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Structural and Genome-wide Analysis Support a Role for SETMAR in Transcriptional Regulation

ABSTRACT: Approximately 50 million years ago, the Hsmar1 transposon entered the primate lineage giving rise to a new protein, a chimeric fusion of a SET domain and the Hsmar1 transposase. This protein, SETMAR or Metnase, is broadly expressed in human tissues and has been shown to retain its ancestral sequence-specific binding to Hsmar1 terminal inverted repeat (TIR) sequences found at the ends of the transposons. Despite the fact that there were estimated to be anywhere from 1500-7000 TIR sites within the human genome, the relevance of SETMAR-TIR interactions was unknown. Here, we report the crystal structure of the SETMAR DNA-binding domain (DBD) complexed with TIR DNA at 2.37 Å. The DBD structure includes two helix-turn-helix motifs (HTH1 and HTH2), which dimerize through HTH1, and confer sequence-specific recognition of the TIR through nucleobase-specific interactions with R371 in HTH1 and R417, H427, S428, and R432 in HTH2. The extent of genome-wide binding was determined by chromatin immunoprecipitation sequencing (ChIP-seq) analysis yielding a total of 7457 SETMAR bound sites. The effect of SETMAR on the transcriptome was assessed by RNA-seq analysis; among the 177 differentially regulated transcripts, a cluster of histones on chromosome 6 were found to be repressed. The dimeric SETMAR structure with each DBD bound to TIR DNA, the presence of eleven TIR sites within the histone gene cluster, and previously reported DNA looping activity are consistent with a direct regulatory mechanism in which SETMAR represses mRNA expression for specific genes through chromatin looping.

ORGANISM(S): Homo sapiens

PROVIDER: GSE103017 | GEO | 2022/02/20

REPOSITORIES: GEO

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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.

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Structural and Genome-wide Analysis Support a Role for SETMAR in Transcriptional Regulation

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