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Molecular Cancer Research 4:635-643 (2006)
© 2006 American Association for Cancer Research

Signaling and Regulation

Nuclear Factor Y Drives Basal Transcription of the Human TLX3, a Gene Overexpressed in T-Cell Acute Lymphocytic Leukemia

Silvia Borghini1, Manuela Vargiolu1, Marco Di Duca2, Roberto Ravazzolo1,3 and Isabella Ceccherini1

1 Laboratorio di Genetica Molecolare and 2 Laboratorio di Fisiopatologia dell'Uremia, Istituto Giannina Gaslini; 3 Dipartimento di Pediatria e CEBR, Università di Genova, Genova, Italy

Requests for reprints: Isabella Ceccherini, Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, L.go Gerolamo Gaslini, 5, 16148 Genova, Italy. Phone: 39-10-5636800; Fax: 39-10-3779797. E-mail: isa.c{at}unige.it.


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Based on a knocked-out mouse model and a few expression studies, TLX3 is regarded as a homeobox gene crucial for the development of the autonomic nervous system. This gene can undergo rearrangements or deregulation, giving rise to T-cell acute lymphocytic leukemia. The present report is focused on the identification of elements and factors playing a role in the TLX3 physiologic expression regulation and therefore likely to be involved in cancer development. In particular, after identifying the transcription start points, we have made use of in vitro transfection assays to show that the 5'-untranslated region of the gene is necessary for the basal promoter activity in cell lines from different origin. By site-directed mutagenesis, two tandem CCAAT boxes have been localized as critical elements of this region. In vivo chromatin immunoprecipitation and electrophoretic mobility shift assays have indicated that nuclear factor Y (NFY) recognizes these sites in all the analyzed cell lines. The physiologic role of such an interaction has been confirmed by a dominant-negative version of the NFY transcription factor that has turned out to decrease both in vitro TLX3 promoter activity and endogenous amount of mRNA. Finally, a consistent in vivo TLX3 expression impairment was also achieved after NFY mRNA knockdown. The full characterization of the TLX3 transcription regulation will ultimately provide crucial elements to define the involvement of this gene in T-cell acute lymphocytic leukemia development. (Mol Cancer Res 2006;4(9):635–43)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
TLX3 (also known as HOX11L2 or Rnx) belongs to the TLX family of homeodomain transcription factors that includes two other members: TLX1 (also known as HOX11) and TLX2 (also known as HOX11L1, Ncx, or Enx). Members of this gene family exhibit restricted, and often overlapping, patterns of expression, including developing neural crest-derived tissues, as shown by in situ hybridization studies (1-4). TLX3 is uniquely expressed in the developing medulla oblongata and is required for proper formation of first-order relay visceral sensory neurons and of most of the (nor)adrenergic centers in the brainstem, especially involved in the physiologic control of cardiovascular and respiratory systems (5). Accordingly, TLX3-deficient mice displayed hypoventilation, sudden apneas, and cyanosis, and all died within 24 hours after birth from a hypothesized central defect in respiratory control, thus having been regarded as a model for congenital central hypoventilation syndrome (6), but extensive screening of this gene failed to detect any mutation in patients (7, 8).

Expression of TLX3 has also been detected in leukemia samples from 20% of children and 13% of adults affected with T-cell acute lymphocytic leukemia (9-12), although this gene has never been involved in normal T-cell differentiation (13). The TLX3 ectopic expression has been associated mainly with the occurrence of chromosomal translocations involving 5q35 (14, 15), where the gene is located (16, 17), but it is still unclear whether the underlying molecular pathogenetic mechanism relies either in the loss of negative regulatory elements on chromosome 5q or in the juxtaposition of positive transcriptional regulatory elements of genes lying on the partner chromosomes. To date, no detailed information is available regarding the biology of the TLX3 gene and cellular pathways in which it is involved, although based on its specific spatiotemporal expression and the correlation between up-regulation and development of T-cell acute lymphocytic leukemia, a role of this gene in either cell cycle, survival, or differentiation can be postulated.

As a first step to identify factors and DNA elements crucial for the transcriptional regulation of the TLX3 gene and able to account for both neuronal and leukemia development, we have focused on its 5'-regulatory region. In particular, we have showed that the 5'-untranslated region (UTR) of TLX3 contains the most important cis-acting elements required for its expression and identified the transcription factor nuclear factor Y (NFY) as the main regulator binding this sequence.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Determination of Transcriptional Start Sites
To identify the transcription start site(s) of TLX3, we took advantage of a protocol for the automated detection of fluorescent primer extension products, adapted from the standard method based on radioactive labeling (18, 19) and already successfully applied to other genes (20, 21).

