Pedro P. Rocha

CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation

Keeping repressed genes repressed Hox genes confer positional identity to cells and tissues. Maintaining precise spatial patterns of Hox gene expression is vital during metazoan development. The transcriptional repressor CTCF is involved in the regulation of chromatin architecture. Narendra et al. show that a CTCF protein binding site insulates regions of active and repressed Hox gene expression from each other. This protects heterochromatin containing repressed Hox genes from the encroaching spread of active chromatin. The CTCF protein appears to organize the active and repressed chromatin regions into distinct architectural domains. Science, this issue p. 1017 A DNA binding protein insulates and protects repressed topological domains in the genome from adjacent active regions. Polycomb and Trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into the repressive domain. CTCF functions as an insulator by organizing Hox clusters into spatially disjoint domains. Ablation of CTCF binding disrupts topological boundaries such that caudal Hox genes leave the repressed domain and become subject to transcriptional activation. Hence, CTCF is required to insulate facultative heterochromatin from impinging euchromatin to produce discrete positional identities.

CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci.

Imaging systems that allow visualization of specific loci and nuclear structures are highly relevant for investigating how organizational changes within the nucleus play a role in regulating gene expression and other cellular processes. Here we present a live imaging system for targeted detection of genomic regions. Our approach involves generating chimaeric transcripts of viral RNAs (MS2 and PP7) and single-guide RNAs (sgRNAs), which when co-expressed with a cleavage-deficient Cas9 can recruit fluorescently tagged viral RNA-binding proteins (MCP and PCP) to specific genomic sites. This allows for rapid, stable, low-background visualization of target loci. We demonstrate the efficiency and flexibility of our method by simultaneously labelling major and minor satellite regions as well as two individual loci on mouse chromosome 12. This system provides a tool for dual-colour labelling, which is important for tracking the dynamics of chromatin interactions and for validating epigenetic processes identified in fixed cells.

4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments

4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or “bait”) that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes.

Breaking TADs: insights into hierarchical genome organization

The 3D organization of chromosomes enables cells to balance the biophysical constraints of the crowded nucleus with the functional dynamics of gene regulation. Physical contacts between genes and their regulatory elements are essential for proper transcriptional control and maintenance of these interactions is critical for preventing aberrations in physiological processes that could manifest as disease states. The first insights into global nuclear organization came from imaging studies using FISH analyses, which demonstrated that chromosomes occupy individual territories in the nucleus with minimal intermingling between them [1]. The development of chromosome conformation capture (3C) in which chromatin fragments in close physical proximity can be detected enabled the characterization of molecular interactions between different loci [2]. When 3C-based techniques incorporated massive parallel sequencing (such as in Hi-C), the description of molecular chromatin interactions at a genome-wide scale was finally possible [3]. Hi-C was the first unbiased approach aimed at capturing all interactions in the nucleus thereby providing a snapshot of nuclear organization at a global scale. The first Hi-C study revealed that each chromosomal territory is further divided into large domains of 5–10 Mb that physically separate two compartments (A and B), which strongly correlate with active and inactive chromatin, respectively [3]. Furthermore, this study demonstrated that interactions between loci in the same compartment occur at a higher frequency than between loci in different com

The origin of recurrent translocations in recombining lymphocytes: a balance between break frequency and nuclear proximity

Translocations occur through the aberrant joining of large stretches of non-contiguous chromosomal regions. The substrates for these illegitimate rearrangements can arise as a result of damage incurred during normal cellular processes, such as transcription and replication, or through the action of genotoxic agents. In lymphocytes many translocations bear signs of having originated from abnormalities introduced during programmed recombination. Although recombination is tightly controlled at different levels, mistakes can occur leading to cytogenetic anomalies that include deletions, insertions, amplifications and translocations, which are an underlying cause of leukemias and lymphomas. In this review we focus on recent studies that provide insight into the origins of translocations that arise during the two lymphocyte specific programmed recombination events: V(D)J and class switch recombination (CSR).

Identification of multi-loci hubs from 4C-seq demonstrates the functional importance of simultaneous interactions.

