Introduction
Heterochromatin protein 1 (HP1) was first identified in
Drosophila as a non-histone component of chromatin [
1]. Mutations in the HP1 gene resulted in suppression of position-effect variegation, a result that implicated HP1 in chromatin structure and gene expression [
2]. Mutation of the gene encoding HP1 in
Drosophila resulted in larval lethality [
3]. Examination of HP1 mutant embryos revealed defects in chromosome segregation and telomere maintenance [
4,
5]. Therefore, HP1 is thought to play an essential role in heterochromatin-dependent processes in
Drosophila. HP1 can also be found in certain euchromatic loci, implying its role in euchromatic regions [
6,
7].
HP1 homologues have been identified in a variety of organisms including yeast, nematodes, insects, chickens, frogs, and mammals [
8]. There are three HP1 isoforms in mammals: HP1α, β and γ [
9,
10]. Each HP1 isoform has a different chromosomal distribution. HP1α is located mainly in heterochromatic regions, HP1β is found in both heterochromatic and euchromatic regions, and HP1γ is located almost exclusively in euchromatic regions [
11‐
13]. The localization of HP1 isoforms to different regions of chromatin implies that each isoform plays a unique role in chromatin structure and transcriptional regulation.
All HP1 family members share a similar structure: an amino-terminal chromodomain (CD), a variable hinge region and a carboxy-terminal chromoshadow domain (CSD) [
8]. HP1 associates with chromatin primarily through the CD, which binds to the histone-fold domain of histone H3 [
13,
14]. This interaction is stimulated by methylation of the H3 histone tail on lysine 9 [
15,
16]. It has therefore been suggested that the repressive effect of H3K9 methylation is mediated, in part, by HP1. HP1α can also interact with histone H1 [
13,
17,
18]. In addition, RNA may play a role in targeting HP1α to pericentric heterochromatin by interacting with the hinge region [
19]. An interaction between HP1α and the histone variant H2A.Z may contribute to the compaction of heterochromatic domains [
20]. However, the mechanism by which different isoforms of HP1 occupy distinct regions of chromatin remains unclear.
Although HP1 associates with chromatin via the CD, the CSD of HP1 can mediate interactions with a number of different proteins [
21]. The CSD can bind HP1 itself, allowing HP1 to hetero- and homo-dimerize [
13]. This interaction is thought to contribute to the compaction of heterochromatic domains. The CSD can also bind the histone methyltransferase SUV39H1, an interaction that may facilitate spreading of heterochromatin to adjacent loci [
22,
23]. The CSD mediates the interaction between HP1 and the co-repressor KAP-1 (TIF1β, KRIP1), which can result in mitotically-heritable gene silencing [
24,
25]. Interaction between HP1α and γ and the TFIID subunit TAF4 (TAF
II130) is also mediated by the CSD and may be responsible for dissociation of TAF4 from promoter regions upon HP1 binding [
26,
27]. The ability of the CSD to associate with such a functionally diverse group of proteins suggests that HP1 exerts its effects on gene expression through a variety of mechanisms.
It has been reported that HP1α expression is reduced in invasive human breast cancer cell lines such as HS578T and MDA-MB-231 compared with non-invasive breast cancer cell lines such as MCF7 and T47D [
28]. Over-expression of HP1α in the invasive cell line MDA-MB-231 reduced its
in vitro invasive potential [
29]. Reducing the expression of HP1α in MCF7 cells increased their invasive potential without affecting their rate of growth [
30]. This data suggests that HP1α acts as a metastasis suppressor in breast cancer cells. In addition, reduction of HP1α expression has been observed in metastatic colon cancer cell lines compared with non-metastatic cell lines, in desmoplastic vs. classic meduloblastoma, and in papillary thyroid carcinoma compared with normal thyroid tissue [
31‐
33].
HP1α is one of many proteins that have been identified as metastasis suppressors. These proteins have roles in diverse cellular functions including cell adhesion and migration as well as cell signaling [
34]. The role of HP1α in epigenetic gene silencing makes it a unique metastasis suppressor. A decrease in HP1α expression could disrupt the epigenetic program of the cell, altering gene expression at a global level. Therefore, it has been hypothesized that decreased expression of HP1α in breast cancer cells leads to deficient epigenetic silencing of genes that promote a metastatic phenotype. We set out to determine the mechanism by which HP1α expression is reduced in highly invasive breast cancer cells.
