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  • br Results br Discussion AP a heterodimeric

    2023-09-15


    Results
    Discussion AP-1, a heterodimeric transcription factor comprising various combinations of FOS, JUN, MAF, ATF, and CREB family proteins, has been known to play a pivotal role in leukocyte development for decades (Liebermann et al., 1998; Valledor et al., 1998). However, its participation in gene regulation via DNA looping during macrophage development has not been previously described. Nevertheless, locus- and gene-specific examples of AP-1-bound DNA loops have been reported (Chavanas et al., 2008; Qiao et al., 2015), supporting a role for AP-1 family proteins in three-dimensional regulation of target genes and the broader, genome-wide participation of AP-1 characterized in the present study. Given its role across diverse cellular differentiation pathways (Eferl and Wagner, 2003; Shaulian and Karin, 2002), we speculate that the composition of the AP-1 transcription factor complex may contribute to re-wiring of chromatin interactions in a cell-type- and tissue-specific manner. However, given the extraordinary number of potential transcription factor combinations that may co-bind at AP-1 consensus motifs (Mechta-Grigoriou et al., 2001), which cannot be determined directly by footprinting methods, future studies aimed at comprehensively mapping this combinatorial landscape would shed significant insight into the precise proteins underlying AP-1-related looping events. The upregulation of macrophage-related genes through both pre-existing DNA loops and through dynamic long-range interactions agrees with previous gene-specific examples of loop-dependent gene regulation within distinct developmental contexts. At the beta-globin locus, one of the best-studied examples of long-range gene regulation (Kim and Dean, 2012), novel loop formation between locus control elements during blood cell development is required and sufficient for appropriate gene activation (Deng et al., 2012, 2014). In contrast, stimulation of IMR90 Desformylflustrabromine hydrochloride mg with tumor necrosis factor alpha (TNF-α) activates enhancers at the promoter-distal anchors of preexisting loops but does not induce large-scale changes to 3D chromatin architecture (Jin et al., 2013). The identification of both static and dynamic loop-based mechanisms in various biological contexts suggests that both phenomena represent important paradigms for dynamic gene regulation. Our data reveal that these two types of regulatory looping mechanisms co-occur at specific loci, forming multi-loop activation hubs at key macrophage regulatory genes. The hubs often connect multiple distal enhancers to a single gene promoter and are associated with strong upregulation of gene transcription. AP-1-bound activation hubs are reminiscent of dynamic chromatin structures found at the beta-globin locus in which multiple distal sites loop to the active beta globin genes during specific stages of erythroid cell development (Tolhuis et al., 2002). Analogous to AP-1 interactions targeting macrophage-specific genes, the beta-globin locus is organized into an “active chromatin hub” that requires regulatory transcription factors such as ELKF, GATA1, LDB1, and FOG1 (Drissen et al., 2004; Song et al., 2007; Vakoc et al., 2005). Indeed, we believe that the multi-loop communities identified herein may have far-reaching implications for how chromosome organization instructs transcription in other cellular contexts and throughout human development. Finally, because Hi-C and other chromatin profiling assays query DNA loops and DNA-protein interactions across cell populations, it is impossible to determine from these datasets whether all loops in a hub exist at the same time or whether we are observing multiple subpopulations of cells with exclusive subsets of DNA looping events. Single-cell Hi-C, while useful for comparison against aggregate cell populations, also struggles to discriminate between these two possibilities, as only a single anchor-to-anchor ligation event is generated per allele by chromosome conformation capture. The prevalence of enhancer-enhancer contacts leads us to speculate that these loops often do form in the same cells. By doing so, activation hubs could increase the local concentrations of enhancers and distally bound transcription factors at gene promoters, contributing to increased transcription. Further development of computational methods and experimental methods to identify multi-loop communities, such as concatemer ligation assay (COLA), should help address some of these pressing questions about chromatin structure and gene regulation (Darrow et al., 2016).