Novel genes in cell cycle control and lipid metabolism with dynamically regulated binding sites for sterol regulatory element-binding protein 1 and RNA polymerase II in HepG2 cells detected by chromatin immunoprecipitation with microarray detection Mehdi Motallebipour1, Stefan Enroth2, Tanel Punga3,*, Adam Ameur2, Christoph Koch4,(cid:2), Ian Dunham4,(cid:3), Jan Komorowski2,5, Johan Ericsson3,§ and Claes Wadelius1
1 Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Sweden 2 Linnaeus Centre for Bioinformatics, Biomedical Centre, Uppsala University, Sweden 3 Ludwig Institute for Cancer Research, Biomedical Centre, Uppsala University, Sweden 4 Wellcome Trust Sanger Institute, Cambridge, UK 5 Interdisciplinary Centre for Mathematical and Computer Modelling, Warsaw University, Poland
Keywords HCFC1; human; intragenic binding; sterol- regulated; transcriptional regulation
Correspondence C. Wadelius, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden Fax: +46 18 471 4808 Tel: +46 18 471 4076 E-mail: claes.wadelius@genpat.uu.se
Sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP- 2) are important regulators of genes involved in cholesterol and fatty acid metabolism, but have also been implicated in the regulation of the cell cycle and have been associated with the pathogenesis of type 2 diabetes, atherosclerosis and obesity, among others. In this study, we aimed to char- acterize the binding sites of SREBP-1 and RNA polymerase II through chromatin immunoprecipitation and microarray analysis in 1% of the human genome, as defined by the Encyclopaedia of DNA Elements consor- tium, in a hepatocellular carcinoma cell line (HepG2). Our data identified novel binding sites for SREBP-1 in genes directly or indirectly involved in cholesterol metabolism, e.g. apolipoprotein C-III (APOC3). The most inter- esting biological findings were the binding sites for SREBP-1 in genes for host cell factor C1 (HCFC1), involved in cell cycle regulation, and for filamin A (FLNA). For RNA polymerase II, we found binding sites at clas- sical promoters, but also in intergenic and intragenic regions. Furthermore, we found evidence of sterol-regulated binding of SREBP-1 and RNA poly- merase II to HCFC1 and FLNA. From the results of this work, we infer that SREBP-1 may be involved in processes other than lipid metabolism.
Present address *Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland (cid:2)QIAGEN Hamburg GmbH, Research & Development, Hamburg, Germany (cid:3)EMBL-European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, UK §UCD Conway Institute, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin, Ireland
(Received 9 October 2008, revised 31 December 2008, accepted 20 January 2009)
doi:10.1111/j.1742-4658.2009.06914.x
Abbreviations ACSL6, acyl-CoA synthetase long-chain family member 6 gene; APOC3, apolipoprotein C-III gene; ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP with microarray detection; DBD, DNA-binding deficient; DOLPP1, dolichyl pyrophosphate phosphatase 1 gene; ENCODE, encyclopaedia of DNA elements; EST, expressed sequence tag; FLNA, filamin A gene; FOXA2, forkhead box A2; H3Ac, acetylation of histone H3; HCFC1, host cell factor C1 gene; HNF, hepatocyte nuclear factor; LCB-DWH, Linnaeus Centre for Bioinformatics data warehouse; LDL, low-density lipoprotein; LDLr, low-density lipoprotein receptor; MAPK, mitogen-activated protein kinase; PCA, principal component analysis; PCG, protein coding gene; PDCR, peroxisomal 2,4-dienoyl CoA reductase 2 gene; RNA-pol II, RNA polymerase II; RPS9, ribosomal protein S9 gene; siRNA, small interfering RNA; SREBP, sterol regulatory element-binding protein; TSS, transcriptional start site; UES, unique enriched spot; USF1, upstream stimulatory factor 1.
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shown that genomic chromatin immunoprecipitation (ChIP) studies of RNA-pol II give an excellent indica- tion of transcriptional activity [27,28].
target genes
Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors crucial for lipid metabolism. There are three well-studied members in this family: SREBP-1a and SREBP-1c, which are iso- forms encoded by the same gene, and SREBP-2. Other splice variants of SREBP-1 have been reported (Swiss-Prot: P36956) [1,2]. SREBPs are synthesized as inactive precursor proteins bound to the endoplasmic reticulum membrane. In response to various signals, the precursor undergoes two proteolytic cleavages, resulting in the release of the active transcription fac- tors, which are translocated to the nucleus and bind [3]. The mature forms of to their SREBPs are modified by phosphorylation [4–9], acet- ylation [10], sumoylation [11] and ubiquitination [8,9,12,13]. These modifications regulate the stability and ⁄ or transcriptional activity of the active transcrip- tion factors.