To address this question we used the MCF7 and HS578T breast cancer cell lines as models of non-invasive and invasive breast cancer, respectively. To study the transcriptional activity of the HP1α gene promoter in these cell lines we sub-cloned the human HP1α gene promoter region into a luciferase reporter construct. Using the Transcription Element Search Software (TESS) database, we identified several highly conserved transcription factor binding motifs in the HP1α promoter region [
35]. We then used site-directed mutagenesis to assess the importance of each motif in the transcriptional activity of the HP1α gene promoter in each cell line. Our study suggests that the transcription factor yin yang 1 (YY1) may be involved in the differential expression of HP1α in MCF7 and HS578T cells. In addition, we demonstrate that YY1 overexpression suppresses HS578T cell migration
in vitro. We conclude that decreased YY1 expression may contribute to the invasive phenotype of metastatic breast cancer cells.
Materials and methods
Cell culture
Cell lines were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA, USA) and 1% penicillin/streptomycin/glutamine mixture (Invitrogen, Carlsbad, CA, USA). Culture medium for the HS578T cell line was supplemented with 0.01 mg/ml bovine insulin (Invitrogen, Carlsbad, CA, USA).
Analysis of the HP1α gene promoter region
The University of California Santa Cruz (UCSC) genome browser [
36] was used to determine which nucleotides in the HP1α promoter region are conserved between human, mouse, rat and chimpanzee. The sequence of the HP1α gene promoter region was entered into the TESS database to identify transcription factor binding motifs [
35]. Motifs with high similarity to a consensus binding site and high sequence conservation between species were noted.
Construction of reporter constructs
Portions of the HP1α gene promoter region were PCR-amplified from a BAC clone [GenBank:AC078778] containing a portion of human chromosome 12. The PCR-amplified fragments were sequenced and ligated into the pGL3 luciferase reporter vector (Promega, Madison, WI, USA) digested with HindIII and XhoI. Primer sequences are available upon request.
Site-directed mutagenesis
The QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) was used according to the manufacturer's instructions. Mutations in transcription factor binding sites were as follows: site YY1.1 was changed from AAATGG to AAGCTT, site YY1.2 was changed from AAAATGGCG to AAAGCTTCG, the E-box site was changed from CACGTG to CGATCG, and site NRF-1.3 was changed from TGCGCAGGCGCA to TGCGCATATGCA. In each case, nucleotides reported to be critical for factor binding were changed. The YY1.2 mutation abolished YY1 binding (see text), and the NRF-1.3 mutation abolished NRF-1 binding as determined by electrophoretic mobility shift assay (EMSA) (data not shown).
Real time RT-PCR
RT-PCR was performed using the Roche LightCycler instrument. Reactions were carried out in LightCycler capillaries (20 μl) (Roche, Indianapolis, IN, USA) and contained SYBR. Green Taq ReadyMix (Sigma, St. Louis, MO, USA), Enhanced avian reverse transcriptase (Sigma, St. Louis, MO, USA) (2 units/reaction), and 250 nM forward and reverse primers. Reactions contained 400 ng of template RNA with the exception of the 28S-specific reactions, which contained 5 ng of template RNA. Reverse transcription was performed at 61°C followed by a 95°C denaturation step. The annealing temperature for all reactions was 60°C.
Western blotting
Concentrations of protein samples were quantified using Bradford reagent (BioRad, Hercules, CA, USA). Samples were run on gels containing 8 or 10% acrylamide (National Diagnostics, Atlanta, GA, USA) and then transferred to nitrocellulose membranes, which were blocked with tris-buffered saline with Tween (TBST) containing 5% milk. The membranes were probed with antibodies against HP1α (Millipore #07-346, Billerica, MA, USA), YY1 (Santa Cruz Biotechnology sc-7341X, Santa Cruz, CA, USA), hnRPA1 (Abcam ab5832, Cambridge, MA, USA), or β-tubulin (Covance TU27 MMS410P, Princeton, NJ, USA). Signal was detected using horseradish peroxidase-conjugated secondary antibody and developed using the BioRad Immun-Star reagents (Hercules, CA, USA).
