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Genes to Cells (2004) 9, 1167-1174. doi:10.1111/j.1365-2443.2004.00804.x
© 2004 Blackwell Publishing or its licensors
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High-throughput screening of genome fragments bound to differentially acetylated histones

Ruri Kaneda1,2, Minoru Toyota3, Yoshihiro Yamashita1, Koji Koinuma1, Young Lim Choi1, Jun Ota1,4, Hiroyuki Kisanuki1, Madoka Ishikawa1, Shuji Takada1, Kazuyuki Shimada2 and Hiroyuki Mano1,4,*

Divisions of
1 Functional Genomics
2 Cardiology, Jichi Medical School, 3311-1 Yakushiji, Kawachigun, Tochigi 329-0498, Japan
3 Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Sapporo 060-8556, Japan
4 CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
Although acetylation-deacetylation of histones contributes to regulation of gene expression, few methods have been available to determine the whole-genome histone acetylation profile in specific cells or tissues. We have now developed a genome-wide screening method, differential chromatin scanning (DCS), to isolate genome fragments embedded in histones subject to differential acetylation. This DCS screening was applied to a human gastric cancer cell line incubated with or without an inhibitor of histone deacetylase (HDAC) activity, resulting in the rapid identification of more than 250 genome fragments. Interestingly, a number of cancer-related genes were revealed to be the targets of HDAC in the cancer cells, including those for tumour protein 73 and cell division cycle 34. Such differential acetylation of histone was also shown to be linked to the regulation of transcriptional activity of the corresponding genes. Among the isolated genome fragments, 94% (32/34) of them were confirmed to be bound to differentially acetylated histones, and the genes corresponding to 78% (7/9) of them exhibited differential transcriptional activity consistent with the level of histone acetylation. With its high fidelity, the DCS method should open a possibility to rapidly compare the genome-wide histone acetylation profiles and to provide novel insights into molecular carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
The genomic DNA of eukaryotes is compactly packaged by complexation with histones, rendering it physically difficult for the nuclear machinery responsible for DNA transcription or replication to access the DNA. However, this three-dimensional structure of genomic DNA is subject to dynamic and rapid regulation by various chemical modifications of the DNA itself or of its surrounding histones. These epigenetic modifications include DNA methylation as well as histone acetylation, methylation, and phosphorylation (Baylin & Herman 2000; Burgess-Beusse et al. 2002; van Leeuwen & Gottschling 2002).

Acetylation of histones is mediated by histone acetyltransferases (HATs) and takes place on the {varepsilon}-amino group of conserved lysine residues located in the NH2-terminal tail of core histones (Carrozza et al. 2003). Such histone modification is tightly linked to transcriptional regulation by either chromatin remodeling or the provision of binding sites for other proteins. HAT activity in cells is rapidly counteracted by the activity of histone deacetylases (HDACs) (Verdin et al. 2003), with the result that the turnover time of histone acetylation is as short as a few minutes (Waterborg 2002).

The importance of histone acetylation in the regulation of gene expression has been demonstrated for a variety of cellular processes including cell differentiation, cell cycle progression, DNA repair, and carcinogenesis (Kouzarides 1999; Yasui et al. 2003). Furthermore, a histone acetylation profile (HAP) of a certain type of acute myeloid leukaemia was shown to be directly related to the sensitivity of the malignancy to chemotherapeutic agents (Grignani et al. 1998; Lin et al. 1998). Elucidation of the HAP of human malignancies may therefore provide a basis for novel approaches to cancer treatment. This notion is supported by the observation that inhibitors of HDAC activity are effective in the treatment of haematological malignancies such as multiple myeloma and myelodysplastic syndrome (Gore et al. 2001; Catley et al. 2003). The means to establish a genome-wide HAP have been lacking, however (Gabrielli et al. 2002).

To characterize the HAP of any given cell or tissue type, we have now developed a new screening method, termed differential chromatin scanning (DCS). Application of the DCS method to a human gastric cancer cell line has readily identified hundreds of target fragments of histone deacetylase (HDAC), including a number of cancer-related genes such as tumour protein p73 (TP73), cell division cycle 34 (CDC34), and bone morphogenetic protein 7 (BMP7).