Until now, SREBPs have generally been analysed for their role in lipid metabolism and the regulation of genes directly involved in these processes. Some studies have indicated a cell cycle-dependent activity for SREBP-1 and have suggested a link to the regulation of cell proliferation and growth [4,6]. Therefore, we hypothesized that genes not directly involved in lipid metabolism could be regulated by this transcription factor. To test this hypothesis, we performed a ChIP study with with microarray detection (ChIP-chip) SREBP-1 in the human hepatocellular carcinoma cell line HepG2. The immunoprecipitated material was hybridized to genomic tiling path microarrays contain- ing the Encyclopaedia of DNA Elements (ENCODE) regions, which cover roughly 1% of the human gen- ome [29]. We also performed ChIP-chip of RNA-pol II in a similar way to identify the correlations between SREBP-1 binding sites and transcriptional activity. As expected, we found target sites for SREBP-1 upstream of the genes involved in lipid metabolism. However, the interesting finding was the binding of SREBP-1 at the 3¢-end, either intragenic or downstream of cell proliferation genes.
Results
Validation of antibody
e.g.
In general, SREBPs are activated in response to decreased levels of cellular sterols. One of the members of this family, SREBP-1c, is also activated in response to insulin signalling, as well as by the liver X receptor-a, a transcription factor involved in the clearance of cho- lesterol [14]. The binding of SREBPs to their target genes is mediated mainly and more efficiently by the SRE, although binding at E-box elements has also been demonstrated [15]. The same study also estab- lished that the different SREBP isoforms have different preferences for and different transactivation ability when associated with the two binding elements. The SRE has been located upstream of several genes involved in lipid and glucose metabolism, e.g. the low- density lipoprotein receptor (LDLr) [16], the insulin gene [17] and granuphilin [18]. A four-nucleotide dele- tion in the promoter of the small heterodimer partner has been implicated in lower birth weights, and has been hypothesized to affect insulin secretion [19]. Later, this mutation was located in an SRE bound by SREBP-1 in humans [20]. This, together with other molecular and genetic studies, has led to the associa- tion of SREBPs with the pathogenesis of several meta- bolic disorders, type 2 diabetes, obesity and syndrome X [21–25].
Antibodies used for ChIP should be tested prior to performing the assay for two reasons: (a) the antibody should be specific in order to obtain reliable results; and (b) the abundance of the studied protein in the desired cell line can be verified. Therefore, we tested the antibody against a panel of cell extracts in two independent western blot assays (data not shown). The antibody was also tested on extracts from small inter- fering RNA (siRNA)-treated HepG2 cells, where we noticed a major decrease in the amount of SREBP-1 in response to SREBP-1 siRNA (Fig. 1A). Impor- tantly, the major bands recognized by the SREBP-1 antibody were reduced in SREBP-1-depleted cells.
the transcriptional
Active SREBP-1 has a high turnover in cells as a result of phosphorylation-dependent ubiquitination and proteasome-mediated degradation. Therefore, pro- teasome inhibitors such as Z-Leu-Leu-Leu-al, also known as MG 132, can be used to inhibit the degrada- tion of SREBP-1 and thereby enhance the enrichment signals. To determine how this inhibitor affects the binding of SREBP-1 to its target sequences, cells were treated with MG 132 or dimethylsulfoxide 4 h prior to
The first stage of the regulation of gene expression occurs at level. This is mainly through the recruitment and regulation of RNA poly- merase II (RNA-pol II) by different constellations of transcription factors and cofactors, which initiate the transcription of mRNA. SREBPs are known to bind p300 ⁄ cAMP response element-binding protein and the activator-recruited cofactor ⁄ Mediator complex to exert their effect on the expression of downstream genes via RNA-pol II [26]. Furthermore, recent studies have
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siRNA C S1
A
ChIP, and the enrichment was detected by PCR (Fig. 1B). For all targets, we verified a higher enrich- ment after inhibitor treatment. The RNA-pol II anti- body was also tested in ChIP-PCR, where we confirmed the enrichment of known target genes (Fig. 1C).
P
ChIP-chip for SREBP-1 and RNA-pol II
WB: SREBP1 (H160)
M
DMSO
B
MG-132 IP IgG Inp
IP IgG
Inp
HMGCS1
LDLr
FDPS
HMGCR
stSG610844
Three independent biological and technical replicates of ChIP were performed for SREBP-1, both before and after treatment with MG 132, for RNA-pol II and for IgG, and the DNA was hybridized to ENCODE arrays without amplification. IgG was used as a nega- tive control and to detect technical false positive spots on the microarray. The reproducibility of our repli- cates was verified by principal component analysis (PCA) of all data (Fig. S1). For each of the factors, a cut-off value for true enrichment was selected and spots with a replicate mean log2 ratio above this cut- off were considered as positive. For each spot, we required at least two of the three replicates to be pres- ent after filtering. In tiling path arrays, depending on the size of the fragments after sonication, one to sev- eral consecutive spots can show enrichment. In these cases, the spot with the highest enrichment was consid- ered and defined as the unique enriched spot (UES).
