Dataset Information

Dissecting the function of the adult ?-globin downstream promoter region using an artificial zinc finger DNA-binding domain.

ABSTRACT: Developmental stage-specific expression of the ?-type globin genes is regulated by many cis- and trans-acting components. The adult ?-globin gene contains an E-box located 60 bp downstream of the transcription start site that has been shown to bind transcription factor upstream stimulatory factor (USF) and to contribute to efficient in vitro transcription. We expressed an artificial zinc finger DNA-binding domain (ZF-DBD) targeting this site (+60 ZF-DBD) in murine erythroleukemia cells. Expression of the +60 ZF-DBD reduced the recruitment and elongation of RNA polymerase II (Pol II) at the adult ?-globin gene and at the same time increased the binding of Pol II at locus control region (LCR) element HS2, suggesting that Pol II is transferred from the LCR to the globin gene promoters. Expression of the +60 ZF-DBD also reduced the frequency of interactions between the LCR and the adult ?-globin promoter. ChIP-exonuclease-sequencing revealed that the +60ZF-DBD was targeted to the adult ?-globin downstream promoter and that the binding of the ZF-DBD caused alterations in the association of USF2 containing protein complexes. The data demonstrate that targeting a ZF-DBD to the adult ?-globin downstream promoter region interferes with the LCR-mediated recruitment and activity of Pol II.

SUBMITTER: Barrow JJ

PROVIDER: S-EPMC3985677 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Dissecting the function of the adult β-globin downstream promoter region using an artificial zinc finger DNA-binding domain.

Barrow Joeva J JJ Li Ying Y Hossain Mir M Huang Suming S Bungert Jörg J

Nucleic acids research 20140204 7

Developmental stage-specific expression of the β-type globin genes is regulated by many cis- and trans-acting components. The adult β-globin gene contains an E-box located 60 bp downstream of the transcription start site that has been shown to bind transcription factor upstream stimulatory factor (USF) and to contribute to efficient in vitro transcription. We expressed an artificial zinc finger DNA-binding domain (ZF-DBD) targeting this site (+60 ZF-DBD) in murine erythroleukemia cells. Expressi ...[more]

PMID: 24497190

Similar Datasets

Project description:Transcription factors are often regarded as having two distinct components: a DNA binding domain (DBD) to allow binding to the regulatory region of a target gene and a trans-acting functional domain (FD) to modulate gene expression. Recently, it is becoming clear that the DBDs of transcription factors alone are incapable of providing sufficient specificity to account for the highly complex genomic structures in eukaryotes. Other regions, outside of the DBD, may play a role in transcription factor DNA binding specificity in vivo. In order to test this hypothesis, we examined a model zinc finger transcription factor, KrÃ¼ppel-like factor 3 (KLF3) with a FD that recruits partner proteins for its repressive activity and a three zinc finger DBD. Previously, we showed that deletion of the entire KLF3 FD reduced DNA binding across the genome. This implied the importance of the FD for in vivo DNA binding specificity. In the current study, we fused the KLF3 FD onto an unrelated, but well characterised Artificial Zinc Finger (AZF) targeting a standard target gene, VEGF-A. We compared the genomic DNA binding profile of this chimeric KLF3 construct, termed KLF3FD-AZF, to that of the AZF alone. Chromatin Immunoprecipitation followed by next generation sequencing was used to assess genomic wide DNA-binding of these AZFs. Remarkably, in these gain-of-function experiments, we again observed that the KLF3 FD is critical for in vivo DNA binding specificity. The addition of KLF3 FD increased the number of binding sites by more than two fold compared to the AZF alone. KLF3 FD also directed more binding to the promoter regions. Further investigations of these acquired sites identified a substantial portion of these sites as the known endogenous KLF3 bound regions, whilst others contain sequences that are similar but not identical to the predicted AZF target sequence. These results clearly demonstrate that the specific localisation of this model transcription factor to target genes is not solely dependent on the DBD and that the regions outside of this domain may also play an important role. ChIP-Seq experiments were performed on four HEK293 cell lines, two each stably expressing AZF or KLF3FD.AZF or KLF3FD only (two biological replicates). Input samples were included as controls.

Project description:Transcription factors are often regarded as having two distinct components: a DNA binding domain (DBD) to allow binding to the regulatory region of a target gene and a trans-acting functional domain (FD) to modulate gene expression. Recently, it is becoming clear that the DBDs of transcription factors alone are incapable of providing sufficient specificity to account for the highly complex genomic structures in eukaryotes. Other regions, outside of the DBD, may play a role in transcription factor DNA binding specificity in vivo. In order to test this hypothesis, we examined a model zinc finger transcription factor, Krüppel-like factor 3 (KLF3) with a FD that recruits partner proteins for its repressive activity and a three zinc finger DBD. Previously, we showed that deletion of the entire KLF3 FD reduced DNA binding across the genome. This implied the importance of the FD for in vivo DNA binding specificity. In the current study, we fused the KLF3 FD onto an unrelated, but well characterised Artificial Zinc Finger (AZF) targeting a standard target gene, VEGF-A. We compared the genomic DNA binding profile of this chimeric KLF3 construct, termed KLF3FD-AZF, to that of the AZF alone. Chromatin Immunoprecipitation followed by next generation sequencing was used to assess genomic wide DNA-binding of these AZFs. Remarkably, in these gain-of-function experiments, we again observed that the KLF3 FD is critical for in vivo DNA binding specificity. The addition of KLF3 FD increased the number of binding sites by more than two fold compared to the AZF alone. KLF3 FD also directed more binding to the promoter regions. Further investigations of these acquired sites identified a substantial portion of these sites as the known endogenous KLF3 bound regions, whilst others contain sequences that are similar but not identical to the predicted AZF target sequence. These results clearly demonstrate that the specific localisation of this model transcription factor to target genes is not solely dependent on the DBD and that the regions outside of this domain may also play an important role.

Dataset Information

Dissecting the function of the adult ?-globin downstream promoter region using an artificial zinc finger DNA-binding domain.

Publications

Dissecting the function of the adult β-globin downstream promoter region using an artificial zinc finger DNA-binding domain.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets