JOurn1al or in.lnerr.3,du.3e Rese-arch
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Regulation of Transcriptional Factor Early B Cell Factor by DNMT1 and Spl
in Osteosarcoma Saos2 Cells
Transcription factors play an essential role in the synthesis of RNA from a DNA template and are therefore
required for gene expression. DNA methyltransferase 1 (DNMT1), which catalyzes the post-replication methylation
of DNA, Early B cell Factor (EBF), a key transcription factor in B cell differentiation and Spl, which binds directly
to DNA and activates transcription, were analyzed. The objective of this study was to investigate the effects
of DNMT1 and Spl on EBF-mediated transcription. It was hypothesized that EBF-mediated transcription would
be repressed by the presence of either DNMT1 or Spl. The promoter of the human Daxx gene (DaxxP) was used
as the promoter for luciferase reporter gene assays in osteosarcoma Saos2 cells. Early B cell factor
enhanced luciferase expression following transfection while a point mutation in the amino acid sequence of
EBF, amino acid 157, eliminated the enhancing effect of EBF on luciferase expression. Similarly, no effects
were observed following DNMT1 transfection. However, when both DNMT1 and EBF were transfected,
luciferase expression increased. Moreover, when Spl was transfected into osteosarcoma cell line Saos2 cells, it
had no effect on the reporter expression while, in separate trials, both Spl and DNMT1 repressed the
enhancing capability of EBF on the reporter gene. However, no effects were observed when a mutated EBF
was transfected. These results show that early B cell factor, EBF, can be regulated by DNMT1 and the
transcription factor Spl.
Synthesis of RNA from DNA template (transcription) is a process mediated by RNA polymerase; however,
RNA polymerase requires other proteins to make the transcript. These other proteins are known as
transcription factors. Transcription factors play an essential role in the synthesis of RNA from a DNA template,
and are therefore required for the eventual expression of proteins. When transcription factors interact with
RNA polymerase or with other transcription factors, they can enhance or repress the expression of the
transcript. Early B cell Factor (EBF), DNA methyltransferase 1 (DNMT1) and Spl were analyzed in this paper.
EBF is a key transcription factor in B cell differentiation; B cells are responsible for production of antibodies once
they interact with antigens. EBF in combination with other transcription factors like E2A and Sox-4 directs a
complex process that eventually leads to the production of recombinases responsible for immunoglobulin
gene rearrangements of immunoglobulin light chains (1). Yu (9) showed that EBF is also responsible for
preventing the expression of genes specific for inducing an erroneous differentiation of lymphocytes into
alternative fates other than becoming B lymphocytes (9), which would result by arresting B cell development at
an early stage. This would cause a decline in the ability of the immune response to react to foreign antigens.
DNMT1 catalyzes the post-replication methylation of DNA. It is the process by which cytosine is converted to
5-methylcytosine and is responsible for maintaining the DNA methylation arrangement throughout cell division
(2,5). DNA methylation protects DNA strands from the attack of endonucleases; moreover, it controls
gene expression. Heavily methylated genes are not expressed (7); on the other hand, non-methylated genes
are actively transcribed. In the human genome, almost 90% of dinucleotides are normally methylated (1). Hodge
et al. (3) showed that expression of DNMT1 induces the methylation of the p53 gene promoter (3). With
this methylation, the inactivation of p53, one of the genetic events required for the transformation of primary
human cells (10) could occur (2); therefore, it could lead to carcinogenesis. In fact, inactivated p53 has
been associated with a variety of human tumors (5). Because of its significance, DNA methylation, an
epigenetic mechanism of gene inactivation, has been proposed as an addition to Knudson's two-hit hypothesis
for oncogenic transformation (4).
Spl, a member of a multigene family of zinc-finger transcription factors (5), directly binds DNA molecules. In
fact, Spl activates transcription in mammalian cells by providing a basal level of transcription that is modulated
by other transcriptional regulating factors (4). This activation of transcription in mammalian cells is done
primarily through interaction with the GC box elements (5). Milutinovic et al. suggests that Spl's basal level
of transcription is modulated by its interaction with different regulatory factors like DNMT1. The purpose of
these studies was to establish the regulatory potential of DNMT1 and Spl over EBF. It was hypothesized that
EBF would be regulated by DNMT1 and Spl.
MATERIALS AND METHODS
Osteosarcoma cell line Saos2 cells, which are p53 deficient cells, were cultured in Dulbecco's modified
Eagle's medium (DMEM) enhanced with 10% fetal bovine serum on a 48-well plate. Each trial was run in
duplicate. To a confluent culture in a 10-cm dish, 1 mL of 0.53mM EDTA and 0.05% trypsin was used to detach
cells from the original plate. 9 mL of Dulbecco's modified Eagle's medium were added to the plate to neutralize
the trypsin. Then 50pL of the cell mixture was transferred to each of the wells in the 48 well plate, and
Dulbecco's modified Eagle's medium was added to each well to cover the bottom of it completely. The 48 well
plate was then incubated in a 37?C incubator for 24 hours.
