GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2008) 13, 53-66. doi:10.1111/j.1365-2443.2007.01144.x
© 2008 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bu, Y.
Right arrow Articles by Ozaki, T.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Bu, Y.
Right arrow Articles by Ozaki, T.

Sp1-mediated transcriptional regulation of NFBD1/MDC1 plays a critical role in DNA damage response pathway

 Youquan Bu1,2, Yusuke Suenaga1, Sayaka Ono3, Tadayuki Koda3, Fangzhou Song2, Akira Nakagawara1,* and Toshinori Ozaki1

1 Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan
2 Department of Biochemistry and Molecular Biology, Chongqing University of Medical Sciences, Chongqing 400016, China
3 Center for Functional Genomics, Hisamitsu Pharmaceutical Co., Inc., Chiba 260-8717, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
NFBD1/MDC1 is a large nuclear protein with an anti-apoptotic potential which participates in DNA damage response. Recently, we have demonstrated that NFBD1 has an inhibitory effect on pro-apoptotic p53 and DNA damage-induced transcriptional repression of NFBD1 plays an important role in p53-dependent apoptotic response. In this study, we have found that NFBD1 promoter region contains canonical Sp1-, STAT-1- and NF-Y-binding sites and finally we have identified Sp1 as a transcriptional activator for NFBD1. The 5'-RACE and bioinformatic analyses revealed that NFBD1 encodes at least four transcriptional variants arising from distinct transcriptional start sites. Luciferase reporter assays using a series of NFBD1 promoter deletion mutants demonstrated that the proximal Sp1-binding site is required for the transcriptional activation of NFBD1. Indeed, the endogenous Sp1 was recruited onto the proximal Sp1-binding site as examined by chromatin immunoprecipitation (ChIP) assay and siRNA-mediated knockdown of the endogenous Sp1 in HeLa cells reduced the expression levels of NFBD1, which renders cells sensitive to adriamycin (ADR). In support of this notion, mithramycin A (MA, Sp1 inhibitor) treatment resulted in a significant down-regulation of NFBD1. Taken together, our present findings suggest that Sp1-mediated transcriptional regulation of NFBD1 plays an important role in the regulation of DNA damage response.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
In response to DNA damage, mammalian cells activate their checkpoint machinery to temporal delay or arrest cell cycle progression and allow rapid recognition of the sites of DNA damage followed by their efficient repair (Kastan & Bartek 2004). In the absence of proper DNA damage response, cells undergo apoptotic cell death or their daughter cells inherit damaged DNA. Thus, DNA checkpoint signaling system plays an important role to maintain genome integrity; however, the precise molecular events behind the cellular protection against DNA damage have been elusive. It has been shown that DNA damage induces conformational alteration in ataxia-telangiectasia-mutated (ATM) protein kinase which is phosphorylated at Ser-1981 and the activated ATM phosphorylates its various substrates including histone H2A variant H2AX (Shiloh 2003). H2AX is rapidly induced to be phosphorylated at Ser-139 ({gamma}H2AX) on chromatin flanking the sites of DNA damage to form nuclear foci (Rogakou et al. 1999; Fernandez-Capetillo et al. 2002; Celeste et al. 2003). {gamma}H2AX then serves as a platform for the recruitment of DNA checkpoint signaling factors as well as multifunctional MRN (MRE11–RAD50–NBS1) complex. The local accumulation of MRN complex facilitates the binding of DNA repair factors (Paulli et al. 2000; Celeste et al. 2002; D’Amours & Jackson 2002). Indeed, the disruption of H2AX results in an induction of genome instability and DNA double-strand break repair defects (Stucki et al. 2005).

NFBD1/MDC1 (nuclear factor with BRCT domain 1/mediator of DNA damage checkpoint protein 1) was initially identified as one of the genes encoding an extremely large nuclear protein with anti-apoptotic function (Nagase et al. 1996; Ozaki et al. 2000). Like many DNA damage checkpoint and repair proteins, NFBD1 contains functional domains including NH2-terminal FHA (forkhead-associated), central PST (proline/serine/threonine-rich) and COOH-terminal tandem BRCT (BRCA1 carboxyl terminus) domains. Subsequent studies demonstrated that NFBD1 also participates in DNA damage response pathway (Goldberg et al. 2003; Stewart et al. 2003; Xu & Stern 2003). According to their results, NFBD1 is hyperphosphorylated by ATM in response to DNA damage and cooperates with {gamma}H2AX to recruit MRN complex to the sites of DNA double-strand breaks. Lee et al. (2005) described that the tandem BRCT repeats interact with {gamma}H2AX, suggesting that NFBD1 acts as a molecular bridge between {gamma}H2AX and MRN complex and is significantly involved in early cellular response to DNA damage. siRNA-mediated knockdown of the endogenous NFBD1 resulted in an increase in sensitivity to irradiation as well as an induction of apoptotic cell death. In support of these results, Lou et al. (2006) reported that NFBD1 knockout mice display chromosome instability, DNA repair defects and radiation sensitivity, which are quite similar to those of H2AX-deficient mice. Recently, we have found that DNA damage-induced transcriptional repression of NFBD1 plays a critical role in the regulation of DNA damage response (Nakanishi et al. 2007). According to our recent results, NFBD1 was associated with latent form of p53 and thereby inhibiting DNA damage-induced phosphorylation of p53. Thus, the expression of NFBD1 appears to be important to maintain genome integrity in response to DNA damage through the regulation of p53; however, the precise molecular mechanisms underlying the regulation of NFBD1 expression remain unclear. Recently, Townsend et al. (2005) found several STAT-1-binding sites exist within the putative NFBD1 promoter region and STAT-1 might be a direct transcriptional activator of the NFBD1 promoter.