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3′Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.

Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination

Thomas-Claudepierre et al. report that mediator facilitates the long-range contacts between acceptor switch regions and the IgH locus enhancers during class switch recombination and their transcriptional activation.

Interpreting 4C-Seq data: how far can we go?

The linear sequence of the genome has been extremely valuable in mapping regulatory elements relative to the genes they control. However, it has become increasingly evident that characterizing the three-dimensional organization of the genome is critical to get a better understanding of long-range regulation. Early studies using fluorescent in-situ hybridization (FISH) revealed that individual chromosomes occupy distinct spaces in the nucleus with minimal intermingling between territories[1]. Recent advances using chromosome conformation capture (3C) techniques have confirmed these findings and further improved the depth at which we can determine the organization of chromosomes and the physical interactions that occur within and between them[2, 3]. Variations of the 3C technique include (i) Hi-C, to capture all pairwise interactions, (ii) 5C, to capture interactions within and between loci of interest and (iii) 4C-Seq, to capture all interactions with a single locus of interest. The choice of technique depends on the biological question being asked and the scale at which this needs to be examined. While Hi-C has been instrumental in characterizing higher-order organization of chromosomes in the nucleus, it lacks the resolution that is required for analysis of specific interactions, such as between enhancers and promoters. This can be achieved with 4C-Seq, which allows interrogation of interactions from a single viewpoint or bait, to the rest of the genome. Several studies have used 4C-Seq to better understand phenomena such as X chromosome inactivation[4], enhancer-promoter interactions[5, 6], organization of antigen receptor loci[7], choice of translocation partners[8, 9] and collinear transcriptional regulation[10]. Here we aim to focus on the current state of the 4C-Seq method and the limitations and challenges of the associated computational analysis. Analysis of 4C-Seq data can be complicated by several technical biases intrinsic to the method. The first bias to consider is that the majority of 4C-seq signal is found on the bait chromosome with lower coverage in trans. This bias does not represent noise but is in agreement with the chromosome territory model, which would predict fewer inter-chromosomal interactions than intra-chromosomal interactions. Second, there is a decrease in signal along the cis chromosome as a function of distance from the bait. Third, similar to other 3C-based techniques, 4C-Seq relies on using restriction enzymes to digest the chromatin, and the frequency of sites in the genome which the enzyme recognizes determines the resolution of the assay. Finally, bias arises from the inverse-PCR amplification step that is required for identification of interacting regions. This can lead to an artificial overrepresentation of regions that amplify with greater efficiency. Thus, when developing methods for analyses of 4C-Seq data it is important to take these issues into account. Current methods provide tools to map 4C-Seq reads, normalize data, identify regions of significant interactions and visualize signal across the genome. These tools have provided a good starting point to characterize chromosomal interactions, however there needs to be improvement in incorporating all of the above inherent biases of 4C. PCR artifacts or identical reads are typically discarded in other genome wide techniques, however in 4C-Seq a distinction cannot be made between repeated amplification of a single captured interaction versus amplification of multiple interactions. To deal with this issue some of the available pipelines transform the data to a binary signal (a score of zero or one) based on the presence or absence of a read at each restriction enzyme fragment[6, 11, 12]. To investigate the extent of the PCR amplification bias a recent study incorporated random barcodes in their experimental strategy and found no selective bias in their usage[13]. This demonstrates that binary transformation removes information that can be obtained from the number of captured interactions at a given restriction enzyme fragment. Hence, it is important to develop methods of analysis that integrate the number of fragments that have interactions with the total number of reads at each of these locations. As mentioned above, the resolution of 4C-Seq is determined by the frequency of restriction enzyme sites in the genome. The resolution achieved from a six bp cutter enzyme can be around 3-4Kb and this can be increased to 200-400 bp using a four bp cutter. Six bp cutters have been used to examine interactions[8, 14] in cis and in trans, while 4bp cutters have only successfully characterized interactions in the region surrounding the bait[5, 6, 10]. The limitations of 4bp cutter derived analysis arise due to low reproducibility of 4C signal between replicates in far-cis and trans. It is thus clear that the choice of restriction enzyme used in the experimental design can directly limit the type of interactions that can be assayed. Due to the polymer nature of chromatin, an interaction between two loci can occur over a region of chromatin in a population of cells. To account for this, a genomic window is typically used to analyze interactions as opposed to dealing with individual fragments.[6, 11, 13, 15]. However, it should be noted that the use of arbitrary window sizes may obscure the boundary of interacting domains that can be detected so it is important to first determine the appropriate size. To accomplish this the first step is establishing the resolution at which interactions are reproducible between replicates. This will vary depending on whether regions are (i) proximal to the bait, (ii) in far-cis, or (iii) in trans (all regions on trans chromosomes can be treated the same way since there is an equal probability of their interacting with the bait). For example, when considering regions that are close to the bait, the window size can be smaller because of increased coverage and reproducibility in this location. Thus, the resolution is highest in regions proximal to the bait. Since coverage and reproducibility depend on linear and spatial separation from the bait, use of the same analytical approach cannot be uniformly applied across the genome. Most 4C based studies look at interactions that occur within ~500kb of the viewpoint because this is where the signal is highest and most reproducible. The combined use of genetics and DNA FISH based approaches have nonetheless revealed that regulatory elements can act over a linear distance of more than 50Mb[16] on the same chromosome and between genes on different chromosomes[17] by being brought into close contact at high frequency in the nucleus. Although DNA FISH is the gold standard when it comes to measuring these types of interactions, it has a low resolution and there is a limit to the number of loci that can be simultaneously analyzed. Furthermore, FISH cannot be used to identify associations in an unbiased manner in the way that 4C-Seq can. But is it possible to reliably identify this type of longer-range interaction by 4C? A number of labs have identified regions with significant interactions in far-cis and trans and ranked the intensity of signal within a sample[8, 12]. In contrast, few studies have quantitatively examined differences in signal of longer-range interactions between different conditions[14]. Because 4C-Seq pipelines used for this purpose have not extensively analyzed reproducibility it is not clear whether it is feasible to reliably perform quantitative analyses of longer-range interactions. Obviously, for analysis of longer-range interactions it is of paramount importance to assess reproducibility as well as to validate associations with DNA FISH using the appropriate controls. Importantly, genetic approaches are additionally required to determine functional relevance. Furthermore, to improve our understanding of the role of chromosomal interactions in regulatory processes it is essential to develop methods that integrate 4C-Seq with other genome-wide data sets. Clearly the field is developing rapidly but it is important that new and improved tools of analysis are developed in tandem.

A Damage-Independent Role for 53BP1 that Impacts Break Order and Igh Architecture during Class Switch Recombination

SUMMARY During class switch recombination (CSR), B cells replace the Igh Cμ or δ exons with another down-stream constant region exon (CH), altering the anti-body isotype. CSR occurs through the introduction of AID-mediated double-strand breaks (DSBs) in switch regions and subsequent ligation of broken ends. Here, we developed an assay to investigate the dynamics of DSB formation in individual cells. We demonstrate that the upstream switch region Sμ is first targeted during recombination and that the mechanism underlying this control relies on 53BP1. Surprisingly, regulation of break order occurs through residual binding of 53BP1 to chromatin before the introduction of damage and independent of its established role in DNA repair. Using chromosome conformation capture, we show that 53BP1 mediates changes in chromatin architecture that affect break order. Finally, our results explain how changes in Igh architecture in the absence of 53BP1 could promote inversional rearrangements that compromise CSR.

Finding the Right Partner in a 3D Genome

The three-dimensional organization of the genome plays a role in controlling legitimate and illegitimate DNA recombination. DNA integrity is frequently compromised as a result of exposure to cytotoxic agents, as well as the normal wear and tear of cellular processes like transcription and replication. DNA double-strand breaks (DSBs) are arguably the most dangerous type of DNA damage as they can lead to chromosomal translocations when their repair joins noncontiguous genomic regions together. Indeed, numerous malignancies have been associated with signature translocations in which an oncogene becomes deregulated through joining with another gene that exerts control over its expression.