IP
IgG
Inp
C
RBM39
GAPDH
stSG610844
As only a few enriched spots could be detected for SREBP-1 without MG 132 treatment, from this point onwards we will only discuss results from ChIP with inhibitor treatment. For SREBP-1, 45 UESs were iden- tified in 1% of the human genome; this number was 143 for RNA-pol II. To corroborate the results, 18 (SREBP-1) and 25 (RNA-pol II) of the positive UESs were randomly selected. For each of the factors, four ChIPs were performed, which, after testing by PCR, were pooled to erase the lot-to-lot variation in enrich- ment. The pooled ChIPs were then used in quantitative PCR with primers targeting the chosen regions. For each factor, four negative regions were also selected to set the background value. For SREBP-1, we found that only seven of the 18 (39%) tested UESs were true positives. The reason for the low frequency of true positives may be the nature of the target sequences bound by SREBP-1 and the design of the arrays, as indicated in the Discussion section. For RNA-pol II, 100% of the chosen UESs were true positives.
New target genes identified
Fig. 1. Pre-ChIP-chip validations. (A) Validation of SREBP-1 anti- body specificity by siRNA. HepG2 cells were transfected with con- the (C) or SREBP-1 (S1) siRNA. Total cell extracts of trol transfected cells were immunoprecipitated with SREBP-1 antibody (H160), resolved by SDS-PAGE and transferred to nitrocellulose membranes, followed by immunoblotting with the same antibody (H160). P, precursor, M, mature. (B) Pre-hybridization ChIP-PCR. Enrichment of known targets for SREBP-1 and RNA-pol II in ChIP with HepG2 cells was verified by semiquantitative PCR. Cells were treated with either MG-132 or dimethylsulfoxide for 4 h, and ChIP was performed with the SREBP-1 antibody. HMGCS1, 3-hydroxy-3- methylglutaryl-coenzyme A synthase 1; LDLr, low-density lipo- protein receptor precursor; FDPS, farnesyl diphosphate synthase; HMGCR, reductase; 3-hydroxy-3-methylglutaryl-coenzyme A stSG610844, Sanger spot ID for a region between A-c globin and G-c globin. (C) ChIP with RNA-pol II antibody. RBM39, RNA binding motif protein 39, also known as RNPC2; GAPDH, glyceraldehyde-3- ID for a phosphate dehydrogenase; stSG610844, Sanger spot region between A-c globin and G-c globin.
Among the UESs identified for SREBP-1 through the ChIP-chip analysis and verification by quantitative PCR, we found genes that, either directly or indirectly,
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were involved in lipid and fatty acid metabolism, as well as genes that were involved in the cell cycle and cell proliferation. To our knowledge, these genes have not been reported previously in any other studies.
Cholesterol and fatty acid metabolism genes
H3Ac (Fig. S3). There are no studies on human DOL- PP1, but mouse DOLPP1 has been demonstrated to be a functional homologue of Saccharomyces cerevisiae CWH8 [30]. The product of this gene is involved in protein N-glycosylation and dephosphorylation of Dol-P-P which participates in lipid intermediate bio- synthesis.
Apolipoproteins are important players in the uptake, transport and catabolism of lipids, and some members of this family are known to be regulated by SREBPs (reviewed in [31]). In this study, we found two new bind- ing sites for SREBP-1, one of which, the verified site, was in the promoter region of the apolipoprotein C-III (APOC3) gene (Fig. S4). Promoter binding coincided with HNF4a, RNA-pol II and H3Ac, whereas the sec- ond target site, located at the 3¢-end of APOC3, over- lapped with binding sites for both HNF4a and FOXA2, together with H3Ac and RNA-pol II. The mature form of apolipoprotein C-III is a part of very low-density lipoprotein, chylomicrons and high-density lipoprotein.
Cell proliferation genes
Approximately 3.5 kb upstream of a longer transcript and in the first intron of a shorter transcript of acyl- CoA synthetase long-chain family member 6 (ACSL6), we found a binding site for SREBP-1 together with RNA-pol II and in close vicinity to forkhead box A2 (FOXA2) and hepatocyte nuclear factor 4a (HNF4a) (Fig. 2). ACSL6 is involved in the activation of long- chain fatty acids. In the second intron of peroxisomal 2,4-dienoyl CoA reductase 2 (PDCR, alias DECR2) and approximately 1 kb upstream of a shorter tran- script, we found another binding site for SREBP-1 together with RNA-pol II and HNF4a in a region with acetylation of histone H3 (H3Ac) (Fig. S2). PDCR plays a role in b-oxidation and metabolism of unsaturated fatty enoyl-CoA esters. Although we have not confirmed the binding site by quantitative PCR, we also found SREBP-1 binding together with RNA- pol II in the promoter region of dolichyl pyrophos- phate phosphatase 1 (DOLPP1) in a broad region of
The common feature for all three genes related to cell proliferation found in this study was that the SREBP-1 located in the defined binding
sites were not
Fig. 2. The ACSL6 gene with binding sites for SREBP-1 and RNA-pol II. A UCSC browser window with the ACSL6 gene on chromosome 5. The first two rows indicate UESs for SREBP-1 and RNA-pol II together with their Sanger spot code. Thereafter follows the map of genes and gene predictions and their transcripts, followed by conservation tracks, CAGE-tag data and indications of Stanford promoter activity. The last rows exhibit data from our laboratory on H3Ac, FOXA2 (HNF3b), HNF4a and USF-1. The confirmed SREBP-1 and RNA-pol II binding site is stSG600984.