In the present study, we have identified the NFBD1 promoter and also found that general transcription factor Sp1 plays a crucial role in the regulation of NFBD1 gene transcription. Furthermore, siRNA-mediated knockdown of the endogenous Sp1 led to a significant down-regulation of NFBD1 in association with an increase in adriamycin (ADR) sensitivity.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Identification of the transcription initiation site(s) of NFBD1 gene

To determine the transcription initiation site(s) of human NFBD1 gene, we have employed a 5'-RACE strategy using two gene-specific primers (GSP1 and 2) and an adapter primer (AP1). Total RNA prepared from human testis was reverse transcribed and PCR-based amplification was performed using the indicated combinations of the primer sets. As shown in Fig. 1A, two distinct PCR products were detected under our experimental conditions. These PCR products were gel-purified and subcloned into pGEM-T Easy plasmid. Sequence analysis of 21 randomly selected clones revealed at least three NFBD1 transcripts exist with distinct transcriptional initiation sites (termed variant II, III and IV). Among 21 clones sequenced, 14, 6 and 1 clones corresponded to variant II, III and IV, respectively. In addition, sequence analysis also indicated that variant IV arises from alternative splicing. To confirm our results and also to search for the additional NFBD1 transcript(s) which could contain the longer 5'-UTR than that of variant II, we performed BLAST search. Sequence comparison demonstrated that several ESTs display complete nucleotide sequence identity to one of the above-mentioned NFBD1 variants, which supported the integrity of our 5'-RACE analysis. Among them, we found out the EST termed BP216855 which contains a longer 5'-UTR of NFBD1 gene (Fig. 1B). To confirm the existence of the longest NFBD1 cDNA (termed variant I) in cells, we carried out RT-PCR analysis using total RNA prepared from A549 and HeLa cells. As expected, RT-PCR using the indicated combinations of primer sets produced the estimated sizes of PCR products; however, the band intensity of PCR products generated by F1/R844 primer set was quite weak (Fig. 1B). It is worth noting that, under our experimental conditions, variants II, III and IV are amplified for 25–30 PCR cycles, whereas more than 40 PCR cycles are required for the detection of variant I. Our present results are summarized in Fig. 1C and suggest that variant II is the major NFBD1 transcript among four variants. For further analysis, we defined the 5'-end of variant II as +1.


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		Figure 1  Identification of the transcription initiation site(s) of NFBD1 gene. (A) 5'-RACE analysis. Upper panel shows the positions of PCR primers (AP1, GSP1 and GSP2) used to amplify NFBD1 cDNA. Filled box indicates the open reading frame (ORF) of NFBD1. Lower panel shows the results of 5'-RACE analysis. Total RNA prepared from human testis was reverse transcribed as described under Experimental procedures, and PCR-based amplification was performed using AP1/GSP1 (lane 1) or AP1/GSP2 (lane 2). Arrows indicate the positions of two distinct PCR products. (B) RT-PCR analysis. Upper panel shows the schematic diagram of variant I, NM-014641 and the EST (BP216855). The positions of PCR primers (F1, F313, R739 and R844) are also indicated and the ORF of NFBD1 is depicted as filled box. Lower panel shows the results of RT-PCR analysis. Total RNA prepared from A549 (lanes 1, 3 and 5) and HeLa cells (lanes 2, 4 and 6) was subjected to RT-PCR using F313/R739 (lanes 1 and 2), F1/R844 (lanes 3 and 4) or F1/R739 (lanes 5 and 6). (C) Schematic representation of the genomic organizations of variant forms of NFBD1 with distinct transcriptional initiation sites. Exons and introns are indicated by filled boxes and thin lines, respectively. Exon 2 contains the initiator codon. The positions of the putative CpG islands are also shown.

 
Identification of the NFBD1 promoter region

To identify the possible promoter region of NFBD1 gene, we have generated a variety of luciferase reporter constructs containing the indicated genomic fragments of NFBD1 gene (Fig. 2, upper panel). HeLa, A549 and U2OS cells were transiently co-transfected with the indicated luciferase reporter constructs together with the Renilla luciferase plasmid (pRL-TK). Forty-eight hours after transfection, cells were lysed and their luciferase activities were measured. As shown in the lower left panel of Fig. 2, a modest increase in luciferase activity was observed in cells transfected with P(–2920/+2133) as compared with that in cells transfected with the empty pGL3-basic plasmid. On the other hand, luciferase activities were undetectable in cells transfected with P(–2920/–436) or with P(+135/+2133). Intriguingly, a significant increase in luciferase activities was detectable in cells transfected with P(–2920/+489) or with P(–1040/+489), suggesting that a genomic region from –1040 to +489 of NFBD1 gene has strong promoter activity. In addition, HeLa cells exhibited the highest promoter activity among three cell lines that we used. These observations were consistent with our expression studies showing that HeLa cells express the endogenous NFBD1 mRNA at higher level as compared with A549 and U2OS cells (Fig. 2, lower right panel).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Figure 2  Identification of the region(s) required for the transcriptional regulation of NFBD1 gene. Upper panel indicates the schematic diagram of the luciferase reporter constructs containing the indicated genomic fragments of NFBD1 gene. The positions relative to the major transcriptional initiation site of NFBD1 gene (+1) are indicated. Lower left panel shows the results of the luciferase reporter assays. A549 (gray box), HeLa (filled box) or U2OS cells (open box) were transiently co-transfected in triplicate in 12-well plates with the indicated luciferase reporter constructs together with the Renilla luciferase reporter plasmid (pRL-TK). Forty-eight hours after transfection, firefly and Renilla luciferase activities were measured by Dual Luciferase Assay System. Data obtained from a representative of at least three independent experiments are shown as fold induction compared to the activity of cells transfected with the empty pGL3-basic luciferase reporter vector alone. The results are presented as mean and SD of triplicates from a representative experiment. Lower right panel shows the results of RT-PCR. Total RNA was prepared from the indicated cell lines and subjected to RT-PCR. GAPDH was used as an internal control.

 
To further delineate the genomic region(s) required for the transcriptional regulation of NFBD1 gene, we generated additional luciferase reporter constructs containing the indicated genomic fragments of NFBD1 gene (Fig. 3, left panel). HeLa cells were transiently co-transfected with the indicated luciferase reporter constructs along with pRL-TK. As shown in the right panel of Fig. 3, P(+44/+489) and P(+271/+489) failed to drive the expression of luciferase, whereas a remarkable enhancement of luciferase activities were observed in cells transfected with the remaining luciferase reporter constructs. Thus, we concluded that NFBD1 promoter exists within the genomic region spanning sequences between –282 and +43.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Figure 3  Delineation of the promoter region of NFBD1 gene. Left panel shows the luciferase reporter constructs used in this study. Right panel shows the results of the luciferase reporter assays. HeLa cells were transiently co-transfected with the indicated luciferase reporter constructs along with pRL-TK. Forty-eight hours after transfection, firefly and Renilla luciferase activities were measured as in Fig. 2.

