ChIP-seq on Zfp961 GFP flox/flox mESCs to verify Zfp961 binding capacity at PBS-Lys elements.
Ontology highlight
ABSTRACT: We performed ChIP-seq assay with anti-GFP antibodies on Zfp961-GFPflox/flox ESCs.We found PBS-Lys-containing endogenous virus K subgroup repeats (ERV-K) were significantly enriched in the strong peak regions (40%, 165 out of 413), compared to their abundance in the mouse genome (~5%), suggesting Zfp961 binding preference towards ERV-K regions. And Zfp961 has a PBS-Lys motif. We performed H3K9me3 ChIP on Zfp961 WT or KO mESCs. We found that Zfp961 recruits H3k9me3 modification at ERVKs, and H3K9me3 level showed a moderate decrease upon Zfp961 deletion.
Project description:We performed ChIP-seq assay with anti-KAP1 antibodies on Zfp961-GFPflox/flox ESCs.We found KAP1 signal decreased at PBS-Lys sites in Zfp961 KO cells compared to WT, suggesting Zfp961 recruiting KAP1 to ERV-K regions. We performed H3K27ac ChIP on Zfp961 WT or KO mESCs. We found that H3k27ac modification at PBS-Lys sites was elevated upon Zfp961 deletion, indicating Zfp961 serving as a repressive transcription factor.
Project description:The ChIP-seq experiments using GFP antibody on ZNF417/ZNF587-GFP overexpressing 293T cells revealed that ZNF417/ZNF587 preferred to bind a PBS-Lys-containing HERVs. Further motif calling analysis showed that both ZNF417 and ZNF587 bind to HERVK PBS-resembled motif.
Project description:To identify ZNF506 genome-wide target sites, we performed ChIP-seq assay and found that ZNF506 binding sites enriched at PBS-Pro-containing ERV subfamilies (ERVPs) and further motif calling analysis showed that ZNF506 binds to PBS-Pro sequences, promoting formation of H3K9me3 modifications at binding regions. And ChIP-seq assay also indicated that the distinction of H3K9me3 signals closed to ZNF506 peak regions between ZNF506 overexpressing (OE) HEK293T cells and ZNF506 Knockout (KO) HEK293T cells was correlated with the different recruitment of corepressor KAP1. The ChIP-seq experiments using GFP antibodies and H3K9me3 antibodies on ZNF506-GFP OE HEK293T cell lines, and ZNF417-GFP OE HEK293T cell lines were used as controls for H3K9me3 ChIP. Also, the ChIP-seq experiments were performed using H3K9me3 antibodies and KAP1 antibodies on ZNF506 KO HEK293T cells and ZNF506 OE HEK293T cells.
Project description:In order to elucidate transcriptional and metabolic networks associated with Lys metabolism, we utilized developing seeds as a system in which Lys synthesis could be stimulated developmentally without application of chemicals and coupled this to a T-DNA insertion knockout mutation impaired in Lys catabolism. This seed-specific metabolic perturbation stimulated Lys accumulation starting from the initiation of storage reserve accumulation. Our results revealed that the response of seed metabolism to the inducible alteration of Lys metabolism was relatively minor, however, that which was observable operated in a modular manner. They also demonstrated that Lys metabolism is strongly associated with the operation of the TCA cycle, whilst largely disconnected from other metabolic networks. In contrast, the inducible alteration of Lys metabolism was strongly associated with gene networks, stimulating the expression of hundreds of genes controlling anabolic processes that are associated with plant performance and vigor, whilst suppressing a small number of genes associated with plant stress interactions. The most pronounced effect of the developmentally-inducible alteration of Lys metabolism was an induction of expression of a large set of genes encoding ribosomal proteins as well as genes encoding translation initiation and elongation factors, all of which are associated with protein synthesis. With respect to metabolic regulation, the inducible alteration of Lys metabolism was primarily associated with altered expression of genes belonging to networks of amino acids and sugar metabolism. The combined data are discussed within the context of network interactions both between and within metabolic and transcriptional control systems.
