September 2004; accepted 3 September 2004

Available online 21 September 2004

Abstract

Induction of murine erythroleukemia (MEL) cell differentiation is accompanied by transcriptional activation of globin genes and

biosynthesis of hemoglobin. In this study, we observed cytoplasmic accumulation of relatively small RNAs of different size (150–600

nt) hybridized to a1 and hmajor globin DNA probes in MEL cells blocked to differentiate by hypomethylating agents (neplanocin A,

3-deazaneplanocin A and cycloleucine). These RNAs lack poly(A) tail and appear to be quite stable. Search within the 3V-endflanking sequences of hmajor globin gene revealed the presence of a B1 repeat element, several ATG initiation codons, a GATA-1

consensus sequence and sequences recognized by AP-1/NF-E2 and erythroid Krqppel-like factor (EKLF) transcription factors. These

data taken together indicate that exposure of MEL cells to hypomethylating agents promotes accumulation of relatively small discrete

RNA transcripts lacking poly(A) tail regardless of the presence or absence of inducer dimethylsulfoxide (DMSO). However, the

relative steady-state level of small RNAs was comparatively higher in cells co-exposed to inducer and each one of the

hypomethylating agents. Although the orientation of these RNAs has not been established as yet, the possibility these small poly(A)�

RNAs which are induced by hypomethylating agents may be involved in the blockade of MEL cell differentiation program is

discussed.

D 2004 Elsevier B.V. All rights reserved.

Keywords: MEL; Cell; Differentiation; Hypomethylating agent; Inducer; Globin gene; Transcription; Neplanocin A; 3-Deazaneplanocin A; Cycloleucine;

Silent DNA; DNA methylation; RNA methylation

1. Introduction

The precise cellular and molecular mechanisms involved

in initiation of commitment of murine erythroleukemia (MEL

or Friend) cells to terminal erythroid maturation are still not

fully elucidated, regardless of the remarkable progress

achieved in this field [1–3]. Earlier studies with metabolic

0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbamcr.2004.09.003

* Corresponding author. Tel.: +30 2310 997658; fax: +30 2310

997645.

E-mail address: ivizir@pharm.auth.gr (I.S. Vizirianakis).

inhibitors (e.g. cordycepin, cycloheximide) revealed that

initiation of commitment to terminal maturation depends on

the synthesis of new RNA transcripts and proteins [1,4,5]. In

addition, erythroid differentiation of MEL cells was shown to

be associated with DNA hypomethylation [6], as well as

transmethylation of RNA [7,8] and other biochemical events

reviewed elsewhere [1–3].

We have shown earlier that induction of erythroid

differentiation of MEL cells is inhibited by N6-methyl-

adenosine (N6mAdo), neplanocin A, 3-deazaneplanocin A

and cycloleucine [7,9], all of which inhibit active

methylation cycle within the cells [10–13]. These agents

ta 1743 (2005) 101–114

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114102

are claimed to have selective inhibitory action on intra-

cellular methylation cycle reactions as reported elsewhere

[7,10–13]. In particular, neplanocin A, 3-deazaneplanocin

A and N6mAdo are inhibitors of S-adenosylhomocysteine

(AdoHcy) hydrolase (E.C. 3.3.1.1) (Ki values for the

isolated AdoHcy hydrolase 2�10�9, 5�10�11 and 2�10�4

M, respectively), whereas cycloleucine is an inhibitor of S-

adenosylmethionine (AdoMet) synthetase (E.C. 2.5.1.6)

(Ki value for the isolated AdoMet synthetase 4.8�10�5 M)

[12,14,15]. The hypomethylating agents block initiation of

commitment and prevent accumulation of hemoglobin in

inducer-treated MEL cells presumably by interrupting a

central process required for commitment to terminal

erythroid maturation [7–9].

Interestingly enough, during the course of our work, we

observed that exposure of MEL cells to each of these

agents, (N6mAdo, neplanocin A, 3-deazaneplanocin A and

cycloleucine), led to cytoplasmic accumulation of discrete

RNAs detected at first by using a mouse genomic DNA

fragment containing the hmajor globin gene as well as its

flanking sequences [7,9]. In the present study, we extended

these observations in order to: (a) analyze the expression

pattern of these RNAs in MEL cells induced to differ-

entiate as well as in cells blocked to commit by the

hypomethylating agents; (b) assess the specificity of such

RNAs with respect to genes other than hmajor globin; (c)

estimate the size of RNA transcripts; and (d) demonstrate

their origin by extensive Northern blot analysis using small

genomic DNA sequences fragments as probes.

Here, we wish to report that: (a) the relatively small

discrete RNA transcripts accumulate in the cytoplasm of cells

exposed to hypomethylating agents in the absence or

presence of the inducer. These RNAs were entirely absent,

however, in MEL cells exposed to neither agent nor

dimethylsulfoxide (DMSO) only; (b) such new RNA tran-

scripts were not detected in the cytoplasmic RNA hybridized

with 32P-labeled probes derived from genes other than globin

including those encoding for transferrin receptor (TfR),

GAPDH, h-actin and ribosomal protein S5 (rpS5); (c)

Northern blot hybridization analysis with probes generated

from different parts of the hmajor globin gene flanking regions

revealed that the detected new RNA transcripts lack poly(A)

tail and it is likely to be encoded by sequences located at the

3V-end flanking sequences of the mouse hmajor globin gene.

This region is enriched in ATG initiation codons, consensus

sequences for transcription factors GATA-1, AP-1/NF-E2

and erythroid Krqppel-like factor (EKLF) as well as it has aB1 repeat element. These data indicate for the first time that

treatment of MEL cells with three hypomethylating agents

that block MEL cell differentiation promotes cytoplasmic

accumulation of relatively small poly(A)� RNAs that are

likely to originate from the 3V-end flanking sequences of

hmajor globin gene. We are in the process of characterizing

these RNAs and investigating whether these molecules

contribute to the blockade of differentiation of MEL cells

exposed to hypomethylating agents.

