The Chimeric Fusion Protein SETMAR Functions as a Chromatin Organizing Factor

Abstract

About 50 million years ago, an Hsmar1 transposon invaded an early primate genome and inserted itself downstream of a SET methyltransferase gene, leading to the birth of a new chimeric protein now called SETMAR. While all other Hsmar1 sequences in the human genome have suffered inactivating mutational damage, the transposase domain of SETMAR has remained remarkably intact, suggesting that it has gained a novel, evolutionarily advantageous function. While SETMAR can no longer transpose itself throughout the genome, it has retained its ancestral sequence-specific DNA binding activity, the importance of which is currently unknown. To investigate this, we performed ChIP-seq to examine SETMAR binding in the human genome. We also utilized RNA-sequencing to assess SETMAR overexpression as well as SETMAR deletion on the human transcriptome. Additionally, we explored SETMAR’s transposase-derived chromatin-looping ability using chromosome-conformation-capture-on-ChIP (4C) in the presence of SETMAR overexpression and performed genome-wide Hi-C to assess the impact of complete SETMAR silencing on global chromatin interactions. ChIP-seq revealed that SETMAR amassed 7,332 unique binding sites, 69% of which included a TIR motif. RNA-sequencing in cells overexpressing SETMAR indicated 177 differentially regulated transcripts, including repression of 17 histone transcripts, suggesting a possible role in chromatin dynamics. RNA-sequencing of parental and SETMAR knockout clones highlighted an average of 5,000 altered transcripts in each cell line, with 343 transcripts significantly differentially expressed in all three knockout clones, many of which participate in embryonic development pathways. 4C analysis in the presence of SETMAR overexpression discovered multiple intrachromosomal looping interactions, and Hi-C analysis of SETMAR knockout cell lines uncovered genome-wide loss of chromatin interactions and disruption of TAD boundaries. The prevalence of SETMAR binding in the human genome combined with its chromatin looping capability and its dramatic effects on the transcriptome suggest a previously undiscovered role for SETMAR as a novel chromatin organizing factor.