Project description:Lysine methylation is abundant on histone proteins, representing a dynamic regulator of chromatin state and gene activity, but is also frequent on many nonhistone proteins, including eukaryotic elongation factor 1 alpha (eEF1A). However, the functional significance of eEF1Amethylation remains obscure and it has remained unclear whether eEF1A methylation is dynamic and subject to active regulation. We here demonstrate, using a wide range of in vitro and in vivo approaches, that the previously uncharacterized human methyltransferase METTL21B specifically targets Lys-165 in eEF1A in an aminoacyl-tRNA- and GTP-dependent manner. Interestingly, METTL21B mediated eEF1A methylation showed strong variation across different tissues and cell lines, and was induced by altering growth conditions or by treatment with certain ER-stress-inducing drugs, concomitant with an increase in METTL21B gene expression. Moreover, genetic ablation of METTL21B function in mammalian cells caused substantial alterations in mRNA translation, as measured by ribosome profiling. A non-canonical function for eEF1A in organization of the cellular cytoskeleton has been reported, and interestingly, METTL21B accumulated in centrosomes, in addition to the expected cytosolic localization. In summary, the present study identifies METTL21B as the enzyme responsible for methylation of eEF1A on Lys-165 and shows that this modification is dynamic, inducible and likely of regulatory importance.
Project description:To understand the underlying cause for reduced lung metastasis, we compared global gene expression profiles of F4/80+ FACS sorted tumor-associated macrophages (TAMs) PyMT;E2f3 f/f (control) and PyMT;Lys Cre:E2f3 f/f (experimental) mice. We compared gene expression profile between TAMs isolated from mammary tumors of PyMT;E2f3 f/f and PyMT;Lys Cre:E2f3 f/f mice
Project description:In order to elucidate transcriptional and metabolic networks associated with Lys metabolism, we utilized developing seeds as a system in which Lys synthesis could be stimulated developmentally without application of chemicals and coupled this to a T-DNA insertion knockout mutation impaired in Lys catabolism. This seed-specific metabolic perturbation stimulated Lys accumulation starting from the initiation of storage reserve accumulation. Our results revealed that the response of seed metabolism to the inducible alteration of Lys metabolism was relatively minor, however, that which was observable operated in a modular manner. They also demonstrated that Lys metabolism is strongly associated with the operation of the TCA cycle, whilst largely disconnected from other metabolic networks. In contrast, the inducible alteration of Lys metabolism was strongly associated with gene networks, stimulating the expression of hundreds of genes controlling anabolic processes that are associated with plant performance and vigor, whilst suppressing a small number of genes associated with plant stress interactions. The most pronounced effect of the developmentally-inducible alteration of Lys metabolism was an induction of expression of a large set of genes encoding ribosomal proteins as well as genes encoding translation initiation and elongation factors, all of which are associated with protein synthesis. With respect to metabolic regulation, the inducible alteration of Lys metabolism was primarily associated with altered expression of genes belonging to networks of amino acids and sugar metabolism. The combined data are discussed within the context of network interactions both between and within metabolic and transcriptional control systems. Experiment Overall Design: Arabidopsis thaliana (WS) seeds of the WT and KD genotype (Zhu and Galili, 2003)were germinated on soil and grown in the greenhouse (23oC). Flowers were marked and at given time intervals following flowering (14DAF to Dry ). Maturing siliques were collected, then seeds were dissected from siliques . Mature seeds were collected at the end of the desiccation period and stored at 4ºC. Two repeats were collected from each time point. Three to five thousands seeds were harvested for each extraction.
Project description:Silencing of endogenous retroviruses (ERVs) is largely mediated by repressive chromatin modifications, such as H3K9me3 and DNA methylation. Their impact on ERV silencing differs in various cell types and, no systematic analyses on the interdependence between H3K9me3 and DNA methylation have been performed in differentiated cells. Here we show that deletion of the H3K9me3 HMTase Setdb1 in mouse embryonic endoderm results in ERV de-repression in only a subset of endoderm cells. We found that de-repression is restricted to visceral endoderm descendants and does not occur in definitive endoderm cells. Deletion of Setdb1 in visceral endoderm progenitors resulted in loss of H3K9me3 and reduced DNA methylation of IAPEz, consistent with up-regulation of this ERV family. In definitive endoderm cells, loss of Setdb1 did not affect H3K9me3 nor DNA methylation, suggesting Setdb1-independent pathways for maintaining these modifications. Importantly, Dnmt1 ko resulted in ERV de-repression in both visceral and definitive endoderm cells, while H3K9me3 was unaltered. Thus, our data suggest a dominant role of DNA methylation over H3K9me3 in ERV silencing in endoderm cells. Our findings suggest, that H3K9me3 is not sufficient for ERV silencing, but rather critical for maintaining high DNA methylation.