2. Materials and methods

2.1. Chemicals and biochemicals

DMSO was purchased from Mallinckrodt, Inc. (St.

Louis, MO, USA). Neplanocin A and 3-deazaneplanocin

A were kindly donated by Dr. V.E. Marquez (National

Cancer Institute, USA), whereas cycloleucine was obtained

from Sigma Chemical Co. (St. Louis, MO, USA). All

restriction enzymes were obtained from New England

Biolabs, Inc. (Hertfordshire, England).

2.2. Cell cultures

Cells employed throughout this study were MEL-745PC-

4A a clone of MEL-745 cells obtained after subcloning and

subsequent testing of clones derived for high degree of

inducibility. All cultures were maintained in Dulbecco’s

modified Eagle’s medium (DMEM) containing 10% (v/v)

fetal calf serum (FCS, Gibco, Long Island, NY) and

antibiotics (penicillin and streptomycin 100 Ag/ml). Cells

were incubated at 37 8C in a humidified atmosphere

containing 5% CO2 and maintained at densities that

permitted logarithmic growth (1�105–1�106 cells/ml). Cell

viability was assessed by trypan blue exclusion as reported

elsewhere [7,8].

2.3. Induction and assessment of differentiation

Cells were incubated with no drug and the inducing agent

in the absence or presence of an inhibitor as indicated in the

text. At certain timed-intervals during incubation, the

proportion of differentiated (hemoglobin-producing cells)

was assessed cytochemically with benzidine–H2O2 solution

as described elsewhere [7,8].

2.4. Assessment of the steady-state level of RNA transcripts

by Northern blot hybridization analysis

Cytoplasmic RNA prepared from control and MEL

cells treated with agents at various timed-intervals, as

indicated in the text, was assessed for the steady-state level

of RNA transcripts coded by hmajor globin, a1-globin,

mouse TfR, h-actin, GAPDH and rpS5 genes, with the use

of [32P]-labeled probes, as previously described [7,8]. The

former three genes are regulated and expressed devel-

opmentally in differentiating cells, while the latter three

are considered housekeeping genes. The probes used were

a 7.3 kb genomic DNA fragment bearing the hmajor globin

gene [7], a ~3 kb SacI/SacI genomic DNA fragment

encoding for the a1-globin mRNA kindly donated by Dr.

N. Anagnou (University of Athens, Greece), a 2.28 kb

DNA fragment encoding for mouse TfR mRNA kindly

donated by Dr. K. Pantopoulos (EMBL, Heidelberg,

Germany), a 426 bp encoding for rat GAPDH [31], a

715 bp cDNA fragment encoding for the full-length of

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 103

rpS5 gene previously isolated in our laboratory [16], and a

350 bp PstI/HindIII cDNA fragment encoding for mouse

h-actin mRNA [7].

2.5. Preparation of DNA hybridization probes from the

different regions of mouse 7.3 kb EcoRI/EcoRI genomic

DNA fragment

Basically, two sets of DNA probes were prepared and

used as hybridization probes in Northern blot analysis of

total cytoplasmic RNA in order to map the area of origin

of new RNA transcripts. Probes designated as C, D, E and

F were generated by the use of polymerase chain reaction

(PCR) and primers as follows: Probe C (length 132 bp):

sense primer 5V-GGCCAATCTGCTCACACAGGA-3V, andantisense primer 5V-GATGTCTGTTTCTGGGGTTGT-3V;Probe D (length 91 bp): sense primer 5V-TGCACCT-GACTGATGCTGAGAAG-3V and antisense primer 5V-CCTGCCCAGGGCCTCACCACCAA-3V; Probe E (length

879 bp): sense primer 5V-CTGCTGGTTGTCTACCCT-TGG-3V and antisense primer 5V-CTGTGGGAATAT-

GGAAGAACC-3V; Probe F (length 224 bp): sense

primer 5V-CTGCTGGTTGTCTACCCTTGG-3V and anti-

sense primer 5V-CCTGAAGTTCTCAGGATCCAC-3V.The probes A, B, G, H, I and J were generated by diges-

tion of 7.3 kb DNA fragment with restriction enzymes as

follows: Probe A is a 191 bp EcoRI/PstI DNA genomic

fragment; Probe B is a PstI/HindIII DNA genomic DNA

fragment; Probe G is a 1028 bp HindIII/HindIII DNA

genomic fragment; Probe H is a 903 bp PstI/XbaI DNA

genomic fragment; Probe I is a 3920 bp XbaI/EcoRI

DNA genomic fragment; and Probe J is a 1429 bp

EcoRV/EcoRI DNA genomic fragment. All probes are

illustrated in Fig. 5.

2.6. DNA methylation analysis at CCGG sites within the 7.3

kb DNA fragment

MEL cells were incubated with each hypomethylating

agent in the absence or the presence of DMSO and at

various incubation times cells were collected by cen-

trifugation at 600�g for 15 min. The genomic DNA was

then isolated by the PUREGENEk DNA isolation kit

(Gentra Systems, Minneapolis, USA). Digestion of DNA

(4–8 Ag/sample) with EcoRI (20 units) was followed by

digestion separately with 20 units of endonucleases

HpaII and MspI (New England Biolabs) carried out in

the appropriate buffers recommended by the manufac-

turer. After incubation at 37 8C overnight to ensure

maximum digestion, DNA samples were subjected to gel

electrophoresis through 1.2% agarose in a Tris acetate/

EDTA buffer. After denaturation, DNA was transferred to

Nylon filters (S&S NYTRAN-N, Schleicher and Schuell,

Dassel, Germany) in 1.5 M NaCl/0.15 M sodium citrate

(pH 7.4) and cross-linked to filters under UV. Hybrid-

ization was performed overnight at 65 8C, by using as

probe the 7.3 kb genomic DNA fragment labeled with

a-[32P]-dCTP (sp. act. 3000 Ci/mmol, IZOTOP, Buda-

pest, Hungary) by random priming (Invitrogen Life

Technologies). The filters were then washed and exposed

to autoradiography films (Kodak, Rochester, NY, USA)

at 70 8C for 1–4 days.

3. Results

3.1. Dose-dependent blockade of cell growth and eryth-

roid differentiation of MEL cells by hypomethylating

agents

It was quite important from the beginning of these

experiments to establish appropriate culture conditions in

order to study the effects of each hypomethylating agent

(neplanocin A, 3-deazaneplanocin A, cycloleucine), on

cell growth and differentiation without killing the cells.

Therefore, exponentially growing cells (MEL-745A-PC-4)

were exposed separately to varying concentrations of each

one of these hypomethylating agents in the absence (Fig.

1A) and/or in the presence of inducer DMSO (Fig. 1B,C).

About 48 h later, the effect of each agent on cell growth

was assessed, whereas the proportion of hemoglobin-

producing cells (benzidine-positive cells) was determined

96 h following treatment. As shown in all three cases, the

agents decreased cell growth either in the absence (Fig.

1A) or in the presence of DMSO (Fig. 1B) in a dose-

dependent fashion but to a different extent. Interestingly,

cycloleucine was 4-log less toxic to the cells as compared

to neplanocin A and 3-deazaneplanocin A. This agent

caused inhibition of cell growth at relatively high

concentration (N10�3 M). Comparatively, all three agents

caused less inhibition of cell growth in the presence of

DMSO rather in the absence of the inducer (Fig. 1B

versus Fig. 1A). Co-treatment of MEL cells with each

hypomethylating agent and DMSO prevented erythroid

differentiation and markedly reduced production of

hemoglobin (Fig. 1C). Neplanocin A was the most potent

inhibitor followed by 3-deazaneplanocin A. Cycloleucine

was much less potent inhibitor of differentiation since it

modulated erythroid differentiation at concentration higher

than 10�4 M. At concentration higher than 10�4 M, 20–

25% of cells continued to differentiate even in the

presence of cycloleucine (see Fig. 1C). These data

enabled us to select the optimum inhibitory concentration

of each agent for our studies (1�10�6 M for neplanocin

A, 1�10�5 M for 3-deazaneplanocin A and 4�10�2 M

for cycloleucine). At these concentrations, all agents

increase the SAH/SAM ratio and inhibit RNA methyl-

ation in MEL cells as we have previously shown [7].

Cells exposed to each one of the three agents under

conditions described remained quite viable in proportion

as shown by the trypan blue exclusion test illustrated in

Table 1 [7].

Table 1

Effect of neplanocin A, 3-deazaneplanocin A and cycloleucine on cell

viability in control and DMSO-treated MEL cells

Treatment Concentration (M) Viability (%)

None – 93

DMSO 0.210 93

Neplanocin A 1�10�6 87

3-Deazaneplanocin A 1�10�5 82.1

Cycloleucine 4�10�2 87.4

DMSO+neplanocin A 0.210+1�10�6 82.8

DMSO+3-deazaneplanocin A 0.210+1�10�5 76.6

DMSO+cycloleucine 0.210+4�10�2 74.9

Exponentially growing MEL-745PC-4A cells were incubated with and

without DMSO, as well as treated separately with neplanocin A, 3-

deazaneplanocin A and/or cycloleucine in the absence or the presence of

DMSO at the concentrations indicated. Cell viability was determined after

72 h incubation by trypan blue exclusion as previously shown [7]. Each

value indicates the mean of two separate experiments.

Fig. 1. Concentration-dependent effects of neplanocin A, 3-deazanepla-

nocin A and cycloleucine on cell growth and differentiation of MEL

cells. MEL-745PC-4A cells were incubated with varying concentrations

of neplanocin A (–.–), 3-deazaneplanocin A (–o–) and/or cycloleucine

(–E–) in the absence (panel A) and presence (panels B and C) of DMSO

(1.5% v/v). Cell growth (panels A and B) was determined after 48 h by

counting the cell number with the use of a hemocytometer under a light

microscope, whereas the proportion of differentiated (hemoglobin-pro-

ducing) cells (panel C) was assessed after 96 h as previously described

[7,8]. Each value represents the mean of two separate experiments.

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114104

3.2. Cytoplasmic accumulation of relatively short RNAs in

MEL cells exposed to hypomethylating agents in the

presence or absence of DMSO

Cytoplasmic RNA isolated from MEL cells exposed to

each one of the three hypomethylating agents in the presence

of DMSO and hybridized with multi-primed 32P-labeled 7.3

kb mouse genomic DNA fragment bearing the hmajor globin

gene and flanking sequences, revealed accumulation of

nascent hmajor globin mRNA as expected as well as of

discrete RNA transcripts of relatively short molecular weight

(Fig. 2A–D). The steady-state level of these RNAs was

higher in cells exposed to each hypomethylating agent in the

presence of DMSO from the beginning as compared to that

seen in cells exposed to the same hypomethylating agent in

the absence of DMSO. These data are in agreement with our

previous observation showing that N 6mAdo promoted

similar pattern of cytoplasmic accumulation of RNA tran-

scripts of hmajor globin gene [9]. Notably, cells exposed to

each hypomethylating agent in the absence of DMSO

produced negligible quantities of nascent hmajor globin

mRNA. These data (Fig. 2A–D) indicate that: (a) all three

hypomethylating agents promote accumulation of discrete

RNA transcripts, an event not seen in cells exposed to

DMSO alone; (b) the level of these short RNAs increased

after 60–72 h exposure to hypomethylating agents only (Fig.

