Aberrant Chromatin Remodeling by APL Fusion Proteins Assessed at the Single-Cell Level: Contribution of Reduced Mobility of Nuclear Coregulators.

Abstract

Acute promyelocytic leukemia (APL) is notable for its specific t(15;17) chromosomal translocation-generated fusion protein, PML-RARα , and a dramatic clinical response to all-trans retinoic acid (ATRA) therapy. To explore the hypothesis that aberrant chromatin remodeling plays a role in PML-RARα-induced leukemogenesis and its reversal by ATRA at the single-cell level, we subcloned the open reading frames of wild type RARα , PML-RARα and PLZF-RARα into the amino-terminus of CFP-LacR. Immunoblot analysis, DNA binding studies, reporter assays and fluorescent microscopic analysis demonstrated that each CFP-LacR tagged construct encoded proteins of the expected size and that each gained the ability to bind to a Lac operator-containing reporter construct and activate transcription in an ATRA-dependent manner; addition of the tag did not interfere with their RARE binding ability or their cellular localization and distribution. Fluorescent microscopic examination of chromosomally integrated Lac operator arrays within the CHO cell line, A03_1, following expression of CFP-LacR-tagged proteins demonstrated that each of the tagged proteins localized to tightly packed arrays of less than half the volume of arrays within A03_1 cells transfected with CFP-LacR alone (p<0.001 for each). Addition of ATRA (10−7 M) normalized the array volume in cells expressing CFP-LacR-RARα but not in cells expressing CFP-LacR-PML-RARα or CFP-LacR-PLZF-RARα . To determine directly whether or not the Lac operon array volume correlated with recruitment of nuclear receptor co-regulators, we imaged A03_1 cells co-expressing CFP-LacR-RARα , CFP-PML-RARα or CFP-LacR-PLZF-RARα and YFP-SMRT or YFP-SRC-1. In the absence of ATRA, YFP-SMRT bound almost exclusively to the arrays within cells expressing CFP-LacR-RARα , CFP-PML-RARα or CFP-LacR-PLZF-RARα , while YFP-SRC-1 did not; rather, CFP-SRC-1 distributed throughout the nucleus in each case. Surprisingly, the concentration of ATRA required to induce dissociation of YFP-SMRT from the arrays and recruitment of YFP-SRC-1 to the arrays (10−7 M) was similar in cells expressing each construct. In contrast, while the mobility of YFP-SRC-1 localized on the CFP-LacR-RARα-bound arrays assessed by its recovery half-life (t1/2) was 17.80 ± 4.31 s, its mobility on the CFP-LacR-PML-RARα-bound arrays or the CFF-LacR-PLZF-RARα-bound arrays was reduced by 47% (t1/2=26.07 ± 8.35 s; p<0.001) and 45% (t1/2=25.80 ± 9.89 s; p<0.001), respectively, indicating that YFP-SRC-1 bound more tightly to both PML-RARα and to PLZF-RARα than to wild type RARα . In addition, while the mobility of YFP-SMRT localized on the CFP-LacR-RARα-bound arrays assessed by its t1/2 (13.57 ± 4.53 s) was similar to the mobility of YFP-SMRT localized on the CFP-LacR-PML-RARα-bound arrays (t1/2=13.50 ± 4.21 s), the mobility of YFP-SMRT on the CFF-LacR-PLZF-RARα-bound arrays was reduced by 68% (t1/2=22.76 ± 7.87 s; p< 0.001) indicating that YFP-SMRT bound more tightly to PLZF-RARα than to either wild type RARα or PML-RARα . Thus, the impaired ability of APL fusion proteins to activate gene transcription in response to ATRA corresponds to their reduced ability to remodel chromatin; aberrant chromatin remodeling by APL fusion proteins at the single-cell level did not correlate with their altered ATRA concentration-dependent associations with key nuclear receptor coregulators rather with their ability to impair the nuclear mobility of these coregulators.