Dataset Information

E4F1 and ZNF148 are transcriptional activators of the A57C and wildtype TERT promoter

ABSTRACT: Point mutations within the TERT promoter are the most common recurrent somatic non-coding mutation identified across different cancer types, including glioblastoma, melanoma, hepatocellular carcinoma and bladder cancer. They are most abundant at C146T and C124T and more rare at A57C, with the latter originally described as a familial case but subsequently shown also to occur somatically. All three mutations create de novo ETS (E-twenty-six specific) binding sites and result in the reactivation of the TERT gene, allowing cancer cells to achieve replicative immortality. Here, we employed a systematic proteomics screen to identify transcription factors preferentially binding to the C146T, C124T and A57C mutations. While we confirmed binding of multiple ETS factors to the mutant C146T and C124T sequences, we identified E4F1 a an A57C-specific binder and ZNF148 as a TERT WT binder that is excluded from the TERT promoter by the C124T allele. Both proteins are activating transcription factors that bind specifically to the A57C and wildtype (at position 124) TERT promoter sequence in corresponding cell lines and upregulate TERT transcription and telomerase activity.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

DISEASE(S): Brain Cancer

SUBMITTER: Dennis Kappei

LAB HEAD: Dennis Kappei

PROVIDER: PXD037776 | Pride | 2023-11-04

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20180328_LAB_BH_U87MG_TERT_146mut.zip	Other
	20180328_LAB_BH_U87MG_TERT_146mut_for01.raw	Raw
	20180328_LAB_BH_U87MG_TERT_146mut_for02.raw	Raw
	20180328_LAB_BH_U87MG_TERT_146mut_for03.raw	Raw
	20180328_LAB_BH_U87MG_TERT_146mut_for04.raw	Raw

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Publications

E4F1 and ZNF148 are transcriptional activators of the -57A > C and wild-type <i>TERT</i> promoter.

Chua Boon Haow BH Zaal Anuar Nurkaiyisah N Ferry Laure L Domrane Cecilia C Wittek Anna A Mukundan Vineeth T VT Jha Sudhakar S Butter Falk F Tenen Daniel G DG Defossez Pierre-Antoine PA Kappei Dennis D

Genome research 20231201 11

Point mutations within the <i>TERT</i> promoter are the most recurrent somatic noncoding mutations identified across different cancer types, including glioblastoma, melanoma, hepatocellular carcinoma, and bladder cancer. They are most abundant at -146C > T and -124C > T, and rarer at -57A > C, with the latter originally described as a familial case, but subsequently shown also to occur somatically. All three mutations create de novo E26-specific (ETS) binding sites and result in activation of th ...[more]

PMID: 37918959

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Project description:The interaction between transcription factors and genomic DNA forms the basis for spatio-temporal control of gene expression. Therefore, these interactions and their impact on disease and cellular fate are extensively studied on a global level, mainly using techniques based on next-generation sequencing. These techniques, however, do not allow an unbiased study of proteins or entire protein complexes that bind to a certain DNA sequence. In recent years, DNA pull-downs followed by quantitative mass spectrometry were introduced to close this gap. Established protocols, however, are based on metabolic labeling techniques or require enormous amounts of cellular material, thus restricting the method to cell lines grown in culture. Furthermore, they require substantial amount of expertise, thus keeping this technique restricted to a limited number of laboratories. Here, we introduce a high-throughput compatible, LC-MS/MS based DNA pull-down that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate, that our method can efficiently identify transcription factors binding to their known consensus DNA motifs when using nuclear extracts from model cell lines. Subsequently, we apply the method to study DNA-protein interactions in primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. We show that the same DNA sequence binds different sets of proteins in an established model cell line as opposed to PBMCs. This stresses the importance of selecting relevant cell extracts for any interaction in question. In-depth nuclear proteomes with absolute quantification of close to 7,000 proteins in K562 cells and PBMCs clearly link these differential interactions to differences in protein abundance. In conclusion, our approach, applicable to primary material and capable of profiling DNA-protein interactions in high-throughput, will likely prove itself as a useful screening platform and will provide invaluable functional data, for example through integration with large scale GWAS.