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

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.

⌬ RS/ZF2 mutant lacking both the RS and ZF2 motifs, with two deletions, one from amino acids 470 to 573 and one from 1,876 to 1,908. The primer sequences to generate the previous mutants are listed in Table 1 . The empty vector backbone pCR3.1 served as control. glutathione S-transferase (GST)-ZNF638 ZF2 and green fl uorescent protein (GFP)-ZNF638 ZF2 constructs expressing only the carboxy-terminal region of ZNF638, from amino acids 1,773 to 1,926, containing the C-terminal ZF domain were previously described ( 2 ). The fi bronectin minigene reporter 7iBi89 plasmid ( 26 ) was obtained from Addgene (14065). This minigene construct contains exons 24 through 28 of the rat fi bronectin gene including introns 24-25 and 25-26 fl anking the alternatively spliced exon 25. The gene cassette is under the control of a human ␤ -actin promoter and contains a human growth hormone polyadenylation signal. To generate a fi bronectin minigene reporter containing C/EBP responsive elements, a 46 bp sequence of the PPAR ␥ 2 promoter containing C/EBP responsive elements (5 ′ -TTTTACTGCAATTTTAAAAAGCAATCAATATTGAACAATCTCT-GCT-3 ′ ) ( 27 ) was inserted between the ␤ -actin promoter and the beginning of the fi bronectin minigene cassette. The C/EBP responsive element sequence was synthesized including Xho I and Bsr GI restriction sites and cloned into the minigene plasmid at those sites (Genewiz). C/EBP ␤ and C/EBP ␦ plasmids were obtained from Addgene, and the C/EBP ␣ construct was a gift of Kai Ge (National Institutes of Health).
Serine/arginine-rich (SR) proteins are a major class of splicing regulators. These proteins contain RNA recognition motifs (RRMs) at the amino terminus and arginine/ serine-rich (RS) repeats at the carboxy terminus ( 13 ). SR proteins reside in discrete subnuclear regions, which represent storage areas for splicing factors serving nearby active transcription sites ( 14,15 ). Furthermore, SR proteins bind to exonic splicing enhancer sequences and recruit spliceosomal ribonucleoproteins (RNPs) and non-RNPs to the pre-mRNA ( 16 ), thereby affecting the selection of splice sites already during the process of transcription. In addition to SR proteins, heterogeneous nuclear ribonucleoproteins (HNRNPs) bind to pre-mRNA transcripts and infl uence alternative splicing (17)(18)(19). It has been shown that pre-mRNA splicing is coupled to transcription both by the function of the C-terminal domain (CTD) of RNA polymerase II ( 3 ) and by a number of cofactors involved in both processes (20)(21)(22)(23), as demonstrated for the metabolic coactivator PPAR gamma coactivator 1-alpha (PGC1 ␣ ), which exerts a dual function when recruited at promoters containing direct repeat-1 responsive elements ( 23 ).
We have recently identifi ed ZNF638 as a novel coactivator with domain homology to PGC1 ␣ and shown that ZNF638 functions as a transcriptional cofactor regulating the early stages of adipocyte differentiation through protein-protein interaction with C/EBP family members at the PPAR ␥ promoter ( 2 ). ZNF638 is composed of RS domains and RRMs, in addition to a homology region to the matrix-bound protein matrin 3 (MH1), two C 2 H 2 zinc fi nger (ZF) motifs (ZF1 and ZF2), a putative DNA binding domain (DBD), and a region with acidic repeats (ARs) ( 24,25 ). This characteristic domain composition of ZNF638, reminiscent of RNA binding proteins and of splicing regulators, and its dotted nuclear localization pattern in adipocytes ( 2 ), led us to hypothesize that ZNF638 may also participate in alternative splicing, as previously demonstrated for the coactivator PGC1 ␣ ( 23 ). In this study, we tested this hypothesis using several experimental approaches, including confocal microscopy, mutagenesis, mass spectrometry analysis, and functional studies.