 
Sp1 and NF-Y bind to the NFBD1 promoter

Using the combination of software such as MATINSPECTOR professional and TFSEARCH software, we have found out putative consensus binding sequences for STAT-1, Sp1 and NF-Y within the NFBD1 promoter regions (Fig. 4). Among them, putative binding sites for Sp1 and NF-Y were evolutionarily well conserved between human and mouse NFBD1 promoter regions (data not shown). To examine whether these transcription factors could bind to the NFBD1 promoter region in cells, we performed chromatin immunoprecipitation (ChIP) assays. Cross-linked chromatin was prepared from HeLa cells and immunoprecipitated with control IgG, anti-Sp1, anti-STAT-1 or with anti-NF-YB. NF-YB is one of NF-Y transcriptional complex (Mantovani 1999). The immunoprecipitated genomic DNA was purified and amplified by PCR using the indicated primer sets (Fig. 5A). As shown in Fig. 5B, ChIP assays demonstrated that Sp1 is recruited onto the distal and proximal Sp1-binding sites (PCR 1 and PCR 3) and NF-YB also binds to the distal and proximal NF-Y-binding sites (PCR 2 and PCR 4). In PCR 1 and PCR 3, we could detect the specific bands immunoprecipitated with anti-NF-YB antibody. Additionally, we could also observe the specific band immunoprecipitated with anti-Sp1 antibody in PCR 4. Since the average size of genomic DNA in sonication was 200–500 bp (data not shown), it might be due to the close localization of Sp1- and NF-Y-binding sites in this region or complex formation between Sp1 and NF-Y in cell nucleus. On the other hand, STAT-1 did not bind to the putative STAT-1-binding site (PCR 1) under our experimental conditions.


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		Figure 4  Nucleotide sequence of the promoter region of NFBD1 gene. The nucleotide sequences of putative transcription factor binding sites are underlined. The underlined bold letters (–615, +1, +88 and +234) indicate the transcription initiation sites of four NFBD1 variants. +1 indicates the position of the major transcription initiation site of NFBD1 gene.

 

Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		Figure 5  Sp1 and NF-YB bind to the NFBD1 promoter in cells. (A) Schematic representation of the positions of primer sets [F(–309)/R(–199), F(–212)/R(–110), F(–156)/R(–34) and F(–62)/R(+43)] used for chromatin immunoprecipitation assay (ChIP). (B) ChIP assay. Sheared chromatin prepared from HeLa cells was immunoprecipitated (IP) with the indicated antibodies (control IgG, anti-Sp1, anti-STAT-1 or anti-NF-YB) and the bound DNA was isolated according to the manufacturer's instructions (Upstate). The immunoprecipitated DNA was used as a template for PCR using the indicated primer sets.

 
The proximal Sp1-binding site is required for the transcriptional activation of NFBD1 gene

To determine whether Sp1 and/or NF-Y could contribute to the transcriptional regulation of NFBD1 gene, mutations were introduced into either Sp1 site or NF-Y site in the P(–282/+43) luciferase reporter construct by site-directed mutagenesis (Fig. 6, left panel). A549, HeLa or U2OS cells were transiently co-transfected with the indicated luciferase reporter constructs along with pRL-TK. Forty-eight hours after transfection, cells were lysed and their luciferase activities were examined. As shown in the right panel of Fig. 6, disruption of the distal and/or proximal NF-Y-binding sites had a negligible effect on the promoter activity of NFBD1 gene. Although mutation of the distal Sp1-binding site had undetectable effect on the promoter activity of NFBD1 gene, disruption of the proximal Sp1-binding site resulted in a significant reduction of the promoter activity of NFBD1 gene.


Figure 6
View larger version (20K):
[in this window]
[in a new window]

  		Figure 6  The proximal Sp1-binding site plays an important role in the regulation of NFBD1 transcription. Site-directed mutations were introduced into the parental P(–282/+43) luciferase reporter construct to disrupt the indicated Sp1- and/or NF-Y-binding sites (left panel). Right panel shows the results of luciferase reporter assay. A549 (gray box), HeLa (filled box) or U2OS cells (open box) were transiently co-transfected with the indicated luciferase reporter constructs together with pRL-TK. Forty-eight hours after transfection, luciferase activities were measured as described in Fig. 2.

 
Next, we examined the possible effect of Sp1 inhibitor mithramycin A (MA) on the transcription of NFBD1 gene. MA is a cell-permeable agent that binds to GC-rich DNA sequence thereby blocking the binding of transcription factors such as Sp1 to GC-specific regions of DNA (Kim et al. 2006). HeLa cells were treated with the indicated concentrations of MA and the expression levels of NFBD1 gene were analyzed by RT-PCR. As shown in Fig. 7A, MA treatment led to a remarkable decrease in the expression levels of NFBD1 gene in a dose-dependent manner. Similar results were also obtained by luciferase reporter assay (Fig. 7B). Consistent with these results, siRNA against Sp1 significantly reduced the expression levels of NFBD1 gene (Fig. 7C). Intriguingly, siRNA-mediated knockdown of endogenous Sp1 in HeLa cells significantly increased sensitivity to ADR and simultaneous expression of exogenous NFBD1 decreased sensitivity to ADR (Fig. 7D). These results strongly suggest that Sp1 plays an important role in the transcriptional regulation of NFBD1 gene through the proximal Sp1-binding site as well as cellular sensitivity to DNA damage.


Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		Figure 7  Sp1 regulates the expression of NFBD1 gene. (A) Mithramycin A (MA) treatment results in the down-regulation of NFBD1. HeLa cells were treated with MA at the indicated concentrations for 24 h. Total RNA was then prepared and subjected to RT-PCR. GAPDH was used as an internal control. (B) Luciferase reporter assay. HeLa cells were transiently co-transfected with P(–282/+43) luciferase reporter construct together with pRL-TK. Twenty-four hours after transfection, cells were treated with the indicated concentrations of MA or left untreated. Twenty-four hours after the treatment with MA, cells were lysed and subjected to luciferase reporter analysis. (C) siRNA-mediated knockdown of Sp1 reduces the expression levels of NFBD1. HeLa cells were transiently transfected with the control siRNA or siRNA against Sp1. Seventy-two hours after transfection, total RNA and whole cell lysates were prepared and subjected to RT-PCR and immunoblotting (IB), respectively. (D) Effect of siRNA-mediated knockdown of the endogenous Sp1 on the sensitivity to DNA damage. HeLa cells (2.5 x 105 cells/well) were transiently transfected with the indicated siRNAs (10 nM) together with or without the expression plasmid for NFBD1 (1 µg). The total amount of plasmid DNA per transfection was kept constant (1 µg) with pcDNA3. Forty-eight hours after transfection, cells were treated with adriamycin (ADR) at a final concentration of 1 µM (filled boxes) or left untreated (gray boxes). Twenty-four hours after ADR treatment, cell viability was examined by MTT assay (left panel). *P < 0.05; **P < 0.01. Right panels show the expression levels of Sp1 and NFBD1 as examined by RT-PCR.