2B,C,D). DMSO enhanced the steady-state level of such

RNAs in each occasion suggesting that these RNAs are

accumulated at higher levels in cells exposed to inducer

regardless of whether erythroid maturation is blocked.

Moreover, these data suggest that DMSO treatment modu-

lates the onset of appearance of these RNAs in cells treated

with hypomethylating agents.

3.3. The detection of short-end RNA transcripts was specific

to bmajor and a1-globin gene DNA probes

To demonstrate whether the appearance of new RNA

transcripts is specific for hmajor globin mRNA, we screened

Fig. 2. Assessment of the effect of neplanocin A, 3-deazaneplanocin A and cycloleucine on the steady-state accumulation of hmajor globin RNA transcripts in

control and differentiating MEL cells. MEL-745PC-4A cells were incubated in DMEM supplemented with FCS (10% v/v) and DMSO (1.5% v/v) (panels A,

E), or exposed separately to neplanocin A (1�10�6 M) (panels B, F), 3-deazaneplanocin A (1�10�5 M) (panels C, G) and/or cycloleucine (4�10�2 M) (panels

D, H) in the absence or presence of DMSO (1.5% v/v). At times indicated (numbers above the panels), cells were removed from culture and total cytoplasmic

RNA was isolated. Ten micrograms of cytoplasmic RNA were electrophoretically separated on 1% agarose gel, transferred and immobilized onto a nylon

membrane (Hybond-N, Amersham) and hybridized at 65 8C with [32P]-labeled genomic DNA fragment coding for hmajor globin gene (7.3 kb), as described

earlier [7]. The filter was then washed at 65 8C and autoradiographed using Kodak XAR-5 film. The autoradiograms obtained are shown above (panels A–D).

Panels E–H show the corresponding ethidium bromide staining patterns of electrophoresed RNA samples (the positions of 28S and 18S rRNAs are indicated).

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 105

Northern blots with DNA probes originated from mouse a1-

globin gene as well as from genes encoding TfR, h-actin,rpS5 and GAPDH. In addition to hmajor probe, hybridization

with a 32P-labeled a1-globin DNA probe also led to the

detection of short-end RNA transcripts whose level was

once again enhanced by DMSO (Fig. 3A–D). In contrast,

the use of DNA probes encoded either by the developmen-

tally regulated TfR gene (Fig. 4), or by the housekeeping

genes h-actin, rpS5 and GAPDH (data not shown) failed to

detect short-end RNA transcripts in MEL cells exposed to

each hypomethylating agent in the absence and/or presence

of DMSO. These findings indicate that the phenomenon of

appearance of small RNA transcripts appears to be unique

for globin genes in MEL cells exposed to hypomethylating

agents.

3.4. The short-end RNAs may originate from the 3V-flankingsequences of bmajor globin gene locus

The appearance of new RNA transcripts in the cytoplasm

of MEL cells exposed to hypomethylating agents prompted

us to investigate their origin. We generated several DNA

probes from the 7.3 kb EcoRI/EcoRI genomic DNA

fragment, as indicated in Fig. 5, and used them accordingly

Fig. 3. Assessment of the effect of neplanocin A, 3-deazaneplanocin A and cycloleucine on the steady-state accumulation of a1-globin RNA transcripts in

control and differentiating MEL cells. MEL-745PC-4A cells were incubated separately in culture with each methylation inhibitor in the absence and presence

of DMSO (1.5% v/v) as indicated under Fig. 2 and the steady-state level of a1-globin mRNA cytoplasmic accumulation was assessed by Northern blot

hybridization analysis using a genomic SacI/SacI DNA fragment (~3 kb) described under Materials and methods. The autoradiograms showing the steady-state

level of a1-globin mRNA and other RNAs hybridized by the same probe are indicated above (panels A–D). Panels E–H show the corresponding ethidium

bromide staining patterns of electrophoresed RNA samples (the positions of 28S and 18S rRNAs are indicated).

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114106

as hybridization probes to demonstrate the origin of these

RNAs. Since the only known gene within the 7.3 kb

genomic DNA fragment was that of hmajor globin we

searched the flanking regions. Extensive Northern blot

hybridization analysis done with the use of several 32P-

labeled DNA fragments revealed that none of the eight

DNA probes generated and used (probes A–H in Fig. 5)

enabled us to detect the small RNA transcripts in MEL cells

exposed to neplanocin A, 3-deazaneplanocin A, or cyclo-

leucine in the absence or presence of DMSO (see Fig. 6 for

probe F; Fig. 7 for probe H; Fig. 8A for probe C; Fig. 8B for

probe D; Fig. 8C for probe E; Fig. 8D for probe G; Fig. 9A

for probe A; Fig. 10A for probe B). Interestingly, however,

we observed that the only two DNA probes used to detect

the new RNA transcripts were those generated from the 3V-

downstream flanking sequences of hmajor globin gene (DNA

probes I and J in Fig. 5) (see Figs. 8E and 10B for probe I;

and Figs. 9B and 10C for probe J). The estimated size of the

detected RNA transcripts ranged from 150 to 600 nt as

shown in Fig. 10. These data indicate that exposure of MEL

cells to hypomethylating agents promotes accumulation of

relatively short RNA transcripts hybridized to DNA

sequences originated from the 3V-downstream region of

hmajor globin gene locus.

3.5. The detected RNA transcripts lack poly(A) tail

To further characterize the detected RNAs, we first asked

if they bear poly(A) tail as the majority of mRNAs by

separating cytoplasmic RNA into poly(A)� and poly(A)+

Fig. 4. Assessment of the effect of neplanocin A, 3-deazaneplanocin A and

cycloleucine on the steady-state accumulation of TfR RNA transcripts in

control and differentiating MEL cells. Membranes bearing the same RNA

samples shown in Fig. 3 were stripped and re-probed with a DNA genomic

fragment encoding for mouse TfR gene in order to assess the cytoplasmic

accumulation of TfR RNA transcripts. The results obtained are shown

above (panels A–D).

