Marjo Malinen

Regulation of multiple insulin-like growth factor binding protein genes by 1α,25-dihydroxyvitamin D3

Recently, insulin-like growth factor binding proteins (IGFBPs) have been found to be primary mediators of the anti-proliferative actions of the nuclear hormone 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], but dependent on cellular context IGFBPs can also have a mitogenic effect. In this study, we performed expression profiling of all six human IGFBP genes in prostate and bone cancer cells and demonstrated that IGFBP1, 3 and 5 are primary 1α,25(OH)2D3 target genes. In silico screening of the 174 kb of genomic sequence surrounding all six IGFBP genes identified 15 candidate vitamin D response elements (VDREs) close to or in IGFBP1, 2, 3 and 5 but not in the IGFBP4 and 6 genes. The putative VDREs were evaluated in vitro by gelshift assays and in living cells by reporter gene and chromatin immuno-precipitation (ChIP) assays. Of these 10 VDREs appear to be functional. ChIP assays demonstrated for each of these an individual, stimulation time-dependent association profile not only with the vitamin D receptor, but also with first heterodimeric partner the retinoid X receptor, other regulatory complex components and phosphorylated RNA polymerase II. Some of the VDREs are located distantly from the transcription start sites of IGFBP1, 3 and 5, but all 10 VDREs seem to contribute to the regulation of the genes by 1α,25(OH)2D3. In conclusion, IGFBP1, 3 and 5 are primary 1α,25(OH)2D3 target genes that in intact cells are each under the control of multiple VDREs.

Regulation of the human cyclin C gene via multiple vitamin D3-responsive regions in its promoter

The candidate human tumor suppressor gene cyclin C is a primary target of the anti-proliferative hormone 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], but binding sites for the 1α,25(OH)2D3 receptor (VDR), so-called 1α,25(OH)2D3 response elements (VDREs), have not yet been identified in the promoter of this gene. We screened various cancer cell lines by quantitative PCR and found that the 1α,25(OH)2D3 inducibility of cyclin C mRNA expression, in relationship with the 24-hydroxylase (CYP24) gene, was best in MCF-7 human breast cancer cells. To characterize the molecular mechanisms, we analyzed 8.4 kb of the cyclin C promoter by using chromatin immunoprecipitation assays (ChIP) with antibodies against acetylated histone 4, VDR and its partner receptor, retinoid X receptor (RXR). The histone 4 acetylation status of all 23 investigated regions of the cyclin C promoter did not change significantly in response to 1α,25(OH)2D3, but four independent promoter regions showed a consistent, 1α,25(OH)2D3-dependent association with VDR and RXR over a time period of 240 min. Combined in silico/in vitro screening identified in each of these promoter regions a VDRE and reporter gene assays confirmed their functionality. Moreover, re-ChIP assays monitored simultaneous association of VDR with RXR, coactivator, mediator and RNA polymerase II proteins on these regions. Since cyclin C protein is associated with those mediator complexes that display transcriptional repressive properties, this study contributes to the understanding of the downregulation of a number of secondary 1α,25(OH)2D3-responding genes.

The Number of Vitamin D Receptor Binding Sites Defines the Different Vitamin D Responsiveness of the CYP24 Gene in Malignant and Normal Mammary Cells

Primary 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3)-responding genes are controlled by the vitamin D receptor (VDR) binding to specific sites (VDREs) that are located within the regulatory regions of these genes. According to previous studies, the gene encoding 25-dihydroxyvitamin D3 24-hydroxylase, CYP24, which is the strongest known 1α,25(OH)2D3-responsive gene, has multiple VDREs that locate within the proximal and the distal promoter. However, it has remained unclear, what is the biological role of these regions and how they participate in the regulation of transcription. In this study, we found a different CYP24 expression profile in normal (MCF-10A) and malignant (MCF-7) human mammary cells. Moreover, CYP24 mRNA showed to be three times more stable in MCF-7 cells than in MCF-10A cells. We studied the mechanism of this difference using expression profiling, quantitative chromatin immunoprecipitation and chromosome conformation capture assays. Interestingly, the number of functional VDREs was higher in MCF-7 cells than in MCF-10A cells. Three functional VDREs in MCF-7 cells are connected to linear mRNA accumulation, whereas only one VDRE seems to lead to stepwise CYP24 mRNA accumulation in MCF-10A cells. The distal VDREs were involved in transcriptional regulation via ligand-dependent, dynamic chromatin looping, which brings cyclically the distal elements together either individually or simultaneously next to the transcription start site. In conclusion, our data suggest that in comparison to normal cells, clearing of 1α,25(OH)2D3 is enhanced in malignant cells due to differences in transcriptional regulation of CYP24 and metabolism of CYP24 mRNA.

Proto-oncogene PIM-1 is a novel estrogen receptor target associating with high grade breast tumors.

