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Wouter de Laat

Publications and source records attributed to Wouter de Laat.

8 recordsLinked to original sources
PubMed2006

Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C).

The spatial organization of DNA in the cell nucleus is an emerging key contributor to genomic function. We developed 4C technology (chromosome conformation capture (3C)-on-chip), which allows for an unbiased genome-wide search for DNA loci that contact a given locus in the nuclear space. We demonstrate here that active and inactive genes are engaged in many long-range intrachromosomal interactions and can also form interchromosomal contacts. The active beta-globin locus in fetal liver preferentially contacts transcribed, but not necessarily tissue-specific, loci elsewhere on chromosome 7, whereas the inactive locus in fetal brain contacts different transcriptionally silent loci. A housekeeping gene in a gene-dense region on chromosome 8 forms long-range contacts predominantly with other active gene clusters, both in cis and in trans, and many of these intra- and interchromosomal interactions are conserved between the tissues analyzed. Our data demonstrate that chromosomes fold into areas of active chromatin and areas of inactive chromatin and establish 4C technology as a powerful tool to study nuclear architecture.

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PubMed2006

CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus.

CTCF (CCCTC-binding factor) binds sites around the mouse beta-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.

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PubMed2004

The active spatial organization of the beta-globin locus requires the transcription factor EKLF.

Three-dimensional organization of a gene locus is important for its regulation, as recently demonstrated for the beta-globin locus. When actively expressed, the cis-regulatory elements of the beta-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 beta-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.

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PubMed2004

Multiple interactions between regulatory regions are required to stabilize an active chromatin hub.

The human beta-globin locus control region (LCR) is required for the maintenance of an open chromatin configuration of the locus. It interacts with the genes and the hypersensitive regions flanking the locus to form an active chromatin hub (ACH) transcribing the genes. Proper developmental control of globin genes is largely determined by gene proximal regulatory sequences. Here, we provide the first functional evidence of the role of the most active sites of the LCR and the promoter of the beta-globin gene in the maintenance of the ACH. When the human beta-globin gene promoter is deleted in the context of a full LCR, the ACH is maintained with the beta-globin gene remaining in proximity. Additional deletion of hypersensitive site HS3 or HS2 of the LCR shows that HS3, but not HS2, in combination with the beta-globin promoter is crucial for the maintenance of the ACH at the definitive stage. We conclude that multiple interactions between the LCR and the beta-globin gene are required to maintain the appropriate spatial configuration in vivo.

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PubMed2003

The beta-globin nuclear compartment in development and erythroid differentiation.

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.

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PubMed2003

Spatial organization of gene expression: the active chromatin hub.

Developmental and tissue-specific expression of higher eukaryotic genes involves activation of transcription at the appropriate time and place and keeping it silent otherwise. Unlike housekeeping genes, tissue-specific genes generally do not cluster on the chromosomes. They can be found in gene-dense regions of chromosomes as well as in regions of repressive chromatin. Depending on the location, shielding against positive or negative regulatory effects from neighboring chromatin may be required and hence insulator and boundary models were proposed. They postulate that chromosomes are partitioned into physically distinct expression domains, each containing a gene or gene cluster with its cis-regulatory elements. Specialized elements at the borders of such domains are proposed to prevent cross-talk between domains, and thus to be crucial in establishing independent expression domains. However, genes and associated cis-acting sequences often do not occupy physically distinct domains on the chromosomes. Rather, genes can overlap and cis-acting sequences can be found tens or hundreds of kilobases away from the target gene, sometimes with unrelated genes in between. Therefore the ability of a gene to communicate with positive cis-regulatory elements rather than the presence of specialized boundary elements appears to be key to establishing an independent expression profile. Our recent finding that active beta-globin genes physically interact in the nuclear space with multiple cis-regulatory elements, with inactive genes looping out, has provided a potential mechanistic framework for this model. We refer to such a spatial unit of regulatory DNA elements as an active chromatin hub (ACH). We propose that productive ACH formation underlies correct gene expression, requiring the presence of protein factors with the appropriate affinities for each other bound to their cognate DNA sequences. Proximity and specificity determines which cis-acting sequences and promoter(s) form an ACH, and thus which gene will be expressed. Other regulatory sequences can interfere with transcription by blocking the appropriate physical interaction between an enhancer and promoter in the ACH. Possible mechanisms by which distal DNA elements encounter each other in the 3D nuclear space will be discussed.

Animals↗
PubMed2002

Looping and interaction between hypersensitive sites in the active beta-globin locus.

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.

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