Unraveling the Hierarchy of cis and trans Factors That Determine the DNA Binding by Peroxisome Proliferator-Activated Receptor gamma
MOLECULAR AND CELLULAR BIOLOGY
2020; 40 (7)
Dynamic transcriptional control of macrophage miRNA signature via inflammation responsive enhancers revealed using a combination of next generation sequencing-based approaches
BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS
2018; 1861 (1): 14?28
Peroxisome proliferator-activated receptor ? (PPAR?) is a nuclear receptor essential for adipocyte development and the maintenance of the alternatively polarized macrophage phenotype. Biochemical studies have established that as an obligate heterodimer with retinoid X receptor (RXR), PPAR? binds directly repeated nuclear receptor half sites spaced by one nucleotide (direct repeat 1 [DR1]). However, it has not been analyzed systematically and genome-wide how cis factors such as the sequences of DR1s and adjacent sequences and trans factors such as cobinding lineage-determining transcription factors (LDTFs) contribute to the direct binding of PPAR? in different cellular contexts. We developed a novel motif optimization approach using sequence composition and chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) densities from macrophages and adipocytes to complement de novo motif enrichment analysis and to define and classify high-affinity binding sites. We found that approximately half of the PPAR? cistrome represents direct DNA binding; both half sites can be extended upstream, and these are typically not of equal strength within a DR1. Strategically positioned LDTFs have greater impact on PPAR? binding than the quality of DR1, and the presence of the extension of DR1 provides a remarkable synergy with LDTFs. This approach of considering not only nucleotide frequencies but also their contribution to protein binding in a cellular context is applicable to other transcription factors.
View details for DOI 10.1128/MCB.00547-19
View details for Web of Science ID 000520043100004
View details for PubMedID 31932484
View details for PubMedCentralID PMC7076253
MicroRNAs are important components of the post-transcriptional fine-tuning of macrophage gene expression in physiological and pathological conditions. However, the mechanistic underpinnings and the cis-acting genomic factors of how macrophage polarizing signals induce miRNA expression changes are not well characterized. Therefore, we systematically evaluated the transcriptional basis underlying the inflammation-mediated regulation of macrophage microRNome using the combination of different next generation sequencing datasets. We investigated the LPS-induced expression changes at mature miRNA and pri-miRNA levels in mouse macrophages utilizing a small RNA-seq method and publicly available GRO-seq dataset, respectively. Next, we identified an enhancer set associated with LPS-responsive pri-miRNAs based on publicly available H3K4 mono-methylation-specific ChIP-seq and GRO-seq datasets. This enhancer set was further characterized by the combination of publicly available ChIP and ATAC-seq datasets. Finally, direct interactions between the miR-155-coding genomic region and its distal regulatory elements were identified using a 3C-seq approach. Our analysis revealed 15 robustly LPS-regulated miRNAs at the transcriptional level. In addition, we found that these miRNA genes are associated with an inflammation-responsive enhancer network. Based on NF?B-p65 and JunB transcription factor binding, we showed two distinct enhancer subsets associated with LPS-activated miRNAs that possess distinct epigenetic characteristics and LPS-responsiveness. Finally, our 3C-seq analysis revealed the LPS-induced extensive reorganization of the pri-miR-155-associated functional chromatin domain as well as chromatin loop formation between LPS-responsive enhancers and the promoter region. Our genomic approach successfully combines various genome-wide datasets and allows the identification of the putative regulatory elements controlling miRNA expression in classically activated macrophages.
View details for DOI 10.1016/j.bbagrm.2017.11.003
View details for Web of Science ID 000423645700002
View details for PubMedID 29133016