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promoter or an upstream region; rather, they were located intragenic or downstream of the 3¢-end (Fig. S5) overlapping with RNA-pol II and H3Ac. In addition, all three genes showed binding of RNA- pol II at the 5¢-end of the gene, which, in some cases, might indicate long-range regulation through looping or regulation of an alternative promoter.
tested whether the binding of SREBP-1 to HCFC1 and FLNA genes was regulated by sterol. For this, HepG2 cells were grown in lipoprotein-deficient medium, either in the absence or presence of cholesterol and 25-hydroxycholestrol, prior to ChIP assay. Binding was verified by PCR using the same set of primers as in quantitative PCR. As demonstrated in Fig. 3, the binding of SREBP-1 to both HCFC1 and FLNA was regulated by the concentration of sterols, with high binding in sterol-depleted cells and low binding in ste- rol-loaded cells. Even more interesting was the finding that the recruitment of RNA-pol II to its binding site close to SREBP-1 was regulated by sterol. As recent data have indicated that there is an almost perfect cor- relation between RNA-pol II binding and active tran- scription, this experiment indicates a sterol-dependent in the HCFC1 and transcriptional start site (TSS) FLNA genes.
To verify the binding of SREBP-1 to the intragenic SRE in HCFC1, an electrophoretic mobility shift
One of the significant findings among these three genes was host cell factor C1 (HCFC1, also known as HCF-1) (Fig. S6), a key regulator of cell prolifera- tion, which has been shown to interact with SP1 transcription factor (SP1), GA-binding protein b, per- oxisome proliferator-activated receptor gamma coacti- vator 1A & B (PGC1a and PGC1b) [32], among others, as well as with different histone-modifying complexes [33,34]. The other important gene found to targeted by SREBP-1 was filamin A (FLNA) be (Fig. S7). Filamins, in addition to remodelling of the actin cytoskeleton, participate in the integration of signals and interact with a large array of proteins in the cell [35]. The third cell proliferation gene possibly ribosomal protein S9 regulated by SREBP-1 was (RPS9) (Fig. S5).
Corroboration of SREBP-1 binding to some of the new target genes
As mentioned previously, a known factor for the acti- vation of SREBP-1 is sterol depletion. Therefore, we
Fig. 4. Binding of SREBP-1 in HCFC1 is through an SRE. A double- stranded oligonucleotide containing the putative binding site of SREBP-1 in the HCFC1 gene was tested in electrophoretic mobility shift assay, together with His-tagged SREBP-1. A protein–DNA complex is detected when the labelled oligonucleotide is incubated with wild-type SREBP-1, but not when incubated with a DBD mutant of SREBP-1. The SREBP-1–DNA complex is specifically supershifted on addition of an His-tag antibody, and the complex is completed when adding the non-labelled oligonucleotide (cold probe). A similar pattern is detected when performing the same experiments with an oligonucleotide containing the SRE from the LDLr promoter.
Fig. 3. Sterol regulation of SREBP-1 and RNA-pol II binding in HCFC1 and FLNA genes. Cells were cultured in the presence or absence of sterols before ChIP was performed. The material was then tested by semiquantitative PCR with primers from the quanti- tative PCR verification. RNAPII, RNA-pol II; HCFC1, host cell fac- tor C1; FLNA, filamin A; LDLr, low-density lipoprotein receptor promoter; LDLrorf, LDLr open reading frame.
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Data mining
assay was performed. Oligonucleotides containing the target sequence for SREBP-1 in either HCFC1 or LDLr were incubated with recombinant wild-type or DNA-binding-deficient (DBD) His-tagged SREBP-1a (Fig. 4). Binding of SREBP-1 to SRE in HCFC1 showed a similar pattern as for the element in LDLr. The specificity could be confirmed by supershifting and competition with cold probes. Thus, we con- cluded that SREBP-1 binds to intragenic sites in the HCFC1 and FLNA genes in a sterol-regulated man- ner, this binding in HCFC1 is specifically through an SRE, and a TSS is activated through the recruitment of RNA-pol II.