 
DNA damage-induced repression of NFBD1 gene expression

As described above, Sp1 plays a critical role in the regulation of NFBD1 transcription and siRNA-mediated knockdown of the endogenous Sp1 resulted in an increase in the sensitivity of HeLa cells to ADR. We then examined whether ADR treatment could affect the promoter activity of NFBD1 gene. For this purpose, HeLa cells were transiently co-transfected with the indicated luciferase reporter constructs together with pRL-TK. Twenty-four hours after transfection, cells were treated with ADR or left untreated. Twenty-four hours after ADR treatment, cells were lysed and their luciferase activities were measured. As shown in Fig. 8, P(–58/+489) displayed almost no response to ADR, whereas the remaining luciferase reporter constructs responded to ADR. These results strongly suggest that ADR treatment decreases the promoter activity of NFBD1 gene and the genomic region spanning –120 and +43 which contains the proximal Sp1-binding site is required at least in part for the ADR-mediated down-regulation of NFBD1 gene. In support of these observations, DNA-binding assays indicated that radio-labeled probe DNA containing the proximal Sp1-recognition site binds to Sp1 prepared from untreated HeLa cells, whereas the radio-labeled DNA binds to Sp1 prepared from HeLa cells exposed ADR to a lesser degree (Supplementary Fig. S1).


Figure 8
View larger version (15K):
[in this window]
[in a new window]

  		Figure 8  ADR-mediated down-regulation of the promoter activity of NFBD1 gene. HeLa cells were transiently co-transfected with the indicated luciferase reporter constructs along with pRL-TK. Twenty-hours after transfection, cells were treated with 1 µM of ADR or left untreated for 24 h and their luciferase activities were then examined as described in Fig. 2.

 
Next, we sought to examine whether the expression levels of NFBD1 and Sp1 genes could be repressed in response to ADR. A549 and HeLa cells were exposed to ADR at a final concentration of 600 nM and 1 µM, respectively. At the indicated time points after ADR treatment, whole cell lysates and total RNA were prepared and subjected to immunoblotting and RT-PCR, respectively. Under our experimental conditions, both cells underwent apoptotic cell death in a time-dependent manner (data not shown). As shown in Fig. 9A, NFBD1 expression at protein as well as mRNA level was down-regulated in response to ADR in a time-dependent manner, which was consistent with our recent results (Nakanishi et al. 2007). On the other hand, the expression levels of Sp1 remained unchanged regardless of ADR treatment. Our antibody against Sp1 recognized two distinct bands on immunoblot in the presence or absence of drug treatment, indicating that Sp1 might be constitutively subjected to chemical modifications such as phosphorylation and/or acetylation. As shown in Fig. 9B, the upper band completely disappeared by the treatment of the anti-Sp1 immunoprecipitates with {lambda}-phosphatase, suggesting that Sp1 is constitutively phosphorylated in cells. To further investigate the phosphorylation status of Sp1, we performed immunoprecipitation/immunoblotting experiments. Equal amounts of whole cell lysates prepared from HeLa cells treated with ADR or CDDP or left untreated were immunoprecipitated with anti-phosphoserine antibody followed by immunoblotting with anti-Sp1 antibody. Intriguingly, the amounts of phospho-Sp1 at Ser increased in response to ADR or CDDP treatment (Fig. 9C).


Figure 9
View larger version (27K):
[in this window]
[in a new window]

  		Figure 9  ADR treatment reduces the expression levels of NFBD1 in association with the decrease in the amounts of Sp1 recruited onto Sp1-binding sites. (A) Expression levels of the endogenous Sp1 and NFBD1 in response to ADR. A549 (left panel) or HeLa cells (right panel) were exposed to ADR at a final concentration of 600 nM or 1 µM, respectively. At the indicated time points after ADR treatment, whole cell lysates and total RNA were prepared and subjected to IB and RT-PCR, respectively. For IB, equality of protein loading was confirmed by the expression of actin. For RT-PCR, GAPDH was used as an internal control. (B) Phosphorylation of Sp1. Whole cell lysates prepared from HeLa cells exposed to ADR or cisplatin (CDDP) were IP with anti-Sp1 antibody. The anti-Sp1 immunoprecipitates were treated with {lambda}-phosphatase and subjected to IB with anti-Sp1 antibody. (C) Immunoprecipitation experiments. HeLa cells were treated with ADR or CDDP. Twenty-four hours after the treatment, whole cell lysates were IP with monoclonal anti-phosphoserine (PSR-45, Sigma) antibody followed by IB with anti-Sp1 antibody. Aliquots of whole cell lysates were IB with anti-actin antibody. (D) ChIP assay. Sheared chromatin was prepared from HeLa cells treated with 1 µM of ADR for 24 h or left untreated and then IP with the indicated antibodies. Genomic DNA was recovered from the immunoprecipitates and PCR-based amplifications were performed using the indicated primer set as shown in Fig. 5B.

 
To address whether ADR treatment could affect the amounts of Sp1 recruited onto the proximal Sp1-binding site of NFBD1 gene, we performed ChIP assay. HeLa cells treated with or without ADR was immunoprecipitated with control IgG, anti-Sp1 or with anti-NF-YB antibody. Chromatin DNA was purified and PCR-based amplification was carried out using the indicated primer set. It is worth noting that ADR treatment inhibits the recruitment of Sp1 as well as NF-YB onto the proximal Sp1-binding site of NFBD1 promoter (Fig. 9D). Similar results were also obtained in HeLa cells exposed to CDDP (Supplementary Fig. S2).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Accumulating evidence strongly suggests that NFBD1 plays an important role in the regulation of early cellular response to DNA damage (Stucki & Jackson 2004). In the present study, we have found for the first time that general transcription factor Sp1 binds to the promoter region of NFBD1 gene and induces its transcription. Upon DNA damage, Sp1 was dissociated from the NFBD1 promoter in later phase thereby reducing the expression levels of NFBD1 gene. Furthermore, siRNA-based depletion of the endogenous Sp1 caused down-regulation of NFBD1 gene in association with an increased sensitivity to ADR. Thus, our present results indicate that Sp1 might be involved at least in part in DNA damage signaling through the transcriptional regulation of NFBD1.