Initial experiments were done to set up the system using variable amounts of RNA (2.5, 5, and 10 µg) extracted from IMR32 cells, which express TLX3, and two different concentrations of fluorescent primer (1 or 10 µmol/L). We run the primer extension reactions together with a molecular weight marker, and this allowed to observe that signals obtained from different primer extension products were always in the same position, with an intensity proportional to the amount of RNA used (data not shown). With 10 µmol/L primer concentration, nonspecific background fluorescence occurred in the form of an increased baseline, thus suggesting 1 µmol/L as the most appropriate concentration for subsequent experiments. To obtain a precise determination of the transcriptional start point(s), we also run the primer extension reactions together with the DNA fragments generated in a sequencing reaction carried out with the same but unlabeled oligonucleotide (20). The peak detected 272 nucleotides upstream of the "ATG" translation start codon (Fig. 1, peak 2 ) showed the highest intensity and was therefore regarded as major transcription start point. According to the notion that the presence of multiple transcription start points is a common feature of TATA-less promoters (22), three additional peaks of minor intensity were detected (Fig. 1, peaks 1, 3, and 4, respectively). Interestingly, the major transcription start point, as well as the start site 1, is located upstream of two CCAAT boxes, whereas start sites 3 and 4 map between them.


Figure 1
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  		FIGURE 1. Determination of the transcriptional start sites of the TLX3 gene. Top box, DNA sequence obtained with the unlabeled primer; middle and bottom boxes, chromatograms of two cell lines (IMR32 and SH-SY5Y, respectively) and have been obtained loading in the same well the sequencing reaction and the primer extension product carried out with the 6-FAM primer. The fluorograms are reversed to match the sequence of the coding strand shown in Fig. 2. Transcription start points can be recognized as additional peaks corresponding to the C nucleotide (complementary to the G nucleotide revealed by 6-FAM fluorescence) laying on the original sequence and marked by arrows. Based on the intensity, peak 2 has been considered the major transcription start point.

 

Figure 2
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  		FIGURE 2. Sequence conservation analysis between human and mouse TLX3 genes. Y axis, percentage of sequence identity detected between human and mouse using the mVISTA software; X axis, reference DNA segment. Black bar arrow above the plot, TLX3 gene; blue rectangles, exons; light blue rectangles, UTRs. SINE and other repetitive elements are also indicated. Conserved regions in which every contiguous subsegment of 100-bp length was at least 60% identical to its paired sequence are highlighted in blue, pink, and light blue for coding, noncoding, and UTR regions, respectively. Box below the plot, 1,311 bp of the 5'-region of human TLX3 selected for the study. Arrows, distal limit of each of the deleted constructs used in the transfection assays. The CCAAT repetitions are boxed. Positions are numbered from the major transcription start point of the gene (+1; red arrow).

 
We could confirm these results in additional experiments done using RNA from SHSY-5Y neuroblastoma cells, which express TLX3, and RNA obtained from a second extraction from IMR32 cells (Fig. 1).

Comparison between Mouse and Human TLX3 Gene Sequences
To characterize crucial elements involved in the transcription regulation of the TLX3 gene, we run computer-assisted analysis finalized to identify evolutionary conserved sequences and putative binding sites for specific factors using mVISTA (http://pipeline.lbl.gov/cgi-bin/gateway2) and MatInspector version 2.2 software (23), respectively. As shown in Fig. 2 , by comparing 5 kb of human and mouse DNA sequences corresponding to the complete TLX3 gene and its 5'-flanking region, we observed an overall degree of conservation of >75% in the 1,300 bp located immediately upstream of the "ATG" translation start codon. In particular, 360 bp, including the 5'-UTR, showed an identity of 98.3%. In this region, MatInspector pointed out the lack of conventional TATA boxes and the presence of two tandem identical CCAAT boxes.