We aimed to discover where the binding sites of RNA- pol II were located in relation to TSSs. As a first step, our data were compared with databases for UCSC Known, ENSEMBL and RefSeq genes. This showed that only 24% (23 and 11 of 143) of RNA-pol II UESs were located within 1 kb from the TSS of protein cod- ing genes (PCGs) (Fig. 5). In the next step, we mapped the UESs in relation to human mRNAs, not consider- ing the spots already mapped to 1 kb distance from Known genes. This procedure was then repeated regarding the spliced expressed sequence tags (ESTs)
Fig. 5. Distribution of RNA-pol II binding in relation to TSS. The distance between the midpoint of UES for RNA-pol II and the TSS of the characterized transcripts. All spots were first mapped according to the data for UCSC Known Genes, ENSEMBL and RefSeq. Those that did not map within 1 kb from TSS were then compared with human mRNA, spliced ESTs and human ESTs, in that order. Spots mapped within 1 kb from TSS in the previous pie chart are masked in the succeeding chart by a black colour.
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Table 1. Number of UESs and overlap with previous data [39] for RNA-pol II. Number of spots for each factor is given either as UES or UES when including one neighbour up ⁄ down (UES + 1n).
No. spots
RNA-pol II
UES
UES + 1n
UES
UES + 1n
Factor ⁄ modification
and human ESTs (Fig. 5). In this manner, we mapped 84% of RNA-pol II UESs within 1 kb of a character- ized transcript. This indicates that 16% (or 23) of RNA-pol II sites in the 1% of the human genome covered by these arrays are at transcripts that need to be identified.
N ⁄ A
RNA-pol II USF1 FOXA2a HNF4a H3Acb
408 90 452 571 513
4 (0.044) 13 (0.029) 25 (0.044) 62 (0.120)
143 31 154 194 N ⁄ A
N ⁄ A 1 (0.032) 7 (0.045) 7 (0.036) N ⁄ A
a Also known as HNF3b. b Histone H3 lysine 9 and 14 acetylation. For H3Ac, all enriched spots have been counted.
Next, we looked at the correlation of our data with the Stanford promoter activity and expression data for HepG2. Stanford promoter activity is an experimen- tally verified prediction of promoters in the human genome through luciferase-based transfection assays [36–38]. In this comparison, we found a correlation between RNA-pol II binding sites and promoter activ- ity in HepG2 cells (Fig. 6A). There was no significant correlation between the promoter activity and the total enrichment signal on the chip. In the comparison of
gene expression in HepG2 cells with RNA-pol II bind- ing, we found that it correlated well with higher gene expression (Fig. 6B).
A
Finally, to identify overlap in binding sites and pos- sible interactions, the results were compared with ChIP-chip data from a previous study on FOXA2, HNF4a, upstream stimulatory factor 1 (USF1) and H3Ac (Table 1) [39]. For FOXA2 and HNF4a, we found that 4.5% and 3.6%, respectively, of the UESs overlapped with RNA-pol II. Furthermore, 43% (62 of 143) of RNA-pol II binding sites overlapped with H3Ac.
Discussion
B
We interrogated 1% of the human genome, as defined by the ENCODE project, for SREBP-1 and RNA- pol II binding in HepG2 cells through ChIP-chip analysis. For SREBP-1, we were able to confirm its binding to regulatory sequences of several novel genes, involved in either lipid metabolism or cell prolifera- tion. For RNA-pol II, the number of binding sites and their position relative to TSS correlated well with stud- ies performed in other tissues. Specifically, we found the binding of SREBP-1 and RNA-pol II to HCFC1 and FLNA genes to be regulated by sterol.
cannot be
spots
that
Fig. 6. RNA-pol II binding correlated with Stanford promoter activ- ity and expression in HepG2 cells. (A) Box-and-whisker plot repre- sents the correlation between Stanford promoter activity in HepG2 cells for the ENCODE region and RNA-pol II data. RNA-pol II bind- ing is highly correlated with promoter activity (P = 0.001997). (B) The expression levels of the genes in the category ‘Known Genes TSS 1k’ and ‘Known Genes TSS 500’ in Fig. 5 (34 genes) were compared with the expression level of all genes in HepG2 cells through the box-and-whisker plot. Binding of RNA-pol II within 1 kb distance of TSS is correlated with the expression of that gene (P = 0.01169).
The surprisingly large number of false positives (11 of 18 tested) for SREBP-1 might be explained by the nature of the sequences bound by this factor. Metabol- ically related genes have been shown to contain more Alu repeats than some other classes of genes, and Alu repeats have been shown to contain many binding sites for SREBPs [40,41]. The repeat sequences can cross- hybridize with other sequences, which might cause positive reproduced and confirmed by quantitative PCR. This problem is prom- inent with the arrays used in this study, as they con- tain repeat elements. Indeed, the false positive spots
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involved in the regulation of other important cellular events, such as the cell cycle.
contain, on average, 25% repeats, whereas the true positives contain 11% repeats. After this correction, we can extrapolate our result for SREBP-1 to the whole genome and predict 1500–2000 binding sites. A recent study found 1141 promoters with putative binding for SREBP-1 in HepG2 cells after insulin and glucose induction [42].
