Identification of p100 target promoters by chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS)

Abstract

The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery. To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100, we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS). From this assay, we determined that a set of promoter fragments, including several factors in the transforming growth factor beta (TGF-β) signaling pathway, exhibited interaction with p100. The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-β signaling pathway in cell lines.

Introduction

Transcriptional regulation involves protein complexes specifically assembled at a given promoter to activate or suppress gene transcription. Transcriptional activation is regulated by coactivators that connect sequence-specific DNA-binding transcription factors to the general transcriptional machinery to initiate gene transcription. Coactivators are broadly divided into two classes according to their function: (1) chromatin remodeling enzymes that promote the DNA binding ability of transcription factors; and (2) adaptors that recruit transcription factors to the transcriptional apparatus.

p100 is a highly homologous protein expressed in Histoplasma capsulatum, Saccharomyces pombe, Mus musculus, Bos taurus and Homo sapiens. Human p100 was first identified as a coactivator of Epstein–Barr virus nuclear protein 2¹ and was subsequently discovered as a coregulator of c-Myb and pim-1.² In mammary epithelial cells, p100 levels increased in response to Prolectin (PRL) during lactation and correlated with the induction of β-casein gene expression.³ In our previous study, we found that p100 functions as a transcriptional coactivator for STAT5-dependent gene regulation and discovered the existence of a positive regulatory loop in PRL-induced transcription in which PRL stabilizes p100.⁴ Other groups have found that p100 serves as a core protein in the STAT6 enhanceosome,^{5, 6} which is composed of STAT6, p100, CBP, RNA pol II and SRC-1.

These studies suggest that p100 may play several important roles in cellular events and that it is likely to be a potential coactivator in distinct signal transduction pathways. Because promoter microarrays are an essential tool for global transcription factor binding studies, we adopted the ChIP-on-chip microarray technique to discover potential transcription complexes that interact with p100.

Materials and methods

Cell culture and transfection

HeLa cells were cultured as previously described.⁶ The HeLa-p100-Flag stable cell line was generated by co-transfecting pSG5-p100-Flag plasmid with hygromycin B resistance gene using the FUGENE6 Transfection Reagent (Roche Diagnostics). The breast cancer cell line MDA-MB-231 was transiently transfected with pSG5-p100-Flag plasmid by using Lipofectamine 2000 (Invitrogen).

Chromatin preparation and chromatin immunoprecipitation

HeLa-p100-Flag stable cells were fixed with crosslinking solution containing 1% formaldehyde (Sigma). The crosslinking reaction was then quenched by adding glycine solution. After washing with cold phosphate buffered saline, cells were resuspended in lysis buffer⁵ at 4 °C for 5 min. After sonication, the lysate was centrifuged at 14 000 r.p.m. for 10 min. The chromatin were immunoprecipitated with anti-Flag M2 agarose (Sigma) or polyclonal rabbit anti-IgG (as negative control) overnight at 4 °C. After washing, the agarose was incubated with elution buffer at 65 °C for 15 min. The eluted samples were incubated at 65 °C overnight to reverse the crosslinking. The samples were then amplified, labeled and hybridized to a chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS) microarray containing 20 000 unique human promoter regions (Aviva Systems Biology, San Diego, CA, USA).

Quantification of gene expression by real-time PCR

Total RNA was isolated from non-transfected and transfected MDA-MB-231 cells using Trizol (Invitrogen) according to the manufacturer's instructions. The RNA was used for cDNA synthesis using oligo(dT) primers and One Step RNA PCR Kit (M-MLV; Takara Biotechnology).

The PCR reactions were performed as previously described.⁶ The cycling conditions (30 cycles) consisted of denaturation at 94 °C for 30 s, annealing at 57.5 °C for 30 s (Smad1, Smad2 and Smad3) or 58.1 °C (Smad4 and Smurf2) or 61.2 °C (transforming growth factor beta 1-induced transcript 1, TGFβ1I1 and transforming growth factor beta 1-induced factor homeobox 1, TGIF1) and elongation at 72 °C for 20 s. The PCR samples were detected on 2% agarose gels.

Western blot analysis

Total cell lysates were collected using ice-cold RIPA lysis buffer (1% NP-40, 0.1% SDS, 300 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 20% glycerol and 0.1 mM sodium orthovanadate). Proteins were separated on an 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and analyzed by western blotting with p100 and β-actin mouse monoclonal antibody (1∶5000 dilution; Sigma) or anti-Flag monoclonal antibody (1∶1000 dilution; Sigma). The results were visualized using chemiluminescent substrate kit (SuperSignal West Pico Trial Kit; Pierce Biochemicals).

Results and discussion

Setting up stable cells overexpressing p100 protein

The eukaryotic expression plasmid containing p100 tagged with Flag peptide was co-transfected with plasmid containing the hygromycin B resistance gene into HeLa cells and then selected with media containing hygromycin B.

Western blot was performed to confirm Flag-tagged p100 expression from p100-stable HeLa cells. Figure 1a shows that while the parental HeLa cells did not express Flag-tagged p100, the stable cell line overexpressed Flag-tagged p100 protein detected by anti-Flag antibody.

Figure 1.