We searched ERα cistromes of MCF-7 breast cancer cells for previously unrecognized ERα targets and identified proto-oncogene PIM-1 as a novel potential target gene. We show that the expression of PIM-1 is induced in response to estradiol in MCF-7 cells and that the induction is mediated by ERα-regulated enhancers located distally upstream from the gene. In keeping with the growth-promoting role of the PIM-1, depletion of the PIM-1 attenuated the proliferation of the MCF-7 cells, which was paralleled with up-regulation of cyclin-dependent protein kinase inhibitor CDKN1A and CDKN2B expression. Analysis of PIM-1 expression between invasive breast tumors and benign breast tissue samples showed that elevated PIM-1 expression is associated with malignancy and a higher tumor grade. In sum, identification of PIM-1 as an ERα target gene adds a novel potential mechanism by which estrogens can contribute to breast cancer cell proliferation and carcinogenesis.

Global SUMOylation on active chromatin is an acute heat stress response restricting transcription

BackgroundCells have developed many ways to cope with external stress. One distinctive feature in acute proteotoxic stresses, such as heat shock (HS), is rapid post-translational modification of proteins by SUMOs (small ubiquitin-like modifier proteins; SUMOylation). While many of the SUMO targets are chromatin proteins, there is scarce information on chromatin binding of SUMOylated proteins in HS and the role of chromatin SUMOylation in the regulation of transcription.ResultsWe mapped HS-induced genome-wide changes in chromatin occupancy of SUMO-2/3-modified proteins in K562 and VCaP cells using ChIP-seq. Chromatin SUMOylation was further correlated with HS-induced global changes in transcription using GRO-seq and RNA polymerase II (Pol2) ChIP-seq along with ENCODE data for K562 cells. HS induced a rapid and massive rearrangement of chromatin SUMOylation pattern: SUMOylation was gained at active promoters and enhancers associated with multiple transcription factors, including heat shock factor 1. Concomitant loss of SUMOylation occurred at inactive intergenic chromatin regions that were associated with CTCF-cohesin complex and SETDB1 methyltransferase complex. In addition, HS triggered a dynamic chromatin binding of SUMO ligase PIAS1, especially onto promoters. The HS-induced SUMOylation on chromatin was most notable at promoters of transcribed genes where it positively correlated with active transcription and Pol2 promoter-proximal pausing. Furthermore, silencing of SUMOylation machinery either by depletion of UBC9 or PIAS1 enhanced expression of HS-induced genes.ConclusionsHS-triggered SUMOylation targets promoters and enhancers of actively transcribed genes where it restricts the transcriptional activity of the HS-induced genes. PIAS1-mediated promoter SUMOylation is likely to regulate Pol2-associated factors in HS.

Cyclical regulation of the insulin-like growth factor binding protein 3 gene in response to 1α,25-dihydroxyvitamin D3

The nuclear receptor vitamin D receptor (VDR) is known to associate with two vitamin D response element (VDRE) containing chromatin regions of the insulin-like growth factor binding protein 3 (IGFBP3) gene. In non-malignant MCF-10A human mammary cells, we show that the natural VDR ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) causes cyclical IGFBP3 mRNA accumulation with a periodicity of 60u2009min, while in the presence of the potent VDR agonist Gemini the mRNA is continuously accumulated. Accordingly, VDR also showed cyclical ligand-dependent association with the chromatin regions of both VDREs. Histone deacetylases (HDACs) play an important role both in VDR signalling and in transcriptional cycling. From the 11 HDAC gene family members, only HDAC4 and HDAC6 are up-regulated in a cyclical fashion in response to 1α,25(OH)2D3, while even these two genes do not respond to Gemini. Interestingly, HDAC4 and HDAC6 proteins show cyclical VDR ligand-induced association with both VDRE regions of the IGFBP3 gene, which coincides with histone H4 deacetylation on these regions. Moreover, combined silencing of HDAC4 and HDAC6 abolishes the cycling of the IGFBP3 gene. We assume that due to more efficient VDR interaction, Gemini induces longer lasting chromatin activation and therefore no transcriptional cycling but monotonically increasing IGFBP3 mRNA. In conclusion, 1α,25(OH)2D3 regulates IGFBP3 transcription through short-term cyclical association of VDR, HDAC4 and HDAC6 to both VDRE-containing chromatin regions.

SUMO ligase PIAS1 functions as a target gene selective androgen receptor coregulator on prostate cancer cell chromatin

Androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in the development and growth of prostate carcinoma. PIAS1 is an AR- and SUMO-interacting protein and a putative transcriptional coregulator overexpressed in prostate cancer. To study the importance of PIAS1 for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the AR-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. Interestingly, PIAS1 depletion also exposed a new set of genes to androgen regulation, suggesting that PIAS1 can mask distinct genomic loci from AR access. In keeping with gene expression data, silencing of PIAS1 attenuated VCaP cell proliferation. ChIP-seq analyses showed that PIAS1 interacts with AR at chromatin sites harboring also SUMO2/3 and surrounded by H3K4me2; androgen exposure increased the number of PIAS1-occupying sites, resulting in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with the pioneer factor FOXA1. Of note, PIAS1 depletion affected AR chromatin occupancy at binding sites enriched for HOXD13 and GATA motifs. Taken together, PIAS1 is a genuine chromatin-bound AR coregulator that functions in a target gene selective fashion to regulate prostate cancer cell growth.

