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The roles of Zbtb11 in embryonic stem cell proliferation and survival

ABSTRACT: Zbtb11 is a transcription factor conserved in vertebrates. Mutations in its human gene cause a form of inherited intellectual disablity, while in zebrafish Zbtb11 mutations lead to impaired hematopoesis and neruonal development. To determine the genes regulated by Zbtb11 we generated an inducible KO ES cell line (Zbtb11 lox/lox, Rosa26 ERt2-Cre) that allows the rapid depletion of Zbtb11 upon treatment with 4-hydroxytamoxyfen (4OHT). We subsequently performed RNA-seq in Zbtb11 KO and control cells to determine differentially expressed genes. Upon Zbtb11 deletion 310 genes changed expression. Integration of the RNA-seq data with Zbtb11 ChIP-seq data (GEO series GSE125047) allowed us to distinguish between genes directly regulated by Zbtb11 and indirect changes. 204 differentially expressed genes were bound by Zbtb11, while 106 did not have Zbtb11 binding sites. Directly regulated genes were significantly enriched in genes with mitochondrial functions, while indirectly controlled genes were enriched for p53 targets. In agreement with these findings Zbtb11 KO led to impaired respiration, proliferation arrest and cell death.

ORGANISM(S): Mus musculus

PROVIDER: GSE130292 | GEO | 2022/12/31

REPOSITORIES: GEO

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Project description:Zbtb11 is an uncharacterised transcription factor that is conserved in vertebrates. Mutations in its human gene cause a form of inherited intellectual disablity, while in zebrafish Zbtb11 mutations lead to impaired hematopoesis and neruonal development. We used a combination of functional genomics approaches to determine its functions. To determine the genomic Zbtb11 binding locations, we carried out ChIP-seq using FLAG antibodies in a cell line with the endogenous Zbtb11 N-terminally tagged with FLAG (Zbtb11 FLAG/FLAG), as well as using an anti-Zbtb11 antibody in a wild type line. To determine the genes regulated by Zbtb11 we generated an inducible KO line (Zbtb11 lox/lox, Rosa26 ERt2-Cre) that allows the rapid depletion of Zbtb11 upon treatment with 4-hydroxytamoxyfen (4OHT). We treated Zbtb11 lox/lox, Rosa26 ERt2-Cre cells either with 4OHT (Zbtb11 KO) or ethanol (EtOH) as control, and subsequently performed directional RNA-seq from samples collected 48 hours post-treatment. As control for the effect of 4OHT on gene expression, we also compared Zbtb11 KO cells to wild type cells treated with 4OHT. The two ChIP-seq approaches showed very good overlap and allowed us to generate a set of high-confidence Zbtb11 binding sites common to both peak sets. We found Zbtb11 preferentially binds to a subset of house-keeping genes among which genes encoding proteins with mitochondrial functions are enriched. Upon Zbtb11 deletion, 154 genes changed expression 48 hours later, the vast majority of them down-regulated. Integration of ChIP-seq and RNA-seq data allowed us to identify the genes directly regulated by Zbtb11, and these were significantly enriched in genes with mitochondrial functions, with respiratory complex I and mitoribosome biogenesis being the overrepresented pathways. Subsequent biochemical experiments confirmed Zbtb11 is required for respiratory complex I assembly and for mitochondrial translation. In agreement with these findings Zbtb11 KO led to impaired respiration, proliferation arrest and cell death.

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The roles of Zbtb11 in embryonic stem cell proliferation and survival

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