Plasmids
Full-length murine ZNF638.pCR3.1 plasmid ( 2 ) was used as a template to generate eight deletion mutants by whole vector mutagenesis (Genewiz). ZNF638-⌬ ZF1 contains a deletion from amino acid 424 to amino acid 456, eliminating the N-terminal ZF motif, the ⌬ RS construct lacks the RS motif present from amino acids 470 to 573, the ⌬ RRM1 has a deletion of the RRM1 motif from amino acids 676 to 751, ⌬ RRM2 has a deletion of the RRM2 motif from amino acids 901 to 981, and ⌬ RRM1-3 has a deletion encompassing all three RRM motifs, from amino acids 676 to 1,077. The ZNF638-⌬ DBD construct has a deletion of the DBD, encompassing amino acids 1,353 to 1,482, the ⌬ AR contains a deletion of the ARs from amino acids 1,538 to 1,770, and ⌬ ZF2 lacks amino acids 1,876 to 1,908, eliminating the C-terminal ZF domain. The ⌬ RS plasmid served as a template to generate the

Immunoprecipitation assays
HEK-293 cells were transfected with GFP-ZNF638 ZF2 .pAcGFP or pAcGFP vector. Immunoprecipitation assays were performed using GFP-trap agarose beads (Chromotek, Allele), according to the manufacturer's instructions. Protein lysates were resolved by SDS-PAGE and transferred to polyvinylidene difl uoride membranes (Millipore). After blocking in TBS with Tween containing 2% nonfat dry milk, membranes were incubated with primary antibodies for 1 h at room temperature or overnight at 4°C and subsequently exposed to secondary HRP-conjugated antibodies for 1 h. Blots were incubated with ECL substrate (Pierce) and exposed to fi lm (Kodak).

Minigene splicing assays
HEK-293 cells were plated on 12-well plates and transfected at 70-80% confl uency with 1 µg of fi bronectin minigene reporter plasmid and either 5 µg of ZNF638.pCR3.1 or pCR3.1 vector. For C/EBP coexpression studies, cells were cotransfected with 1 µg fi bronectin minigene plasmid containing C/EBP responsive elements and with 1 µg of C/EBP ␣ , C/EBP ␤ , or C/EBP ␦ plasmids or vector, and 3 µg of vector control or ZNF638. Twenty-four hours after transfection, cells were harvested, and the extracted mRNA (RNeasy, Qiagen) was reverse transcribed using anchored oligo dT primer (Transcriptor First Strand cDNA Synthesis Kit, Roche). Ratio of exon inclusion (EI) to exon skipping (ES) was assessed by real-time PCR. The EI primers specifi cally amplifi ed the longer isoform including the alternatively spliced exon 25,

Differentiation assays
The 10T1/2 cells were electroporated (Amaxa) with either control, full-length ZNF638, or mutant plasmids, induced to differentiate in culture medium supplemented with 5 µg/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 10 µM dexamethasone, and 10 µM troglitazone for 2 days and subsequently maintained in culture medium supplemented with 5 µg/ml insulin ( 28 ). Gene expression was assayed by real-time PCR 3 days after induction of differentiation, following RNA extraction (TRIzol) and reverse transcription (Roche). The sequences of the primers used were the following: for 36B4, forward 5 ′ -GCTTCATTGTG- To quantify the extent of lipid accumulation, cells were stained with Oil Red O at day 6 of differentiation, and the dye was extracted with isopropanol and absorbance measured at 520 nm, as described previously ( 29,30 ).

Immunofl uorescence
U2OS cells were plated on chamber slides (Labtek) and transfected with either vector, full-length ZNF638, or one of the eight ZNF638 deletion mutants. Twenty-four hours after transfection, cells were fi xed in 4% paraformaldehyde, permeabilized with 0.1% (w/v) Triton X-100 in PBS, blocked with 1% nonfat dry milk, and incubated with primary antibody for 1 h at room temperature or overnight at 4°C and with secondary antibodies for 1 h at room temperature. DAPI (4',6-diamidino-2-phenylindole, Invitrogen, D1306) was used for nuclear counterstaining. Confocal imaging was performed with an LSM 5 live DuoScan (Zeiss) laser scanning microscope with a 63× oil objective, using ZEN 2009 acquisition software (Zeiss).