Luciferase and β-galactosidase assays
Cells were plated in 35 mm culture dishes (105 cells/dish). On the following day cells were transfected with plasmid DNA (0.5 μg total) using 1.5 μl TransIT-LT1 reagent (Mirus, Madison, WI, USA) per plate. Each plate was transfected with 0.25 μg of luciferase reporter plasmid and 0.25 μg of the CMV-β-galactosidase plasmid. The final concentration of plasmid DNA was 0.5 ng/μl. After 6 to 8 hours cells were washed with phosphate-buffered saline (PBS) and re-fed. The next day plates were washed with PBS and cells were harvested using 0.4 ml Triton/Gly-Gly lysis buffer (25 mM Gly-Gly, pH 7.8; 15 mM MgSO4; 4 mM ethylene glycol tetraacetic acid (EGTA); 1% Triton X-100; 1 mM dithiothreitol (DTT)). Luciferase assays were performed by adding 50 μl of cell lysate to 300 μl of luciferase reaction solution (27 mM Gly-Gly, pH 7.8; 16 mM MgSO4; 0.1 mg/ml BSA; 1 mM DTT; 1 mM ATP). The sample was put in a luminometer (EG&G Berthold Lumat LB 9507, Oak Ridge, TN, USA), which injected 100 μl of 1 mM D-luciferin into each sample and measured the light emission for 10 seconds. β-galactosidase assays were carried out by adding 50 μl of cell lysate to 500 μl of Lac-Z reaction buffer (100 mM sodium phosphate, pH 6.95; 10 mM KCl; 1 mM MgSO4; 0.21% β-mercaptoethanol), followed by addition of 100 μl of o-nitrophenyl-β-D-galactopyranoside (ONPG) (4 mg/ml). The reactions were stopped by addition of 1 M Na2CO3 and quantified by measuring the absorbance at 420 nm. Transfection and reporter assays were performed at least three times and the result of a representative experiment is shown.
siRNA knockdown experiments
MCF7 cells were plated in 60 mm dishes (105 cells/dish). The following day the cells were transfected with siRNA using Oligofectamine reagent (Invitrogen, Carlsbad, CA, USA). siRNA oligos were diluted to 500 nM with Opti-MEM (Invitrogen, Carlsbad, CA, USA). Oligofectamine reagent (2.8 μl/plate) was diluted approximately 1:100 in Opti-MEM and allowed to stand at room temperature for five minutes. siRNA and Opti-MEM dilutions were then mixed at a 1:1 ratio and allowed to stand at room temperature for 20 minutes before adding to plates of cells. The final concentration of siRNA in each plate was 50 nM. The following day the plates were washed with PBS and re-fed with DMEM containing 10% fetal bovine serum. Five days after transfection RNA and protein were isolated using TRI Reagent (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. siRNA oligos were purchased from Dharmacon and were specific for luciferase (sense strand 5'-CUUACGCUGAGUACUUCGA-3') or YY1 (oligo 59: sense strand 5'-AAGAUGAUGCUCCAAGAAC-3'; oligo OT: sense strand 5'-CAUAAAGGCUGCACAAAGA-3').
Electrophoretic mobility shift assay
Probes were prepared by end-labeling each oligo (wild type probe: 5'-GCGCAAAACTCGCCATTTTACTACACG-3' and its complementary sequence; YY1 mutant probe: 5'-GCGCAAAACTCGAAGCTTTACTACACG-3' and its complementary sequence) with γ-32P ATP using T4 polynucleotide kinase (Promega, Madison, WI, USA). Radiolabeled probes were purified using Quick Spin Columns (Roche, Indianapolis, IN, USA). A 6% TBE (tris, boric acid, edta)-polyacrylamide gel was poured and allowed to polymerize overnight. Nuclear extract (25 μg) was incubated on ice for 15 minutes in EMSA buffer (6 mM Tris, pH 8.0; 6 mM MgCl2; 150 mM NaCl; 1 mM DTT) containing 10 μg/ml BSA, 10 μg/ml poly (dI-dC), 50 μg/ml salmon sperm DNA (Invitrogen, Carlsbad, CA, USA). One μg of antibody against AcK9H3 (Upstate #06-599) or YY1 (Santa Cruz sc-7341X) was then added to the appropriate samples, and all samples were incubated at room temperature for 30 minutes. One μl of the appropriate radiolabeled probe was then added and the samples (10 μl total volume) were incubated at room temperature for 30 minutes. Samples were run on a 6% polyacrylamide gel (pre-run for 30 minutes) at 80V for approximately 1.5 hours. The gel was dried and exposed to film.
Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (ChIP) assay was carried out as previously described [
37] with the exception of the sonication step. The chromatin was sonicated in a dry ice/ethanol bath for 10 minutes at amplitude of 40% to generate DNA fragments between 150 and 350 bp in length. Samples included either 2 μg normal mouse IgG, 0.2 μl nuclear respiratory factor-1 (NRF-1) antibody [
38] or 4 μg YY1 antibody (Santa Cruz sc-1703). PCR was performed using 5 μl of the ChIP-enriched DNA in a 25 μl reaction. The reaction was carried out for 34 cycles with an annealing temperature of 60°C. PCR products were stained with SYBR Green I nucleic acid gel stain (Invitrogen, Carlsbad, CA, USA) and run on a 2% low-melt agarose gel. The gel was visualized using a Typhoon scanner (GE Healthcare, Piscataway, NJ, USA) and the intensity of each band was quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA). Primer sequences are available on request.
Cell invasion/migration assays
HS578T cells were split 1:6 and transfected the next day with 6 μg of either an empty vector (CMV-empty) or a YY1 overexpression vector (pCDNA3 HA-YY1) using TransIT-LT1 reagent (Mirus, Madison, WI, USA). After 72 hours the cells were treated with Versene-EDTA (Cambrex Bio Science, East Rutherford, NJ, USA) and were resuspended in migration buffer (DMEM without phenol red (Mediatech, Manassas, VA, USA), 1% BSA, 1 μM MgCl2, 0.2 μM MnCl2). Migration transwells (Corning, Lowell, MA, USA) and matrigel invasion chambers (BD Biosciences, Franklin Lakes, NJ, USA) were prepared according to the manufacturer's protocols. 105 cells were used for each migration assay and 5 × 104 cells for each invasion assay. Twenty-four hours later the migration and invasion chambers were cleaned with cotton-tip applicators and stained for 15 minutes in crystal violet solution (diluted 1:5 in dH2O from a stock of 2 mg/ml in methanol). The membranes were destained in dH2O and allowed to dry overnight. The stain was eluted from the migration membranes using 10% acetic acid, and the eluate was read at OD 600 nm in a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The invasion membranes were removed, and mounted on slides using Permount (Fisher Scientific, Pittsburgh, PA, USA). The cells in 8 to 10 high-power fields (200× magnification) were counted and averaged for each membrane.
Discussion
The development and progression of cancer is due to changes in gene expression that result in the ability of cancer cells to proliferate autonomously, resist apoptosis, evade the immune system, and metastasize to distant sites [
42]. In past decades, much work in the cancer field has focused on identifying genetic alterations that suppress or promote these phenotypes. It has become increasingly apparent, however, that disruption of proper epigenetic mechanisms also contributes significantly to cancer development [
43]. For example, expression of HP1 in prostate cancer was found to be altered compared with normal prostate tissue [
44]. It is therefore not surprising that HP1 has been linked to cancer progression in humans [
45]. Although the molecular mechanism by which HP1α suppresses the invasive potential of breast cancer cells is unclear, it is important to understand the mechanism of reduced HP1α expression in invasive breast cancer cells.
In a previously published study, the DNA sequence of the HP1α gene in MCF7 and MDA-MB-231 was compared in order to identify any polymorphisms that may account for differential expression [
40]. This study found no differences in the sequence in the HP1α gene between these two cell lines. In addition, no difference in the pattern of DNA methylation was found in the HP1α gene promoter region. The investigators therefore hypothesized, as we did, that the difference in HP1α expression is attributable to differences in transcription factor occupancy at the promoter region in different cell lines [
40].
In the previous study, as in our study, luciferase reporter assays were used to determine how HP1α expression is differentially regulated at the level of transcription. One of the sequence motifs that was identified by this group was an E-box site (noted in Figure
1b). Mutation of this E-box site in the HP1α gene promoter construct resulted in a reduction in differential transcriptional activity between MCF7 and MDA-MB-231 cells [
40]. In agreement with their data, we found that mutation of the same E-box site in our CbxU283 construct caused a small increase in promoter activity in MCF7 cells (Figure
2b, M4). However, we did not find that mutation of the E-box site had a differential effect on promoter activity of the CbxU283 construct between MCF7 cells and MDA-MB-231 cells (data not shown) or HS578T cells (Figure
2b).
