Targeted locus amplification to detect molecular markers in mantle cell and follicular lymphoma.

Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell lymphoma (MCL) and follicular lymphoma (FL). However, about 20% of MCL and 40% of FL patients lack a reliable molecular marker, being thus not eligible for MRD studies. Recently, targeted locus amplification (TLA), a next-generation sequencing (NGS) method based on the physical proximity of DNA sequences for target selection, identified novel gene rearrangements in leukemia. The aim of this study was to test TLA in MCL and FL diagnostic samples lacking a classical, PCR-detectable, t(11; 14) MTC (BCL1/IGH), or t(14; 18) major breakpoint region and minor cluster region (BCL2/IGH) rearrangements. Overall, TLA was performed on 20 MCL bone marrow (BM) or peripheral blood (PB) primary samples and on 20 FL BM, identifying a novel BCL1 or BCL2/IGH breakpoint in 16 MCL and 8 FL patients (80% and 40%, respectively). These new breakpoints (named BCL1-TLA and BCL2-TLA) were validated by ASO primers design and compared as MRD markers to classical IGH rearrangements in eight MCL: overall, MRD results by BCL1-TLA were superimposable (R Pearson = 0.76) to the standardized IGH-based approach. Moreover, MRD by BCL2-TLA reached good sensitivity levels also in FL and was predictive of a primary refractory case. In conclusion, this study offers the proof of principle that TLA is a promising and reliable NGS-based technology for the identification of novel molecular markers, suitable for further MRD analysis in previously not traceable MCL and FL patients.

Genetic Screening Test to Detect Translocations in Acute Leukemias by Use of Targeted Locus Amplification.

BACKGROUND: Over 500 translocations have been identified in acute leukemia. To detect them, most diagnostic laboratories use karyotyping, fluorescent in situ hybridization, and reverse transcription PCR. Targeted locus amplification (TLA), a technique using next-generation sequencing, now allows detection of the translocation partner of a specific gene, regardless of its chromosomal origin. We present a TLA multiplex assay as a potential first-tier screening test for detecting translocations in leukemia diagnostics. METHODS: The panel includes 17 genes involved in many translocations present in acute leukemias. Procedures were optimized by using a training set of cell line dilutions and 17 leukemia patient bone marrow samples and validated by using a test set of cell line dilutions and a further 19 patient bone marrow samples. Per gene, we determined if its region was involved in a translocation and, if so, the translocation partner. To balance sensitivity and specificity, we introduced a gray zone showing indeterminate translocation calls needing confirmation. We benchmarked our method against results from the 3 standard diagnostic tests. RESULTS: In patient samples passing QC, we achieved a concordance with benchmarking tests of 81% in the training set and 100% in the test set, after confirmation of 4 and nullification of 3 gray zone calls (in total). In cell line dilutions, we detected translocations in 10% aberrant cells at several genetic loci. CONCLUSIONS: Multiplex TLA shows promising results as an acute leukemia screening test. It can detect cryptic and other translocations in selected genes. Further optimization may make this assay suitable for diagnostic use.

Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping.

Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.

A common genetic variant within SCN10A modulates cardiac SCN5A expression.

Variants in SCN10A, which encodes a voltage-gated sodium channel, are associated with alterations of cardiac conduction parameters and the cardiac rhythm disorder Brugada syndrome; however, it is unclear how SCN10A variants promote dysfunctional cardiac conduction. Here we showed by high-resolution 4C-seq analysis of the Scn10a-Scn5a locus in murine heart tissue that a cardiac enhancer located in Scn10a, encompassing SCN10A functional variant rs6801957, interacts with the promoter of Scn5a, a sodium channel-encoding gene that is critical for cardiac conduction. We observed that SCN5A transcript levels were several orders of magnitude higher than SCN10A transcript levels in both adult human and mouse heart tissue. Analysis of BAC transgenic mouse strains harboring an engineered deletion of the enhancer within Scn10a revealed that the enhancer was essential for Scn5a expression in cardiac tissue. Furthermore, the common SCN10A variant rs6801957 modulated Scn5a expression in the heart. In humans, the SCN10A variant rs6801957, which correlated with slowed conduction, was associated with reduced SCN5A expression. These observations establish a genomic mechanism for how a common genetic variation at SCN10A influences cardiac physiology and predisposes to arrhythmia.

The pluripotent genome in three dimensions is shaped around pluripotency factors.

It is becoming increasingly clear that the shape of the genome importantly influences transcription regulation. Pluripotent stem cells such as embryonic stem cells were recently shown to organize their chromosomes into topological domains that are largely invariant between cell types. Here we combine chromatin conformation capture technologies with chromatin factor binding data to demonstrate that inactive chromatin is unusually disorganized in pluripotent stem-cell nuclei. We show that gene promoters engage in contacts between topological domains in a largely tissue-independent manner, whereas enhancers have a more tissue-restricted interaction profile. Notably, genomic clusters of pluripotency factor binding sites find each other very efficiently, in a manner that is strictly pluripotent-stem-cell-specific, dependent on the presence of Oct4 and Nanog protein and inducible after artificial recruitment of Nanog to a selected chromosomal site. We conclude that pluripotent stem cells have a unique higher-order genome structure shaped by pluripotency factors. We speculate that this interactome enhances the robustness of the pluripotent state.

Robust 4C-seq data analysis to screen for regulatory DNA interactions.