Recently, it has been shown that a significant defect in the cell cycle checkpoint is detectable in STAT-1-deficient cells in association with down-regulation of NFBD1 (Townsend et al. 2005). According to their results, NFBD1 might be a direct transcriptional target of STAT-1. Under our experimental conditions, however, STAT-1 could not be recruited onto the promoter region of NFBD1 gene. In support of these observations, {gamma}-interferon treatment induced the phosphorylation of STAT-1 in HeLa cells; however, we did not detect transcriptional activation of NFBD1 gene in response to {gamma}-interferon (data not shown). In addition, siRNA-mediated knockdown of the endogenous STAT-1 in HeLa cells had undetectable effects on the expression levels of NFBD1 (data not shown). This discrepancy might be due to the different cell systems and/or the existence of the as yet unidentified transcriptional activator(s) for NFBD1.

Our extensive luciferase reporter analysis using systematically designed NFBD1 promoter constructs demonstrated that the genomic fragment spanning –282 and +43 relative to the major transcriptional initiation site of NFBD1 gene is required for the transcriptional activation of NFBD1 gene. Within this region, we found out the putative Sp1- and NF-Y-binding sites. As described (Chu & Ferro 2005), Sp1 which contains a zinc finger DNA-binding domain at its COOH-terminal portion, recognizes GC boxes in promoter regions of its target genes and plays an important role in the regulation of a wide variety of cellular processes including cell cycle regulation and apoptotic cell death. The ubiquitous transcription factor NF-Y is a trimetric complex composed of NF-YA, NF-YB and NF-YC, each of which is required for CCAAT consensus sequence-binding and transactivation (Bellorini et al. 1997; de Silvio et al. 1999; Mantovani, 1999; Motta et al. 1999). According to our present results, site-directed disruption of the proximal Sp1-binding site significantly reduced the promoter activity of NFBD1 gene, whereas the mutations introduced into NF-Y-binding site had undetectable effects on the promoter activity of NFBD1 gene. Consistent with these results, siRNA-mediated knockdown of the endogenous Sp1 or MA treatment resulted in a decrease in the expression levels of NFBD1. Intriguingly, ADR treatment led to a reduction of NFBD1 expression levels in association with a remarkable decrease in the amounts of Sp1 recruited onto the Sp1-binding site. Collectively, our present results suggest that Sp1 is significantly involved in the ADR-mediated transcriptional regulation of NFBD1.

It is worth noting that, under normal conditions, Sp1 as well as NF-YB was efficiently recruited onto the promoter region of NFBD1 gene containing the putative Sp1-binding sites but not the canonical NF-Y-binding sites (left panels of Fig. 5B). In these cases, PCR primer set was designed to amplify the genomic region outside the putative NF-Y-binding sites. However, it is possible that the anti-NF-YB immunoprecipitates might contain slightly longer genomic fragments bearing the putative NF-Y-binding site as well as the neighboring Sp1-binding site and thereby generating the estimated size of PCR products. Alternatively, Roder et al. (1999) described that NF-Y has an ability to interact physically with Sp1. Our preliminary experiments indicated that the endogenous Sp1 is co-immunoprecipitated with the endogenous NF-YB (data not shown). In support of these results, our gel retardation assay using HeLa nuclear extracts revealed that the radio-labeled probe DNA (spanning from –120 to +43) forms a specific complex including Sp1 and NF-YB (data not shown). Furthermore, it has been shown that NF-Y binds to p300/CBP as well as P/CAF with intrinsic histone acetyltransferase (HAT) activity and facilitates transcription within chromatin by recruiting them (Wade et al. 1997; Li et al. 1998). In addition, Huang et al. (2005) found that Sp1 forms a multiprotein complex including p300, P/CAF and NF-Y. Thus, it is likely that Sp1 might cooperate with NF-Y to transactivate NFBD1 gene through the efficient recruitment of co-activators such as p300/CBP and/or P/CAF. Further studies should be necessary to address this issue.

As described above, ADR treatment caused a transcriptional repression of NFBD1 gene, which was accompanied with a remarkable dissociation of Sp1 from the Sp1-binding site. The expression levels of Sp1 remained unchanged regardless of ADR treatment. It has been demonstrated that Sp1 is subjected to post-translational modifications such as O-linked glycosylation, phosphorylation and acetylation (Jackson & Tjian 1988; Jackson et al. 1990; Huang et al. 2005). Feng & Kan (2005) described that phosphorylation of the NH2-terminal region of Sp1 enhances its transactivation function, whereas phosphorylation of Sp1 at Thr-579 results in its inactivation. Based on our present results, Sp1 was constitutively phosphorylated regardless of ADR treatment. Of note, immunoprecipitation analysis with anti-phosphoserine antibody demonstrated that phospho-Sp1 at Ser is induced in response to ADR or CDDP, suggesting that specific phosphorylation of Sp1 at as yet unidentified Ser residue(s) might contribute to the dissociation of Sp1 from the Sp1-binding site of NFBD1 gene in response to DNA damage. Alternatively, acetylation of Sp1 mediated by p300 and/or P/CAF enhances its transcriptional activity (Huang et al. 2005). Although it is still unknown whether ADR treatment could influence the acetylation status of Sp1, it is possible that ADR treatment might cause the conformational changes of multiprotein complex including Sp1, and thereby affecting their functional as well as physical interaction.

According to our present results, siRNA-mediated knockdown of endogenous Sp1 resulted in a significant down-regulation of NFBD1 gene in association with the increased sensitivity to ADR. These results were consistent with the previous observations showing that NFBD1 protects cells from apoptotic cell death induced by ionizing radiation (Peng & Chen 2003; Xu & Stern 2003). In addition, EGF treatment which sensitizes cells against ionizing radiation, resulted in a decrease in the DNA-binding activity of Sp1 (Gueven et al. 2001). Furthermore, an increased DNA-binding activity of Sp1 was observed in ADR-resistant cells (Borellini et al. 1990). Taken together, our current results suggest that Sp1-mediated transcriptional regulation of NFBD1 gene plays an important role in DNA damage response pathway.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell cultures

Human cervical carcinoma HeLa and human osteosarcoma U2OS cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and penicillin (100 IU/mL)/streptomycin (100 µg/mL). Human non-small cell lung carcinoma A549 cells were grown in RPMI 1640 medium with the same supplements. Cells were maintained at 37 ºC in a water-saturated atmosphere of 5% CO2 in air. Where indicated, cells were treated with adriamycin (ADR) or cisplatin (CDDP).

