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. 2014 Sep;55(9):1886-96.
doi: 10.1194/jlr.M047555. Epub 2014 Jul 14.

The adipogenic transcriptional cofactor ZNF638 interacts with splicing regulators and influences alternative splicing

Chen Du  1 , Xinran Ma  1 , Sunitha Meruvu  1 , Lynne Hugendubler  1 , Elisabetta Mueller  1
Affiliations

The adipogenic transcriptional cofactor ZNF638 interacts with splicing regulators and influences alternative splicing

Chen Du et al. J Lipid Res. 2014 Sep.

Abstract

Increasing evidence indicates that transcription and alternative splicing are coordinated processes; however, our knowledge of specific factors implicated in both functions during the process of adipocyte differentiation is limited. We have previously demonstrated that the zinc finger protein ZNF638 plays a role as a transcriptional coregulator of adipocyte differentiation via induction of PPARγ in cooperation with CCAAT/enhancer binding proteins (C/EBPs). Here we provide new evidence that ZNF638 is localized in nuclear bodies enriched with splicing factors, and through biochemical purification of ZNF638's interacting proteins in adipocytes and mass spectrometry analysis, we show that ZNF638 interacts with splicing regulators. Functional analysis of the effects of ectopic ZNF638 expression on a minigene reporter demonstrated that ZNF638 is sufficient to promote alternative splicing, a function enhanced through its recruitment to the minigene promoter at C/EBP responsive elements via C/EBP proteins. Structure-function analysis revealed that the arginine/serine-rich motif and the C-terminal zinc finger domain required for speckle localization are necessary for the adipocyte differentiation function of ZNF638 and for the regulation of the levels of alternatively spliced isoforms of lipin1 and nuclear receptor co-repressor 1. Overall, our data demonstrate that ZNF638 participates in splicing decisions and that it may control adipogenesis through regulation of the relative amounts of differentiation-specific isoforms.

Keywords: adipocyte differentiation; minigene reporter; nuclear speckles; transcriptional coactivator.

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Figures

Fig. 1.
Fig. 1.
ZNF638 colocalizes with the splicing factor ASF/SF2 in nuclear speckles. Subcellular localization of transiently expressed ZNF638 in U2OS cells detected by indirect immunofluorescence and by overlay with DAPI as nuclear counterstaining (A). Double immunofluorescence staining of transiently expressed ZNF638 in U2OS cells and overlay with endogenous ASF/SF2, a marker for nuclear speckles (B). A, B: Confocal imaging; scale bars: 10 µm.
Fig. 2.
Fig. 2.
The RS domain and the C-terminal ZF motif are required for ZNF638 localization to nuclear speckles. Schematic illustration of the domains present in full-length ZNF638 and schematic representation of ZNF638 deletion mutants analyzed in this study (A). NLS, nuclear localization signal. Subcellular localization of ectopically expressed full-length ZNF638 and ZNF638 deletion mutants in U2OS cells, detected by indirect immunofluorescence staining and confocal imaging (B). Scale bar, 10 µm.
Fig. 3.
Fig. 3.
ZNF638 interacts with factors involved in pre-mRNA splicing present in differentiating adipocytes. Graphical representation of the clustering of the 172 novel ZNF638 interacting proteins present in differentiating adipocytes identified by mass spectrometry analysis (A). Interacting proteins purified from nuclear extracts obtained from differentiating 3T3-L1 cells using a GST-ZNF638ZF2 fusion protein were resolved by SDS-PAGE and subjected to mass spectrometry analysis. Coimmunoprecipitation assays were performed to validate mass spectrometry data (B). GFP-ZNF638ZF2 fusion protein was expressed in HEK-293 cells, and endogenous interacting proteins were detected by Western blot after GFP immunoprecipitation. Asterisk indicates nonspecific band.
Fig. 4.
Fig. 4.
ZNF638 modifies splicing of minigene reporter transcripts. Schematic representation of the fibronectin minigene reporter and of the assay performed (A). EI-specific primers (filled arrows) and ES primers (empty arrows) were used to quantify the mRNA isoforms retaining the alternative spliced exon 25 (black boxes) and the short isoform skipping the alternatively spliced exon. Gray boxes indicate constitutively retained exons; thin lines, introns; and thick lines, spliced isoforms. Quantification of the ratio of EI to ES in HEK-293 cells transiently expressing the minigene plasmid with ZNF638 or vector control (B). Mean ± SEM. Statistical analysis was performed on three independent experiments. ** P < 0.01. Western blot of ZNF638 expression levels from a representative experiment (C). β-actin was used as loading control. Schematic representation of the fibronectin minigene containing C/EBP responsive elements in the promoter (D). Ratio of EI over ES in HEK-293 cells transiently expressing the minigene containing C/EBP binding sites, in the presence or absence of ZNF638 and C/EBP factors (E). Experiments were repeated at least three times. A representative experiment is shown. Mean ± SEM; n.s., not statistically significant; # P < 0.05, ## P < 0.01, ### P < 0.001. Western blot analysis to assess protein levels of transiently expressed ZNF638, C/EBPα, C/EBPβ, and C/EBPδ in a representative experiment. β-actin was used as loading control (F).
Fig. 5.
Fig. 5.
Requirements of ZNF638 domains in adipocyte differentiation. Quantification of lipid accumulation in 10T1/2 cells expressing vector, full-length ZNF638, or deletion mutants at day 6 after induction of differentiation measured through quantification of Oil Red O extracted from stained cells (A). mRNA levels of adipocyte markers in 10T1/2 cells expressing either vector, full-length ZNF638, or deletion mutants, after 3 days of differentiation (B). Ratio of alternatively spliced isoforms during adipocyte differentiation in 10T1/2 cells expressing either vector, full-length ZNF638, or deletion mutants, after 3 days of induction of differentiation (C). A, B, C: Mean ± SEM. Full-length ZNF638 compared with vector control: # P < 0.05, ## P < 0.01. Mutants compared with full-length ZNF638: * P < 0.05, ** P < 0.01.
Fig. 6.
Fig. 6.
Proposed models of ZNF638’s mode of action on alternative splicing. Sequestration model (A). ZNF638 may affect splicing decisions by altering the balance between available inhibitory and activating splicing factors competing for their binding to the pre-mRNA. Via sequestration of HNRNP proteins like HNRPA1, ZNF638 may prevent their binding to splice sites on the pre-mRNA, increasing the availability of activating splicing factors, such as SF2. Facilitator model (B). The loading of ZNF638 on the promoters of its target genes via C/EBP responsive elements may facilitate the recruitment of SR proteins to the CTD of the polymerase II and to the nearby nascent pre-mRNA. A, B: Purple exon is an alternatively spliced exon. Pol II, RNA polymerase II.
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