Eukaryotic transcription can be regulated over tens or even hundreds of kilobases. We show that such long-range gene regulation in vivo involves spatial interactions between transcriptional elements, with intervening chromatin looping out. The spatial organization of a 200 kb region spanning the murine beta-globin locus was analyzed in expressing erythroid and nonexpressing brain tissue. In brain, the globin cluster adopts a seemingly linear conformation. In erythroid cells the hypersensitive sites of the locus control region (LCR), located 40-60 kb away from the active genes, come in close spatial proximity with these genes. The intervening chromatin with inactive globin genes loops out. Moreover, two distant hypersensitive regions participate in these interactions. We propose that clustering of regulatory elements is key to creating and maintaining active chromatin domains and regulating transcription.
Efficient transcription of genes requires a high local concentration of the relevant trans-acting factors. Nuclear compartmentalization can provide an effective means to locally increase the concentration of rapidly moving trans-acting factors; this may be achieved by spatial clustering of chromatin-associated binding sites for such factors. Here we analyze the structure of an erythroid-specific spatial cluster of cis-regulatory elements and active beta-globin genes, the active chromatin hub (ACH; ref. 6), at different stages of development and in erythroid progenitors. We show, in mice and humans, that a core ACH is developmentally conserved and consists of the hypersensitive sites (HS1-HS6) of the locus control region (LCR), the upstream 5' HS-60/-62 and downstream 3' HS1. Globin genes switch their interaction with this cluster during development, correlating with the switch in their transcriptional activity. In mouse erythroid progenitors that are committed to but do not yet express beta-globin, only the interactions between 5' HS-60/-62, 3' HS1 and hypersensitive sites at the 5' side of the LCR are stably present. After induction of differentiation, these sites cluster with the rest of the LCR and the gene that is activated. We conclude that during erythroid differentiation, cis-regulatory DNA elements create a developmentally conserved nuclear compartment dedicated to RNA polymerase II transcription of beta-globin genes.
CTCF (CCCTC-binding factor) binds sites around the mouse -globin locus that spatially cluster in the erythroid cell nucleus. We show that both conditional deletion of CTCF and targeted disruption of a DNA-binding site destabilize these long-range interactions and cause local loss of histone acetylation and gain of histone methylation, apparently without affecting transcription at the locus. Our data demonstrate that CTCF is directly involved in chromatin architecture and regulates local balance between active and repressive chromatin marks. We postulate that throughout the genome, relative position and stability of CTCF-mediated loops determine their effect on enhancer-promoter interactions, with gene insulation as one possible outcome. Chromatin insulators are DNA sequences that confer autonomous expression on genes by protecting them against inadvertent signals coming from neighboring chromatin. CTCF (CCCTC-binding factor) is the prototype vertebrate protein exhibiting insulator activity that can act as an enhancer blocker or as a barrier against repressive forces from nearby heterochromatin in vitro (Defossez and Gilson 2002; RecillasTarga et al. 2002). In vivo, CTCF binds to the imprinting control region of the H19/insulin-like growth factor (Igf2) locus, where it acts as a methylation-sensitive enhancer blocker (Bell and Felsenfeld 2000;Hark et al. 2000). Moreover, CTCF-binding sites have been foundand its insulator activity has been anticipated-at the imprinting center that determines choice of X inactivation (Chao et al. 2002), at boundaries of domains that escape X inactivation (Filippova et al. 2005), and at sites flanking CTG/CAG repeats at the DM1 locus (Filippova et al. 2001). CTCF was first defined as an insulator protein when it was found to be required for the enhancerblocking activity of a hypersensitive site 5Ј of the chicken -globin locus (5ЈHS4) (Bell et al. 1999). A similar CTCF-dependent insulator site was subsequently found at the 3Ј end of the locus and both sites coincide with erythroid-specific transitions in DNase I sensitivity of chromatin (Saitoh et al. 2000). Such observations suggested that CTCF partitions the genome in physically distinct domains of gene expression. The molecular mechanism underlying CTCF's insulating activity is still unknown.CTCF-binding sites also flank the human and mouse -globin locus (Fig. 1A), which contains a number of developmentally regulated, erythroid-specific -globin genes and an upstream locus control region (LCR) required for high -globin expression levels. In mice, three CTCF-binding sites have been identified upstream (HS-85, HS-62, and HS5) and one downstream (3ЈHS1) of the locus (Farrell et al. 2002;Bulger et al. 2003). Previously, we applied chromosome conformation capture (3C) technology (Dekker et al. 2002) to study long-range DNA interactions between these and other sites in the -globin locus. In erythroid cells, the CTCF-binding sites (including HS-85; see below) were found to participate in spatial interactions between the LCR and th...
One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex-in particular, the binding of the negative regulators Eto2 and Mtgr1-are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences.[Keywords: ChIP sequencing; transcription factor complexes; development; differentiation; erythropoiesis; long-range interactions] Supplemental material is available at http://www.genesdev.org.