Apolipoprotein C-III is a component of very low- density lipoprotein, chylomicrons and high-density lipoprotein. The triglyceride levels in blood are directly regulated by apolipoprotein C-III, so that over- or underexpression of this protein results in higher or lower triglyceride levels [47]. SREBP-1 has been indi- cated as a regulator of other members of the APO gene family, but this is the first time that its binding in vivo at the APOC3 gene has been shown. Binding of HNF4a in the promoter of APOC3 has been reported, where it functions as a positive regulator of expression [31,48]. According to our data, both HNF4a and SREBP-1 bind together with RNA-pol II in the pro- moter of APOC3, and therefore may have a synergistic function. HNF4a and SREBPs are known to interact in vivo and SREBP-1 inhibits some of the HNF4a tar- get genes, except when these are involved in lipid metabolism [49,50]. Enhanced levels of triglycerides in blood are a major factor in metabolic disorders, such as type 2 diabetes, obesity and atherosclerosis. There- fore, this finding may add to our understanding of the pathogenesis of these diseases.
The 143 binding sites for RNA-pol II detected in this study can roughly be extrapolated to 14 300 bind- ing events for the whole human genome, 84% of which are located within 1 kb of a characterized transcript. Using the same antibody, Brodsky et al. [43] located 102 binding sites in the ENCODE regions for HeLa cells, with 62% at the first exons of a known gene. In human fibroblast cells IMR90, 12 000 binding sites were found for the preinitiation complex subunit TFIID in the whole human genome, where 87% of the binding sites were positioned within 2.5 kb from an mRNA [27]. Of these, 83% were sites within 500 bp of the TSS. Thus, the number of binding events for RNA-pol II and their positions relative to TSS in HepG2 cells seem to correlate well with HeLa and IMR90 cells. The difference in absolute numbers between these studies might be a result of tissue speci- ficity, the type and resolution of the arrays used, the size of the fragments after sonication and the chosen cut-offs.
Some of our reported binding sites for RNA-pol II are located further along the gene or at the 3¢-exons of the genes. Two examples are the binding sites for HCFC1 and FLNA. This is not surprising, as CAGE- tag analysis has revealed at least 1000 of such instances in the mouse genome [44]. In addition, treatment of MCF-7 cells with 17b-oestradiol has been shown to lead to the activation of TSS at the 3¢-end of genes [45]. The function of RNA-pol II binding at the 3¢-sites of genes is not completely understood, but three main categories can be assessed: (a) alternative TSS for the same gene; (b) antisense transcription affecting the gene where it is transcribed, a down- stream gene, or both [46]; and (c) other functional transcripts. These bindings may often occur together with other transcription factors, e.g. SREBP-1 (as presented in this study), HNF4a and FOXA2. The last two transcription factors are involved in the activation of a large set of genes, and they have also been demonstrated to occupy intragenic sites or sites located far from TSS [39].
In a recent publication, HCFC1 has been demon- strated to interact with E2Fs in a cell cycle-dependent manner [51]. Specifically, HCFC1 has been shown to interact with E2F2 bound to its target genes during the G1 ⁄ S phase and to recruit histone H3 lysine 4-meth- yltransferases, which, in turn, activate the E2F2 target genes cyclin A, E2F1 and p107. Here, we have shown sterol-regulated binding of SREBP-1, together with RNA-pol II, to an intragenic site close to the TSS of a shorter transcript from the HCFC1 gene. Bengoechea- Alonso & Ericsson [4] have demonstrated that siRNA- mediated knockdown of SREBP-1 in human HeLa, U2OS and MCF-7 cells leads to an accumulation of cells in late G1 phase, prior to the G1 ⁄ S transition. Thus, if the binding of SREBP-1 results in the regulation of HCFC1, it would add HCFC1 to the list of cell cycle regulatory genes targeted by SREBP-1. The cyclin- dependent kinase inhibitor p21waf1 ⁄ CIP1, another cell cycle regulatory gene, is activated by SREBP-1 in liver [52]. Moreover, a ChIP-chip study with promoter arrays in HepG2 has indicated that insulin-induced SREBP-1 binds at the promoter of 60 genes involved in cell cycle regulation [42]. In the same study, the promoter of the host cell factor C1 regulator 1, a regulator of HCFC1 subcellular localization, was found to be occupied by SREBP-1, further strengthening our results.
Through the ChIP-chip study presented here, several possible novel targets of SREBP-1 with important cellular functions have been revealed. These findings not only show that SREBP-1 directly regulates several aspects of lipid metabolism, but also that this factor is
He et al. [53] demonstrated that FLNa interacts with the insulin receptor in HepG2 cells and inhibits insulin signalling through the p42 ⁄ 44 mitogen-activated protein
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(Santa Cruz Biotechnology). In addition, a normal rabbit IgG was used as control (12-370) (Upstate, Temecula, CA, USA).
siRNA
HepG2 cells were transfected with control or SREBP-1 siRNA (Ambion, Austin, TX, USA) using SilentFect (Bio-Rad, Hercules, CA, USA), as described previously [4]. Whole-cell lysates were prepared 48 h after transfection and immunoprecipitated with SREBP-1 antibody (H160). The immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The levels of SREBP-1 in the samples were estimated by western blotting.
