A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.     

Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation

In complex genomes, insulators set up chromatin domain boundaries and protect promoters from inappropriate activation by enhancers from neighboring genes. The Drosophila Abdominal-B locus uses insulator elements to organize its large regulatory region into several body segment-specific chromatin domains. This organization leads to a problem in enhancer-promoter communication, that is, how do distal enhancers activate the Abd-B promoter when there are several insulators in between? This issue is partially resolved by the Promoter Targeting Sequence, which can overcome the enhancer blocking effect of an insulator. In this study, we describe a new Promoter Targeting Sequence, PTS-6, from the Abd-B 3' regulatory region. PTS-6, comprised of approximately 200 bp, was found to bypass both homologous Abdominal-B insulators, such as Fab-7 and Fab-8, and a heterologous insulator, suHw. Most importantly, it also overcomes a combination of two insulators such as Fab-7/Fab-8. Thus, PTS-6 could, in principle, target remote enhancers that are separated from the Abd-B promoter by multiple insulators. In addition, PTS-6 selectively targets the distal enhancer to only one transgenic promoter, and it strongly facilitates Abd-B enhancers. These results suggest that promoter targeting is necessary for long-range enhancer-promoter communication in Abd-B, and PTS elements could be a common occurrence in large, complex genetic loci.     

Effect of TNFα on activities of different promoters of human apolipoprotein A-I gene

Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1β and TNFα. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNFα-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNFα on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNFα leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5′-regulatory region (apoA-I promoter switching) in the cells treated by TNFα. The MEK1/2-ERK1/2 cascade and nuclear receptors PPARα and LXRs are important for TNFα-mediated apoA-I promoter switching.     

Genomic context modulates insulator activity through promoter competition.

Chromatin insulators regulate gene expression by preventing inappropriate enhancer-promoter interactions. Our previous study showed that insulators do not merely function as rigid blockers, rather their activities are quantitative and selective. We have investigated the factors and mechanisms that determine the effectiveness of the suHw insulator in transgenic Drosophila. We show that the suHw-mediated blockage of the AE1 enhancer from a downstream promoter depends on the ability of the promoter to compete for AE1. Promoters that are highly competitive for the enhancer are blocked less effectively. Moreover, blockage of AE1 from its cognate ftz promoter can range from virtually complete to non-detectable, depending on the property of the neighboring upstream promoter. A highly competitive neighboring promoter enhances the suHw-mediated blockage, whereas a less competitive promoter reduces the insulator effectiveness. The influence on insulator effectiveness by both the interacting and the neighboring competing promoters correlates with their ability to compete for the enhancer, which was previously shown to depend on core promoter sequences. Our findings suggest a mechanism at the level of gene organization that modulates insulator effectiveness through promoter competition. The dependence of insulator function on its cis contexts may provide it with more regulatory flexibility while imposing organizational restraints on eukaryotic gene complexes.     

Quantitative assessment of higher-order chromatin structure of the INK4/ARF locus in human senescent cells

Somatic cells can be reset to oncogene-induced senescent (OIS) cells or induced pluripotent stem (iPS) cells by expressing specified factors. The INK4/ARF locus encodes p15(INK4b) , ARF, and p16(INK4a) genes in human chromosome 9p21, the products of which are known as common key reprogramming regulators. Compared with growing fibroblasts, the CCCTC-binding factor CTCF is remarkably up-regulated in iPS cells with silencing of the three genes in the locus and is reversely down-regulated in OIS cells with high expression of p15(INK4b) and p16(INK4a) genes. There are at least three CTCF-enriched sites in the INK4/ARF locus, which possess chromatin loop-forming activities. These CTCF-enriched sites and the p16(INK4a) promoter associate to form compact chromatin loops in growing fibroblasts, while CTCF depletion disrupts the loop structure. Interestingly, the loose chromatin structure is found in OIS cells. In addition, the INK4/ARF locus has an intermediate type of chromatin compaction in iPS cells. These results suggest that senescent cells have distinct higher-order chromatin signature in the INK4/ARF locus.     

Integration of embryonic and fetal skeletal myogenic programs at the myosin light chain 1f/3f locus

The genetic control of skeletal muscle differentiation at the onset of myogenesis in the embryo is relatively well understood compared to the formation of muscle during the fetal period giving rise to the bulk of skeletal muscle fibers at birth. The Mlc1f/3f (Myl1) locus encodes two alkali myosin light chains, Mlc1f and Mlc3f, from two promoters that are differentially regulated during development. The Mlc1f promoter is active in embryonic, fetal and adult fast skeletal muscle whereas the Mlc3f promoter is upregulated during fetal development and remains on in adult fast skeletal muscle. Two enhancer elements have been identified at the mammalian Mlc1f/3f locus, a 3′ element active at all developmental stages and an intronic enhancer activated during fetal development. Here, using transgenesis, we demonstrate that these enhancers act combinatorially to confer the spatial, temporal and quantitative expression profile of the endogenous Mlc3f promoter. Using double reporter transgenes we demonstrate that each enhancer can activate both Mlc1f and Mlc3f promoters in vivo, revealing enhancer sharing rather than exclusive enhancer-promoter interactions. Finally, we demonstrate that the fetal activated enhancer contains critical E-box myogenic regulatory factor binding sites and that enhancer activation is impaired in vivo in the absence of myogenin but not in the absence of innervation. Together our observations provide insights into the regulation of fetal myogenesis and the mechanisms by which temporally distinct genetic programs are integrated at a single locus.