MCO Molecular and Clinical Oncology 2049-9450 2049-9469 D.A. Spandidos 10.3892/mco.2014.468 mco-03-02-0408 Articles Characterization of regulatory sequences in alternative promoters of hypermethylated genes associated with tumor resistance to cisplatin IBRAHIM-ALOBAIDEMOHAMMED A.1 ABDELSALAMABDELSALAM G.2 3 ALOBYDIHYTHAM4 RASULKAKIL IBRAHIM5 ZHANGRUIWEN6 SRIVENUGOPALKALKUNTE S. 1 Department of Biomedical Sciences and Cancer Biology Research Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA Department of Mathematics, Statistics and Physics, College of Arts and Sciences, Qatar University, Doha, Qatar Department of Statistics, Faculty of Economics and Political Sciences, Cairo University, Giza 12613, Egypt Biomedica, LLC, Sterling Heights, MI 48310, USA Weill Cornell Medical College and Hamad Medical Corporation, Doha, Qatar Department of Pharmaceutical Sciences and Cancer Biology Research Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA Correspondence to: Professor Kalkunte Srivenugopal, Department of Biomedical Sciences and Cancer Biology Research Center, School of Pharmacy, Texas Tech University Health Sciences Center, 1406 S. Coulter Street, Amarillo, TX 79106, USA kalkunte.srivenugopal@ttuhsc.edu 032015 27112014 3 2 408 414 7102014 23102014 Copyright © 2015, Spandidos Publications 2015

The development of cisplatin resistance in human cancers is controlled by multiple genes and leads to therapeutic failure. Hypermethylation of specific gene promoters is a key event in clinical resistance to cisplatin. Although the usage of multiple promoters is frequent in the transcription of human genes, the role of alternative promoters and their regulatory sequences have not yet been investigated in cisplatin resistance genes. In a new approach, we hypothesized that human cancers exploit the specific transcription factor-binding sites (TFBS) and CpG islands (CGIs) located in the alternative promoters of certain genes to acquire platinum drug resistance. To provide a useful resource of regulatory elements associated with cisplatin resistance, we investigated the TFBS and CGIs in 48 alternative promoters of 14 hypermethylated cisplatin resistance genes previously reported. CGIs prone to methylation were identified in 28 alternative promoters of 11 hypermethylated genes. The majority of alternative promoters harboring CGIs (93%) were clustered in one phylogenetic subclass, whereas the ones lacking CGIs were distributed in two unrelated subclasses. Regulatory sequences, initiator and TATA-532 prevailed over TATA-8 and were found in all the promoters. B recognition element (BRE) sequences were present only in alternative promoters harboring CGIs, but CCAAT and TAACC were found in both types of alternative promoters, whereas downstream promoter element sequences were significantly less frequent. Therefore, it was hypothesized that BRE and CGI sequences co-localized in alternative promoters of cisplatin resistance genes may be used to design molecular markers for drug resistance. A more extensive knowledge of alternative promoters and their regulatory elements in clinical resistance to cisplatin is likely to usher novel avenues for sensitizing human cancers to treatment.

 alternative promoters cisplatin resistance CpG islands DNA methylation transcription factor-binding sites bioinformatics Introduction

The success of platinum drugs in the treatment of various types of cancer has been challenged by the hamper of intrinsic and acquired resistance. Cell and molecular biology research over several decades has provided insight into this problem and has demonstrated that alterations in drug influx and efflux, detoxification via glutathione and glutathione transferases and enhanced DNA repair (1–3) are all involved, either singly or in combination, in cisplatin resistance. However, it remains puzzling that genomic expression patterns of platinum-sensitive/-resistant determinants in several cell lines, including squamous cell carcinoma of the head and neck, bladder and ovarian cancers, do not always correlate with the results of biochemical analyses (4–6). Additionally, epigenetic changes in the DNA methylation profiles during cancer development are associated with the acquisition of cisplatin resistance (6–9). However, the problem of platinum resistance at the genomic and epigenomic levels has not been clearly defined. Therefore, it is paramount to gain a better understanding of gene expression machinery components that control platinum resistance genes. A promising approach to these questions appears to be the characterization of regulatory elements, CpG islands (CGIs) and transcription factor-binding sites (TFBS) in alternative promoters of genes that have been validated and their association with cisplatin resistance proven.