Three-dimensional organization of a gene locus is important for its regulation, as recently demonstrated for the -globin locus. When actively expressed, the cis-regulatory elements of the -globin locus are in proximity in the nuclear space, forming a compartment termed the Active Chromatin Hub (ACH). However, it is unknown which proteins are involved in ACH formation. Here, we show that EKLF, an erythroid transcription factor required for adult -globin gene transcription, is also required for ACH formation. We conclude that transcription factors can play an essential role in the three-dimensional organization of gene loci. The mouse -globin locus contains multiple -like globin genes, arranged from 5Ј to 3Ј in order of their developmental expression (Fig. 1A). The adult-type  maj -gene is transcribed at a very low level during primitive erythropoiesis in the embryonic yolk sac, but becomes expressed at high levels around day 11 of gestation (E11) when definitive erythropoiesis commences in the fetal liver (Trimborn et al. 1999). The -globin locus control region (LCR) is essential for efficient globin transcription (Grosveld et al. 1987;Bender et al. 2000). It consists of a series of DNaseI hypersensitive sites (HS) located ∼50 kb upstream of the  maj promoter (Fig. 1A). We have shown that the -globin locus forms an Active Chromatin Hub (ACH) in erythroid cells (Tolhuis et al. 2002). The ACH is a nuclear compartment dedicated to RNA polymerase II transcription, formed by the cis-regulatory elements of the -globin locus with the intervening DNA looping out. The ACH consists of the HS of the LCR, two HS located ∼60 kb upstream of the embryonic y-globin gene (5ЈHS-62/-60) and 3ЈHS1 downstream of the genes. In addition, the actively expressed globin genes are part of the ACH (Carter et al. 2002;Tolhuis et al. 2002). In erythroid precursors that do not express the globin genes yet, a substructure of the ACH, called a chromatin hub (CH) (Patrinos et al. 2004) is found, which excludes the genes and the HS at the 3Ј site of the LCR (Palstra et al. 2003).Expression of the  maj -gene requires the presence of the erythroid Krüppel-like transcription factor EKLF, the erythroid-specific member of the Sp/XKLF-family (Miller and Bieker 1993). EKLF −/− mice die of anemia around E14, because of a deficit in -globin expression (Nuez et al. 1995;Perkins et al. 1995). The -globin locus contains a number of EKLF-binding sites, in particular in the LCR and the  maj -globin promoter (Perkins 1999;Bieker 2001). Because  maj -globin expression depends on the presence of EKLF, we were interested in determining whether EKLF is involved in the formation of the ACH. Results and DiscussionWe used chromatin conformation capture (3C) technology (Dekker et al. 2002) to investigate the three-dimensional conformation of the mouse -globin locus in the absence of EKLF. Cells from E12.5 EKLF −/− and wild-type fetal livers were cross-linked with formaldehyde, followed by restriction enzyme digestion of the DNA. The samples were ligated ...
Pigmentation of skin, eye, and hair reflects some of the most evident common phenotypes in humans. Several candidate genes for human pigmentation are identified. The SNP rs12913832 has strong statistical association with human pigmentation. It is located within an intron of the nonpigment gene HERC2, 21 kb upstream of the pigment gene OCA2, and the region surrounding rs12913832 is highly conserved among animal species. However, the exact functional role of HERC2 rs12913832 in human pigmentation is unknown. Here we demonstrate that the HERC2 rs12913832 region functions as an enhancer regulating OCA2 transcription. In darkly pigmented human melanocytes carrying the rs12913832 T-allele, we detected binding of the transcription factors HLTF, LEF1, and MITF to the HERC2 rs12913832 enhancer, and a long-range chromatin loop between this enhancer and the OCA2 promoter that leads to elevated OCA2 expression. In contrast, in lightly pigmented melanocytes carrying the rs12913832 C-allele, chromatin-loop formation, transcription factor recruitment, and OCA2 expression are all reduced. Hence, we demonstrate that allelic variation of a common noncoding SNP located in a distal regulatory element not only disrupts the regulatory potential of this element but also affects its interaction with the relevant promoter. We provide the key mechanistic insight that allele-dependent differences in chromatin-loop formation (i.e., structural differences in the folding of gene loci) result in differences in allelic gene expression that affects common phenotypic traits. This concept is highly relevant for future studies aiming to unveil the functional basis of genetically determined phenotypes, including diseases.
The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIPSequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping.
In the International Visible Trait Genetics (VisiGen) Consortium, we investigated the genetics of human skin color by combining a series of genome-wide association studies (GWAS) in a total of 17,262 Europeans with functional follow-up of discovered loci. Our GWAS provide the first genome-wide significant evidence for chromosome 20q11.22 harboring the ASIP gene being explicitly associated with skin color in Europeans. In addition, genomic loci at 5p13.2 (SLC45A2), 6p25.3 (IRF4), 15q13.1 (HERC2/OCA2), and 16q24.3 (MC1R) were confirmed to be involved in skin coloration in Europeans. In follow-up gene expression and regulation studies of 22 genes in 20q11.22, we highlighted two novel genes EIF2S2 and GSS, serving as competing functional candidates in this region and providing future research lines. A genetically inferred skin color score obtained from the 9 top-associated SNPs from 9 genes in 940 worldwide samples (HGDP-CEPH) showed a clear gradual pattern in Western Eurasians similar to the distribution of physical skin color, suggesting the used 9 SNPs as suitable markers for DNA prediction of skin color in Europeans and neighboring populations, relevant in future forensic and anthropological investigations.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-015-1559-0) contains supplementary material, which is available to authorized users.
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