After 24 hours of being seeded, cells were subjected to transfection with different DNA constructs by using
the procedure for transfection with Qiagen Effectene reagent. First, the specific amount of the DNA to be
transfected was added to an eppendorf tube (0.2 pg of each desired plasmid per well). Since duplicates of each
well were performed, then the amount of the desired DNA construct was doubled. Then, 36 pL of Quiagen's EC
Buffer were added to each tube and mixed by pipetting three times. Next, 4pL of Quiagen's enhancer was added
to the DNA-buffer mixtures, and they were incubated for 5 minutes at room temperature. Buffer and enhancer
were added in order to provide favorable salt conditions for effective DNA condensation required for DNA
transfection. Subsequently, 6.5 pL of Quiagen's Effectene were added to the DNA mixture to form an effectene-
DNA complex that facilitates DNA transfection. Incubation of these complexes was done at room temperature for
While the DNA mixture to be transfected was being incubated, cells in the 48 wells plate were washed. First, the
old media was suctioned, and the wells were washed with Dubelcco's Phosphate Buffered Saline (D-PBS). The D-
PBS was removed, and 230 pL of fresh DMEM were added to each well. After the cells were washed, the
Effectene-DNA complexes to be transected were added directly to the cells by adding 23 pL of the desired complex
to each well. Finally, the transfected cells were incubated for 24 hours at 370C.
Twenty-four hours after transfection, the medium was removed from each well. The cells were washed once with
D-PBS. The D-PBS was removed after washing the cells. This was followed by addition of enough passive
luciferase lysing buffer to cover the bottom of each cell, 70mL were used. Immediately after the lysing buffer
was added, the plate was incubated at -800C for at least one hour. After that time, the cells were ready for
analysis of gene expression.
Lumat LB 9507
The Lumat LB 9507 instrument, shown in Fig. 1, was used to determine the luciferase activity of the cell lysates.
This instrument allows measurement of luminescence in cells, using a system of automatic injectors to add
two different substrates to measure the activity of two different luciferase proteins. First, luciferase assay
substrate was added to measure the activity of firefly luciferase, and then, Stop & Glo was added to measure
the activity of sea pansy luciferase (Renilla luciferase).
Figure 1. Lumat LB 9507. DLReady instrument.
Gene Expression Assay
In order to determine the regulatory potential of DNMT1 and Spl on EBF, reporter gene assays were constructed.
The basis for this type of assay is the procedure for assaying expression of transfected DNA. DNA molecules
require two sequences in order to be expressed. One sequence regulates the transcription of the DNA molecule.
This sequence is known as a promoter, as shown in Fig 2. The other required sequence is the part of the DNA
that encodes the protein produced after DNA transcription and mRNA translation. This sequence is known as
the coding region (Fig 2). The function of the promoter is to either activate or repress the expression of the
coding region. In these studies, DaxxP was used as a promoter for the expression of luciferase reporter gene and
the effects of coexpression of EBF, DNMT1 and Spl on the reporter gene expression.
RNA polymcrasc and Transcriplion factors
* Reporter Protein
Figure 2. DNA transcription and RNA translation of a reporter gene
Dual Luciferase Reporter Gene Assay
The Lumat LB 9507 apparatus was used to analyze the lysates of the cells for gene expression. After placing
the lysates in the freezer at -800C for at least an hour, the cell lysates were thawed at room temperature.
luciferase assay substract and Stop & Glo were used to measure the expression of both firefly luciferase and
sea pansy luciferase respectively. Firefly luciferase activity was normalized against sea pansy luciferase activity.
5mL of each cell lyaste from each individual transfection were added into 12 x 75 mm tubes and introduced into
the Lumat LB 9507 apparatus, so that the expression of reporter gene could be measured.
RESULTS AND DISCUSSION
Plasmids Transfected into Osteosarcoma Cell Lines Saos2 cells and the
Folds Increase of Expression of Luciferase when Co-Transfected with
Different Types of Plasmids Coding for Indicated Transcription Factors.
Plasmids Fold Increase
DaxxP-Luc & EBF 15.11
DaxxP-Luc & EBF H157A 1.90
DaxxP-Luc & Spl 1.19
DaxxP-Luc, EBF & Spl 9.91
DaxxP-Luc, EBF H157A & Spl 1.89
DaxxP-Luc & DNMT1 0.80
DaxxP-Luc, EBF & DNMT1 7.56
DaxxP-Luc, EBF H157A & DNMT1 1.72
DaxxP-Luc, DNMT1 & Spl 1.27
DaxxP-Luc, EBF, Spl & DNMT1 7.83
DaxxP-Luc, EBF H157A, Spl &
DaxxP was used as the promoter sequence for directing the expression of firefly luciferase. Early B cell factor
was transfected along with DaxxP-luciferase DNA sequence. EBF showed an enhancement of firefly
luciferase expression (Fig 3). This might indicate that EBF transcription factor interacts with RNA polymerase
to enhance its capability to bind the DNA strand. In a separate independent trial, it was observed that a
point mutation in the amino acid sequence of EBF (H157A), amino acid 157, which was replaced for alanine instead
of histidine, eliminated the ability of EBF to enhance the firefly luciferase expression (Fig 3). This could mean that
the site of interaction between EBF and DaxxP is the region around amino acid 157 of EBF, or that this region is
part of the active domain of EBF. Consequently, the mutation would either prevent it from attaching to the
DaxxP promoter, or to RNA polymerase. It could also mean that the mutation would stop the mutated EBF
from interacting with other transcriptional factors so as prevent enhancement of the expression of the
14 m Da'.P
1-- DauP - EBF
12 Da.AP * EBFHI57A
Figure. 3. Effects of the EBF protein and its mutant, EBF H157A, on the expression of the luciferase
gene by interaction with the DaxxP promoter in osteosarcoma Saos2 cells. Fold increase of
luciferase gene expression vs. plasmids transfected into Saos2 cells.