Biochemical purifi cation of ZNF638 interacting proteins present in adipocytes and mass spectrometry analysis
Purifi cation of the GST-ZNF638 ZF2 fusion protein from BL21 bacterial cells and preparation of nuclear extracts from differentiating 3T3-L1 cells 2 days after induction with 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 1 M dexamethasone (Fluka), and 5 g/ml insulin (Sigma) were performed as previously described ( 2 ). ZNF638 interacting proteins purifi ed from nuclear lysates obtained from differentiating 3T3-L1 adipocytes were separated by SDS-PAGE and stained with Coomassie blue. Prominent bands were excised and subjected to mass spectrometry analysis (Taplin and the ES primers amplifi ed the short isoform, skipping exon 25, as previously described ( 31 ). The EI and ES primers utilized were the following: EI forward 5 ′ -CCGTCATCCCAGAGGTGCCCCA-3 ′ , EI reverse 5 ′ -GGAGGGACGGCCGTTTGCTGTG-3 ′ ; and ES for- The following realtime PCR conditions were used: 10 min 95°C, 35 cycles of 10 s at 95°C, 30 s at 67°C, and 30 s at 72°C. PCR products corresponding to EI or ES were further confi rmed by sequencing (Genewiz). Statistical analysis was performed by the two-sided Student's t -test with unequal variance on three replicates as indicated. A P value < 0.05 was considered statistically signifi cant.

The adipogenic cofactor ZNF638 localizes in nuclear bodies enriched in splicing factors
We previously identifi ed ZNF638 as a transcriptional cofactor implicated in the regulation of PPAR ␥ expression and adipocyte differentiation through interaction with the C/EBP family of transcription factors. In addition, we showed that ZNF638 is localized in the nucleus of differentiating 3T3-L1 cells in a punctate pattern ( 2 ). To better characterize the identity of the nuclear bodies in which ZNF638 resides, we performed immuofl uorescence staining using antibodies against ZNF638 and the splicing factor ASF/SF2, which served as a marker for nuclear speckles ( 32 ). Confocal imaging analysis of U2OS cells transiently expressing ZNF638 revealed that ZNF638 colocalizes with endogenous ASF/SF2 in nuclear speckles ( Fig. 1A, B ).

The RS and the C-terminal ZF domains are required for ZNF638's localization to nuclear speckles
To investigate the contribution of the domains present in ZNF638 to the speckled localization observed, we generated deletion mutants, as shown in the schematic representation in Fig. 2A . Indirect immunofl uorescence staining of the ectopically expressed full-length ZNF638 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 immunofl uorescence staining and confocal imaging (B). Scale bar, 10 µm. and mutants followed by confocal imaging demonstrated that the ablation of either the RS domain (ZNF638-⌬ RS) or the C-terminal ZF domain (ZNF638-⌬ ZF2), or of both domains (ZNF638-⌬ RS/ZF2), abolished ZNF638's localization to speckles ( Fig. 2B ). These fi ndings indicate that the RS domain and the C-terminal ZF domain are required for ZNF638's localization to nuclear speckles.

ZNF638 interacts with regulators of pre-mRNA splicing present in adipocytes
Given the punctate nuclear localization of ZNF638 and the evidence that speckles are nuclear bodies enriched in pre-mRNA splicing factors ( 14 ), we hypothesized that ZNF638 may complex with spliceosomal proteins. To assess this, we performed biochemical purifi cation of ZNF638 interacting proteins from differentiating 3T3-L1 adipocytes using a GST fusion protein expressing the region containing the ZF domain present at the carboxy terminus (GST-ZNF638 ZF2 ) required for speckled localization. Mass spectrometry analysis identifi ed 172 novel ZNF638 interactors ( Fig. 3A ), and their clustering according to function revealed that 38% of these interacting proteins have been previously shown to be either associated with constitutive and alternative splicing, reported in interchromatin granule clusters or present in early or late spliceosomal complexes ( 33-36 ) ( Fig. 3A , Table 2 ). Mass spectrometry analysis also revealed that the novel ZNF638 interacting proteins identifi ed are involved in transcription, translation, nucleic acid binding, and metabolism, according to their annotation in the Uniprot database ( Fig. 3A ). Furthermore, we detected ZNF638 peptide sequences, which were not part of the ZNF638-GST fusion protein used for affi nity purifi cation (data not shown). To validate the results obtained via mass spectrometry analysis, we performed coimmunoprecipitation assays. As shown in Fig. 3B , ectopically expressed GFP-ZNF638 ZF2 was able to immunoprecipitate endogenous HNRNPA1, HNRNPA2B1, HNRNPLL, NONO, and PABP1 . Taken together, our data we assessed the effect of ZNF638 on splicing of the fi bronectin minigene when a C/EBP responsive element cassette was introduced in the fi bronectin minigene reporter ( Fig. 4D ). As shown in Fig. 4E, F , low amounts of ectopically expressed ZNF638 were able to increase the ratio of EI to ES only when C / EBP ␣ , C/EBP ␤ , or C/EBP ␦ were coexpressed, suggesting that loading of ZNF638 on promoters enhances its action on splicing. These data indicate that ZNF638 is able to infl uence alternative splicing of a minigene reporter and that this process is facilitated in the presence of C/EBP responsive elements and C/EBP proteins.