Histone demethylase GASC1 - a potential prognostic and predictive marker in invasive breast cancer

BackgroundThe histone demethylase GASC1 (JMJD2C) is an epigenetic factor suspected of involvement in development of different cancers, including breast cancer. It is thought to be overexpressed in the more aggressive breast cancer types based on mRNA expression studies on cell lines and meta analysis of human breast cancer sets. This study aimed to evaluate the prognostic and predictive value of GASC1 for women with invasive breast cancer.MethodsAll the 355 cases were selected from a cohort enrolled in the Kuopio Breast Cancer Project between April 1990 and December 1995. The expression of GASC1 was studied by immunohistochemistry (IHC) on tissue microarrays. Additionally relative GASC1 mRNA expression was measured from available 57 cases.ResultsIn our material, 56% of the cases were GASC1 negative and 44% positive in IHC staining. Women with GASC1 negative tumors had two years shorter breast cancer specific survival and time to relapse than the women with GASC1 positive tumors (p=0.017 and p=0.034 respectively). The majority of GASC1 negative tumors were ductal cases (72%) of higher histological grade (84% of grade II and III altogether). When we evaluated estrogen receptor negative and progesterone receptor negative cases separately, there was 2 times more GASC1 negative than GASC1 positive tumors in each group (chi2, p= 0.033 and 0.001 respectively). In the HER2 positive cases, there was 3 times more GASC1 negative cases than GASC1 positives (chi2, p= 0.029). Patients treated with radiotherapy (n=206) and hormonal treatment (n=62) had better breast cancer specific survival, when they were GASC1 positive (Cox regression: HR=0.49, p=0.007 and HR=0.33, p=0.015, respectively). The expression of GASC1 mRNA was in agreement with the protein analysis.ConclusionsThis study indicates that the GASC1 is both a prognostic and a predictive factor for women with invasive breast cancer. GASC1 negativity is associated with tumors of more aggressive histopathological types (ductal type, grade II and III, ER negative, PR negative). Patients with GASC1 positive tumors have better breast cancer specific survival and respond better to radiotherapy and hormonal treatment.

SUMOylation modulates the transcriptional activity of androgen receptor in a target gene and pathway selective manner

Androgen receptor (AR) plays an important regulatory role in prostate cancer. ARs transcriptional activity is regulated by androgenic ligands, but also by post-translational modifications, such as SUMOylation. To study the role of AR SUMOylation in genuine chromatin environment, we compared androgen-regulated gene expression and AR chromatin occupancy in PC-3 prostate cancer cell lines stably expressing wild-type (wt) or doubly SUMOylation site-mutated AR (AR-K386R,K520R). Our genome-wide gene expression analyses reveal that the SUMOylation modulates the AR function in a target gene and pathway selective manner. The transcripts that are differentially regulated by androgen and SUMOylation are linked to cellular movement, cell death, cellular proliferation, cellular development and cell cycle. Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis. Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression. De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions. Taken together, our data indicate that SUMOylation does not simply repress the AR activity, but it regulates ARs interaction with the chromatin and the receptors target gene selection.

Hypoxia-inducible factor 1-induced G protein-coupled receptor 35 expression is an early marker of progressive cardiac remodelling

AIMSnG protein-coupled receptor 35 (GPR35) has been characterized to be one of the genes that are up-regulated in human heart failure. Since mechanisms controlling GPR35 expression are not known, we investigated the regulation of GPR35 gene and protein expression in cardiac myocytes and in the mouse models of cardiac failure.nnnMETHODS AND RESULTSnIn cardiac myocytes, GPR35 gene expression was found to be exceptionally sensitive to hypoxia and induced by hypoxia-inducible factor-1 (HIF-1) activation. HIF-1-dependent regulation was established by genetic (HIF-1/VP16, Inhibitory Per/Arnt/Sim domain protein) and chemical [desferrioxamine (DFO)] modulation of the HIF-1 pathway and further confirmed by mutation analysis of the GPR35 promoter and by demonstrating direct binding of endogenous HIF-1 to the gene promoter. Hypoxia increased the number and density of GPR35 receptors on the cardiomyocyte cell membranes. Chemical GPR35 agonist Zaprinast caused GPR35 activation and receptor internalization in cardiac myocytes. In addition, overexpressed GPR35 disrupted actin cytoskeleton arrangement and caused morphological changes in cultured cardiomyocytes. GPR35 gene and protein expressions were also induced in mouse models of cardiac failure; the acute phase of myocardial infarction and during the compensatory and decompensatory phase of pressure-load induced cardiac hypertrophy.nnnCONCLUSIONSnCardiac expression of GPR35 is regulated by hypoxia through activation of HIF-1. The expression of GPR35 in mouse models of cardiac infarction and pressure load suggests that GPR35 could be used as an early marker of progressive cardiac failure.