Instead, we identified transcription factor binding motifs that were not identified in the previous study, including binding motifs for NRF-1 and YY1. Although our mutagenic analysis was not exhaustive, our data suggests that NRF-1 and YY1 are both important positive regulators in the expression of HP1α. Mutating both the distal NRF-1 and YY1 sites (NRF-1.3 and YY1.2) resulted in a drastic reduction in promoter activity (Figure
2b, M6). This result is supported by previously reported NRF-1 knockdown experiments [
39], and by the YY1 knockdown data in this study (Figure
3b). YY1 has been implicated in the expression of many genes with numerous functions [
46]. However, this is the first report to show that YY1 regulates HP1α expression. The role of YY1 as a positive regulator of HP1α expression implicates YY1 as an important gene in maintaining the cellular epigenetic program.
Our mutagenic analysis of the HP1α gene promoter region also revealed a possible role for YY1 in differential expression between MCF7 and HS578T cells. We found that the level of YY1 RNA and protein is higher in MCF7 cells than in HS578T cells (Figure
1c, d). Our ChIP data show that YY1 occupancy at the HP1α promoter region is much lower in HS578T cells than in MCF7 cells (Figure
5). Taken together, our data suggests that this difference in YY1 occupancy contributes to the difference in HP1α expression between the MCF7 and HS578T cell lines.
YY1 is a member of the GLI-Kruppel family of zinc-finger transcription factors [
47]. YY1 is known to be essential for development because knockout of YY1 in mice results in peri-implantation lethality [
48]. YY1 can act as an activator or repressor of transcription depending on its interaction with other factors [
49,
50]. Multiple studies have implicated YY1 in the development and progression of cancer [
51,
52]. Some of the most compelling data implicating YY1 in tumorigenesis are regarding its role in apoptosis. YY1 negatively regulates p53 by facilitating its interaction with Mdm2 (and its human orthologue, Hdm2), leading to p53 ubiquitination [
53,
54]. This mechanism is thought to increase the cell's resistance to apoptosis in response to genotoxic stress [
54]. In addition, depletion of YY1 can sensitize cells to apoptotic stimuli through p53-independent pathways [
46]. The fact that YY1 expression can render a cell resistant to apoptosis may explain why levels of YY1 are increased in certain types of cancer such as osteosarcoma, non-melanoma skin cancer, and acute myeloid leukemia [
55‐
57].
Our data suggest that YY1 acts as a suppressor of migration in breast cancer cells. This is not the first report to implicate YY1 in invasion and metastasis. YY1 has been implicated in the metastatic progression of lung cancer cells due to its regulation of the putative metastasis suppressor HLJ1 [
58]. YY1 has also been implicated in the negative regulation of the chemokine receptor CXCR4 [
59], which has been implicated in the ability of breast cancer cells to metastasize to bone [
60]. In addition, our results indicate that decreased YY1 expression can result in decreased expression of HP1α, which could contribute to the development of an invasive phenotype.
By definition, metastasis suppressor genes affect the metastatic process without affecting tumorigenesis [
34]. As mentioned previously, YY1 has been proposed to play a number of roles in the process of tumorigenesis, and can therefore not be regarded as a pure metastasis suppressor. YY1 may play a role at several different points in cancer development and progression. High levels of YY1 may be advantageous to transformed cells during the early stages of cancer development, principally by reducing the tendency toward apoptosis. However, reduced expression of YY1 may be advantageous to metastasizing cells. Interestingly, high levels of YY1 are observed in high-grade prostate cancer, but prostate tumors with areas of low YY1 expression show a high rate of recurrence [
61]. It is possible that decreased expression of YY1 allows sub-populations of cells within these tumors to become more highly invasive. By exploring the role of YY1 in migration and invasion, our study adds another layer to the complex role that YY1 may play in the development of metastatic disease.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JGL and NT designed the research. JGL, MK, and CNP performed the research. JGL, MK, CNP, and NT analyzed the data. JGL and NT wrote the paper.