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Regulation of Transcriptional Factor Early B Cell Factor by DNMT1 and Sp1 in Osteosarcoma Saos2 Cells

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Regulation of Transcriptional Factor Early B Cell Factor by DNMT1 and Sp1 in Osteosarcoma Saos2 Cells
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Rios, Jólian
Liao, Diaqing
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Gainesville, Fla.
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JOurn1al or in.lnerr.3,du.3e Rese-arch


2 - No. en i:er..'C :ecen i:er -,.,,.",.



Regulation of Transcriptional Factor Early B Cell Factor by DNMT1 and Spl
in Osteosarcoma Saos2 Cells

J6tian Rios


ABSTRACT


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.



INTRODUCTION


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


Cell Culture


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.





Transfection


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

10 minutes.



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.



Cell Harvesting


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).









.do


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


Promoter
Promoter Transcription
mRNA

Translation


* 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





Table 1.

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 1.00


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 &
1.65
DNMT1



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

firefly luciferase.





16

14 m Da'.P
1-- DauP - EBF
12 Da.AP * EBFHI57A

10

8

6

4

2

0

Plasmid



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 *� i I-%n1t1














0-

Plasmid


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.




12
i DaxxP
DaxxP + Spl
10 1 DaxxP + Spl + EBF
[E2 DaxxP + Spl + EBFH157A

8-


6-


4


2-


0


Plasmid







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

(Fig 6).





10
-M D*KxP
DaxxP * ONTM1 * Spl
DaxxP + DNTMI * Sp1 * EBF
8 - DaxxP + DNTM1 * Sp1 * EBFH1S7A






4-



2-





Plasmid


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.




CONCLUSION



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.









ACKNOWLEDGEMENTS


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.






REFERENCES


1. Brena RM, Auer H, Kornacker K, Hackanson B, Rava A, Byrd J, Plass C. 2006. Accurate quantification of

DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer

platform. Nucl. Acids Res. 34(3):17.

2. Goll MG, Bestor TH. 2005. -Eukaryotic cytosine methyltransferases. Ann. Rev. Biochem. 74:481-514.

3. Hodge DR, Peng B, Cherry JC, Hurt EM, Fox SD, Kelley JA, Munroe DJ, Farrar WL. 2005. Interleukin 6 supports

the maintenance of p53 tumor suppressor gene promoter methylation. Cancer Res. 65(11):4673-4682.

4. Jones PA. and Laird PW. 1999. Cancer epigenetics comes of age. Nature Genet. 21: 163-167.

5. Milutinovic S, Brown SE, Zhuang Q, Szyf M. 2004. DNA methyltransferase 1 knock down induces gene expression

by a mechanism independent of DNA methylation and histone deacetylation. J. Biol. Chem. 279(27):27915-27927.

6. Ohlsson C, Kley N, Werner H, LeRoith D. 1998. p53 Regulates Insulin-Like Growth Factor-I (IGF-I)

Receptor Expression and IGF-I-Induced Tyrosine Phosphorylation in an Osteosarcoma Cell Line: Interaction

between p53 and Sp.1. Endocr. 139:1101 - 1107.

7. Pogribny IP, Pogribna M, Christman JK, James SJ. 2000. Single-site methylation within the p53 promoter

region reduces gene expression in a reporter gene construct: possible in vivo relevance during Tumorgenesis.

Cancer Res60:588-594.

8. Thiery J, Dorothee G, Haddada H, Echchakir H, Richon C, Stancou R, Vergnon I, Benard J, Mami-Chouaib F,

Chouaib S. 2003. Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type

p53 function. J. Immun. 170: 5919-5926.

9. Yu D, Allman D, Goldschmidt MH, Atchison ML, Monroe JG, Thomas-Tikhonenko A. 2003. Oscillation between

B-lymphoid and myeloid lineages in Myc-induced hematopoietic tumors following spontaneous silencing/

reactivation of the EBF/Pax5 pathway. Blood. 101(5):1950-1955.

10. Zhao LY, Colosimo AL, Liu Y, Wan Y, Liao D. 2003. Adenovirus E1B 55-kilodalton oncoprotein binds to Daxx

and eliminates enhancement of p53-dependent transcription by Daxx. J. Virol. 77(21):11809-11821.








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PAGE 1

Journal of Undergraduate Research Volume 8, Issue 2 2 November/December 2006Regulation of Transcriptional Factor Early B Cell Factor by DNMT1 and Sp1 in Osteosarcoma Saos2 CellsJlian Rios ABSTRACTTranscription 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 Sp1, which binds directly to DNA and activates transcription, were analyzed. The objective of this study was to investigate the effects of DNMT1 and Sp1 on EBF-mediated transcription. It was hypothesized that EBF-mediated transcription would be repressed by the presence of either DNMT1 or Sp1. 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 Sp1 was transfected into osteosarcoma cell line Saos2 cells, it had no effect on the reporter expression while, in separate trials, both Sp1 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 Sp1.INTRODUCTIONSynthesis 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 Sp1 were analyzed in this paper. EBF is a key transcription factor in B cell differentiation; B cells are responsible for production of antibodies once

PAGE 2

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 Knudsons two-hit hypothesis for oncogenic transformation (4). Sp1, a member of a multigene family of zinc-finger transcription factors (5), directly binds DNA molecules. In fact, Sp1 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 Sp1s 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 Sp1 over EBF. It was hypothesized that EBF would be regulated by DNMT1 and Sp1.MATERIALS AND METHODSCell Culture Osteosarcoma cell line Saos2 cells, which are p53 deficient cells, were cultured in Dulbeccos modified Eagles 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 Dulbeccos modified Eagles medium were added to the plate to neutralize the trypsin. Then 50L of the cell mixture was transferred to each of the wells in the 48 well plate, and Dulbeccos modified Eagles 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.

