SUMMARY The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.

SUMMARY Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are formed in regions containing active genes and clusters of architectural protein binding sites. Transcription of most genes is repressed after temperature stress in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. Heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies in the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be quickly reconfigured.

Long-range interactions between distant regulatory elements, such as enhancers, and their target genes underlie the specificity of gene expression in many developmentally regulated gene families. NLI/Ldb1, a widely expressed nuclear factor, is a potential mediator of long-range interactions. Here, we show that NLI/Ldb1 and erythroid-binding partners GATA-1/SCL/LMO2 bind in vivo to the beta-globin locus control region (LCR). The C-terminal LIM interaction domain of NLI is required for formation of the complex on chromatin. Loss of the LIM domain converts NLI into a dominant-negative inhibitor of globin gene expression, and knockdown of NLI by using shRNA results in failure to activate beta-globin expression. Kinetic studies reveal that the NLI/GATA-1/SCL/LMO2 complex is detected at the beta-globin promoter coincident with RNA Pol II recruitment, beta-globin transcription, and chromatin loop formation during erythroid differentiation, providing evidence that NLI facilitates long-range gene activation.

CTCF sites are abundant in the genomes of diverse species but their function is enigmatic. We used chromosome conformation capture to determine long-range interactions among CTCF/cohesin sites over 2 Mb on human chromosome 11 encompassing the β-globin locus and flanking olfactory receptor genes. Although CTCF occupies these sites in both erythroid K562 cells and fibroblast 293T cells, the long-range interaction frequencies among the sites are highly cell type specific, revealing a more densely clustered organization in the absence of globin gene activity. Both CTCF and cohesins are required for the cell-type-specific chromatin conformation. Furthermore, loss of the organizational loops in K562 cells through reduction of CTCF with shRNA results in acquisition of repressive histone marks in the globin locus and reduces globin gene expression whereas silent flanking olfactory receptor genes are unaffected. These results support a genome-wide role for CTCF/cohesin sites through loop formation that both influences transcription and contributes to cell-type-specific chromatin organization and function.chromatin loops | insulator | CTCF | globin | transcription E ukaryotic chromatin is dynamically organized to form distinct transcriptionally active or silent domains (1). Chromatin insulators are proposed to play a role in the establishment of such domains within which proper enhancer-gene interactions occur and improper ones are excluded (2). Insulators can function as boundary elements between active and silent chromatin domains and can also interfere with enhancer-gene interaction when placed between them. Evidence also suggests that insulators are involved in higher-order chromatin organization by clustering together with other insulators. Such "insulator bodies" can be visualized at the nuclear periphery of certain cells in Drosophila and are thought to coalesce through the interaction of proteins such as Su(Hw) and Drosophila CTCF (3, 4).In mammalian cells, insulators are bound by CTCF, a protein with 11 zinc fingers through which it can bind to a range of DNA sequences, to itself, and to nuclear structural proteins such as nucleophosmin and matrix attachment regions (MARs) (2). Although insulators would be expected to reside primarily in intergenic regions where they could provide boundary or enhancer blocking activity, genome scans of CTCF enrichment show that CTCF sites are overrepresented in genes and promoter regions, with only 41-56% at intergenic locations (5). In Drosophila, between about 49% and 64% of insulator sites are located in intergenic regions (4). Thus, although CTCF sites are proposed to be insulators, their in vivo functions may not be limited to insulation. Recently, cohesin has been reported to colocalize at most sites of CTCF enrichment and to play a role in transcriptional insulation, but the significance of this joint occupancy to higher-order chromatin structures is unknown (6).In the Igf2/H19 locus, CTCF binding to the imprinting control region on the maternal allele is required to loop ...