ChIP
kinase (MAPK) pathway (also known as extracellular signal-regulated kinase 1 ⁄ 2), but not through the AKT pathway. This inhibitory effect was specific for insulin and insulin growth factor 1, but not epidermal growth factor or serum. Furthermore, it was demonstrated that the C-terminal fragment of FLNa inhibited the interac- tion between filamin and the insulin receptor, and potentiated the phosphorylation of MAPK, as well as in response to the transcriptional activity of Elk-1, insulin signalling [53]. It is known that insulin activates SREBP-1 in cells. In our ChIP-chip study, we demon- strated a binding site for SREBP-1, together with RNA- pol II, approximately 1 kb upstream of a shorter mRNA for the FLNA gene (Fig. S7), which might correspond to the C-terminus of FLNa. Based on these insulin signalling could data, we hypothesize that the in turn, promotes activate SREBP-1, which, transcription of the shorter fragment of FLNa. This C-terminal fragment of FLNa then potentiates the transcriptional activity of Elk-1, to regulate insulin signalling further. Moreover, the C-terminal fragment of FLNa has been shown to interact with transcription factors in the nucleus and to inhibit their activity [54]. Promoting the transcription of this fragment might be a way for SREBP-1 to down-regulate the activity of other transcription factors.
For the ChIP-chip experiments, HepG2 cells were treated with the proteasome inhibitor Z-Leu-Leu-Leu-al (MG 132) (Sigma-Aldrich) at a final concentration of 25 lm, or with dimethyl sulfoxide, which is the MG 132 solvent, 4 h prior to the start. The ChIP assay was performed as described previously [39]. Briefly, cells were washed with NaCl ⁄ Pi and cross-linked with formaldehyde at a final concentration of 0.37%. After lysis of the nuclei, the chromatin was soni- cated at 200 W for 30 min, at intervals of 25 s on and off, using a Bioruptor (Diagenode, Lie` ge, Belgium), to generate fragments of 200–500 bp. The sonicated chromatin was pre- cleared and immunoprecipitated overnight, followed by incubation with protein G–agarose beads (Roche, Man- nheim, Germany), and several washes. The DNA–protein complex was eluted, followed by reversal of cross-linking and proteinase-K treatment overnight. The DNA was puri- followed by fied through phenol–chloroform extraction, ethanol precipitation.
From this study, we can conclude that SREBP-1 might regulate its target genes through binding in pro- moters of well-known as well as new transcripts, and that it does not only regulate the expression of genes involved in lipid metabolism, but may also play a role in cell proliferation. As we found several binding sites out- side of classical promoters, this study emphasizes the value of performing unbiased genome-wide studies, not restricting the analysis to known promoters. Hence, SREBP-1 is a suitable candidate for true whole-genome studies in order to obtain a better understanding of its role in metabolism and cell proliferation, as well as in major human metabolic disorders.
Materials and methods
Cells and antibodies
carcinoma)
In sterol depletion studies for verification of the results, cells were grown in lipoprotein-deficient medium in the absence or presence of cholesterol and 25-hydroxycholesterol (50 and 5.0 lgÆmL)1, respectively) to suppress the activa- tion of endogenous SREBPs. The ChIP assays were per- formed as described previously [8], with the following modifications: (a) cells were cross-linked in 1% formalde- hyde for 10 min at room temperature; (b) 90 lg of precle- ared chromatin was immunoprecipitated with 3 lg of anti-SREBP-1 (H160), RNA-pol II (8GW16) or control (haemagglutinin) antibodies. The PCR conditions were optimized to remain in the linear range of amplification. Primer sequences are given in Tables S1 and S2.
Microarray design and hybridization
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The design of the arrays used for this study has been described previously [39]. The arrays are PCR-based with HepG2 (human hepatocellular cells were cultured in RPMI1640 supplemented with 10% non-heat inactivated fetal bovine serum and 1% l-glutamine (Sigma- Aldrich, Steinheim, Germany). Antibodies used in this study were raised against SREBP-1 (H160, SC-8984) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the non-phos- phorylated C-terminal heptapeptide repeat on the largest subunit of RNA-pol II (8WG16, MMS-126R) (Covance, Emeryville, CA, USA) and haemagglutinin (Y-11, SC-805)
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1–1.5 kb long amplicons, which were allowed to contain repetitive elements. The final array presents a 75% coverage of the ENCODE regions.
input DNA were
were averaged and a standard deviation was calculated. The background value was calculated as 2 · standard deviation + average. For each factor and primer, the quantitative value was then divided by the background value to obtain the fold enrichment over background. Fold enrichments of 1.5 or more were considered to be over background and thus positive.