Rapid amplification of cDNA ends (RACE) analysis

The 5'-RACE analysis was carried out using reagents in the SMARTTM RACE cDNA amplification kit (Clontech Laboratories, Mountain View, CA), following the general guidelines provided by the kit. For 5'-RACE, the anti-sense NFBD1 oligodeoxynucleotides (GSP1, 5'-GCCTCTACAGTGGCACCTCTTCTG-3'; GSP2, 5'-CATCAGTGTCGCTGTCGATGAAGC-3') corresponding to positions 711–735 and 973–997 downstream from the initiation codon were synthesized. Total RNA was reverse-transcribed using a modified lock-docking oligonucleotide (dT) primer and BD SMART II A oligonucleotide to obtain first-strand cDNA. PCR-based amplification was performed using the gene-specific primer (GSP1 or GSP2) and adaptor primer (AP1). PCR products were gel-purified using QIAquick gel extraction kit (Qiagen, Valencia, CA), cloned into pGEM-T Easy Vector (Promega, Madison, WI) and sequenced using an Applied Biosystems 3730 DNA analyzer (Applied Biosystems, Foster City, CA) to determine the transcription start site(s).

RNA isolation and RT-PCR

Total RNA was prepared from the indicated cells using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. cDNA was generated from 3 to 5 µg of total RNA using SuperScript II reverse transcriptase and random primers following the manufacturer's conditions (Invitrogen). PCR-based amplification was carried out under standard conditions with rTaq DNA polymerase (Takara, Ohtsu, Japan). Regions of NFBD1 and GAPDH cDNAs were amplified using the following primer sets: NFBD1, 5'-AGCAACCCCAGTTGTCATTC-3' and 5'-AGCGCTGCTGAGACTTCTTC-3'; GAPDH, 5'-ACCTGACCTGCCGTCTAGAA-3' and 5'-TCCACCACCCTGTTGCTGTA-3'. To control for the integrity and uniformity of the sample preparation, GAPDH mRNA was amplified. PCR products were separated by 2.0% agarose gel electrophoresis and stained with ethidium bromide.

Immunoblot analysis

Cells were washed in ice-cold PBS and lysed in lysis buffer containing 25 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1% Triton X-100 and protease inhibitor mixture (Sigma, St. Louis, MO) at 4 ºC for 30 min followed by brief sonication. After centrifugation at 10 000 g at 4 ºC for 10 min to remove insoluble materials, supernatants were collected and their protein concentrations were measured by Bio-Rad protein assay system (Bio-Rad Laboratories, Hercules, CA). Equal amounts of whole cell lysates were boiled in an SDS-sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% β-mercaptoethanol and 0.01% bromophenol blue) for 5 min, separated by 6%–8% SDS-PAGE and electro-transferred onto Immobilon-P membranes (Millipore, Bedford, MA). After saturation, the membranes were blocked with TBS-T containing 5% non-fat dry milk at room temperature for 1 h and subsequently incubated with polyclonal anti-NFBD1 (Abcam, Cambridge, UK), polyclonal anti-Sp1 (Upstate, Lake Placid, NY) or with polyclonal anti-actin (20–33, Sigma) antibody, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Cell Signaling, Beverly, MA). After washing with TBS-T, the membranes were developed using an enhanced chemifluorescence detection system (Amersham Biosciences, Uppsala, Sweden).

Cloning of the NFBD1 promoter region

To amplify the genomic DNA fragment encompassing a 5'-flanking region, exon 1 and intron1 of human NFBD1 gene, we amplified regions corresponding to –2920 to –436, –1040 to +489 and +136 to +2133 using primer sets: A1, 5'-ACCGAGCTCGCCCTGATTAGTCGATGC-3' and 5'-CTAGCTAGCTGGAGAACAGCCCGTAG-3'; A2, 5'-GGGGTACCAAGAGCGAAAGTCCGTCTC-3' and 5'-CTAGCTAGCACCGAGCCTACTGATGAAG-3'; A3, 5'-GGGGTACCTTGGGTGCGACTGGGC-3' and 5'-CTAGCTAGCGATCTGGGAAGGATACACATT-3', respectively. Underlined nucleotides in the oligonucleotide listed above were SacI, NheI or KpnI restriction sites. The PCR products were gel-purified using the QIAquick gel extraction kit (Qiagen) and digested completely with SacI and PacI, PacI and EcoRI or with EcoRI and NheI. The luciferase reporter plasmid termed P(–2920/+2133) was constructed by joining the above-mentioned three restriction fragments, subsequently cloning into SacI and NheI restriction sites of pGL3-basic luciferase reporter vector (Promega) and validated by sequencing.

Site-directed mutagenesis

The luciferase reporter constructs including P(–282/+43)-Sp1M1, P(–282/+43)-Sp1M2, P(–282/+43)-Sp1M1/2, P(–282/+43)-NFYM1, P(–282/+43)-NFYM2 and P(–282/+43)-NFYM1/2 were generated by GeneTailor Site-Directed Mutagenesis System (Invitrogen) on the basis of the parental construct P(–282/+43) according to the manufacturer's instructions. The following primer sets were used: P(–282/+43)-Sp1M1, 5'-GCGCGGGAAGTGGGTGGTGGATGGGCAAGCGGT-3' and 5'-CCACCACCCACTTCCCGCGCAGTTCCAAAC-3'; P(–282/+43)-Sp1M2, 5'-CCTATTTGAACTCTAAGGGGATGGGGCTTTGGG-3' and 5'-CCCCTTAGAGTTCAAATAGGTGGTGTCTCC-3'; P(–282/+43)-NFYM1, 5'-GTTGTCCCTTGGAGCTGCCCTTTCGACGTGCAT-3' and 5'-GGGCAGCTCCAAGGGACAACCCACTACCGC-3'; P(–282/+43)-NFYM2, 5'-GAAAGACAGCGGCAGAAGCCTTTCAGCAAATAAG-3' and 5'-GGCTTCTGCCGCTGTCTTTCACAACCGCAG-3'. The mutations were verified by DNA sequencing.

Luciferase reporter gene analysis

Cells were seeded in triplicates into 12-well plates 24 h before transfection at a density of 5 x 104 cells per well. Cells were transiently co-transfected with 100 ng of the indicated reporter plasmids, 10 ng of pRL-TK plasmid (Promega) encoding Renilla luciferase and 390 ng of the empty plasmid pcDNA3 (Invitrogen) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were lysed with passive lysis buffer and their luciferase activities were measured using the Dual-luciferase assay system (Promega) according to the manufacturer's protocol. The transfection efficiency was standardized against Renilla luciferase activity.