The RS motif and the C-terminal ZF domain are necessary for the proadipogenic function of ZNF638
Given our previous characterization of ZNF638 as a coregulator of adipocyte differentiation, we assessed the requirement of each domain of ZNF638 for this process. We therefore ectopically expressed either vector, full-length ZNF638, or each deletion mutant in the mesenchymal cell line 10T1/2 and induced their differentiation. As shown in Fig. 5A, B , while 10T1/2 cells expressing full-length ZNF638 showed increased lipid accumulation and induced classic adipocyte markers, cells expressing ZNF638 mutants lacking either the RS, the C-terminal ZF, or both domains showed a decreased ability to induce adipocyte differentiation compared with full-length ZNF638, even though these mutants were expressed at the same levels as full-length ZNF638.
Given that alternatively spliced isoforms of lipin1 and NCoR have been previously shown to be differentially regulated during the adipogenic process ( 8, 10 ), we determined whether their levels were altered in 10T1/2 cells expressing ZNF638 or its deletion mutants. As shown in Fig. 5C , we observed an altered ratio of lipin1 ␤ /lipin1 ␣ and of NcoR /NcoR in cells expressing the mutants lacking the RS or the C-terminal ZF domains compared with cells expressing full-length ZNF638. Overall, these data demonstrate that the RS and the C-terminal ZF domains are necessary for the proadipogenic function of ZNF638 and for the regulation of alternatively spliced isoforms present in adipocytes.

DISCUSSION
The ZF protein ZNF638 is a multidomain protein initially cloned in the mid-1990s whose function has remained unknown for more than a decade. We have recently characterized ZNF638 as a transcriptional cofactor involved in adipocyte differentiation acting in cooperation with C/EBP ␤ and C/EBP ␦ to regulate the expression of the nuclear receptor PPAR ␥ in adipocytes ( 2 ). In the present study, we have provided novel evidence that ZNF638 localizes in nuclear regions enriched in splicing factors, that it interacts with splicing regulatory proteins present in adipocytes, and that it participates in the modulation of alternative splicing of a pre-mRNA transcript derived from a minigene reporter.
indicate that ZNF638 can complex with factors implicated in alternative splicing expressed in differentiating adipocytes.