Functional Characterization of the Promoter Activity of the Human TLX3 Gene
We have cloned 1,311 bp of the 5'-region adjacent to the ATG of the human TLX3 gene mentioned above into the pGL3-basic vector, upstream of the reporter firefly luciferase gene, as a first step to study the promoter activity by transient cellular transfections. Because TLX3 is specifically expressed in neural crest-derived tissues, we used as cell recipients two neuroblastoma cell lines that express the gene, SHSY-5Y and IMR32, and two cell lines from different lineages that do not express the gene, the adenocarcinoma MCF-7 and the osteosarcoma SaOS-2. The construct displayed well-detectable transcriptional activity in all the tested cell lines, ranging from 25% in MCF-7 to 80% in SaOS-2 relative to a control plasmid containing the SV40 promoter, thus confirming that the fragment under analysis contains regulatory sequence of the human TLX3 gene (data not shown). To establish the contribution of the cis-acting elements identified by computer-assisted analysis to the promoter activity, we transfected six deletion constructs obtained by removing DNA fragments of increasing lengths at the distal end of the region under investigation. As shown in Fig. 3 , only the deletion mapping at +98 affected transcription, leading to statistically significant loss of detectable activity of the reporter gene, whereas the removal of shorter fragments had no negative effect. Actually, the sequence +32/+98 was sufficient to guarantee a level of luciferase expression comparable with that obtained transfecting the whole 1,311-bp 5'-region in all the tested cell lines (Fig. 3, top construct), thus suggesting that crucial elements for the transcriptional activation of TLX3 localize in this proximal sequence, a region showing a very high level of conservation during evolution, as already observed (Fig. 2). The sequence +32/+98 is characterized by the presence of two "CCAAT" boxes whose site-directed mutagenesis reduced the promoter activity to <50% of that displayed by the wild-type (WT) 1,311-bp sequence in all the analyzed cell lines (Fig. 4 ). A further reduction was observed when both sites were mutated, suggesting that transcription factors binding these sites do have an additive effect.


Figure 3
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  		FIGURE 3. Functional analysis of the 5'-regulatory sequence of human TLX3. Transient transfections of constructs containing progressively deleted fragments of the promoter fused to firefly luciferase gene were carried out in four different cell lines. Y axis, nucleotide position, relative to the major start point, of the 5'-end of each construct; X axis, relative luciferase activity corrected for luciferase activity of the promoter-less pGL3 vector. Asterisk, the only deletion construct that induced, in all the four cell lines under analysis, a statistically significant decrease in the reporter gene expression levels with respect to the full-length reporter-promoter construct (–1,039 bp). In particular, the following probabilities (P) were estimated: 0.00023 (SHSY-5Y), 0.019 (IMR32), 0.022 (SaOS-2), and 0.00021 (MCF-7).

 

Figure 4
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  		FIGURE 4. Site-directed mutagenesis of the CCAAT boxes. A. Transcriptional luciferase activity driven by mutated constructs, relative to the activity of the WT construct (100%), with numerical values. Decreases in the reporter gene expression due to presence of single or double mutations were all highly statistically significant. B. Sequence diversity of the three mutant constructs tested. In particular, the distal CCAAT box 1 site is mutated in MUT1, the proximal CCAAT box 2 site is mutated in MUT2, and both CCAAT boxes are mutated in MUT3.

 
Interestingly, the two CCAAT boxes, responsible for a basal transcriptional activity in TLX3-expressing and nonexpressing cells, are located in the 5'-UTR, a gene region that therefore seems necessary for inducing TLX3 expression.

NFY Binding the +32/+98 Sequence
Matrix sequences of the CCAAT boxes in the proximal 5'-regulatory region of the TLX3 gene were indicated as peculiar of NFY binding following MatInspector analysis. To investigate the possible in vivo interaction between NFY and the above sequence, we did chromatin immunoprecipitation assays using formaldehyde cross-linked and sonicated chromatin from SHSY-5Y, MCF-7, and IMR32 cells. PCR results (Fig. 5 ) showed that an antibody specific for the A subunit of NFY effectively immunoprecipitated the TLX3 regulatory sequence under analysis (–95/+189) from all these cell lines. Control assays, done in SHSY-5Y cells both without antibody and in the presence of different unrelated antibodies ({alpha}-p53, {alpha}-E2F-1, {alpha}-ß-actin, and normal mouse IgG), and in MCF-7 cells using {alpha}-ß-actin did not show any significant DNA amplified from the same region. Finally, an amplification product was obtained also from SHSY-5Y and MCF-7 nucleoproteins precipitated by an {alpha}-acetyl-histone H4 antibody, thus suggesting that this region is characterized by an open configuration chromatin even in cells that do not express TLX3 (Fig. 5).


