Robert-Jan Palstra

Looping and Interaction between Hypersensitive Sites in the Active β-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.

The β-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 β-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 β-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 β-globin genes.

The genome-wide dynamics of the binding of Ldb1 complexes during erythroid differentiation

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.

HERC2 rs12913832 modulates human pigmentation by attenuating chromatin loop formation between a long-range enhancer and the OCA2 promoter

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.

Maintenance of Long-Range DNA Interactions after Inhibition of Ongoing RNA Polymerase II Transcription

A relationship exists between nuclear architecture and gene activity and it has been proposed that the activity of ongoing RNA polymerase II transcription determines genome organization in the mammalian cell nucleus. Recently developed 3C and 4C technology allowed us to test the importance of transcription for nuclear architecture. We demonstrate that upon transcription inhibition binding of RNA polymerase II to gene regulatory elements is severely reduced. However, contacts between regulatory DNA elements and genes in the β-globin locus are unaffected and the locus still interacts with the same genomic regions elsewhere on the chromosome. This is a general phenomenon since the great majority of intra- and interchromosomal interactions with the ubiquitously expressed Rad23a gene are also not affected. Our data demonstrate that without transcription the organization and modification of nucleosomes at active loci and the local binding of specific trans-acting factors is unaltered. We propose that these parameters, more than transcription or RNA polymerase II binding, determine the maintenance of long-range DNA interactions.

Chapter 4 β‐Globin Regulation and Long‐Range Interactions

Transcriptional activation in higher eukaryotes frequently involves the long-range action of a number of regulatory DNA elements. One of the main questions in transcriptional regulation is how cis-regulatory elements communicate with the promoter of a gene over large distances. There has been a lively debate in recent years whether this communication takes place via a noncontact mechanism (linking, tracking) or via a contact mechanism (looping). The demonstration that the major regulatory element of the beta-globin locus, the locus control region (LCR), is in close proximity to the active beta-globin genes validates the contact model for long-range activation. Here, we will review the beta-globin locus as a model system to study long-range activation, briefly describe the different models for long-range activation, and summarize the recent findings that the LCR of the beta-globin locus is in close proximity to the active promoters. Although it is now firmly established that looping takes place within the beta-globin locus (and other loci), it is not clear how these long-range contacts are established and what the precise role is of the LCR. We will argue that the main action of the LCR takes place at the promoter and open reading frame of the gene itself and we will discuss key rate-limiting steps in transcriptional activation and the possible mechanisms by which they are influenced by the LCR.

Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development

The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP‐Sequencing 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.

Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions

Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1–8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.

Three-dimensional organization of gene expression in erythroid cells

The history of globin research is marked by a series of contributions seminal to our understanding of the genome, its function, and its relation to disease. For example, based on studies on hemoglobinopathies, it was understood that gene expression can be under the control of DNA elements that locate away from the genes on the linear chromosome template. Recent technological developments have allowed the demonstration that these regulatory DNA elements communicate with the genes through physical interaction, which loops out the intervening chromatin fiber. Subsequent studies showed that the spatial organization of the beta-globin locus dynamically changes in relation to differences in gene expression. Moreover, it was shown that the beta-globin locus adopts a different position in the nucleus during development and erythroid maturation. Here, we discuss the most recent insight into the three-dimensional organization of gene expression.

β-Globin Active Chromatin Hub Formation in Differentiating Erythroid Cells and in p45 NF-E2 Knock-out Mice

Expression of the β-globin genes proceeds from basal to exceptionally high levels during erythroid differentiation in vivo. High expression is dependent on the locus control region (LCR) and coincides with more frequent LCR-gene contacts. These contacts are established in the context of an active chromatin hub (ACH), a spatial chromatin configuration in which the LCR, together with other regulatory sequences, loops toward the active β-globin-like genes. Here, we used recently established I/11 cells as a model system that faithfully recapitulates the in vivo erythroid differentiation program to study the molecular events that accompany and underlie ACH formation. Upon I/11 cell induction, histone modifications changed, the ACH was formed, and the β-globin-like genes were transcribed at rates similar to those observed in vivo. The establishment of frequent LCR-gene contacts coincided with a more efficient loading of polymerase onto the β-globin promoter. Binding of the transcription factors GATA-1 and EKLF to the locus, although previously shown to be required, was not sufficient for ACH formation. Moreover, we used knock-out mice to show that the erythroid transcription factor p45 NF-E2, which has been implicated in β-globin gene regulation, is dispensable for β-globin ACH formation.