With its high fidelity, DCS enables us a genome-wide screening of DNA fragments embedded in histones with differential acetylation level between a given pair of samples.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 Supplementary material
 References
 
The DCS method

The DCS procedure is schematically shown in Fig. 1. We verified the screening ability of this method by identifying HDAC targets in the genome of the human gastric cancer cell line MKN28. The cells were treated with trichostatin A (TSA), a specific inhibitor of HDAC activity, for use as a ‘tester’ sample, whereas MKN28 cells not subjected to TSA treatment were used as a ‘driver’. Inhibition of HDAC activity by TSA would be expected to increase the acetylation level of target histones compared with that apparent in the driver sample. We thus attempted to rapidly isolate genomic fragments bound to histones that were acetylated only in the tester sample.



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  		Figure 1  The DCS method. DNA fragments bound to acetylated (Ac) histones are purified by immunoprecipitation (IP) and subjected to TAG adaptor ligation (green bars) and PCR amplification. The tester DNA is then digested with XmaI, ligated to the first subtraction adaptor (red bars), and annealed with an excess amount of the driver DNA. Given that only the tester-specific fragments self-anneal, PCR with the first subtraction primer selectively amplifies these fragments. The products are subjected to a second round of subtraction PCR with the second subtraction adaptor and primer to ensure the fidelity of subtraction.

 
After the cross-linking of DNA to histones with the use of formaldehyde, both tester and driver cells were separately lysed and subjected to mild DNA shearing by sonication for a short period. Complexes of DNA and acetylated histones were then specifically immunoprecipitated with antibodies to acetylated histone H3, after which the DNA fragments were released from such complexes into solution.

The nonspecific binding of residual RNA was minimized by treating the DNA solution with RNase A, and the DNA fragments were then rendered blunt-ended. The DNA was digested maximally with RsaI to obtain fragments with a relatively uniform size of several hundred base pairs. A TAG adaptor was ligated to both ends of the DNA fragments, and subsequent PCR amplification with a TAG primer yielded amplicons with an XmaI/SmaI site at each end.

The tester DNA was then digested with XmaI (thereby generating cohesive ends), whereas the driver DNA was digested with SmaI (generating blunt ends). The tester DNA was ligated with the first subtraction adaptor (Toyota et al. 1999) through its cohesive ends and was then annealed to an excess amount of the driver DNA. Under this condition, DNA fragments present only in the tester sample undergo self-annealing and thereby generate a binding site for the first subtraction primer at both ends. Subsequent PCR amplification with this primer thus selectively amplified the tester-specific DNA fragments.

To exclude DNA fragments that possess endogenous (probably nonspecific) binding sites for the first subtraction primer, we digested the first subtraction products with XmaI and ligated the resulting molecules with the second subtraction adaptor. A second round of subtraction amplification was then performed with the second subtraction primer, yielding DNA fragments that were associated with acetylated histones specifically in the tester cells.

HDAC targets in a cancer cell line

From the products of the second round of subtraction PCR, we randomly selected 288 DNA fragments and determined their nucleotide sequence. The size of the genome fragments was  50 bp in 265 clones (mean size, ~270 bp), and these DNA sequences were subjected to subsequent analyses. The sequences were first screened with the BLAT search program (Kent 2002) against the nucleotide sequence database assembled as of July 2003 by the Genome Bioinformatics Group of the University of California at Santa Cruz (http://genome.ucsc.edu/). Among the 265 DNA fragments examined, 200 sequences showed > 95% identity to the human genome sequence and 198 of these were located either within a protein-coding gene (demonstrated or predicted) or in the vicinity (within 5 kbp) of such a gene (Table 1 and Table S1).


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  		Table 1  Genome fragments isolated through the DCS screening
 
To verify the fidelity of the tester-specific amplification by our DCS method, we chose 34 DNA fragments and quantified their abundance in the immunoprecipitates prepared from both tester and driver cells with the antibodies to acetylated histone H3. The amount of each DNA fragment relative to that of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was determined by quantitative real-time polymerase chain reaction (PCR). As shown in Table 1, selective amplification by DCS was proved to be highly reliable, with 32 out of 34 clones exhibiting tester-specific precipitation (tester/driver ratio  1.5).

TP73 as a target of HDAC

Interestingly, certain of these 32 clones corresponded to sequences either within or close to human cancer-related genes, including those for TP73, CDC34, and BMP7. One of the clones (10sBKR_D10) was, for instance, mapped to a region located ~1.2 kbp downstream of the last exon of TP73 (GENBANK accession number, NM_005427 [GenBank] ) (Fig. 2A). TP73 is a close homologue of a well-known tumour suppressor, TP53. The TP73 gene is localized at 1p36 locus which is subject to recurrent loss of heterozygosity in various human cancers (Kaghad et al. 1997). An array of isoforms for TP73 are generated in vivo through alternative splicing mechanism of its mRNA, and those forms exert a variety of (sometimes, opposite) actions on tumour growth (Benard et al. 2003).



