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.
Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N–bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.
Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila melanogaster Kc cells. We found that Pc associates with large genomic regions of up to approximately 150 kb in size, hereafter referred to as 'Pc domains'. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 Lys27 and is generally transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains.
Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. Here, we adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. Our results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and we demonstrate that a topological rather than a sequence-based mechanism is responsible for this constraint. Our results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture.
Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD, MIM 242900), an incompletely penetrant autosomal recessive disorder. Using human, Drosophila and mouse models, we show that the proteins encoded by SMARCAL1 orthologs localize to transcriptionally active chromatin and modulate gene expression. We also show that, as found in SIOD patients, deficiency of the SMARCAL1 orthologs alone is insufficient to cause disease in fruit flies and mice, although such deficiency causes modest diffuse alterations in gene expression. Rather, disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD.
Postzygotic mutations are DNA changes acquired from the zygote stage onwards throughout the lifespan. These changes lead to differences in DNA sequence among cells of an individual, potentially contributing to the etiology of complex disorders. Here we compared whole genome DNA sequence data of two monozygotic twin pairs, 40 and 100 years old, to detect somatic mosaicism. DNA samples were sequenced twice on two Illumina platforms (13X and 40X read depth) for increased specificity. Using differences in allelic ratios resulted in sets of 1,720 and 1,739 putative postzygotic mutations in the 40‐year‐old twin pair and 100‐year‐old twin pair, respectively, for subsequent enrichment analysis. This set of putative mutations was strongly (p < 4.37e–91) enriched in both twin pairs for regulatory elements. The corresponding genes were significantly enriched for genes that are alternatively spliced, and for genes involved in GTPase activity. This research shows that somatic mosaicism can be detected in monozygotic twin pairs by using allelic ratios calculated from DNA sequence data and that the mutations which are found by this approach are not randomly distributed throughout the genome.
Maintenance of cell identity and cell fate depends on the tight regulation of gene expression patterns in correct time and space. Two families of proteins, the trithorax group (trxG) and the Polycomb group (PcG), use epigenetic mechanisms to faithfully ensure that designated genes are maintained on or off throughout the life of the organism. This maintenance function is imperative to allow the proper development of an organism from a single cell to an organized combination of multifunctional cells. Here, we briefly review the advances achieved in recent years aimed at understanding how members of PcG and trxG function (for more in-depth reviews, see Otte and Kwaks 2003;Pirrotta et al. 2003;Lund and van Lohuizen 2004;Valk-Lingbeek et al. 2004). Particularly, we will discuss methods that can be employed to uncover additional target genes regulated by PcG and/or trxG families. Additionally, we will focus on recent results linking PcG regulation with X-inactivation and with stem cell biology.
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