It has been well established that alternative promoters are involved in the transcription of nearly half of all eukaryotic genes and are considered one of the main molecular characteristics of eukaryotic genomes (10, 11). Overall, alternative promoters have been suggested to add significant flexibility and greater diversity to the regulation of gene expression (10–16). The functional significance of alternative promoters and their role in disease development and progression remains unclear, except for a few well characterized genes, such as the tumor suppressor p53, lymphoid enhancer-binding factor 1, insulin growth factor 2 and guanine nucleotide-binding protein α -stimulating genes, where these sequences are aberrantly activated, developmentally regulated or silenced (11). In addition, alternative promoters have been shown to regulate the expression of paired box homeotic gene 6 in various tissue types and play crucial roles in the development of the eye, brain, olfactory system and endocrine pancreas (17).

The TFBS and the CGIs targeted for DNA methylation, significantly affect the functional activities of alternative promoters (6–9, 18, 19). To date, the role and molecular characteristics of alternative promoters have not been investigated in platinum drug resistance. The present study mapped the distribution of TFBS and CGIs in 48 alternative promoters involved in the regulation of 14 hypermethylated genes, known to be associated with the development of cisplatin resistance.

 Materials and methods <sec> <title>Alternative promoters of cisplatin resistance genes

A total of 14 genes known to contribute to cisplatin resistance and to be specifically hypermethylated in resistant cell lines, but not in the sensitive parental cell lines (9), were selected for this study. The genes were screened for the presence of alternative promoters and regulatory elements (CGIs and TFBS) within these sequences. The chromosomal map locations of cisplatin genes were searched in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) and the alternative promoters for each gene were searched in the Transcriptional Regulatory Element Database (TRED; https://cb.utdallas.edu/cgi-bin/TRED/tred.cgi?process=home). The length of the promoter sequence was set to 1,000 base pairs (BPS), which were numbered along the sequence of the promoter relative to the transcription start site (TSS). The sequence of bps upstream the TSS was numbered with a negative sign, whereas downstream bases were identified with a positive sign.

 Search for regulatory elements in the alternative promoters

The alternative promoters of 14 cisplatin resistance genes were analyzed for TFBS, namely, TATA-8 (TATAWAWR) and TATA-532 (HWHWWWWR, excluding HTYTTTWR, CAYTTTWR, MAMAAAAR and CTYAAAAR) elements, initiator (INR; YYANWYY), CCAAT and its inverted sequence TAACC, B recognition element (BRE; SSRCGCC) and downstream promoter element (DPE; RGWCGTG) sequences (20, 21). CGIs in alternative promoters were searched in a 100-bp window (N=100) moving across the sequence at 1-bp intervals. The parameter sets used to search for CGIs in the alternative promoters were as follows: observed/expected (O/E) CpG ≥0.6 and %GC >55% (22–25). The O/E ratio for CpG was calculated according to the equation reported by Gardiner-Garden and Frommer (22).

 Construction of phylogenetic tree

To investigate the evolutionary associations among the 48 alternative promoter sequences and to group the related sequences into specific categories, a phylogenetic tree was constructed using the Phylogenetic Tree application in the MATLAB Bioinformatics toolbox to probe the interrelationships and linking the tree nodes. The alternative promoter sequences and their derivatives were drawn using a hierarchical diagram.

 Statistical analysis

The promoter sequences were analyzed using Perl, C++ and Excel software. The independence of each promoter element was examined using the Fisher's exact probability test.

 Results <sec> <title>Hypermethylated genes in acquired cisplatin resistance

Chang et al (9) published an elegant study of specific genes that selectively undergo methylation in cisplatin-resistant cell lines, but not in their cisplatin-sensitive isogenic parent cell counterparts. The authors found that a set of 14 genes were silenced in the cisplatin-resistant cells and confirmed that promoter methylation, as determined by sodium bisulfite sequencing, accounted for the lack of gene expression. A number of these genes were also reactivated by azacytidine treatment of resistant cells (9). We used this panel of methylated genes for analysis of alternative promoters and their regulatory characteristics in cisplatin resistance. A list of these genes and their designations is provided in Table I. We also mapped the locations of these genes to their chromosomes and these sites are represented in Table I.

 Genomic context of alternative promoters of cisplatin hypermethylated genes

A search through TRED identified 48 promoters for the 14 hypermethylated cisplatin resistance genes reported by Chang et al (9). These promoters were analyzed to identify the CGIs and TFBS (Fig. 1 and Table II). The number of identified alternative promoters for each gene varied between 2 and 6 (Fig. 1A) and were located on 9 chromosomes (Fig. 1B). Chromosome 1 and the long arm ‘q’ were found to host a higher number of genes and alternative promoters compared to other chromosomes and short arms (Fig. 1B). Of the 14 cisplatin resistance genes, 5 (35.71%) were located on chromosome 1, while of all the promoters investigated, 18 (37.5%) were located on this chromosome. However, the distributions of alternative promoters and genes were almost equal on forward and reverse strands of the chromosomes: 6 genes (43%) were located on the reverse strands and 8 genes (57%) on the forward strands (Fig. 1C); 26 promoters (54%) were on the reverse strands and 22 (46%) on the forward strands (Fig. 1D).