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  		Figure 2  Identification of TP73 as a target of HDAC activity. (A) one of the clones (10sBKR_D10; red rectangle) identified by DCS screening was mapped to chromosome 1p36.32, located ~1.2 kbp downstream of the last exon of TP73. Exons are denoted by black boxes, arrows indicate the direction of transcription, and green triangles depict distance markers of 5 kbp. (B) the amount of DNA corresponding to the 10sBKR_D10 sequence relative to the amount of that derived from GAPDH was measured by real-time PCR in the immunoprecipitates prepared with antibodies to acetylated histone H3 from MKN28 cells treated (+) or not (–) with TSA. (C) the amounts of TP73 mRNAs relative to that of GAPDH mRNA in MKN28 cells treated or not with TSA were determined by real-time RT-PCR.

 
Thus, it was intriguing to examine whether, in this gastric caner cell line, histone acetylation takes place on the TP73 gene, and thereby regulates its transcriptional activity. Quantity of the genome fragment corresponding to the 10sBKR_D10 clone was measured by the real-time PCR method, revealing that its tester/driver ratio was 15.03 (Fig. 2B). Therefore, the extent of histone acetylation in this genome region in the tester cells was 15.03 times that in the driver cells. We also quantified the mRNA amount of TP73 genes in both tester and driver cells by real-time reverse transcription (RT)-PCR method. As demonstrated in Fig. 2C, acetylation of the 10sBKR_D10 region was accompanied with a profound activation in the transcription level of TP73. These lines of evidence indicate that the TP73 gene is epigenetically regulated by HDAC in a gastric cancer cell line.

Other targets of HDAC

One of our clones (10sBKR_E11) was mapped to a region which contains the first intron and the second exon of the granzyme M (GZMM) gene (GENBANK accession number, NM_005317 [GenBank] ), and is also located ~5 kbp downstream of the last exon of CDC34 (GENBANK accession number, NM_004359 [GenBank] ) (Fig. 3A). Therefore, it was possible that histone acetylation of this region may affect the transcription of both genes. We first quantified the tester/driver ratio for the relative amount of the 10sBKR_E11 fragment in the acetylated histone H3 immunoprecipitates. As shown in Fig. 3B, histone at this region was more heavily acetylated in the tester cells compared to the driver cells.



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  		Figure 3  Identification of CDC34 and GZMM as targets of HDAC activity. (A) one of the clones (10sBKR_E11; red rectangle) identified by DCS screening was mapped to chromosome 19p13.3, spanning the first intron and second exon of GZMM and located ~5 kbp downstream of the last exon of CDC34. The position of the 10sBKR_E11 in the genome is schematically shown as in Figure 2A. (B) The amount of DNA corresponding to the 10sBKR_E11 sequence relative to the amount of that derived from GAPDH was measured by real-time PCR. (C) the amounts of CDC34 and GZMM mRNAs relative to that of GAPDH mRNA in MKN28 cells treated or not with TSA were determined by real-time RT-PCR.

 
To examine how histone acetylation in this area affected the transcriptional activity of CDC34 and GZMM, we measured the amounts of the corresponding mRNAs in both tester and driver cells by real-time RT-PCR. As demonstrated in Fig. 3C, histone acetylation up-regulated the expression of both CDC34 and GZMM, although the extent of gene activation was greater for the latter than for the former. These data suggest that the histone acetylation status of a given genomic locus may regulate the transcription of distinct genes in a differential manner. Our data do not exclude the possibility, however, that histones in other regions of CDC34 or GZMM were also acetylated in a TSA-dependent manner and that these regions possess an enhancer activity greater than that of the 10sBKR_E11 region.

CDC34 encodes a ubiquitin E2 ligase, and its activity is essential for G1- to S-phase transition in cell cycle (Pagano et al. 1995). CDC34 catalyses ubiquitination of the inhibitors for cyclin-dependent kinases (CDKI) which are thereby subject to proteolysis. Therefore, it was interesting to find that CDC34 gene was a target of HDAC in a cancer cell line.

We selected seven additional genes for quantification of the relative amounts of their mRNAs. Of the total of nine genes examined, seven genes were preferentially expressed in the TSA-treated cells compared with the non-treated cells (tester/driver ratio  1.5) (Table 1), consistent with the notion that histone acetylation results in the recruitment of transcription factors and consequent transcriptional activation.