Chromatin immunoprecipitation assay (ChIP)

ChIP assay was performed according to the protocol provided by Upstate. In brief, HeLa cells were treated with 1% formaldehyde at 37 ºC for 15 min. Cells were then washed twice in ice-cold PBS and resuspended in 200 µL of SDS-sample buffer containing protease inhibitor mixture. The suspension was sonicated on ice until cross-linked chromatin was sheared to an average DNA fragment length of 200–500 bp. After centrifugation, soluble cross-linked chromatin was diluted in immunoprecipitation buffer and precleared with 20 µL of protein A-agarose beads blocked with sonicated salmon sperm DNA for 30 min at 4 ºC under rotation. The protein A-agarose beads were then removed by centrifugation and the chromatin solution was immunoprecipitated with preimmune serum, polyclonal anti-NF-YB (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal anti-Sp1 or with polyclonal anti-STAT-1 antibody (Santa Cruz Biotechnology) overnight at 4 ºC. The immunoprecipitates were eluted with 100 µL of elution buffer and de-cross-linked by heating at 65 ºC for 6 h. Proteinase K was then added to the reaction mixtures and incubated at 45 ºC for 1 h. DNAs of the immunoprecipitates and control input DNAs were purified using a QIAquick PCR Purification kit (Qiagen) and analyzed by standard PCR using the following primer sets: F(–309), 5'-ACACAGCCCCTCTATCCGTTGC-3' and R(–199), 5'-CAAGGGACAACCCACTACCG-3'; F(–212), 5'-TGGGTTGTCCCTTGGAGC-3' and R(–110), 5'-TGTCTCCCAGGCTGCTGA-3'; F(–156), 5'-GCCTTCAATTACCGTCTC-3' and R(–34), 5'-CGCTGTCTTTCACAACCG-3'; F(–62), 5'-TCGACTGGCTGCGGTTGT-3' and R(+43), 5'-GCCACCAGTAACGGTCGC-3'.

Phosphatase treatment

Whole cell lysates prepared from HeLa cells were immunoprecipitated with polyclonal anti-Sp1 antibody at 4 ºC overnight. The immunoprecipitates were washed extensively with the lysis buffer and then incubated with 2000 units of {lambda}-phosphatase (New England Biolabs, Herts, UK) in the presence of 2 mM MnCl2 at 30 ºC for 2 h. The bound dephosphorylated proteins were eluted in SDS sample buffer, resolved by 10% SDS-PAGE and analyzed by immunoblotting with polyclonal anti-Sp1 antibody.

RNA interference

The small interfering RNA against Sp1 and the nonspecific siRNA were purchased from Santa Cruz Biotechnology. Sp1 or control siRNA was transfected into the indicated cells using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer's instructions. Cells were collected and subjected to subsequent analysis 72 h after transfection.

Cell survival assay

For cell survival assay, HeLa cells transiently transfected with an Sp1-specific siRNA for 48 h were seeded in 96-well plates at a concentration of 5 x 103 cells per well and allowed to grow in standard culture medium. Forty-eight hours after the treatment with ADR, cell viability was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay-based cell counting kit-8 (Dojindo, Kumamoto, Japan). The percentage of cell survival was determined by estimating the value of untreated cells as 100%.


   Acknowledgements
 
We thank Dr T. Kamijo for critical reading of the manuscript. We also thank Ms Y. Nakamura for her excellent technical assistance. This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare of Japan for the Third Term Comprehensive Control Research for Cancer, a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Uehara Memorial Foundation, Japan and Hisamitsu Pharmaceutical Co. Bu Y.Q. is a recipient of China state scholarship fund award.


   Footnotes
 
Communicated by: Takeo Kishimoto

* Correspondence: E-mail: akiranak{at}chiba-cc.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Bellorini, M., Lee, D.K., Dantonel, J.C., Zemzoumi, K., Roeder, R.G., Tora, L. & Mantovani, R. (1997) CCAAT binding NF-Y-TBP interactions: NF-YB and NF-YC require short domains adjacent to their histone fold motifs for association with TBP basic residues. Nucleic Acids Res. 25, 2174–2181.[Abstract/Free Full Text]

Borellini, F., Aquino, A., Josephs, S.F. & Glazer, R.I. (1990) Increased expression and DNA-binding activity of transcription factor Sp1 in doxorubicin-resistant HL-60 leukemia cells. Mol. Cell. Biol. 10, 5541–5547.[Abstract/Free Full Text]

Celeste, A., Fernandez-Capetillo, O., Kruhlak, M.J., Pilch, D.R., Staudt, D.W., Lee, A., Bonner, R.F., Bonner, W.M. & Nussenzweig, A. (2003) Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat. Cell Biol. 5, 675–679.[CrossRef][Medline]

Celeste, A., Petersen, S., Romanienko, P.J., et al. (2002) Genomic instability in mice lacking histone H2AX. Science 296, 922–927.[Abstract/Free Full Text]

Chu, S. & Ferro, T.J. (2005) Sp1: regulation of gene expression by phosphorylation. Gene 348, 1–11.[CrossRef][Medline]

D’Amours, D. & Jackson, S.P. (2002) The Mre11 complex: at the crossroads of DNA repair and checkpoint signaling. Nat. Rev. Mol. Cell Biol. 3, 317–327.[CrossRef][Medline]

de Silvio, A., Imbriano, C. & Mantovani, R. (1999) Dissection of the NF-Y transcriptional activation potential. Nucleic Acids Res. 27, 2578–2584.[Abstract/Free Full Text]

Feng, D. & Kan, Y.W. (2005) The binding of the ubiquitous transcription factor Sp1 at the locus control region represses the expression of β-like globin genes. Proc. Natl. Acad. Sci. USA 102, 9896–900.[Abstract/Free Full Text]

Fernandez-Capetillo, O., Chen, H.T., Celeste, A, Ward, I., Romanienko, P.J., Morales, J.C., Naka, K., Xia, Z., Camerini-Otero, R.D., Motoyama, N., Carpenter, P.B., Bonner, W.M., Chen, J. & Nussenzweig, A. (2002) DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1. Nat. Cell Biol. 4, 993–997.[CrossRef][Medline]