ZNF638 modulates alternative splicing decisions on a minigene reporter
In order to test the hypothesis that ZNF638 may participate in the regulation of pre-mRNA splicing, we investigated the effects of coexpressing ZNF638 with a minigene reporter containing a genomic exon cassette derived from the rat fi bronectin gene. This reporter system is used for the quantifi cation of alternative splicing through measurements of an alternatively spliced exon ( Fig. 4A ) ( 26 ). When ZNF638 was coexpressed with the minigene reporter, we observed an increase in the ratio of EI to ES, as compared with the ratio observed in control cells ( Fig. 4B,  C ). Exon inclusion and exclusion were further confi rmed by direct sequencing of PCR products (data not shown).
Given our previously published data demonstrating a function for ZNF638 as a coactivator of C/EBP proteins at C/EBP responsive elements present in the PPAR ␥ promoter, 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 identifi ed by mass spectrometry analysis (A). Interacting proteins purifi ed from nuclear extracts obtained from differentiating 3T3-L1 cells using a GST-ZNF638 ZF2 fusion protein were resolved by SDS-PAGE and subjected to mass spectrometry analysis. Coimmunoprecipitation assays were performed to validate mass spectrometry data (B). GFP-ZNF638 ZF2 fusion protein was expressed in HEK-293 cells, and endogenous interacting proteins were detected by Western blot after GFP immunoprecipitation. Asterisk indicates nonspecifi c band.  Our systematic analysis of ZNF638 interactors in adipocytes revealed that ZNF638 complexes with 16 factors shown to be part of the early prespliceosomal complex, including U2SURP, SRSF7, TDRD3, SF3B1, FUS, DDX46, DDX5, DHX9, ELAVL1, ILF3, NCBP1, RBM39, and the HNRNP proteins A1, A2/B1, A3, and U ( 34 ). These interactions suggest an early association of ZNF638 with the splicing machinery and its participation in splice site selection during prespliceosomal assembly ( 4 ). Particularly noteworthy is the interaction of ZNF638 with HNRNPA1, HNRNPA2B1, and HNRNPLL, which have been critically implicated in alternative splicing (37)(38)(39)(40)(41)(42). Given that HNRNPA1 functions by antagonizing the splicing factor ASF/SF2 on splice site selection ( 37 ), it is possible that ZNF638 may affect the relative local abundance of HNRNPA1 through binding and sequestration, shifting the cellular balance in favor of the splicing factor SF2, as proposed in Fig. 6A .
Our mass spectrometry analysis has also revealed that the C-terminal portion of ZNF638 interacts with other ZNF638 proteins present in adipocytes, suggesting that either multiple ZNF638 molecules may be recruited to protein complexes formed by ZNF638 or that ZNF638 may be able to di-or multimerize. Direct verifi cation of the functional signifi cance of these fi ndings in adipocyte differentiation will require further investigation.
The present data indicating that ZNF638 affects alternative splicing of a minigene containing C/EBP binding sites combined with the previous reported function of ZNF638 in transcription through interaction with C/EBP proteins at C/EBP responsive elements ( 2 ) suggest that ZNF638 may act as a dual function regulator. This double role is consistent with emerging evidence indicating that cofactors can participate in both transcription and splicing, as described for p52, RNA binding motif protein 39, RNA binding motif protein 14, and PGC1 ␣ ( 20-23 ). It is plausible that when loaded on a promoter through C/EBP responsive elements, ZNF638 could facilitate the recruitment of the splicing factor(s) required for splice choice to the CTD of the largest subunit of RNA polymerase II ( 3,43 ) and to the proximity of the nascent pre-mRNA ( Fig. 6B ).  5. Requirements of ZNF638 domains in adipocyte differentiation. Quantifi cation of lipid accumulation in 10T1/2 cells expressing vector, full-length ZNF638, or deletion mutants at day 6 after induction of differentiation measured through quantifi cation 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. This model is supported by the identifi cation through our mass spectrometry analysis of SRSF4, a splicing factor previously shown to be involved in alternative splice site selection during pre-mRNA processing ( 44,45 ), as a novel ZNF638 interactor. This putative dual function of ZNF638 is reminiscent of the splicing role played by the coactivator PGC1 ␣ when loaded on the promoter of its target genes ( 23 ). through their function as splicing activation domains via interactions with spliceosomal components or whether their role in adipocyte differentiation is through the recruitment of cofactors involved in transcription.
Through mutagenesis and confocal microscopy analysis, we showed that the RS domain and the C-terminal ZF motif are required for ZNF638's localization in nuclear bodies enriched in splicing factors. While it has been previously recognized that RS domains can confer speckle targeting (46)(47)(48), our data indicate that also the U1/matrin-like ZF (C-X 2 -C-X (12,16) -H-X 5 -H) ( 49 ) present at the C terminus of ZNF638 is required for speckle localization. Interestingly, this type of ZF motif has been previously identifi ed in other proteins present in nuclear speckles and shown to be required for their localization, as in the case of SF3A2 and SF3A3 ( 50 ). These observations suggest a possible contribution of matrin-like ZFs in targeting proteins to nuclear speckles.
Our structure-function analysis has revealed that the RS and the C-terminal ZF domains required for speckle localization are also necessary for the differentiation function of ZNF638. While it is well established that RS domains play a role in splicing ( 51 ), the requirement of SR domains in adipocyte differentiation has been only recently identifi ed through the analysis of the RS domain in the function of the CDC-like kinase 1) kinase in adipocyte differentiation ( 52 ). Further studies will determine whether the RS and C-terminal ZF domains present in ZNF638 are necessary for the generation of adipocyte-specifi c isoforms 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.