Among the 136 clones that mapped within or close to known genes, only eight pairs were assigned to the same genes. Given the high fidelity of our DCS screening, these data suggest that HDAC complexes simultaneously act at hundreds of independent target histones within the same cell. To visualize directly the genome-wide HAP, we mapped to human chromosome figures our genomic clones whose chromosomal positions are known (Fig. 4). The HDAC targets were distributed widely throughout the human genome, although some ‘hot spots’ for deacetylation were apparent. Seven of our DCS clones were, for instance, mapped to the same 8q24.3 locus. Detailed mapping indicated that all these clones were located within a region spanning 2.5 Mbp. Therefore, it may be possible that regional alterations of chromatin structure lead to a coordinated transcriptional regulation of genes within the affected region.



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  		Figure 4  Chromosomal distribution of HDAC targets. The genome fragments (red dots) isolated by the DCS method were mapped to human chromosomes. The MKN28 cell line was established from a female and therefore does not contain the Y chromosome.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 Supplementary material
 References
 
We have developed a rapid and highly reliable method for the selective isolation of genome fragments bound to histones whose level of acetylation differs between samples. Application of the DCS method to MKN28 cell line has readily revealed that many cell proliferation-associated genes are epigenetically regulated in cancer cells, including those for BMP7, TP73, GBP, AXUD1, CDC34, NGFR, RGS11 (see Table 1), JUNB, SIDEA, and MAFG (see Table S1).

Recently, similar genome-wide comparison methods have been developed by coupling chromatin immunoprecipitation (ChIP) with either comparative genomic hybridization (CGH) or analysis with a DNA microarray containing thousands of CpG islands or promoter fragments (Weinmann et al. 2002; Ballestar et al. 2003; Odom et al. 2004). However, the CGH analysis is not able to provide information on the precise genome sequences differentially immunoprecipitated, and the CpG array can only identify genome fragments that contain CpG islands. Interestingly, the majority of our clones identified by DCS do not satisfy the criteria of CpG islands (for instance, none of the clones corresponding to TP73, CDC34-GZMM, or BMP7 was rich in the CpG sequence). Additionally, screening of whole promoter fragments is still hampered by the incomplete human genome annotation and by its inability to investigate the enhancer regions. The emerging number of non-coding RNAs also makes it difficult to precisely map ‘promoter’ sequences in human genome. Therefore, for the identification of HAT-HDAC targets, our DCS method is likely to provide a non-biased means of genome-wide screening.

Although the study in this manuscript has revealed only the HDAC targets in a cancer cell line, the DCS method should be useful to directly identify HAT targets as well. For instance, a DCS comparison between chemotherapy-sensitive cancer cells and chemotherapy-resistant ones would enable to isolate genes whose associated histones are acetylated in a chemoresistance-dependent manner. In addition, DCS method can be applied to other non-malignant cells/tissues. We could indeed identify HDAC targets in cardiomyocytes (R.K., personal communication).

Furthermore, modification of our method may open up the possibility of a general ChIP-based subtraction strategy. For instance, DCS performed with antibodies to a given histone- or DNA-binding protein, rather than with antibodies to acetylated histone, should readily allow the isolation of target genomic fragments differentially recognized by such proteins.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
Supplementary material
 References
 
Cell culture

MKN28 cells were obtained from the Japanese Collection of Research Bioresources (http://cellbank.nihs.go.jp/) and were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% foetal bovine serum. For preparation of the tester sample, cells were incubated for 24 h with 300 nM TSA (Wako, Tokyo, Japan). Satoh et al. (2002) have already demonstrated that, in MKN28 cells, histones of several genes become acetylated in a TSA-dependent manner.