PAGE 3

Transfection 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 g 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 L of Quiagens EC Buffer were added to each tube and mixed by pipetting three times. Next, 4L of Quiagens 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 L of Quiagens Effectene were added to the DNA mixture to form an effecteneDNA complex that facilitates DNA transfection. Incubation of these complexes was done at room temperature for 10 minutes. 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 Dubelccos Phosphate Buffered Saline (D-PBS). The DPBS was removed, and 230 L 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 L of the desired complex to each well. Finally, the transfected cells were incubated for 24 hours at 37C. Cell Harvesting 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 -80C 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).

PAGE 4

Figure 1. Lumat LB 9507. DLReady instrument. Gene Expression Assay In order to determine the regulatory potential of DNMT1 and Sp1 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 Sp1 on the reporter gene expression. 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 -80C 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

PAGE 5

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 Table 1. 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 1.00 DaxxP-Luc & EBF 15.11 DaxxP-Luc & EBF H157A 1.90 DaxxP-Luc & Sp1 1.19 DaxxP-Luc, EBF & Sp1 9.91 DaxxP-Luc, EBF H157A & Sp1 1.89 DaxxP-Luc & DNMT1 0.80 DaxxP-Luc, EBF & DNMT1 7.56 DaxxP-Luc, EBF H157A & DNMT1 1.72 DaxxP-Luc, DNMT1 & Sp1 1.27 DaxxP-Luc, EBF, Sp1 & DNMT1 7.83 DaxxP-Luc, EBF H157A, Sp1 & DNMT1 1.65 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

PAGE 6

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 firefly luciferase. 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 EBFs 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.

PAGE 7

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 Sp1 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 Sp1 does not enhance RNA polymerases activity to increase luciferase expression, or that it has no interact with the DaxxP promoter. In addition, since Sp1 transcription factor requires other transcription factors to actively interact with DNA, the absence of other transcription factors would prevent Sp1s interaction with DNA. Then when Sp1 was transfected with EBF, it repressed EBFs enhancing potential of the luciferase reporter gene (Fig 5) just as DNMT1 repressed EBF (Fig 4). In addition, when Sp1 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.

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Figure 5. Effects of Sp1 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. Sp1 and DNMT1 were also cotransfected with DaxxP but no major difference could be observed as compared to control (Fig 6). Thus DNMT1 and Sp1 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, Sp1 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 (Fig 6). Figure 6. Effects of Sp1 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.CONCLUSIONThese 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 Sp1. Therefore, in the presence of DNMT1 or Sp1, EBFmediated transcription is reduced. As a potential functional consequence, DNMT1 and Sp1 may regulate EBF during B-cell differentiation. Further studies are necessary to investigate how DNMT1 and Sp1 regulate EBF function in B cell development or other biological contexts.

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ACKNOWLEDGEMENTSThis 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. REFERENCES1. Brena RM, Auer H, Kornacker K, Hackanson B, Rava A, Byrd J, Plass C. 2006. Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform. Nucl. Acids Res. 34(3):17. 2. Goll MG, Bestor TH. 2005. Eukaryotic cytosine methyltransferases. Ann. Rev. Biochem. 74:481-514. 3. Hodge DR, Peng B, Cherry JC, Hurt EM, Fox SD, Kelley JA, Munroe DJ, Farrar WL. 2005. Interleukin 6 supports the maintenance of p53 tumor suppressor gene promoter methylation. Cancer Res. 65(11):4673-4682. 4. Jones PA. and Laird PW. 1999. Cancer epigenetics comes of age. Nature Genet. 21: 163167. 5. Milutinovic S, Brown SE, Zhuang Q, Szyf M. 2004. DNA methyltransferase 1 knock down induces gene expression by a mechanism independent of DNA methylation and histone deacetylation. J. Biol. Chem. 279(27):27915-27927. 6. Ohlsson C, Kley N, Werner H, LeRoith D. 1998. p53 Regulates Insulin-Like Growth Factor-I (IGF-I) Receptor Expression and IGF-I-Induced Tyrosine Phosphorylation in an Osteosarcoma Cell Line: Interaction between p53 and Sp.1. Endocr. 139:1101 1107. 7. Pogribny IP, Pogribna M, Christman JK, James SJ. 2000. Single-site methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during Tumorgenesis. Cancer Res60:588-594. 8. Thiery J, Dorothe G, Haddada H, Echchakir H, Richon C, Stancou R, Vergnon I, Benard J, Mami-Chouaib F, Chouaib S. 2003. Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function. J. Immun. 170: 5919-5926. 9. Yu D, Allman D, Goldschmidt MH, Atchison ML, Monroe JG, Thomas-Tikhonenko A. 2003. Oscillation between B-lymphoid and myeloid lineages in Myc-induced hematopoietic tumors following spontaneous silencing/ reactivation of the EBF/Pax5 pathway. Blood. 101(5):1950-1955. 10. Zhao LY, Colosimo AL, Liu Y, Wan Y, Liao D. 2003. Adenovirus E1B 55-kilodalton oncoprotein binds to Daxx and eliminates enhancement of p53-dependent transcription by Daxx. J. Virol. 77(21):11809-11821.

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