Extensive and functional overlap of the STAT6 and RXR cistromes in the active enhancer repertoire of human CD14+ monocyte derived differentiating macrophages

https://doi.org/10.1016/j.mce.2017.07.034Get rights and content

Highlights

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    Extensive overlap between IL-4-activated STAT6 and lipid sensing RXR cistromes in human differentiating macrophages.

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    RXR/STAT6 co-peaks are associated with IL-4 responsive genes.

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    Combined activation of STAT6 and RXRs lead to the complex crosstalk at gene and enhancer level enhancing IL-4 responsiveness.

Abstract

Macrophages are able to differentiate into classically polarized (M1) or alternatively polarized (M2) states upon encountering pro-inflammatory cytokines such as interferon (IFN) γ or anti-inflammatory cytokines such as interleukin (IL) -4/IL-13, respectively. Moreover, macrophages are known to regulate lipid metabolism via multiple members of the nuclear hormone receptor family, including the retinoid X receptors (RXR). It has been also documented that cytokines are able to modulate macrophage responses to lipid signals but the nature of these interactions and the underlying mechanisms of these processes especially at the level of the chromatinized genome are not well understood. Previous work from our laboratory suggested that STAT6 is a facilitator of nuclear receptor mediated transcriptional activity acting at the genome level. This prompted us to investigate genome-wide DNA binding events and the development of cistromes in human CD14+ monocyte-derived macrophages upon exposure to IL-4. We determined the impact of IL-4 on the PU.1, RXR and STAT6 cistromes within the active enhancer regions marked by H3K27-acetylation using chromatin immunoprecipitation followed by deep sequencing and integrated bioinformatics analyses. We found that about 2/3rd of the IL-4 induced STAT6 peaks co-localized with RXR peaks. These STAT6/RXR co-peaks differed at least in part from the non-overlapping RXR peaks regarding the most enriched de novo transcription factor binding motifs. Interestingly, RXR-binding was not regulated at the STAT6/RXR co-bound enhancers following IL-4 stimulation, but differential enhancer interactions were observed between the IL-4/STAT6 and RXR signaling pathways acting in a gene selective manner. Our results suggest that there is a novel, so far uncharacterized cistromic crosstalk between RXR and STAT6 that is likely to contribute to the formation of the active enhancer repertoire, transcriptome and differential signal-specific gene regulation of polarized macrophages.

Section snippets

Background

Macrophages are immune cells that take part in both the initiation as well as the resolution phase of inflammation, depending on the surrounding cytokine milieu. If macrophages are exposed to inflammatory cytokines such as interferon (IFN) gamma or tumor necrosis factor (TNF) alpha then classically polarized (aka M1) cells form, while anti-inflammatory cytokines such as interleukin (IL) -4 or IL-13 give rise to alternatively polarized (M2) macrophages (Gordon and Taylor, 2005, Lawrence and

Monocyte isolation and differentiation, cell culture and treatments

Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Buffy coats were obtained from the Regional Blood Bank. Monocyte separation was carried out using CD14 MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. Monocytes were cultured and differentiated to macrophages by their attachment to cell culture plate in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, penicillin and streptomicyn for the indicated time (Liu et al.,

Investigation of IL-4-modulated gene expression signature in primary human monocyte-derived differentiating macrophages

In order to investigate the potential interaction between IL-4/STAT6 and RXR signaling in human differentiating macrophages (diffMQ), we induced the differentiation of human peripheral blood-derived CD14+monocytes to macrophages by their attachment to the cell culture plate. First, we validated the experimental cell model as depicted in Fig. 1A by testing IL-4 responsiveness using RNA-seq methodology to confirm that the cells were able to differentiate towards the M2 phenotype (Fig. 1B). We

Conclusions

Taken together, herein we provided and described several novel functional interactions deduced from extensive mapping of both cistromic as well as transcriptomic events induced by IL-4 alone or in combination with LG268 in primary human macrophage-like cells. These include (i) the cistromic maps of PU.1, RXR and STAT6 in primary macrophage like cells, (ii) the cistromic overlap between STAT6 and RXR, (iii) the lack of NR motif within these co-peaks, (iv) the gene and/or enhancer-specific

Competing interests

No competing interests are declared.

Authors’ contributions

ZC and ZSN designed and performed experiments, analyzed the data and wrote the paper, TSCS and AK performed experiments, SS, JF-D, DJ, AH, GN and LS analyzed data, and LN designed experiments, wrote the paper, obtained funding and directed the research.

Accession numbers

Sequencing data have been submitted to GEO database under accession: GSE100889.

Acknowledgements

This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-1-2012-0001 ‘National Excellence Program’. Work in the Nagy laboratory is supported by a grant from the Hungarian Scientific Research Fund (OTKA K100196), and TÁMOP-4.2.2/A-11/1/KONV-2012-0023 implemented through the New Hungary Development Plan co-financed by the European Social Fund and the European Regional Development Fund. Z.S.N. was

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