Figure 5
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  		FIGURE 5. In vivo interaction between NFY and 5'-regulatory region of the TLX3 gene. Chromatin immunoprecipitation assays done on chromatin derived from SHSY-5Y, IMR32, and MCF-7 cells. Primers specific for –95/+189 sequence were used to amplify DNA from complexes immunoprecipitated with an {alpha}-NFYA subunit antibody ({alpha}-NFY). INPUT, fragmented DNA before immunoprecipitation; NoAb, no antibody immunoprecipitation control; {alpha}-acH4, antibody against hyperacetylated histone H4; {alpha}-act, antibody against ß-actin.

 
To clarify whether NFY binds both CCAAT boxes, electrophoretic mobility shift assays (EMSA) were done using nuclear extracts prepared from IMR32 cells. When two oligonucleotides spanning regions corresponding to either CCAAT boxes 1 and 2 were incubated with IMR32 nuclear extract, one major complex was detected in both cases (Fig. 6, lanes 1 and 5 ). The sequence-specific nature of this interaction was confirmed through abolition of the complex by a 200-fold molar excess of unlabeled WT competitor (Fig. 6, lanes 4 and 6). Oligonucleotides bearing the mutations tested in transfection experiments had no effect in the analysis of CCAAT box 1 (Fig. 6, lane 3) but displayed an effect on CCAAT box 2 although weaker than the WT competitor (Fig. 6, lane 7). However, in both cases, preincubation with an antibody specific for NFY subunit A induced the supershift of the retarded bands, showing the involvement of NFY in binding the two CCAAT boxes (Fig. 6, lanes 2, 8, and 9). The specificity of such interactions is well proven by the observation that the supershifted bands were competed by a 200-fold molar excess of the unlabeled WT oligonucleotides (data not shown for CCAAT box 1; Fig. 6, lane 10 for CCAAT box 2). Because the mutant CCAAT box 2 oligonucleotide partially competed with the probe (Fig. 6, lane 11), to confirm the specificity of the interaction between NFY and the CCAAT box 2 sequence, we incubated the reaction mixture with an unrelated oligonucleotide containing the consensus binding site for Sp1 (Fig. 6, lane 12). Differently from what expected in nonspecific interaction between DNA, proteins, and antibody, the unrelated Sp1 oligonucleotide was not able to destabilize this complex, confirming the NFY-specific recognition of the CCAAT box 2.


Figure 6
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  		FIGURE 6. In vitro interaction between NFY and CCAAT boxes. EMSA assays were done incubating nuclear extracts from IMR32 cells with biotin-labeled double-stranded oligonucleotide containing either CCAAT box 1 or 2. WT, WT competitor 200x; MUT, mutated competitor 200x; Sp1, competitor 200x representing the Sp1 consensus binding sequence; Ab, antibody {alpha}-NFYA; SS, supershifted band; asterisk, major complex.

 
Effect of a Dominant-Negative Version of NFYA
We did experiments in which the complete 5'-regulatory region under analysis (–1,039LUC) or the identified minimal sequence (+32LUC) was cotransfected in SHSY-5Y neuroblastoma cells and MCF-7 adenocarcinoma cells together with an expression plasmid containing {Delta}NFYA13m19, which is characterized by a NFY dominant-negative activity already described (24). As shown in Fig. 7A , overexpression of {Delta}NFYA13m29 resulted in marked reduction of luciferase activity induced by both the long and the short promoter constructs with respect to control cotransfections done using the {Delta}NFYA13 WT construct.


Figure 7
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  		FIGURE 7. Effect of NFY dominant-negative protein on the TLX3 gene transactivation. A. Luciferase activity of either –1040LUC or +31LUC sequences was measured on lysates from SHSY-5Y and MCF-7 cells cotransfected with expression plasmid containing WT ({Delta}NFYA13) or dominant-negative NFYA ({Delta}NFYA13m29). Fold induction versus activity derived from transfection of pGL3-basic vector alone. *, P ≤ 0.05, significant levels of difference between WT and mutant NFY constructs. B. Western blot obtained using 4 µg lysates from cells transfected with either the empty expression vector, the expression plasmid containing WT ({Delta}NFYA13), or the dominant-negative NFYA ({Delta}NFYA13m29). {alpha}-act, antibody specific for ß-actin. C. Endogenous levels of TLX3 mRNA were measured by real-time PCR on cDNA obtained from SHSY-5Y cells transfected with the above plasmids. Columns, TLX3 transcript quantity relative to that of mock-transfected cells. Only the mutant construct has resulted to significantly decrease the TLX3 transcript (*, P < 0.01).