DNMT1 did not have the effect that EBF had on the expression of the firefly luciferase gene (Fig 4). In fact, it
seemed that it had no specific effect on the reporter gene. This means that DNMT1 requires some
other transcriptional factors so as to maintain DNA methylation through cell division. When DNMT1 and EBF
were transfected together, an increment in firefly luciferase expression was observed (Fig 4). However,
this increment was less than when EBF was transfected alone (Fig 3). Therefore, DNMT1 seemed to repress the
EBF enhancing capability for the expression of reporter gene. That would suggest that DNMT1 binds EBF and
prevent it from enhancing the reporter gene, or that DNMT1 inhibits EBF's ability to bind to the promoter
sequence. Furthermore, DNMT1 lost the ability to repress EBF when this last transcriptional factor is mutated on
its amino acid 157 (Fig 4). This reduction of the expression of luciferase protein seemed to correspond to
the reduction of its expression when mutated EBF was transfected alone.
S L. I i uaxxr - U I MI *ï¿½ i I-%n1t1
Figure 4. Effects of DNMT1 protein on the expression of the luciferase gene in conjunction with the
DaxxP promoter, EBF and EBF H157A in osteosarcoma Saos2 cells. Fold increase of luciferase
gene expression vs. plasmids transfected into Saos2 cells.
When Spl was transfected into osteosarcoma cell line Saos2 cells with DaxxP as the promoter sequence
and luciferase as the reporter, it had no effect on firefly luciferase expression (Fig 5). This could mean that Spl
does not enhance RNA polymerase's activity to increase luciferase expression, or that it has no interact with
the DaxxP promoter. In addition, since Spl transcription factor requires other transcription factors to actively
interact with DNA, the absence of other transcription factors would prevent Spl's interaction with DNA. Then
when Spl was transfected with EBF, it repressed EBF's enhancing potential of the luciferase reporter gene (Fig 5)
just as DNMT1 repressed EBF (Fig 4). In addition, when Spl was transfected with mutated EBF H157A (Fig 5)
there was no regulatory potential observed since the expression of the firefly luciferase in this trial was the same
as in the trial where EBF H157A was transfected alone.
DaxxP + Spl
10 1 DaxxP + Spl + EBF
[E2 DaxxP + Spl + EBFH157A
Figure 5. Effects of Spl protein on the expression of the luciferase gene in conjunction with the
DaxxP promoter, EBF, and EBF H157A in osteosarcoma Saos2 cells. Fold increase of luciferase
gene expression vs. plasmids transfected into Saos2 cells.
Spl and DNMT1 were also cotransfected with DaxxP but no major difference could be observed as compared
to control (Fig 6). Thus DNMT1 and Spl do not interact with the DaxxP promoter, and as previously stated they
do not affect the expression of firefly luciferase. Furthermore, when both were transfected with EBF in a separate
well (Fig 6), the regulatory potential of EBF over the expression of the reporter gene was reduced. This reduction
on expression was also seen in when DNMT1 and EBF were transfected together (Fig 4). Therefore, Spl had
no further effect on the expression of the luciferase. In addition, as expected, when both were transfected
with mutated EBF, no major difference as compared to the control expression of luciferase activity was observed
DaxxP * ONTM1 * Spl
DaxxP + DNTMI * Sp1 * EBF
8 - DaxxP + DNTM1 * Sp1 * EBFH1S7A
Figure 6. Effects of Spl and DNMT1 proteins on the expression of the luciferase gene by interacting
with DaxxP promoter, EBF, and EBF H157A in osteosarcoma Saos2 cells. Fold increase of luciferase
gene expression vs. plasmids transfected into Saos2 cells.
These results show that early B cell factor, EBF appears to regulate the Daxx promoter, and the impact of EBF can
be regulated by other factors such as DNMT1 and Spl. Therefore, in the presence of DNMT1 or Spl, EBF-
mediated transcription is reduced. As a potential functional consequence, DNMT1 and Spl may regulate EBF during
B-cell differentiation. Further studies are necessary to investigate how DNMT1 and Spl regulate EBF function in B
cell development or other biological contexts.
This study was conducted in the laboratory of Dr. Daiqing Liao in the Department of Anatomy and Cell Biology in
the University of Florida College of Medicine. I am gratefulfor the guidance, encouragement and constant
assistance of Dr. Liao and members of his laboratory. The author was awarded a scholarship for this study from
the University of Florida University Scholars Program.
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