Electrophoretic mobility shift assay
The immunoprecipitated DNA and one-quarter of the labelled with cyanine-5 and total cyanine-3, respectively (GE Healthcare, Chalfont St Giles, UK) with the Bioprime Labelling system (Invitrogen, Temecula, CA, USA). The labelled DNA was purified with Microspin G50 columns (GE Healthcare) and com- bined before ethanol precipitation, together with human Cot-1 DNA. The resulting pellet was then resuspended in hybridization buffer and hybridized to the array pre- blocked with human Cot-1 and salmon sperm DNA. After 48 h of incubation, the slides were washed and scanned in a GenePix 4000 B scanner (Axon Instruments, Molecular Dynamics, Sunnyvale, CA, USA). lower
Data analysis
Recombinant 6 · His–SREBP-1a, either wild-type or DBD, was used in electrophoretic mobility shift assays with 32P- labelled probes containing the SRE-1 sequence from the LDLr promoter or the potential SREBP binding element in HCFC1. The oligonucleotides for the potential SREBP binding element in HCFC1 were as follows: upper strand, 5¢-ATGGTGCTCACCTCACCTGG-3¢; strand, 5¢- TCCCAGGTGAGGTGAGCACCA-3¢. The electrophoretic mobility shift assay reactions were carried out in binding buffer consisting of 15 mm Hepes ⁄ KOH (pH 7.8), 5% glyc- erol, 40 mm KCl, 2 mm MgCl2, 2 lg of single-stranded sal- mon sperm DNA, 10 lg of bovine serum albumin and 5 mm of dithiothreitol. Where indicated, His-tag antibodies or unlabelled competitor DNA was included in the assay. The data were analysed within the Linnaeus Centre for Bio- informatics data warehouse (LCB-DWH) [55]. After remov- ing flagged spots, the arrays were background corrected and print-tip loess normalized. Enriched spots were detected as described in [39]. The detected spots were anno- tated with their genomic location in HG18.
Acknowledgements
accession number GSE7307,
The detected regions were annotated against PCGs (UCSC Known, ENSEMBL and RefSeq), human mRNA, spliced ESTs and human ESTs, in that order [56]. Expres- sion data for HepG2 cells were collected from the National Center for Biotechnology Information Gene Expression and Omnibus database the P values were calculated using a two-sided t-test with the null hypothesis that the distribution should be as all the data. The promoter comparison was performed using the Stanford promoter analysis data from the ENCODE project [29]. All correlations are Pearson correlations.
Quantitative PCR for verification
CW was supported by the Swedish Research Council for Science and Technology, the Diabetes Association, the Cancer Foundation, the Markus Borgstro¨ m Foun- dation, and the Family Ernfors Fund and Novo Nordisk. JE was supported by grants from the Swedish Research Council and the Novo Nordisk Foundation. JE is a Research Fellow of the Royal Swedish Acad- emy of Sciences through a grant from the Knut and Alice Wallenberg Foundation. JK was supported by the Swedish Foundation for Strategic Research, The Knut and Alice Wallenberg Foundation, Uppsala University and the Swedish University for Agricultural Sciences.
References
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The following supplementary material is available: Fig. S1. Principal component analysis of the ChIP-chip data. Fig. S2. The PDCR gene with binding sites SREBP-1 and RNA-pol II. Fig. S3. The DOLPP1 gene with binding sites for SREBP-1 and RNA-pol II. Fig. S4. SREBP-1 and RNA-pol II UESs APOA1–APOA5 region. Fig. S5. The RPS9 gene with binding sites for SREBP- 1 and RNA-pol II. Fig. S6. The HCFC1 gene with binding sites SREBP-1 and RNA-pol II. Fig. S7. The FLNA gene with binding sites SREBP-1 and RNA-pol II. Table S1. List of primers for SREBP-1 semiquantita- tive PCR and quantitative PCR. Table S2. List of primers for RNA-pol II semiquanti- tative PCR and quantitative PCR.
54 Loy CJ, Sim KS & Yong EL (2003) Filamin-A frag- ment localizes to the nucleus to regulate androgen receptor and coactivator functions. Proc Natl Acad Sci USA 100, 4562–4567.
This supplementary material can be found in the
online version of this article.
55 Ameur A, Yankovski V, Enroth S, Spjuth O & Komo- rowski J (2006) The LCB Data Warehouse. Bioinfor- matics (Oxford, England) 22, 1024–1026.
56 Rada-Iglesias A, Ameur A, Kapranov P, Enroth S, Komorowski J, Gingeras TR & Wadelius C (2008) Whole-genome maps of USF1 and USF2 binding and histone H3 acetylation reveal new aspects of promoter
Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corre- sponding author for the article.
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