Goldberg, M., Stucki, M., Falck, J., D’Amours, D., Rahman, D., Pappin, D., Barte, J. & Jackson, S.P. (2003) MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature 421, 952–956.[CrossRef][Medline]

Gueven, N., Keating, K.E., Chen, P., Fukao, T., Khanna, K.K., Watters, D., Rodemann, P.H. & Lavin, M.F. (2001) Epidermal growth factor sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia. J. Biol. Chem. 276, 8884–8891.[Abstract/Free Full Text]

Huang, W., Zhao, S., Ammanamanchi, S., Brattain, M., Venkatasubbarao, K. & Freeman, J.W. (2005) Richostatin A induces transforming growth factor β type II receptor promoter activity and acetylation of Sp1 by recruitment of PCAF/p300 to a Sp1–NF–Y complex. J. Biol. Chem. 280, 10047–10054.[Abstract/Free Full Text]

Jackson, S.P. & Tjian, R. (1988) O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell 55, 125–133.[Medline]

Jackson, S.P., MacDonald, J.J., Lees-Miller, S.P. & Tjian, R. (1990) GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell 63, 155–165.[CrossRef][Medline]

Kastan, M.B. & Bartek, J. (2004) Cell-cycle checkpoints and cancer. Nature 432, 316–323.[CrossRef][Medline]

Kim, C.S., Choi, H.S., Hwang, C.K., Song, K.Y., Lee, E.K., Law, P.Y., Wei, L.N. & Loh, H.H. (2006) Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the µ opioid receptor (MOR) gene. Nucleic Acids Res. 34, 6392–6403.[Abstract/Free Full Text]

Lee, M.S., Edwards, R.A., Thede, G.L. & Glover, J.N. (2005) Structure of the BRCT repeat domain of MDC1 and its specificity for the free COOH-terminal end of the {gamma}-H2AX histone tail. J. Biol. Chem. 280, 32053–32056.[Abstract/Free Full Text]

Li, Q., Herrler, M., Landsberger, N., Kaludov, N., Ogryzko, V.V., Nakatani, Y. & Wolffe, A.P. (1998) Xenopus NF-Y pre-sets chromatin to potentiate p300 and acetylation-responsive transcription from the Xenopus hsp70 promoter in vivo. EMBO J. 17, 6300–6315.[CrossRef][Medline]

Lou, Z., Minter-Dykhouse, K., Franco, S., Gostissa, M., Rivera, M.A., Celeste, A., Manis, J.P., van Deursen, J., Nussenzweig, A., Paull, T.T., Alt, F.W. & Chen, J. (2006) MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. Mol. Cell 2, 187–200.[Medline]

Mantovani, R. (1999) The molecular biology of the CCAAT-binding factor NF-Y. Gene 239, 15–27.[CrossRef][Medline]

Motta, M.C., Carreti, G., Badaracco, G.F. & Mantovani, R. (1999) Interactions of the CCAAT-binding trimer NF-Y with nucleosomes. J. Biol. Chem. 274, 1326–1333.[Abstract/Free Full Text]

Nagase, T., Seki, N., Ishikawa, K., Tanaka, A. & Nomura, N. (1996) Prediction of the coding sequences of unidentified human genes V: The coding sequences of 40 new genes (KIAA0161–KIAA0200) deduced by analysis of cDNA clones from human cell line KG-1.DNA Res. 3, 17–24.[Abstract]

Nakanishi, M., Ozaki, T., Yamamoto, H., Hanamoto, T., Kikuchi, H., Furuya, K., Asaka, M., Delia, D. & Nakagawara, A. (2007) NFBD1/MDC1 associates with p53 and regulates its function at the crossroad between cell survival and death in response to DNA damage J. Biol. Chem. 282, 22993–23004.[Abstract/Free Full Text]

Ozaki, T., Nagase, T., Ichimiya, S., Seki, N., Ohira, M., Nomura, N., Takada, N., Sakiyama, S., Weber, B.L. & Nakagawara, A. (2000) NFBD1/KIAA0170 is a novel nuclear transcriptional transactivator with BRCT domain. DNA Cell Biol. 19, 475–485.[CrossRef][Medline]

Paulli, T.T., Rogakou, E.P., Yamazaki, V., Kirchgessner, C.U., Gellert, M. & Bonner, W.M. (2000) A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895.[CrossRef][Medline]

Peng, A. & Chen, P.L. (2003) NFBD1, like 53BP1, is an early and redundant transducer mediating Chk2 phosphorylation in response to DNA damage. J. Biol. Chem. 278, 8873–8876.

Roder, K., Wolf, S.S., Larkin, K.J. & Schweizer, M. (1999) Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1. Gene, 234, 61–69.[CrossRef][Medline]

Rogakou, E.P., Boon, C., Redon, C. & Bonner, W.M. (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916.[Abstract/Free Full Text]

Shiloh, Y. (2003) ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer 3, 155–168.[CrossRef][Medline]

Stewart, G.S., Wang, B., Bignell, C.R., Taylor, A.M. & Elledge, S.J. (2003) MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421, 961–966.[CrossRef][Medline]

Stucki, M. & Jackson, S.P. (2004) MDC1/NFBD1: a key regulator of the DNA damage response in higher eukaryotes. DNA Repair 3, 953–957.[CrossRef][Medline]

Stucki, M., Clapperton, J.A., Mohammad, D., Yaffe, M.B., Smerdon, S.J. & Jackson, S.P. (2005) MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123, 1213–1226.[CrossRef][Medline]

Townsend, P.A., Cragg, M.S., Davidson, S.M., McCormick, J., Barry, S., Lawrence, K.M., Knight, R.A., Hubank, M., Chen, P.L., Latchman, D.S. & Stephanou, A. (2005) STAT-1 facilitates the ATM activated checkpoint pathway following DNA damage. J. Cell Sci. 118, 1629–1639.[Abstract/Free Full Text]

Wade, P.A., Pruss, D. & Wolffe, A.P. (1997) Histone acetylation: chromatin in action. Trends Biochem.Sci. 22, 128–132.[CrossRef][Medline]

Xu, X. & Stern, D.F. (2003) NFBD1/MDC1 regulates ionizing radiation-induced focus formation by DNA checkpoint signaling and repair factors. FASEB J. 17, 1842–1848.[Abstract/Free Full Text]

Received: 28 June 2007
Accepted: 15 October 2007





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bu, Y.
Right arrow Articles by Ozaki, T.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Bu, Y.
Right arrow Articles by Ozaki, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS