Abstract
Zinc finger proteins constitute the largest family of transcription regulators in eukaryotes. These factors are involved in diverse processes in many tissues, including development and differentiation. We report here the characterization of the zinc finger protein ZNF638 as a novel regulator of adipogenesis. ZNF638 is induced early during adipocyte differentiation. Ectopic expression of ZNF638 increases adipogenesis in vitro, whereas its knockdown inhibits differentiation and decreases the expression of adipocyte-specific genes. ZNF638 physically interacts and transcriptionally cooperates with CCAAT/enhancer-binding protein (C/EBP) β and C/EBPδ. This interaction leads to the expression of peroxisome proliferator-activated receptor γ, which is the key regulator of adipocyte differentiation. In summary, ZNF638 is a novel and early regulator of adipogenesis that works as a transcription cofactor of C/EBPs.
Highlights
Adipogenesis is a process in which an undifferentiated mesenchymal cell becomes fully competent in storing lipids and secreting adipokines
Zinc Finger Factor ZNF638 Is Induced during Early Adipogenesis—To identify novel zinc finger factors involved in adipogenesis, we performed an in silico screen for molecules with a similar domain composition to PGC-1␣
This study has shown that the zinc finger factor ZNF638 is a novel regulator of the early phases of adipocyte differentiation
Summary
Plasmids—ZNF638 cDNA was generated from a mouse cDNA library obtained from 3T3-L1 cells at day 2 of differentiation utilizing primers 5Ј-ACAGCCACCATGTCGAGACCCAGGTTTAATCC-3Ј (forward) and 5Ј-CCCGGGTCACCTAGAGCTTCTTTCTTCAGTC-3Ј (reverse) and cloned into the pCR2.1-TOPO TA vector (Invitrogen). FLAG-ZNF638 was constructed utilizing primers 5Ј-CCATGGACTACAAGGACGACGACGACGAGACCCAGGTTTAATC-3Ј (forward) and 5Ј-CGCCTTGTCGTCGTCGTCCTTGTAGTCTGGTGGCTGTAAGGC-3Ј (reverse) and cloned into pCR3.1 at the AflII and AgeI sites. FLAG-⌬DBD-ZNF638 was prepared using primers 5Ј-GTTACAAACCCTGAAACTGAATTAGCAGTATCTGAC-3Ј (forward) and 5Ј-CTGTTTCCTGAGACTGTTTTGTTATTGGCTTTTCTCTTATTGCTGC (reverse). Anti-FLAG M2 affinity gel (Sigma) or GFP-Trap beads (Chromotek-GFP-Trap, Allele Biotechnology) were used. For FLAG immunoprecipitation assays, nuclear extracts were obtained from transfected HEK-293 cells. 40 l of anti-FLAG M2 beads were added to 200 l of nuclear lysates and incubated overnight at 4 °C. For GFP immunoprecipitation assays, transfected HEK-293 cells were lysed, and 30 l of GFP-Trap beads were added to the lysate and incubated for 2 h at 4 °C. RNA was precipitated with 0.5 volume of isopropyl alcohol, washed with
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