 
Moreover, we investigated the effect of the same dominant-negative version of NFY on the endogenous level of TLX3 mRNA. After transfection of SHSY-5Y cells with both {Delta}NFYA13 expression plasmids, comparable levels of mutant and WT NFY proteins, clearly higher than those associated with the empty vector transfection, were induced (Fig. 7B). Subsequently, RNA extraction, cDNA synthesis, and real-time PCR allowed to reveal a relative level of TLX3 mRNA in the dominant-negative {Delta}NFYA13m29-transfected cells dramatically lower than that obtained with the empty vector (Fig. 7C). On the other hand, as expected, because NFY works as a trimer, the {Delta}NFYA13 WT construct does not have any effect on the TLX3 transcription. Taken together, these observations confirm the functional role played by NFY in the transcription regulation of TLX3.

Effect of NFYA Knockdown
To definitely confirm the in vivo role of NFY in TLX3 gene regulation, we carried out the knockdown of NFY subunit A by a specific small interfering RNA pool. As shown in Fig. 8 , these small interfering RNAs, transfected into SHSY-5Y, hindered the synthesis of the targeted transcription factor determining a specific effect on both mRNA (Fig. 8A) and protein (Fig. 8B). As expected, when the NFYA expression was reduced, the level of TLX3 expression was significantly diminished with respect to that obtained after the transfection of a control small interfering RNA pool.


Figure 8
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  		FIGURE 8. NFYA knockdown. A. NFYA and TLX3 transcript levels in SHSY-5Y cells were normalized to the transcript level of ß2-microglobulin and expressed as fold to the mock-transfected SHSY-5Y. Columns, mean of four experiments each done in triplicate; bars, SD. *, P < 0.05; **, P < 0.01 (t test). B. Western blot of NFYA protein level in 4 µg lysates from cells transfected with either siNFYA or siControl. Filter was reprobed with {alpha}-ß-actin antibody as control of total lysates quantity.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
TLX3 is a homeobox gene that plays a fundamental role during the development of nervous tissues derived from neural crest cells. Deregulated expression of this gene is also detected in several patients affected with T-cell acute lymphocytic leukemia. Thus far, nothing is known about the regulation of TLX3 expression both under physiologic conditions, during embryogenesis, and in pathology, during leukemia onset.

In the current study, we have focused on mechanism(s) regulating transcription of the human TLX3 gene and showed a pivotal role of the 5'-UTR. A similar location of crucial binding sites for transcription factors has already been reported for other genes as exemplified by the human plasminogen gene (25), the A {gamma}-globin gene (26), and the human monocyte chemoattractant protein-1 receptor gene (27).

The most critical regulatory site of the human TLX3 5'-UTR seems to be recognized by the transcription factor NFY, a complex composed of three subunits (NFYA, NFYB, and NFYC) that are highly conserved through evolution among eukaryotes (28) and all necessary for DNA binding (29). NFY binds CCAAT motifs in regulatory regions of many constitutively active genes and, in spite of its ubiquitous pattern of expression, of cell type–specific genes. In addition, NFY activity has been shown to be modulated during differentiation (30, 31), proliferation (32), and senescence (33). Although NFY recognition sites are usually located at a relatively fixed position in the promoter, between 80 and 100 bp upstream of the transcription start site (28), an increasing number of exceptions has been described (34); in particular, NFY binding 5'-UTR has already been reported for proper regulation of human CD34 gene (35).

EMSA experiments have shown that NFY specifically binds two tandem CCAAT boxes in the 5'-UTR of the TLX3 gene. Both site-directed mutagenesis of these sites and forced expression of a dominant-negative version of NFY had a dramatic effect on the activity of the luciferase reporter gene driven by the 5'-regulatory region of TLX3. These in vitro results were confirmed in vivo at the endogenous TLX3 gene level. In particular, NFY binding the 5'-UTR of the TLX3 gene was unequivocally shown by chromatin immunoprecipitation analysis in three cell lines, both neuronal and not neuronal, and NFY-dependent TLX3 gene transcription was strengthened by down-regulation of its mRNA under two different conditions: forced expression of a dominant-negative NFYA and endogenous NFYA knockdown.

