ASXL1 Represses Retinoic Acid Receptor-mediated Transcription through Associating with HP1 and LSD1

Sang-Wang Lee,Yang-Sook Cho,Jung-Min Na,Ui-Hyun Park,Myengmo Kang,Eun-Joo Kim,Soo-Jong Um

doi:10.1074/jbc.m109.065862

Sang-Wang Lee, Yang-Sook Cho + Show 5 more

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https://doi.org/10.1074/jbc.m109.065862

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Abstract

We previously suggested that ASXL1 (additional sex comb-like 1) functions as either a coactivator or corepressor for the retinoid receptors retinoic acid receptor (RAR) and retinoid X receptor in a cell type-specific manner. Here, we provide clues toward the mechanism underlying ASXL1-mediated repression. Transfection assays in HEK293 or H1299 cells indicated that ASXL1 alone possessing autonomous transcriptional repression activity significantly represses RAR- or retinoid X receptor-dependent transcriptional activation, and the N-terminal portion of ASXL1 is responsible for the repression. Amino acid sequence analysis identified a consensus HP1 (heterochromatin protein 1)-binding site (HP1 box, PXVXL) in that region. Systematic in vitro and in vivo assays revealed that the HP1 box in ASXL1 is critical for the interaction with the chromoshadow domain of HP1. Transcription assays with HP1 box deletion or HP1alpha knockdown indicated that HP1alpha is required for ASXL1-mediated repression. Furthermore, we found a direct interaction of ASXL1 with histone H3 demethylase LSD1 through the N-terminal region nearby the HP1-binding site. ASXL1 binding to LSD1 was greatly increased by HP1alpha, resulting in the formation of a ternary complex. LSD1 cooperates with ASXL1 in transcriptional repression, presumably by removing H3K4 methylation, an active histone mark, but not H3K9 methylation, a repressive histone mark recognized by HP1. This possibility was supported by chromatin immunoprecipitation assays followed by ASXL1 overexpression or knockdown. Overall, this study provides the first evidence that ASXL1 cooperates with HP1 to modulate LSD1 activity, leading to a change in histone H3 methylation and thereby RAR repression.

Highlights

18 JOURNAL OF BIOLOGICAL CHEMISTRY various physiological processes such as cell differentiation, proliferation, and development
The diverse regulation of nuclear receptor (NR) transcriptional activity may predict the presence of other ligand-dependent corepressors that may be linked to chromatin modifications other than histone ing domain; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; HcRed, red fluorescent protein; siRNA, small interfering RNA; RA-response elements (RAREs), RA response element; m, murine; RA, retinoic acid; IP, immunoprecipitation; Western blotting (WB), Western blot; Reverse Transcription (RT), reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; ETP, Enhancer of Trithorax and Polycomb; sh, small hairpin; AD, activation domain
Our previous data showed that ASXL1, binding to the AF-2 AD core of retinoic acid receptor (RAR) or Gal4-RXR (or RXR) in the presence of RA, enhances RA-induced transactivation of RAR/RXR by cooperating with SRC-1 in certain mammalian cells (NIH3T3, HeLa, and MCF-7; Ref. 43)

Summary

Introduction

18 JOURNAL OF BIOLOGICAL CHEMISTRY various physiological processes such as cell differentiation, proliferation, and development (reviewed in Refs. 1, 2). Transcriptional regulation by RARs (and RXRs) involves the binding and recruitment of corepressors and coactivators to target gene promoters depending on ligand availability [5]. Transfections into HEK293 cells and luciferase reporter assays indicated that ASXL1 strongly inhibited the RA-induced transcriptional activity of Gal4-RAR (or Gal4-RXR; Fig. 1B) and RAR (or RXR) in a dose-dependent manner (Fig. 1C), ASXL1 alone had no significant effect on basal promoter activity in the absence of RA (data not shown).

Results

Conclusion