Unknown,Transcriptomics,Genomics,Proteomics

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ChIP-seq of ZFP30-HA and KAP1 in mouse 3T3-L1 cells during adipogenic differentiation

ABSTRACT: 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.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Petra Catalina Schwalie

PROVIDER: E-MTAB-6817 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:KAP1 is overexpressed in breast cancer. To determine KAP1 regulated genes, we performed microarray analysis of gene expression in KAP1 depleted breast cancer cells MDA-MB-231LN. The transcriptional regulator TRIM28/KAP1 plays an important role in development, stem cell self-renewal, chromatin organization and the DNA damage response. KAP1 is an essential co-repressor for KRAB zinc finger proteins (KRAB-ZNFs). Though KRAB-ZNFs represent the largest family of human transcription factors, their biological functions are largely unknown. Using the conserved zinc fingers linker region (ZnFL) as antigen, we have developed a ZnFL antibody that recognizes multiple KRAB-ZNFs. We showed that KAP1 and many KRAB-ZNFs were overexpressed in human breast cancers and breast cancer cell lines. In addition, an active SUMOylated form of KAP1 was markedly increased in breast cancer cells. Furthermore, KAP1 depletion in breast cancer cell lines reduced cell proliferation and inhibited tumor growth and metastasis of tumor xenografts. Conversely, KAP1 overexpression stimulated cell proliferation and tumor growth. KAP1 knockdown led to down-regulation of genes previously linked to tumor progression and metastasis, including PTGS2/COX2, EREG, CD44, MMP1 and MMP2. Interestingly, KAP1 depletion or genomic deletion led to dramatic down-regulation of multiple KRAB-ZNF proteins due in part to their increased degradation. KAP1-dependent stabilization of KRAB-ZNFs required a direct KRAB-ZNF-KAP1 interaction. These results establish KAP1 as a positive regulator of multiple KRAB-ZNFs and an important factor in the development of breast cancer. 7 total samples were analyzed. Stable sublines of MDA-MB-231LN cells expressing control non-targeting shRNA (Scr, 3 biological replicates) and two different shRNAs against KAP1 (KAP1-3, 2 biological replicates and KAP1-4, 2 biological replicates) from doxycycline-inducible pTRIPZ vector were cultured in the presence of 0.5 ug/ml doxycycline for 7 days to induce shRNA expression. Cells were lysed and total RNA was isolated using mirVana miRNA isolation kit (Ambion) in the WVU Genomics Core Facility.

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ChIP-seq of ZFP30-HA and KAP1 in mouse 3T3-L1 cells during adipogenic differentiation

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