Can the complex transcriptional response of monocytic cells to 1α,25-dihydroxyvitamin D3 treatment be elucidated by predicting gene targets of transcription factors downstream of the vitamin D receptor, and are downstream factors important to the modulation of cardiovascular disease risk by vitamin D?

1α,25-dihydroxyvitamin D3 (1,25D) deficiency has been associated with adverse cardiovascular outcomes in patients, and as such has been researched heavily in the context of cardiovascular disease, including atherosclerosis. Monocytes are heavily involved in atheroma progression, and treatment of monocytic cells with 1,25D provokes a widespread and extended transcriptomic response, which could subsequently modulate the development of cardiovascular disease. 1,25D mediates its transcriptomic effects via its corresponding nuclear receptor, vitamin D receptor (VDR). It is widely accepted that VDR cannot mediate all of these changes alone, and a network of transcription factors downstream of VDR must also act to alter gene expression of 1,25D-target genes. However, it is unclear which transcription factors are involved, what the specific gene targets of these transcription factors are, and whether they are important to cardiovascular disease.

The TEPIC workflow was used to leverage RNA sequencing, FAIRE sequencing, and VDR ChIP-sequencing data to predict transcription factor binding at differentially expressed genes following 1,25D treatment. Transcription factors predicted to be bound by VDR were subsequently assigned to differentially expressed genes, and a transcription factor network centred on VDR was created. ChIP sequencing data for a subset of these identified transcription factors was used to validate transcription factor binding predictions, along with RNA-sequencing of 1,25D-treated THP-1 cells after siRNA-mediated transcription factor knockdown. Genomic knockout of VDR using CRISPR-Cas9 in THP-1 cells was followed by RNA sequencing to confirm VDR-dependency of the entire 1,25D response.

Direct VDR target genes were identified in THP-1 cells at both early (2.5h, 4h) and late (24h) time points after 1,25D treatment. Among these were 47 transcription factors whose expression was significantly upregulated following 1,25D treatment. These transcription factors were subset into early and late targets of VDR, and the gene targets for each transcription factor were identified by transcription factor binding site prediction. A transcription factor network was constructed and the gene targets of CEBPA and ETS1 after 1,25D treatment were validated using ChIP sequencing data. The dependence of the 1,25D response on CEBPA was also shown by siRNA-mediated CEBPA knockdown followed by RNA sequencing of THP-1 cells. Pathways related to modulation of inflammation were significantly overrepresented among the gene targets of ETS1, which included many inflammatory cytokines and chemokines, indicating the importance of downstream transcription factors in modulating inflammation in the cardiovascular system. Genomic knockout of VDR completely ablated the transcriptional response to 1,25D in THP-1 cells.

There exists a complex network of transcription factors downstream of, and dependent on, VDR which are required to facilitate the widespread transcriptional response of THP-1 cells to 1,25D treatment. Downstream factors such as ETS1 are important in mediating the anti-inflammatory effects of 1,25D.