 CGIs and TFBS

CGIs were identified in 28 alternative promoters of 11 cisplatin resistance genes, while 3 genes, namely the C4-binding protein β(C4BPB), chromosome 8 open reading frame 4 (C8orf4) and cytidine deaminase (CDA) genes, exhibited no CGIs (Table II). As shown in Table II, the INR and TATA-532 sequences prevailed over TATA-8 sequences. We also observed that the alternative promoters of cisplatin resistance genes were rich in TATA-8 and TATA-532 sequences, although these varied significantly in their frequencies. For example, the four promoters of opsin 3 (OPN3), namely 44257, 2157, 114168 and 118881, which lack CGIs, contained 13–35 TATA-532 elements. Our analysis also demonstrated that each of two promoters, melanoma cell adhesion molecule (MCAM)-117653 (lacked CGI) and S100P-31251 (harbored two CGIs), contained 10 TATA-8 sequences. Furthermore, the alternative promoters that harbored CGIs contained TATA-532 sequences in a wide range, from 1 to 22. The CCAAT and its inverted sequence TAACC, BRE and DPE sequences were not as frequent as INR and TATA-532 sequences. Another interesting observation was associated with BRE sequences: we noticed that the identified 48 BRE sequences were present in 20 promoters of 9 cisplatin genes that harbored CGIs and were absent in other alternative promoters that lacked CGIs.

 Phylogenetic tree analysis of alternative promoter sequences

A phylogenetic tree was created from a set of 48 alternative promoter sequences of the 14 cisplatin resistance genes (Fig. 2). Two main clustering patterns were observed, namely A and B, which may be further divided into subclasses and secondary classes within each category. The first main class (A) included 38 alternative promoters and the second main class (B) included 10 promoters. Furthermore, the first main class (A) may be divided into two subclasses, namely C and D; subclass C contained 30 alternative promoters, of which 26 contained CGIs. Groups E and F were identified in subclass C, which were represented by 19 and 11 alternative promoters, respectively. Group E included 18 alternative promoters with CGIs and 1 promoter (S100P-117263) without CGIs; group F included 8 alternative promoters with CGIs: SAT-43140,-43141 and-115065, TUBB2A-37272, G0S2-119612 and-1833 and LAMB3-2377 and-2378. The small subclass (D) contained 8 promoters, with 7 promoter sequences lacking CGIs: TM4SF1 −30388, −30390 and −30389, OPN3 −2157 and −44257 and C8norf4 −39909 and −121250. Of the 10 alternative promoters in class B, 9 did not have CGIs: C4BPB −113384, −1813 and −1814, LAMB3 −113646, B4GALT1 −118955, MCAM −117653, OPN3 −114168 and −118881 and TM4SF1 −118807. The remaining promoter in this class, TUBB2A-113646, was characterized by the presence of 22 sequences of TATA-532 type and harbored CGIs. The evolutionary distances between the two major classes A and B and subclasses C and D were in the range of 0.0125 and 0.03125, respectively, exhibiting weak evolutionary ties between them; however, stronger ties were observed among the alternative promoters in group E and F within subclass C, which were composed mainly from alternative promoters with CGIs (93%). The alternative promoters that lacked CGIs were distributed in two unrelated groups, B and D.

