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Microarray analysis of altered gene expression in ERβ-overexpressing HEK293 cells

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Abstract

Estrogen receptors (ERs), ERα and ERβ, mediate estrogen actions in a broad range of target tissues. With the introduction of microarray techniques, a significant understanding has been gained regarding the interplay between the ERα and ERβ in breast cancer cell lines. To gain a more comprehensive understanding of ERβ-dependent gene regulation independent of ERα, we performed microarray analysis on HEK293/mock and HEK293/ERβ cells. A total of 332 genes was identified as ERβ-upregulated genes and 210 identified as ERβ-downregulated genes. ERβ-induced and ERβ-repressed genes were involved in cell–cell signaling, morphogenesis, and cell proliferation. The ERβ repressive effect on genes related to proliferation was further studied by proliferation assays, where ERβ expression resulted in a significant decrease in cell proliferation. To identify primary ERβ target genes, we examined a number of ERβ-regulated genes using chromatin immunoprecipitation assays for regions bound by ERβ. Our results showed that ERβ recruitment was significant to regions associated with 12 genes (IL1RAP, TMSB4X, COLEC12, ENPP2, KLRC1, RERG, RGS16, TNNT2, CYR61, FER1L3, FAM108A1, and CYP4X1), suggesting that these genes are likely to be ERβ primary target genes. This study has provided novel information on the gene regulatory function of ERβ independent of ERα and identified a number of ERβ primary target genes. The results of Gene Ontology analysis and proliferation assays are consistent with an antiproliferative role of ERβ independent of ERα.

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Abbreviations

IL1RAP:

Interleukin 1 receptor accessory protein

TMSB4X:

Thymosin, beta 4, X-linked

COLEC12:

Collectin sub-family member 12

ENPP2:

Ectonucleotide pyrophosphatase/phosphodiesterase 2

KLRC1:

Killer cell lectin-like receptor subfamily C, member 1

RERG:

RAS-like, estrogen-regulated, growth inhibitor

RGS16:

Regulator of G-protein signaling 16

TNNT2:

Troponin T-type 2

CYR61:

Cysteine-rich, angiogenic inducer, 61

FER1L3:

Fer-1-like 3, myoferlin

FAM108A1:

Family with sequence similarity 108, member A1

CYP4X1:

Cytochrome P450, family 4, subfamily X, polypeptide 1

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Acknowledgments

This study was supported by grants from the Swedish Cancer Fund and from KaroBio AB. Jan-Åke Gustafsson is a shareholder and consultant of KaroBio AB. We are grateful to the Bioinformatic and Expression Analysis core facility at the Karolinska Institute (BEA, www.bea.ki.se) for performing the Affymetrix assays.

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Authors and Affiliations

  1. Department of Biosciences and Nutrition, NOVUM, Karolinska Institutet, 141 57, Huddinge, Sweden

    Chunyan Zhao, Milica Putnik, Jan-Åke Gustafsson & Karin Dahlman-Wright

Authors
  1. Chunyan Zhao
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  2. Milica Putnik
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  3. Jan-Åke Gustafsson
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  4. Karin Dahlman-Wright
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Corresponding author

Correspondence to Chunyan Zhao.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Table 1

The differentially expressed genes regulated by ERβ. Description: This is a table listing all of the 332 genes identified as ERβ-upregulated genes and 210 as ERβ-downregulated genes by a factor of 1.5 or more. (XLS 1632 kb)

Supplementary Table 2

ERE sequence similarity search results and genomic locations for ERβ binding regions of ERβ primary target genes. Description: This is a table listing ERE-like sequences and their genomic location and percent similarity to a perfect ERE for eight genes. (DOC 32 kb)

Supplementary Table 3

Primer sequences for real-time PCR. Description: This is a table listing primer sequences for 11 genes used in real-time PCR. (XLS 26 kb)

Supplementary Table 4

Primer sequences for real-time PCR following ChIP analysis. Description: This is a table listing primer sequences for 12 genes used in real-time PCR following ChIP analysis. (XLS 19 kb)

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Zhao, C., Putnik, M., Gustafsson, JÅ. et al. Microarray analysis of altered gene expression in ERβ-overexpressing HEK293 cells. Endocr 36, 224–232 (2009). https://doi.org/10.1007/s12020-009-9233-8

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  • DOI: https://doi.org/10.1007/s12020-009-9233-8

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