Fig. 5. Map of the different DNA probes originated from the 7.3 kb EcoRI/EcoRI genomic DNA fragment and used for Northern blot hybridization studies.

The mouse genomic 7.3 kb EcoRI/EcoRI DNA fragment is illustrated along with the different segments generated and used as probes to map the area of origin

of new RNA transcripts detected upon incubation of MEL cells with methylation inhibitors. The hmajor globin gene with the three exons and two introns is

shown at the 5V-end of this genomic fragment. The probes designated C, D, E and F shown at the bottom were generated by PCR, whereas these named as A, B,

G, H, I and J were generated by digestion of 7.3 kb DNA fragment with the restriction enzymes indicated at the top.

Fig. 6. Northern blot hybridization analysis of cytoplasmic RNA by using

the exon-2 DNA sequences of hmajor globin gene as probe. The same

membranes shown in Fig. 4 were stripped and re-hybridized with the

radiolabeled probe F shown in Fig. 5 which contains the full-length of

exon-2 of hmajor globin gene. The results shown in panels A–D indicate no

detection of discrete small weight RNA transcripts.

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 107

Fig. 7. Northern blot hybridization analysis of cytoplasmic RNA with

probes encoding the exon-3 and the 3V-UTR DNA sequences of hmajor

globin gene. The same membranes shown in Fig. 6 were stripped and re-

hybridized with the radiolabeled probe H shown in Fig. 5 which contains

the full-length of exon-3 and 3V-UTR sequences of hmajor globin gene. The

results shown in panels A–D indicate no detection of discrete small weight

RNA transcripts.

Fig. 8. Northern blot hybridization analysis of cytoplasmic RNA by using

each one of the probes C, D, E, G and I generated from the 7.3 kb genomic

DNA fragment. MEL-745PC-4A cells were incubated in DMEM supple-

mented with 10% v/v FCS with DMSO, or exposed separately to

neplanocin A, 3-deazaneplanocin A and/or cycloleucine in the absence or

the presence of DMSO as indicated in Fig. 2. At times indicated (numbers

above the panels), cells were removed from culture and total cytoplasmic

RNAwas isolated. Ten micrograms of cytoplasmic RNAwere then assessed

by Northern blot hybridization analysis by using the following probes that

have previously been indicated in Fig. 5: in Panel A the probe C

corresponds to the 5V-UTR region of hmajor globin gene; in Panel B the

probe D encodes the exon-1 of hmajor globin gene; in Panel C the probe E

encodes the exon-2 and the intron-2 of hmajor globin gene; in Panel D the

probe G corresponds to the 5V-UTR, the exon-1, the intron-1, the exon-2

and part of the intron-2 region of hmajor globin gene; in Panel E the probe I

spans throughout the 3V-flanking sequences of hmajor globin gene. Panel F

shows the corresponding ethidium bromide staining patterns of electro-

phoresed RNA samples. The positions of 28S and 18S rRNAs are also

indicated by the arrows. Note that the detection of the discrete small weight

RNA transcripts upon exposure of control and/or DMSO-treated MEL cells

to each one of the methylation inhibitors is achieved only with the use of

probe I shown in panel E.

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114108

fractions by oligo(dT)-cellulose chromatography. While

three known nascent mRNAs encoding for hmajor globin,

TfR and GAPDH were detected in the poly(A)+ RNA

fraction of cytoplasmic RNA (Fig. 11B) as expected, to our

surprise the small RNAs were found exclusively in the

poly(A)� RNA fraction (Fig. 11A). Therefore, we reason

that these poly(A)� RNA transcripts must represent RNA

species accumulated in the cytoplasm of MEL cells exposed

to hypomethylating agents.

3.6. DNA sequences located downstream of bmajor globin

gene locus contain unique cis-elements and consensus

binding sites for GATA-1, NF-E2 and EKLF transcription

factors

The data presented thus far revealed that the short

RNAs are hybridized with the probes I and J that originate

from the 3V-end flanking sequences of hmajor globin gene.

This observation prompted us to examine the sequence

characteristics of this region illustrated in Fig. 5. The entire

Fig. 9. Northern blot hybridization analysis with the probes A and J

encoding sequences that are located in distant 5V- and 3V-flanking sequencesof hmajor globin gene locus. MEL-745PC-4A cells were incubated in culture

with either DMSO alone, or with DMSO in the presence of either

neplanocin A, 3-deazaneplanocin A and/or cycloleucine for 60 h as

indicated in Fig. 2. Untreated cells were used as control experiment.

Constant amount of cytoplasmic RNA (10 Ag) derived from each sample

was assessed by Northern blot hybridization analysis using the following

probes illustrated in Fig. 5. Panel A: probe A was derived from the 5V-flanking sequences of hmajor globin gene. Panel B: probe J spans 3V-flanking sequences far away of hmajor globin gene. Both probe A and J

detected nascent hmajor globin mRNA, but only probe J detected small

molecular weight RNAs. Corresponding ethidium bromide staining pattern

of electrophoresed RNA samples is shown in panel C. Note that the

detection of the discrete small weight RNA transcripts upon exposure of

DMSO-treated MEL cells to each one of the three methylation inhibitors is

achieved only with the use of probe J shown in panel B.

