This article is available online at http://www.jlr.org CCAAT/enhancer binding protein (C/EBP)  and C/EBP ␦ , and by cofactors ( 1 ), such as the recently characterized zinc fi nger protein ZNF638, which regulates the expression of the master controller of adipogenesis, PPAR ␥ , in conjunction with C/EBP proteins ( 2 ). The gene expression program occurring during differentiation includes the engagement and cooperation of these tissue-selective factors and cofactors with components of the RNA polymerase II machinery at active transcription sites, initiation of transcription followed by mRNA processing, through RNA capping, polyadenylation, and removal of noncoding intronic sequences, prior to protein translation ( 3 ). The process of splicing gives rise to the mature mRNA though the sequential succession of several complexes, starting from the precomplex in which sites of splicing are chosen, to the catalytic removal of introns executed by complex C components ( 4 ). During differentiation and development, tissue-specifi c enrichment of splicing factors ensures that alternative splicing is achieved to generate tissue-specifi c, temporally and developmentally regulated isoforms required to confer the specifi c phenotype ( 4-6 ). It has been shown that the relative abundance or activity of splicing regulators that have opposing effects determines the use of competing splice sites, ultimately controlling the exon composition of tissue-specifi c isoforms ( 4, 5 ). In adipocytes, alternatively spliced isoforms such as those of the insulin receptor ( 7 ), lipin ( 8 ), mitochondrial oxodicarboxylate carrier ( 9 ), nuclear receptor co-repressor 1 (NCoR) ( 10 ), preadipocyte factor 1 ( 11 ), and mechanistic target of rapamycin ( 12 ) have been shown to play a role in the differentiation process.Abstract Increasing evidence indicates that transcription and alternative splicing are coordinated processes; however, our knowledge of specifi c factors implicated in both functions during the process of adipocyte differentiation is limited. We have previously demonstrated that the zinc fi nger protein ZNF638 plays a role as a transcriptional coregulator of adipocyte differentiation via induction of PPAR ␥ 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 purifi cation 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 suffi cient 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 fi nger domain required for speckle localization are necessary for the adipocyte differentiation function of ZNF638 and for t...