The DCS method

Both formaldehyde treatment of cells and immunoprecipitation with antibodies to acetylated histone H3 were performed with the ChIP assay kit (#06-599; Upstate Biotechnology). Sonication of DNA was performed in one 10-s pulse to minimize the extent of DNA shearing (the resulting DNA fragments should migrate as a smear corresponding to a size of 10–20 kbp in gel electrophoresis). Immunoprecipitation was achieved with protein A-Sepharose beads (Sigma) suspended in TE (10 mM Tris-HCl, 7.4, and 0.1 mM EDTA) containing 0.2 mg/mL tRNA (Roche Biochemicals) and 0.5 mg/mL bovine serum albumin (New England Biolabs), instead of with the protein A beads supplied with the kit. DNA fragments were recovered from the immunoprecipitates and treated with RNase A to remove residual RNA. The DNA fragments were digested with RsaI (New England Biolabs), and ligated to the TAG adaptor (5'-CCACCGCCATCCGAGCCTTTCTGCCCGGG-3'/3'-GAAAGACGGGCCC-5'). After PCR amplification with the TAG primer (5'-CCACCGCCATCCGAGCCTTTCTGC-3'), the tester DNA and the driver DNA were digested with XmaI and SmaI, respectively. Only the tester DNA (0.5 µg) was ligated to the first subtraction adaptor (5'-GTGAGGGTCGGATCTGGCTGGCTC-3'/3'-CGACCGAGGGCC-5'). The adaptor-ligated tester DNA was then annealed with 40 µg of the driver DNA at 67 °C for 20–24 h before PCR amplification with the first subtraction primer (5'-GTGAGGGTCGGATCTGGCTGGCTC-3'). After digestion of single-stranded DNA with mung bean nuclease (New England Biolabs), the amplified products were subjected to digestion with XmaI followed by a second round of subtraction PCR with the second subtraction adaptor (5'-GTTAGCGGACACAGGGCGGGTCAC-3'/3'-GCCCAGTGGGCC-5') and second subtraction primer (5'-GTTAGCGGACACAGGGCGGGTCAC-3'). The final products were digested with XmaI and ligated into pBlueScript (Stratagene) for nucleotide sequencing. The detailed protocol for the DCS method is available as a supplemental protocol online.

DNA quantification

Genome fragments immunoprecipitated by antibodies to acetylated histone H3 were subjected to PCR with a QuantiTect SYBR Green PCR Kit (Qiagen). The amplification protocol comprised incubations at 94 °C for 15 s, 62 °C for 30 s, and 72 °C for 60 s. Incorporation of the SYBR Green dye into PCR products was monitored in real time with an ABI PRISM 7700 sequence detection system (PE Applied Biosystems), thereby allowing determination of the threshold cycle (CT) at which exponential amplification of PCR products begins. The CT values for DNA molecules corresponding to the GAPDH gene and genome fragments of interest were used to calculate the abundance of the latter fragments relative to that of the former. The oligonucleotide primers for PCR were 5'-TCGGTGCGTGCCCAGTTGAACC-3' and 5'-ATGCGGCTGACTGTCGAACAGGAG-3' for GAPDH, 5'-ACCAGTCGCTGCTTTTAAATAAGG-3' and 5'-GCTAGGAGCTTCCCCTACTAACAT-3' for 10sBKR_D10, and 5'-CTGATGTGCGTTTTGAAGGACT-3' and 5'-GAAACTTTTAGGCTGATACGTGTG-3' for 10sBKR_E11.

mRNA quantification

Total RNA was prepared from the tester or driver cells with an RNeasy Mini column (Qiagen), treated with RNase-free DNase (Qiagen), and subjected to reverse transcription with PowerScript reverse transcriptase (BD Biosciences Clontech) with an oligo(dT) primer. Portions of the resulting cDNAs were subjected to PCR with a QuantiTect SYBR Green PCR Kit. The amplification protocol comprised incubations at 94 °C for 15 s, 65 °C for 30 s, and 72 °C for 60 s. The annealing temperature of PCR was changed to 60 °C for the cDNA of CDC34 and 66 °C for that of GZMM. The oligonucleotide primers for PCR were 5'-AGAACATCATCCCTGCCTCTACT-3' and 5'-ATATTTGGCAGGTTTTTCTAGACG-3' for GAPDH, 5'-GACGAGGACACGTACTACCTTCA-3' and 5'-GTAGGTGACTCGGCCTCTGTAG-3' for TP73, 5'-AAGATGTGGCACCCTAACATCTAC-3' and 5'-AGGGAGATCACACTCAGGAGAAT-3' for CDC34, and 5'-ATGTGTAACAACAGCCGCTTCT-3' and 5'-CTTGAAGATGTCAGTGCAGACC-3' for GZMM.


   Supplementary material
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
References
 
The following supplementary material is available from: http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC804/GTC804sm.htm. Appendix S1 The detailed protocol of the DCS method. Table S1 All genome fragments isolated through the DCS screening.


   Acknowledgements
 
We thank Dr Kohei Miyazono for his helpful suggestions. This work was supported in part by a Grant-in-Aid for Research on the Second-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor, and Welfare of Japan; a grant from Mitsubishi Pharma Research Foundation; a grant from Takeda Science Foundation; and a grant from Sankyo Foundation of Life Science.


   Footnotes
 
Communicated by: Fuyuki Ishikawa

* Correspondence: E-mail: hmano{at}jichi.ac.jp


   References
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 Introduction
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 Discussion
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Received: 21 August 2004
Accepted: 16 September 2004




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