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 109

nucleotide sequence of the BALB/c mouse h-globincomplex spans almost 55.9 kb as had been previously

reported by Shehee et al. [17], (accession number

X14061). The 7.3 kb genomic DNA fragment used as

hybridization probe to detect the new RNA transcripts in

MEL cells exposed to hypomethylating agents corresponds

to sequences spanning from 36,884 bp to 44,192 bp (the

numbers refer to the sequence data deposited at Gen/EMBL

with accession number X14061). This region contains the

hmajor globin gene (38,207–39,683 bp) and a B1 repeat

element (42,382–42,545 bp) along with two short direct

repeats (SDR) located at the 5V-end (5V SDR: 42,367–42,381bp) and 3V-end (3V SDR: 42,546–42,560 bp) of B1 element

[17] (see Fig. 12). This B1 repeat element is being

transcribed by RNA polymerase III and it has been

previously identified by hybridization to human Alu repeat

elements [18]. More interestingly, however, detailed struc-

tural analysis enable us to reveal in the region upstream of

the B1 repeat element the presence of consensus binding

sites for the transcription factors GATA-1 (binding site

AGATAAGG at position 42,283– 42,290 bp) as well as for

AP-1/NF-E2 (binding site TGCTGACTCA at position

42,463–42,272 bp) (see Fig. 12). In addition to these two

consensus sequences for GATA-1 and AP-1/NF-E2 tran-

scription factors, another binding site for EKLF has been

revealed internally; the B1 element at the 42,528–42,537 bp

region (binding site CCCCCCCCCA) (Fig. 12). The

identified GATA-1 binding site is identical to the one

previously found within the promoter of hmajor globin gene

(at position 38,076–38,083 bp; promoter site �216 to �209

bp) (Fig. 12), which it is known to play a crucial role in the

transcriptional activation of hmajor globin gene [19].

Furthermore, we identified another identical GATA-1 bind-

ing site located within the bh3 gene (at position 31,153–

31,160 bp). In addition, several ATG initiation codons

localized around the GATA-1 and AP-1/NF-E2 binding sites

were identified in the 3V-downstream sequences of hmajor

globin gene, the most of which found to be at their 3V-endsequences near the B1 repeat element (Fig. 12). The

existence of so many structural elements within the 3V-endflanking sequences of hmajor globin gene tend to suggest that

this DNA region is probably involved in the appearance of

small RNAs detected upon treatment of MEL cells with

hypomethylating agents.

3.7. DNA methylation analysis within the region of 7.3 kb

genomic DNA fragment in MEL cells exposed to hypome-

thylating agents

Since we employed hypomethylating agents, it was

reasonable to investigate whether exposure of MEL cells

to these agents alters the methylation status of CpG sites

located within the region of interest. The 7.3 kb genomic

DNA fragment contains a CCGG consensus methylation

sequence at position 40,523 bp that is recognized by the

methylation-sensitive restriction isoschizomer enzymes

HpaII and MspI. HpaII does not cut when the internal

cytosine is methylated, whereas MspI does so regardless

of whether the cytosine is methylated or not. Thus, we

applied restriction enzyme analysis complemented by

molecular hybridization as previously shown [16]. If

sequence CCGG was being methylated, then MspI

produces two fragments of 3641 and 3663 bp, in contrast

to HpaII which leaves the fragment with intact size (7.3

kb). DNA extracted from control, DMSO- and/or hypo-

methylating agent-treated cells was at first digested with

EcoRI and then with MspI and/or HpaII and was

analyzed by molecular hybridization, as shown in Fig.

13. The results have shown that the CCGG site remained

methylated and it is not affected by either neplanocin A

or 3-deazaneplanocin A (Fig. 13). Similar results were

obtained upon treatment of MEL cells with cycloleucine

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114110

(data not shown). These data indicate that methylation at

the specific CCGG site examined is not altered. There-

fore, the appearance of small RNAs induced by the

hypomethylating agents in MEL cells is not related to

changes in the level of DNA methylation of the region

tested. It is likely, however, that changes to other DNA

methylation sequences may be related to the blockade of

differentiation program of MEL cells by hypomethylating

agents with the possible involvement of transcription

factors or with specific changes in chromatin superfine

structure.

4. Discussion

Despite the plethora of data accumulated during the last

years concerning the in vitro MEL cell differentiation

program, the precise molecular mechanism(s) which govern

this process are as yet not fully delineated. Furthermore, the

detailed mechanism by which RNA and DNA metabolic

inhibitors like cordycepin [4,5], 3-deazaadenosine [20], S-

5V-isobutylthioadenosine and 5V-methylthioadenosine [21]

block differentiation are still not clear. The data presented in

this study indicate for the first time that three agents

(neplanocin A, 3-deazaneplanocin A and cycloleucine),

known to inhibit DNA and RNA methylation by blocking

the active methylation cycle induced cytoplasmic accumu-

lation of discrete RNAs of relatively small molecular

weight. The appearance of four to five different size RNAs

(150–600 nt) lacking the poly(A) tail in MEL cells exposed

to three different hypomethylating agents suggest that their

accumulation is likely to result from unique, although

unknown, effects of these agents. The observations, how-

ever, that the steady-state level of these RNAs was higher in

cells co-exposed to hypomethylating agents and DMSO

indicate that the inducer of differentiation favors the

accumulation of such RNAs. Although it is not clear at

the moment whether DMSO/hypomethylating agent-treated

cells become committed to terminal maturation without

hemoglobin synthesis or both commitment to terminal

maturation and hemoglobin production are blocked, it is

essential to ask the following questions: (a) How specific

the accumulation of these RNAs is and where do these

poly(A)� RNA species originate from? (b) Are these RNAs

authentic products resulting from transcription of DNA

sequences located within the h-globin gene family complex,

or are abnormal products coming from mis-processed

Fig. 10. Detection of discrete small RNA transcripts in cytoplasmic RNA

prepared from methylation inhibitor-treated MEL cells with probes

corresponding to 3V-flanking sequences of hmajor globin gene. MEL-

745PC-4A cells were incubated in culture with DMSO or exposed

separately to neplanocin A, 3-deazaneplanocin A and/or cycloleucine in

the absence or presence of DMSO for 60 h as indicated in Fig. 2. Untreated

cells were also used as control experiment. A constant amount of

cytoplasmic RNA (10 Ag) derived from each sample was assessed by

Northern blot hybridization analysis using probe B originated from 5V-flanking sequences, and probe I and J originated from the 3V-flankingsequences of hmajor globin gene shown in Fig. 5. The results obtained are