NFY has been shown to exert a cell type–specific activity of gene expression regulation (30). However, after observing comparable luciferase activities in cell lines from different origin in our transfection experiments, the tandem CCAAT boxes can be regarded as unique proximal cis-acting elements sufficient for NFY-mediated transcription of TLX3 in all the analyzed cell lines. NFY might constitutively bind the two CCAAT boxes, located in the TLX3 5'-UTR, with a major role of either attracting the basal transcription machinery or, as a result of its association with histone acetyltransferases (36), maintaining the chromatin structure in an open configuration. Results from our chromatin immunoprecipitation assays suggest that this is the case also in TLX3 nonexpressing cells (MCF-7), thus confirming the constitutive nature of the NFY binding to the identified regulatory region. Distal elements, possibly favoring the access of repressively acting factors to the promoter, would then account for the TLX3-specific expression during embryogenesis. Such a hypothesis could also explain the ectopic TLX3 expression in leukemia, which would be due to dislocation of the gene and of its basal constitutively active promoter with respect to those cis-elements normally driving its cell-specific transcription. Long-range regulation is increasingly recognized as a means by which developmentally significant transcription control occurs (37, 38). In addition, a recent report has provided a functional mapping of such a kind of regulation for TLX1 (39), a gene belonging to the same family and that, similarly to TLX3, is abnormally expressed in a subset of T-cell acute lymphocytic leukemia patients (40). Therefore, for both genes, a mechanism driving their ectopic expression can be postulated which would rely in either the loss of distal negative regulation elements on the chromosome regions where they are located or the juxtaposition of positive transcriptional regulatory elements of genes lying on the other involved partner chromosomes. The failure to detect gross cytogenetic abnormalities in some T-cell acute lymphocytic leukemia patients characterized by the expression of TLX1 or TLX3 has suggested that an alternative activation mechanism may exist (10, 41), thus enforcing the hypothesis of deletion or mutation of dominant distal negative cis-acting regulators.

In conclusion, we have reported robust evidence on how the basal expression of the human TLX3 gene is achieved. Further investigations will be needed to clarify the mechanism(s) both conferring cell and developmental stage specificity to TLX3 expression and eventually predisposing to its involvement in leukemia.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Primer Extension Analysis
Total RNA was extracted from IMR32 and SHSY-5Y neuroblastoma cells by both Trizol reagent (Invitrogen, Carlsbad, CA) and RNeasy columns (Qiagen, Hilden, Germany). Primer 5'-GAGGCGGGCAGAGACGGGTATGTGGCG-3', designed downstream of the ATG first codon, was labeled at the 5'-terminus with the fluorochrome 6-FAM. Total RNA (2.5, 5, or 10 µg) from IMR32 cells was hybridized with the above primer (1 or 10 µmol/L final concentration) in H2O by heating at 70°C for 5 minutes and subsequently incubating at 30°C for 15 minutes. For the extension reactions, annealing solutions were combined with 800 units Moloney murine leukemia virus reverse transcriptase and its specific buffer (Clontech, Mountain View, CA), deoxynucleotide triphosphates each at 5 mmol/L final concentration, 40 units RNase inhibitor (BD Clontech), and 150 ng/µL actinomycin D. cDNA syntheses were run at 42°C for 2 hours and stopped by heating 5 minutes at 95°C. EDTA (1 µL; 0.5 mmol/L, pH 8.0) supplemented with 5 ng RNase A/DNase free was then added for 30 minutes at 37°C to remove RNA, potentially affecting the electrophorectic mobility of the synthesized cDNA. Reactions were phenol extracted and each was subdivided into four aliquots before ethanol precipitation. Samples were air dried and redissolved in formamide. Primer extension reactions were loaded, in a same gel well, together with either a molecular weight marker (ROX 500, AB) or the DNA fragments generated in a sequencing reaction carried out with the same but unlabeled oligonucleotide. Runs of the extended products were done in an ABI PRISM 3130 DNA sequencer (Applied Biosystems, Foster City, CA) and data thus generated were analyzed by using GeneScan and Sequence Analysis, respectively.

The experiment was repeated using 10 µg RNA from both IMR32 (a different extraction) and SHSY-5Y cells with 1 µmol/L final concentration of primer.

