Transcription and Chromatin Organization of a Housekeeping Gene Cluster Containing an Integrated β-Globin Locus Control Region

The activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with transcription factories, and long-range interactions via chromatin looping. To investigate the relative importance of these events in the regulation of gene expression, we targeted the human β-globin LCR in two opposite orientations to a gene-dense region in the mouse genome containing mostly housekeeping genes. We found that each oppositely oriented LCR influenced gene expression on both sides of the integration site and over a maximum distance of 150 kilobases. A subset of genes was transcriptionally enhanced, some of which in an LCR orientation-dependent manner. The locus resides mostly at the edge of its CT and integration of the LCR in either orientation caused a more frequent positioning of the locus away from its CT. Locus association with transcription factories increased moderately, both for loci at the edge and outside of the CT. These results show that nuclear repositioning is not sufficient to increase transcription of any given gene in this region. We identified long-range interactions between the LCR and two upregulated genes and propose that LCR-gene contacts via chromatin looping determine which genes are transcriptionally enhanced

Determining long-range chromatin interactions for selected genomic sites using 4C-seq technology: From fixation to computation

Chromosome Conformation Capture (3C) and 3C-based technologies are constantly evolving in order to probe nuclear organization with higher depth and resolution. One such method is 4C-technology that allows the investigation of the nuclear environment of a locus of choice. The use of Illumina next generation sequencing as a detection platform for the analysis of 4C data has further improved the sensitivity and resolution of this method. Here we provide a step-by-step protocol for 4C-seq, describing the procedure from the initial template preparation until the final data analysis, interchanged with background information and considerations

Beta-globin active chromatin Hub formation in differentiating erythroid cells and in p45 NF-E2 knock-out mice

Expression of the beta-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 beta-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 beta-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 beta-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 beta-globin gene regulation, is dispensable for beta-globin ACH formatio

Quantitative analysis of chromosome conformation capture assays (3C-qPCR)

Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7-9 days

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

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

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

4C technology: Protocols and data analysis

Chromosome conformation capture (3C) technology and its genome-wide derivatives have revolutionized our knowledge on chromatin folding and nuclear organization. 4C-seq Technology combines 3C principles with high-throughput sequencing (4C-seq) to enable for unbiased genome-wide screens for DNA contacts made by single genomic sites of interest. Here, we discuss in detail the design, application, and data analysis of 4C-seq experiments. Based on many hundreds of different 4C-seq experiments, we define criteria to assess data quality and show how different restriction enzymes and cross-linking conditions affect results. We describe in detail the mapping strategy of 4C-seq reads and show advanced strategies for data analysis

Variegated gene expression caused by cell-specific long-range DNA interactions

Mammalian genomes contain numerous regulatory DNA sites with unknown target genes. We used mice with an extra β-globin locus control region (LCR) to investigate how a regulator searches the genome for target genes. We find that the LCR samples a restricted nuclear subvolume, wherein it preferentially contacts genes controlled by shared transcription factors. No contacted gene is detectably upregulated except for endogenous β-globin genes located on another chromosome. This demonstrates genetically that mammalian trans activation is possible, but suggests that it will be rare. Trans activation occurs not pan-cellularly, but in 'jackpot' cells enriched for the interchromosomal interaction. Therefore, cell-specific long-range DNA contacts can cause variegated expression