 Discussion

We selected a panel of genes specifically known to undergo methylation in cisplatin-resistant cell lines (9) and characterized the alternative promoters and regulatory sequences associated therewith. The exact mechanisms by which these genes contribute to cisplatin resistance has not been fully elucidated; however, a number of these were shown to be inducible by cisplatin treatment in sensitive cells and silenced in sensitive isogenic cells (9). Recent findings support a prominent role for alternative promoters in cell type and human tissue type-specific gene expression (26). Considering that the transcription machinery utilizes alternative promoters for regulating differential transcription (10, 16) and the aberrant use of one alternative promoter over another may result in disease, including cancer (11), we hypothesized that cisplatin resistance may be mediated by a differential usage of alternative promoters with variable regulatory sequences, TFBS and CGIs. Transcription factors and their binding sites in a given promoter are key elements in controlling the rate and extent of mRNA synthesis (19, 27). However, the interaction between transcription factors and cis-regulatory modules, which contain the TFBS in promoter sequences, has not been clearly determined (27–33). Seven types of TFBS, namely INR, TATA-8, TATA-532, BRE, DPE, CCAAT and TAACC are well recognized (20, 21). Our results demonstrated that 11 alternative promoters have TATA-8 sequences and 47 promoters contain TATA-532 sequences. It has been reported that ∼76% of human core promoters lack TATA-like elements (20) and only 10–20% of promoters contain the TATA sequences (34). Of note, our study was not restricted to core promoters, which are located ∼40 bp up-and downstream of the TSS (35), but included the entire length of 1,000 bp that included 700 bp upstream and 300 bp downstream of TSS, as given by TRED. Furthermore, our results demonstrated that CCAAT, TAACC, BRE and DPE sequences were not as frequent as TATA sequences. Seventeen alternative promoters (35.4%) contained CCAAT; 19 (39.6%) contained TAACC, 20 (41.6%) contained BRE and one (2%) contained DPE. The percentage obtained for CCAAT was similar to that reported previously, reflecting its ubiquity in mammalian promoters (21, 36). As the mechanism associated with cisplatin resistance is multifactorial (1–3), our results suggest that the genes encoding cisplatin resistance harbor more than one option to initiate their transcripts to enhance the efficiency of mRNA production.

It has been well documented that CGIs are prone to DNA methylation and this epigenetic mechanism is associated with gene silencing, initiation and maintenance of malignancy and drug resistance (6–9, 37–39). Hypermethylation in CGIs and subsequent gene inactivation contributes to cisplatin resistance (9), as described earlier in this study. In the present study, we demonstrated that 11 out of the 14 genes reported by Chang et al (9) have 28 promoters harboring CGIs. However, the remaining 3 genes, namely C4BPB, C8orf4 and CDA, lacked discernible CGIs, raising the possibility of other mechanisms. The majority of alternative promoters harboring CGIs (93%) were clustered in one phylogenetic subclass, indicating relatively strong evolutionary ties among them. Thus, it may be hypothesized that the use of promoters with less frequent DNA methylation hotspots may assist the genes in escaping the epigenetic modifications and enable continued and efficient expression. Furthermore, we demonstrated that BRE and CGI sequences co-localized in the alternative promoters of cisplatin resistance genes (Table II) and this property may be utilized to design molecular markers for resistance to cisplatin and/or drugs. Such an approach will involve designing specific primers for amplifying DNA sequences that include BRE and a downstream segment of alternative promoter and a short exon region of the gene in question. A more detailed understanding of the functionality of the alternative promoters in cisplatin resistance is likely to have other applications; for example, it may be possible to design novel anticancer drugs that interact with specific promoter sequences, e.g., G-rich sequences which may form DNA G-quadruplexes (40–42). In addition, future research may make use of novel synthetic biology methods to build molecular models of various components of alternative promoters (43–46) to overcome drug resistance and enable more effective and personalized cancer therapies.

 Acknowledgements

This study was supported in part by a grant from the Cancer Prevention and Research Institute of Texas (CPRIT; no. RP130266) to K.S.S. We would like to thank Ibtisam Ismael Alobaidi for technical assistance.

 Abbreviations TFBS

transcription factor-binding sites

 CGI

CpG island

 CGI

TSS

 CGI

transcription start site

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Analysis of 48 alternative promoters for the selected 14 genes associated with cisplatin resistance. (A) Number of alternative promoters for each gene. (B) Map locations of the 14 cisplatin genes and their alternative promoters on the chromosomes and the chromosome arms (p or q). (C) Number of cisplatin resistance genes on the p and q arms of the reverse and forward strands. (D) Number of alternative promoters of cisplatin genes on the reverse and forward strands of the p and q arms of chromosomes. ALCAM, activated leukocyte cell adhesion molecule; B4galt1, UDP-Gal: βGlcNAc β1,4-galactosyltransferase, polypeptide 1; C4BPB, C4-binding protein β; C8orf4, chromosome 8 open reading frame 4; CDA, cytidine deaminase; CRIP1, cysteine-rich protein 1; G0S2, G0/G1 switch 2; LAMB3, laminin β3; MCAM, melanoma cell adhesion molecule; OPN3, opsin 3; S100P, S100 calcium-binding protein P; SAT1, spermidine/spermine N1 acetyltransferase 1; TM4SF1, transmembrane 4 L six family member 1; TUBB2A, tubulin β2A class IIa.

Phylogenetic tree generated for the 48 DNA sequences of the alternative promoters of 14 cisplatin resistance genes.

Description of functional activity and map locations for 14 genes associated with cisplatin resistance.