shown in Panel A for probe B, in Panel B for probe I and in Panel C for

probe J. Panel D shows the corresponding ethidium bromide staining

pattern of electrophoresed RNA samples. The arrows show the molecular

weight of different size RNA ladder markers for comparative study. The

position of 28S and 18S rRNAs is also indicated. Note that the detection of

the discrete small weight RNA transcripts upon exposure of DMSO-treated

MEL cells to each one of the three methylation inhibitors is achieved only

with the use of probes I and J as illustrated in panels B and C.

Fig. 11. The discrete RNA transcripts detected in methylation inhibitor-

treated MEL cells lack poly(A) tail. MEL-745PC-4A cells were incubated

in culture with either DMSO alone, or with DMSO in the presence of

neplanocin A for 72 h as indicated in Fig. 2. Untreated cells served as a

control experiment. Total cytoplasmic RNA derived from such treated cells

was then separated into the poly(A)� and the poly(A)+ RNA fractions using

oligo(dT)-cellulose column as previously shown [7]. Northern blot hybrid-

ization analysis then carried out and the results obtained are shown above.

In panel A, hybridization was performed at 65 8C with [32P]-labeled 7.3 kb

EcoRI/EcoRI genomic DNA fragment. Panel B: after the stripping of the

membrane shown in panel A, hybridization was repeated simultaneously

with three different [32P]-multi-primed labeled probes encoding the exon-2

of hmajor globin (probe F in Fig. 5), the TfR and the GAPDH genes in order

to assess the relative purity of the poly(A)+ RNA fraction. Note, as shown

in panel A, that the detection of the discrete small weight RNAs upon

exposure of DMSO-treated MEL cells to neplanocin Awas possible only in

the samples of total cytoplasmic RNA (lane 3) and exclusively in poly(A)�

RNA fraction (lane 4), suggesting that small RNAs lack poly(A) tail.

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 111

globin-like RNA precursors which are unable to be

polyadenylated in the presence of hypomethylating agents?

(c) What is their orientation (sense or antisense), and why do

they lack poly(A) tail? (d) To what extent do these RNAs

share structural homology? Finally, it would be very

interesting to investigate whether these RNA species may

be responsible for blockade of MEL cell differentiation seen

following treatment with hypomethylating agents. Unfortu-

nately, the answers to some of these questions are not

presented in this paper at this stage, since additional work is

needed to further characterize these RNAs and then

investigate their role in differentiation. The fact that these

RNA species were detected only by using DNA hybrid-

ization probes originated from a- and h-globin genes

indicates that these RNAs are exclusively related to the

globin gene family and not to other non-globin genes tested

(TfR, h-actin, GAPDH, rpS5). We cannot rule out, however,

the possibility that these short RNA transcripts may

originate from other regions of the mouse genome that

contain sequences structurally homologous to those being

present within the 3V-end flanking sequences of hmajor

globin gene.

Previous work has shown the presence of antisense RNA

molecules of hmajor globin gene in the cytoplasm of MEL

cells as well as in erythroid spleen cells and reticulocytes

from anemic mice [22,23]. These antisense globin RNA

transcripts are detected in full size or as truncated molecules.

They are complementary to the spliced hmajor globin mRNA

synthesized by the mouse erythroid cells, and they are

implicated in a mechanism that gives rise to new sense-

strand hmajor globin mRNA by the involvement of an RNA-

dependent RNA polymerase [23]. The fact that the detected

poly(A)� RNA species presented in this paper do not

hybridize with DNA probes derived exclusively from the

hmajor globin coding sequences, it tends to suggest that they

represent a different group of RNA molecules, although we

do not know yet their orientation (sense or antisense). On

the contrary, we know that these poly(A)� RNAs are

detected in Northern blots only with DNA probes derived

from downstream DNA sequences of hmajor globin gene,

and for this reason, they do not seem to be generated from

premature globin-like RNA molecules attacked by specific

endoribonucleases as those recently characterized and being

involved in the cleavage of h- and a-globin mRNAs

intracellularly [24,25].

The Northern blot hybridization experiments of cyto-

plasmic RNAs isolated from MEL cells exposed to

hypomethylating agents and presented in this paper have

revealed that besides the transcriptional activation of hmajor

gene, DNA sequences located at 3V-flanking region may be

also selectively activated. A careful look at this DNA locus

indicated the presence of two consensus sequences for

GATA-1 and AP-1/NF-E2 transcription factors. These

sequences are located upstream of a previously identified

B1 repeat element, the mouse equivalent of the Alu element

seen in human DNA [17,26]. The fact that preliminary

results from electrophoretic mobility binding assays by

using DNA fragments from this region have shown binding

of several transcription factors (data not shown), tends to

suggest that the identified sequences may have some

functional role in transcription, a result that needs further

verification. Alternatively, the presence in the 3V-down-stream sequences of hmajor globin gene of the consensus

Fig. 12. Diagrammatic presentation of unique DNA sequences located within the 3V-flanking sequences of mouse hmajor globin gene. Detailed examination of

sequences located at the 3V-flanking region, (position 42,201–42,759 bp at the sequence deposited at Gen/EMBL with accession numberX14061), of hmajor

globin gene revealed the presence of several consensus sequences for the binding of the NF-E2, GATA-1 and EKLF transcription factors shown in boxes at the

lower right side of the panel. In addition, this area has several ATG initiation codons (shown in bold and italics) and a B1 element whose sequence is

underlined. The sequences of SDR that are located at the 5V- and 3V-end of B1 element is shown in lowercase letters. Finally, the GATA-1 consensus sequence

known to exist within the promoter region of hmajor globin gene (position �220 to �208 bp), which is identical to that identified at the 3V-flanking sequences isalso illustrated in a large box at lower left panel.