(a) The human p100 protein was efficiently overexpressed in HeLa cells transfected with recombinant plasmids containing p100-Flag. Total cell lysates from parental HeLa cells or stable cells overexpressing p100 (HeLa-p100) were separated on an 8% SDS-PAGE gel and analyzed by western blotting with anti-p100 antibody (upper panel), anti-Flag antibody (middle panel) or anti-β-actin antibody (lower panel). (b) Smad1, Smad2, Smad3, Smad4, Smurf2, TGFβ1I1 and TGIF1 mRNA levels were measured by real-time PCR in parental HeLa cells and HeLa-p100 stable cells. (c) The p100 protein was efficiently overexpressed in MDA-MB-231 cells transfected with recombinant plasmid containing p100-Flag. Equal amounts of total cell lysates from pSG5-p100-Flag transfected or untransfected parental MB-231 cell line were separated on an 8% SDS-PAGE gel followed by immunoblotting with anti-Flag antibody (upper panel), anti-p100 antibody (middle panel) or anti-β-actin antibody as a control (lower panel). (d) Smad1, Smad2, Smad3, TGFβ1I1, TGIF1, Smurf2 and Smad4 mRNA levels were detected by real-time PCR. (e) The PCR product was detected on a 2% agarose gel. The results shown are representative of three independent experiments. TGFB1I1, transforming growth factor beta 1-induced transcript 1; TGIF1, transforming growth factor beta 1-induced factor homeobox 1; SDS-PAGE, SDS–polyacrylamide gel electrophoresis.

Transforming growth factor beta (TGF-β) signal pathway target genes identified by ChIP-GLAS

We used microarrays containing 20 000 putative human promoters to detect the promoters with high binding affinity to p100. The complete ChIP-GLAS statistical data analysis is shown in the supplementary material. To eliminate background noise, we only considered those gene promoters showing a more than twofold relative binding value.

On the basis of the above results, we found that a set of promoter fragments of genes involved in the TGF-β signaling pathway exhibited interaction with p100 in vitro. We set a P value of <0.05, which corresponds to a relative binding value of 2.0 or more, as a threshold for p100-binding candidate promoters. Using this threshold, we identified 16-targeted promoters that were involved in the TGF-β signaling pathway, including TGFβ1I1, the four Smads (Smad1–4) and other transcription factors or regulators.^{7, 8, 9} This suggested that p100 might be a transcriptional coactivator involved in the TGF-β signaling pathway (Table 1). To verify the effects of p100 on the expression of the related members of the TGF-β signaling pathway, we performed real-time PCR to detect the mRNA expression in parental HeLa cells and HeLa-p100 stable cells. As shown in Figure 1b, p100 overexpression enhanced Smad1, Smad4, Smurf2 and TGIF1 mRNA expression, which is consistent with the ChIP-GLAS results. However, there was no significant difference in Smad2, Smad3 and TGFβ1I1 mRNA expression.

Table 1. List of transcription factors and regulators with promoters exhibiting high p100 binding affinity in the TGF-β signaling pathway.

Nucleotide accession	ID	Ratio	P value	Name	Description
NM_022739	Amine#10693	4.790	0.04	SMURF2	SMAD-specific E3 ubiquitin protein ligase 2
NM_006022	Amine#18644	4.578	0.03	TGFB1I4	TSC22 domain family, member 1
NM_015927	Amine#18772	4.168	0.03	TGFB1I1	TGF-β1-induced transcript 1
NM_005902	Amine#18626	3.829	0.05	SMAD3	SMAD, mothers against DPP homolog 3 (Drosophila)
NM_173211	Amine#13690	3.701	0.05	TGIF1	TGFB-induced factor 1
NM_020429	Amine#10170	3.688	0.05	SMURF1	SMAD-specific E3 ubiquitin protein ligase 1
NM_005359	Amine#18590	2.909	0.05	SMAD4	SMAD, mothers against DPP homolog 4 (Drosophila)
NM_005901	Amine#06888	2.675	0.05	SMAD2	SMAD, mothers against DPP homolog 2 (Drosophila)
NM_005900	Amine#06887	2.497	0.05	SMAD1	SMAD, mothers against DPP homolog 1 (Drosophila)

Abbreviations: DPP, decapentaplegic protein; TGF, transforming growth factor.

It was recently reported that p100 expression level is higher in breast cancer cells than in normal breast tissue.¹⁰ Therefore, we used the breast cancer cell line MDA-MB-231 to further investigate the effect of p100 on the expression of related target genes in the TGF-β pathway. As shown in Figure 1d and 1e, while Smad3 mRNA expression level was only slightly increased, Smad1, Smad2, TGIF1, Smad4, TGFβ1I1 and Smurf2 mRNA expression levels were increased remarkably in MDA-MB-231 cells with ectopically expressed p100 compared with parental 231 cells. These data are consistent with the ChIP-GLAS results and indicate that ectopic p100 expression enhanced the expression of certain TGF-β signaling pathway members in the breast cancer cell line MDA-MB-231. Thus, p100 is a potential regulator in the TGF-β signaling pathway. Considering the roles of Smad1-4, Smurf2, TGFβ1I1 and TGIF1 in the TGF-β signaling pathway and recent studies demonstrating that p100 upregulation is related to breast cancer,¹⁰ prostate cancer ¹¹ and colon cancer,¹² further understanding of p100 in the regulation of the TGF-β pathway may help elucidate the underlying mechanisms of tumorigenesis.