Reporter Gene Constructs
Reporter constructs were prepared using the pGL3-basic vector (Promega, Madison, WI), which contains firefly luciferase as reporter gene. Cloning of the 1,311-bp sequence lying upstream of the ATG first codon of TLX3 was done as described previously (7). Progressive deletions of the promoter sequence were obtained by exonuclease III digestion using the Erase-a-Base System according to the manufacturer's instructions (Promega).

Site-directed mutagenesis has been achieved by PCR (42) using the WT construct as template to obtain single mutated constructs from which, finally, a construct with both mutated CAAT boxes has been generated.

Oligonucleotides used for site-directed mutagenesis were Mut 1 (+31/54) sense 5'-AGCCGGGAGCCTCTGAGAGGAGAG-3' and Mut 2 (+63/86) sense 5'-TGATCGCAGCCTTTGGCTGCGGCA-3'. Mutated nucleotides are represented in bold. Each construct was controlled by direct sequencing, and MatInspector analysis (23) was run both to check the loss of the specific putative binding site and to exclude the appearance of any additional different site.

Assay for the Promoter Activity
A total of 105 cells were plated on 35-mm Petri dishes 1 day before transfection. Constructs under analysis and Renilla luciferase reporter plasmid pRL-CMV (Promega), used as a transfection efficiency control, were cotransfected using Fugene 6 (Roche, Basel, Switzerland). The promoter-less pGL3-basic vector and the pGL3-promoter vector, containing the SV40 promoter, were used as negative and positive controls, respectively. Constructs (90 fmol) under analysis were mixed with 10 fmol pRL-CMV and added to 1 µL Fugene 6. In cotransfection experiments, 1 µg expression vectors containing {Delta}NFYA13 WT or {Delta}NFYA13m29 dominant negative (kind gifts of Roberto Mantovani, Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano, Italy; ref. 24) was added to the DNA solution.

For the IMR32 neuroblastoma cell line, constructs under analysis (375 fmol) and Renilla luciferase reporter plasmid pRL-CMV (150 fmol) were cotransfected using polyethylenimine method. After transfection, cells were incubated for 2 hours at 37°C and polyethylenimine solution was then substituted with fresh complete medium.

After 24 hours (48 hours for cotransfections), transfected cells were lysed with PLB 1x buffer (Promega). Luciferase activities in cell lysates were determined using Promega protocol (Dual Luciferase Reporter Assay System) and luminometer (Turner Designs, Sunnyvale, CA). Each construct was transfected in duplicate and each test was repeated at least thrice.

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described previously (43) from IMR32 cells using the following solutions: solution A (10 mmol/L HEPES, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride), BLS solution (20 mmol/L HEPES, 1.5 mmol/L MgCl2, 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.2 mmol/L EDTA, 20 mmol/L NaCl), and BHS solution (20 mmol/L HEPES, 1.5 mmol/L MgCl2, 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.2 mmol/L EDTA, 0.9 mol/L NaCl). EMSA assays were done using LightShift chemiluminescent EMSA kit (Pierce, Rockford, IL) following the manufacturer's instructions. Briefly, binding reactions were carried out for 20 minutes at room temperature in binding buffer 1x containing 50 ng/µL poly(deoxyinosinic-deoxycytidylic acid), 50 mmol/L KCl, and 5% glycerol using 20 fmol biotin-end-labeled target DNA and 10 µg nuclear extracts. For competition and supershift assays, unlabeled target DNA (4 pmol) or 1 µL anti-NFYA subunit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were added, respectively. DNA-protein complexes were run in nondenaturing polyacrylamide gels, transferred onto a positively charged nylon membrane (Hybond-N+), UV cross-linked, and finally detected using horseradish peroxidase–conjugated streptavidin.

Double-stranded oligonucleotides used in EMSA experiments were CCAAT box 1 sense 5'-AGCCGGGAGCCAATGAGAGGAGAG-3' and CCAAT box 2 sense 5'-TGATCGCAGCCAATGGCTGCGGCA-3'. Mutated versions are the same used in transfection assays.

Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation assays were done on SHSY-5Y, IMR32, and MCF-7 cells following the protocol provided by the manufacturer (Upstate Biotechnology, Lake Placid, NY). Briefly, after cross-linking obtained by incubation with formaldehyde at 1% final concentration for 10 minutes at 37°C and subsequent quenching with 125 mmol/L glycine, cells were lysed in SDS buffer in the presence of the protease inhibitors cocktail (Roche) and 0.5 mmol/L phenylmethylsulfonyl fluoride. Samples were sonicated and supernatants containing fragmented chromatin were precleared by adding salmon sperm-DNA protein A-agarose beads. A little portion of the supernatants was kept as "input" material. The remaining cleared chromatin was incubated overnight with 5 µg of the following antibodies: anti-NFYA subunit (Santa Cruz Biotechnology) to study NFY-DNA interaction, anti-acetyl-histone H4 (Upstate Biotechnology) to study chromatin condensation state, normal mouse IgG (Upstate Biotechnology), anti-p53 (Santa Cruz Biotechnology), anti-E2F-1 (Santa Cruz Biotechnology), and anti-ß-actin (Sigma, St. Louis, MO), of the same isotype of the anti-NFY as negative controls. Finally, an overnight incubation with no antibody was also done. Genomic sequences of interest were amplified by PCR using primers forward (–95/–77) 5'-TCGGGCTCTAATATCCGCC-3' and reverse (+189/+171) 5'-CCTCTCGTCGCACTGAAAC-3'.

Analysis of Endogenous TLX3 mRNA
SHSY-5Y neuroblastoma cells (1 x 106) were transfected by Fugene 6 with 6 µg expression vectors containing {Delta}NFYA13 WT or {Delta}NFYA13m29 dominant negative (24). The same quantity of empty expression vector was transfected as negative control. Forty-eight hours later, total RNA was extracted using RNeasy Mini kit and DNase treatment (Qiagen) following manufacturer's instructions. Spectrophotometer-quantified RNA (500 ng) from each sample was retrotranscribed using Advantage RT-for-PCR kit (BD Clontech). Real-time quantitative PCR was carried out using inventoried Assays-on-Demand provided by Applied Biosystems (Hs.00253271_m1 for TLX3 and Hs.99999907_m1 for ß2-microglobulin, used as endogenous reference) using an ABI Prism 7700 Sequence Detection System (Applied Biosystems). After having ascertained that efficiencies of TLX3 and ß2-microglobulin are approximately equal, changes in mRNA amount of TLX3 were quantified by using the comparative CT Method (Sequence Detection System Chemistry Guide, Applied Biosystems). Real-time PCR amplification was done in triplicate and repeated thrice.

Small Interfering RNA–Based NFYA Knockdown
SHSY-5Y neuroblastoma cells were transfected in 60-mm Petri dishes using 15 µL LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) and 500 pmol siNFYA pool or siControl pool (Dharmacon, Lafayette, CO) according to the manufacturer's instructions. After growing in antibiotic-free medium for 48 hours, total RNA was extracted by Qiagen Mini columns, retrotranscribed, and analyzed by quantitative real-time reverse transcription-PCR as described above (Assay-on-Demand used for NFYA Hs.00242929_m1).

Western Blot
SHSY-5Y cells were transfected with the NFY expression constructs or small interfering RNAs as described above. Forty-eight hours later, cells were lysed and the total amount of proteins was quantified. Each sample (4 µg) were resolved on an SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and hybridized with {alpha}-NFYA or {alpha}-ß-actin and finally with rabbit {alpha}-mouse IgG-horseradish peroxidase. Detection was achieved using chemiluminescence reagent enhanced chemiluminescence plus (Amersham, Munich, Germany).

Statistical Analysis
Statistical differences in the levels of reporter gene expression driven by each progressively deleted portion of the TLX3 5'-flanking region, with respect to an entire sequence of 1,311 bp, were determined by applying the Student's t test. The same test was also used to compare means of different experimental points. In particular, statistical significance was assessed for (a) the preventing effect of the mutated CCAAT boxes in driving reporter gene expression, (b) the in vitro and in vivo effects of the NFY dominant-negative construct, and (c) the TLX3 expression decrease induced by the NFY knockdown.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. Roberto Mantovani for the kind gift of expression plasmids containing {Delta}NFYA13 WT and {Delta}NFYA13m29 dominant negative, Dr. Fabio Pastorino for the ß-actin antibody, and Loredana Velo for excellent secretarial assistance.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: Compagnia di San Paolo and Italian Telethon grant GGP04257 (I. Ceccherini).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Present address for M. Vargiolu: U.O. Genetica Medica, Policlinico S. Orsola-Malpighi, Bologna, Italy.

Received 11/27/05; revised 6/23/06; accepted 7/ 5/06.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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