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114112

binding sequences for GATA-1 and AP-1/NF-E2 tran-

scription factors in the vicinity of B1 repeat element may

suggest some specific role in the expression pattern of hmajor

gene during development. This is an attractive direction,

since it is well known that these two transcription factors

have crucial roles in the regulation of globin gene

expression through binding in consensus DNA sequences

located within the locus control region (LCR) and promoter

site of the genes [27–29]. Although no data exists to support

such an assumption, this hypothesis is further strengthened

by recent findings that implicate the function of B1 repeat

family in complex regulatory elements involved in post-

transcriptional control of gene expression in the mouse

genome [30]. In particular, the insertion of a B1 repeat

element in the 3V untranslated region of the rabbit h-globingene conferred growth- and transformation-dependent tran-

scriptional regulation of this gene after its transfection into

the cells. This effect was attributed to the function of B1

repeat element that cooperated with other regulatory

sequences in a way that somehow modulated the RNA

polymerase II transcription machinery [30]. In the light of

these structural elements being involved in the 3V-down-stream region of hmajor globin gene, the data presented

suggest, but by no means prove, that the detected small size

RNAs stem from locally transcriptionally activated DNA

sequences. Unfortunately, no conventional transcriptional

units have been detected in these 3V-flanking sequences

regardless of the existence of the B1 element, of several

ATG initiation codons and consensus sequences for the

binding of the transcription factors AP-1/NF-E2, GATA-1

and EKLF. Therefore, we cannot rule out the possibility that

transcriptional units might be located somewhere else within

the mouse genome which is activated by hypomethylating

agents and produce such RNAs which share structural

complementarities with the 3V-downstream sequences of

hmajor globin gene used as probes for detection.

Fig. 13. Analysis of methylation of CCGG sites located within the 7.3 kb genomic DNA fragment in MEL cells exposed to neplanocin A and 3-

deazaneplanocin A. MEL-745PC-4A cells were incubated in culture with neplanocin A and 3-deazaneplanocin A in the absence or presence of DMSO as

described under Fig. 2. At times indicated (numbers above the panels), cells were removed from culture and nuclear DNA was isolated. Constant amount of

DNA (4–8 Ag) was digested first with EcoRI and then by either MspI or HpaII. The DNA digestion products were separated in 1.2% agarose gel, transferred

onto Hybond-N membrane and hybridized with [32P]-multi-primed labeled 7.3 kb genomic DNA fragment as indicated under Materials and methods. Panel A:

digestion of DNA with both EcoRI and HpaII followed by hybridization. Note that the EcoRI/EcoRI 7.3 kb genomic DNA fragment remained intact upon

HpaII digestion as shown by the arrowhead. Panel C: digestion of DNAwith both EcoRI andMspI and hybridization. In this caseMspI digestion produced two

fragments from the EcoRI/EcoRI 7.3 kb genomic DNA fragment at the expected size (3641 and 3663 bp) as shown by the arrowhead. The first lane in each

panel (A, C) represents the results obtained with the DNA isolated from control untreated MEL cells and digested with only EcoRI. The corresponding

ethidium bromide staining patterns of digested DNAs are illustrated in panel B (EcoRI and HpaII digestion) and panel D (EcoRI and MspI digestion). Size of

molecular weight DNA markers are indicated in the left of each panel.

I.S. Vizirianakis, A.S. Tsiftsoglou / Biochimica et Biophysica Acta 1743 (2005) 101–114 113

Experiments in our laboratory are underway to clone and

characterize these RNA transcripts and then demonstrate if

the identified consensus sequences for the GATA-1 and AP-

1/NF-E2 are indeed functional. Moreover, it would be of

great interest to delineate how DNA regions, which are silent

in untreated MEL cells, are somehow activated, and how this

process leads to the blockade of erythroid differentiation

program. Since the appearance of these short poly(A)�

RNAs is seen exclusively in cells exposed to hypomethylat-

ing agents, but not in control cells, we tend to conclude that

their production may be due to the direct action of

hypomethylating agents on chromatin transcriptional state,

or alternatively, that these agents may exert their action

indirectly on chromatin by modulating the active methyl-

ation cycle. The latter focuses on data that strongly support

the notion of a close relationship between DNA methylation

state and chromatin conformation status in a way that affects

gene transcription and represents a research area that has

received a lot of attention in recent years [31,32]. Although

at present, we have no clue as to what these small RNAs do,

we may ask whether these RNAs act at the transcriptional or

even posttranscriptional level to abrogate erythroid differ-

entiation. This may be a novel mechanism for regulating

gene expression posttranscriptionally. Alternatively, the

detected poly(A)� RNAs might be encoded by genes that

belong to the family of non-coding RNAs (ncRNAs) which

are well known to have important roles in processes like

transcriptional regulation, RNA maturation, stability and

translation as well as protein degradation and translocation

[33,34]. In any case, however, these processes impinge on

the crucial molecular mechanisms that govern the initiation

of MEL differentiation program and justify our attempt to

isolate, clone and characterize the detected poly(A)� RNAs

presented in this paper.

Acknowledgements

We would like to thank Mr. Sotirios Tezias for technical

help and Dr. Aristidis Kritis for sequence alignment at the

initial phase of this work. This work was supported in part

by the EKVAN Project from the Greek Secretariat Science

and Technology awarded to Dr. A.S. Tsiftsoglou.

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