Regulatory DNA elements can control the expression of distant genes via physical interactions. Here we present a cost-effective methodology and computational analysis pipeline for robust characterization of the physical organization around selected promoters and other functional elements using chromosome conformation capture combined with high-throughput sequencing (4C-seq). Our approach can be multiplexed and routinely integrated with other functional genomics assays to facilitate physical characterization of gene regulation.

3D chromatin conformation correlates with replication timing and is conserved in resting cells.

Although chromatin folding is known to be of functional importance to control the gene expression program, less is known regarding its interplay with DNA replication. Here, using Circular Chromatin Conformation Capture combined with high-throughput sequencing, we identified megabase-sized self-interacting domains in the nucleus of a human lymphoblastoid cell line, as well as in cycling and resting peripheral blood mononuclear cells (PBMC). Strikingly, the boundaries of those domains coincide with early-initiation zones in every cell types. Preferential interactions have been observed between the consecutive early-initiation zones, but also between those separated by several tens of megabases. Thus, the 3D conformation of chromatin is strongly correlated with the replication timing along the whole chromosome. We furthermore provide direct clues that, in addition to the timing value per se, the shape of the timing profile at a given locus defines its set of genomic contacts. As this timing-related scheme of chromatin organization exists in lymphoblastoid cells, resting and cycling PBMC, this indicates that it is maintained several weeks or months after the previous S-phase. Lastly, our work highlights that the major chromatin changes accompanying PBMC entry into cell cycle occur while keeping largely unchanged the long-range chromatin contacts.

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.

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.

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 ß-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 ß-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.

Epigenetic regulation of CIITA expression in human T-cells.

In humans, T-cells accomplish expression of MHC-II molecules through induction of CIITA upon activation. Here we show that CIITA promoter accessibility in T-cells is epigenetically regulated. In unstimulated T-cells, CIITA-PIII chromatin displays relative high levels of repressive histone methylation marks (3Me-K27-H3 and 3Me-K20-H4) and low levels of acetylated histones H3 (Ac-H3) and H4 (Ac-H4). These repressive histone marks are replaced by histone methylation marks associated with transcriptional active genes (3Me-K4-H3) and high levels of Ac-H3 and Ac-H4 in activated T-cells. This is associated with concomitant recruitment of RNA polymerase II. In T-leukemia cells, devoid of CIITA expression, similar repressive histone methylation marks and low levels of acetylated histone H3 correlated with lack of CIITA expression. This in contrast to CIITA expressing T-lymphoma cells, which display high levels of Ac-H3 and 3Me-K4-H3, and relative low levels of the 3Me-K27-H3 and 3Me-K20-H4 marks. Of interest was the observation that the levels of histone acetylation and methylation modifications in histones H3 and H4 were also noted in chromatin of the downstream CIITA-PIV promoter as well as the upstream CIITA-PI and CIITA-PII promoters both in normal T-cells and in malignant T-cells. Together our data show that CIITA chromatin in T-cells expressing CIITA display similar histone acetylation and methylation characteristics associated with an open chromatin structure. The opposite is true for T-cells lacking CIITA expression, which display histone modifications characteristic of condensed chromatin.

The inactive X chromosome adopts a unique three-dimensional conformation that is dependent on Xist RNA.

Three-dimensional topology of DNA in the cell nucleus provides a level of transcription regulation beyond the sequence of the linear DNA. To study the relationship between the transcriptional activity and the spatial environment of a gene, we used allele-specific chromosome conformation capture-on-chip (4C) technology to produce high-resolution topology maps of the active and inactive X chromosomes in female cells. We found that loci on the active X form multiple long-range interactions, with spatial segregation of active and inactive chromatin. On the inactive X, silenced loci lack preferred interactions, suggesting a unique random organization inside the inactive territory. However, escapees, among which is Xist, are engaged in long-range contacts with each other, enabling identification of novel escapees. Deletion of Xist results in partial refolding of the inactive X into a conformation resembling the active X without affecting gene silencing or DNA methylation. Our data point to a role for Xist RNA in shaping the conformation of the inactive X chromosome at least partially independent of transcription.

Integrated transcript and genome analyses reveal NKX2-1 and MEF2C as potential oncogenes in T cell acute lymphoblastic leukemia.

To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements.

High-resolution identification of balanced and complex chromosomal rearrangements by 4C technology.

Balanced chromosomal rearrangements can cause disease, but techniques for their rapid and accurate identification are missing. Here we demonstrate that chromatin conformation capture on chip (4C) technology can be used to screen large genomic regions for balanced and complex inversions and translocations at high resolution. The 4C technique can be used to detect breakpoints also in repetitive DNA sequences as it uniquely relies on capturing genomic fragments across the breakpoint. Using 4C, we uncovered LMO3 as a potentially leukemogenic translocation partner of TRB@. We developed multiplex 4C to simultaneously screen for translocation partners of multiple selected loci. We identified unsuspected translocations and complex rearrangements. Furthermore, using 4C we detected translocations even in small subpopulations of cells. This strategy opens avenues for the rapid fine-mapping of cytogenetically identified translocations and inversions, and the efficient screening for balanced rearrangements near candidate loci, even when rearrangements exist only in subpopulations of cells.

Transcription and chromatin organization of a housekeeping gene cluster containing an integrated beta-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 beta-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.

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 beta-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.

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.

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.

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 formation.

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.