E-MTAB-6817
ChIP-seq of ZFP30-HA and KAP1 in mouse 3T3-L1 cells during adipogenic differentiation
We show that a hitherto poorly characterized KRAB domain-containing zinc-finger (ZF) transcription factor, ZFP30, positively regulates adipogenesis. We demonstrate ZFP30's function in murine in vitro and in vivo models, as well as in human stromal vascular fraction cells. We reveal through mechanistic studies that ZFP30 directly targets and activates Pparg2 by binding a retrotransposon-derived enhancer, suggesting a process of adipogenic exaptation. We further show that ZFP30 recruits the co-regulator KRAB-associated protein 1 (KAP1), which, surprisingly, acts as a ZFP30 co-activator in this adipogenic context.  As neither the commercial ZFP30 antibodies nor four batches of customized ZFP30 antibodies recognized it specifically (data not shown), we performed ChIP-seq in 3T3-L1 cells expressing HA-tagged ZFP30 in a tetracycline-inducible manner, as also previously employed for ZEB1 (Gubelmann et al., 2014). The mRNA expression of exogenous Zfp30 was induced to a similar level as the endogenous Zfp30 by adjusting the amount of Doxycycline (data not shown), to avoid potential artefacts due to protein overexpression. To further characterize its role in adipogenesis and as a ZFP30 partner, we performed KAP1 ChIP-seq in undifferentiated (day 0) and differentiated (day 2) 3T3-L1 cells, as described above for HA-ZFP30. We first confirmed the high enrichment obtained with the employed KAP1 antibody.
We show that a hitherto poorly characterized KRAB domain-containing zinc-finger (ZF) transcription factor, ZFP30, positively regulates adipogenesis. We demonstrate ZFP30's function in murine in vitro and in vivo models, as well as in human stromal vascular fraction cells. We reveal through mechanistic studies that ZFP30 directly targets and activates Pparg2 by binding a retrotransposon-derived enhancer, suggesting a process of adipogenic exaptation. We further show that ZFP30 recruits the co-regulator KRAB-associated protein 1 (KAP1), which, surprisingly, acts as a ZFP30 co-activator in this adipogenic context.  As neither the commercial ZFP30 antibodies nor four batches of customized ZFP30 antibodies recognized it specifically (data not shown), we performed ChIP-seq in 3T3-L1 cells expressing HA-tagged ZFP30 in a tetracycline-inducible manner, as also previously employed for ZEB1 (Gubelmann et al., 2014). The mRNA expression of exogenous Zfp30 was induced to a similar level as the endogenous Zfp30 by adjusting the amount of Doxycycline (data not shown), to avoid potential artefacts due to protein overexpression. To further characterize its role in adipogenesis and as a ZFP30 partner, we performed KAP1 ChIP-seq in undifferentiated (day 0) and differentiated (day 2) 3T3-L1 cells, as described above for HA-ZFP30. We first confirmed the high enrichment obtained with the employed KAP1 antibody.