Dissecting The Mechanisms Of DDB1 In Promoting Adipogenesis

Adipogenesis is orchestrated by a cascade of transcriptional factors;however,how the transcriptional cascade is initiated remains unclear.Here we show that the cascade is turned on by damaged DNA binding protein 1(DDB1)can conform complexes with the positive transcription elongation factor b(P-TEFb)and ELK1 which mediated release of ELK1 downstream gene promoter-proximally paused RNA polymerase ?(Pol ?).DDB1 was first identified as a member of the UV-induced damage protein complex DDB1/DDB2.It is now well-characterized as a subunit of the Cullin4-DDB1 E3 ubiquitin ligase complex which recognizes and ubiquitinates substrates through a subset of WD40-containing proteins.Consistent with that previous studies have shown that the Cullin4-DDB1-WDTC1 complex inhibits adipogenesis,we find that depletion of Cullin4A(CUL4A)or Cullin4B(CUL4B)promotes adipogenesis in both 3T3-L1 and primary preadipocytes,and that adult Cul4a or Cul4b knockout mice are prone to develop diet-induced obese on high-fat-diet feeding.However,we were surprised to find that lack of DDB1 significantly inhibits adipogenesis in both 3T3-L1 and primary preadipocytes.Furthermore,Ddbl+/-mice show delayed postnatal development in white adipose tissues and are lean on high-fat-diet feeding.These results suggest that DDB1 works independently of Cullin4 complex to promote adipogenesis.Mechanistically,upon stimulation,we demonstrate that DDB1 is required for the transcription of immediate-early response genes(IEGs)during adipogenesis.We find DDB1 and ELK1 indeed share the binding sites on their common downstream genes,which provides structural basis for DDB1 to regulate the transactivation activity of ELK1.We then use chromatin immunoprecipitation sequencing analysis(chip-seq)demonstrated that transcription of IEGs in adipogenesis is regulated by Pol ? pausing.Under basal state,Pol II binds and pauses at the proximal promoters of the IEGs without transcriptional activity.Upon stimulation,DDB1 binds the positive transcription elongation factor b(P-TEFb)and complexes with ELK1 to release paused Pol ? on the proximal promoters of IEGs in adipogenesis,thereby initiating the transcriptional cascade.Our findings have thus revealed the molecular mechanism by which P-TEFb-DDB1-ELK1 initiated transcriptional cascade in adipogenesis.We have also uncovered a Cullin4-independent role of DDB1 in preparing preadipocytes for differentiation and a physiological significance of Pol ? pausing in fine-tuning adipogenesis.