Genes GenBank ID Description Map location
ALCAM NM_001627 Activated leukocyte cell adhesion molecule 3q13.1, forward strand
B4GALT1 NM_001497 UDP-Gal: βGlcNAc β1,4-galactosyltransferase, polypeptide 1 9p13, reverse strand
C4BPB NM_000716 C4-binding protein β 1q32, forward strand
C8orf4 NM_020130 Chromosome 8 open reading frame 4 8p11.2, forward strand
CDA NM_001785 Cytidine deaminase 1p36.2-p35, forward strand
CRIP1 NM_001311 Cysteine-rich protein 1 (intestinal) 14q32.33, forward strand
G0S2 NM_015714 G0/G1 switch 2 1q32.2-41, forward strand
LAMB3 NM_000228 Laminin β3 1q32, reverse strand
MCAM NM_006500 Melanoma cell adhesion molecule 11q23.3, reverse strand
OPN3 NM_014322 Opsin 3 1q43, reverse strand
S100P NM_005980 S100 calcium-binding protein P 4p16, forward strand
SAT1 NM_002970 Spermidine/spermine N1 acetyltransferase 1 Xp22.1, forward strand
TM4SF1 NM_014220 Transmembrane 4 L six family member 1 3q21-q25, reverse strand
TUBB2A NM_001069 Tubulin β2A class IIa 6p25, reverse strand

TFBS and CGIs in the 48 alternative promoters of 14 genes associated with cisplatin resistance.

Genesa Promoter ID INR T8 T532 CCAAT TAACC BRE DPE CGIs
ALCAM 114029 5 0 5 1 0 1 1 +
29583 7 0 7 1 0 1 0 +
29584 11 0 8 0 0 0 0 +
B4GALT1 42895 6 0 1 0 2 4 0 +
113955 5 0 18 0 0 0 0 −
C4BPB 1813 12 0 15 0 1 0 0 −
1814 11 0 11 0 1 0 0 −
113384 12 0 15 0 1 0 0 −
C8orf4 39909 10 0 10 0 0 0 0 −
121250 10 0 10 0 0 0 0 −
CDA 279 12 0 5 0 0 0 0 −
114145 12 0 5 0 0 0 0 −
CRIP1 12302 4 0 1 0 1 2 0 +
12303 0 0 1 0 0 1 0 +
113817 4 0 1 0 1 2 0 +
G0S2 1832 2 0 7 0 0 1 0 +
1833 3 0 13 0 1 1 0 +
119612 3 0 13 0 1 1 0 +
LAMB3 2377 7 1 6 0 1 0 0 +
2378 9 1 8 0 1 0 0 +
2379 13 1 13 3 3 0 0 −
113034 6 0 7 1 1 0 0 −
MCAM 7410 4 0 0 0 0 3 0 +
7411 4 0 2 0 2 0 0 +
117653 5 10 45 0 0 0 0 −
OPN3 2156 7 0 3 1 0 5 0 +
44257 12 0 13 0 0 0 0 −
2155 7 0 3 1 0 5 0 +
2157 14 0 28 1 1 0 0 −
114168 8 2 27 2 1 0 0 −
118881 13 1 35 1 0 0 0 −
S100P 31250 6 0 4 0 0 0 0 +
31251 4 10 50 0 0 0 0 +
117263 3 0 4 0 0 0 0 −
SAT1 43141 6 0 9 1 1 3 0 +
43140 6 0 9 1 1 3 0 +
115065 6 0 9 1 1 3 0 +
TM4SF1 30388 8 0 15 2 0 0 0 −
30389 8 0 14 2 0 0 0 −
30390 7 0 14 2 0 0 0 −
30391 10 0 2 0 2 0 0 +
30392 11 0 6 0 0 1 0 +
118807 7 1 33 2 0 0 0 −
TUBB2A 37269 6 1 1 0 0 3 0 +
37270 6 1 1 0 0 3 0 +
37271 6 1 1 0 0 3 0 +
37272 10 0 14 1 0 0 0 +
113646 8 0 22 0 0 2 0 +

For gene description, see Table I. TFBS: T8, TATAWAWR; T532, HWHWWWWR (excluding HTYTTTWR, MAMAAAAAR and CTYAAAAR); BRE, SSRCGCC; DPE, RGWCGTG; INR, YYANWYY; CCAAT and its inverted sequence TAACC (20, 21). Parameter sets used to search for CGIs in the alternative promoters: observed/expected CpG ≥0.6 and %GC >55% (22–25). + and − indicate presence and absence of CGIs. TFBS, transcription factor-binding sites; CGIs, CpG islands; INR, initiator; T8, TATA-8; T532, TATA-532; BRE, B recognition element; DPE, downstream promoter element .