Cell specialization is the defining hallmark of metazoans and results from differentiation of precursor cells. Differentiation is characterized by growth arrest of proliferating cells followed by expression of specific phenotypic traits. This process is essential throughout development and for adult tissue maintenance. For example, improper cellular differentiation in adult tissues can lead to human diseases such as leukemia [1
]. For this reason, identifying mechanisms involved in differentiation is not only essential to understand biology, but also to develop effective strategies for prevention, diagnosis and treatment of cancer. Suzuki et al
. recently defined the underlying transcription network of differentiation in the THP-1 leukemia cell line [3
]. Using several powerful genomics approaches, this study challenges the traditional views that transcriptional activators acting as master regulators mediate differentiation. Instead, differentiation is shown to require the concerted up- and down-regulation of numerous transcription factors. This study provides the first integrated picture of the interplay between transcription factors, proximal promoter activity, and RNA transcripts required for differentiation of human leukemia cells.
Although extremely powerful, several observations indicate that implementation of new technologies will be required to gain a full appreciation of how cells differentiate. First, gene expression is controlled by a complex array of regulatory DNA elements. Each gene may be controlled by multiple elements and each element may control multiple genes [4
]. Second, the functional organization of genes and elements is not linear along chromosomes. For example, a given element may regulate distant genes or genes located on other chromosomes without affecting the ones adjacent to it [4
]. Third, gene regulation is known to involve both local and long-range chromatin structure changes [6
]. Although the role of histone and DNA modifications is increasingly well described, relatively little is known about the function of spatial chromatin organization in the regulation of genes. Interestingly, recent studies show that control DNA elements can mediate long-range cis
regulation by physically interacting with target genes [8
]. These studies indicate that genomes are organized into dynamic three-dimensional networks of physical DNA contacts essential for proper gene expression (Figure ). Therefore, mapping the functional (physical) connectivity of genomes is essential to fully identify the mechanisms involved in differentiation, and might provide important diagnostic and prognostic signatures of human diseases.
Figure 1 Capturing spatial chromatin organization in vivo with 3C/5C technologies. (a) Current model of genome organization in the interphase nucleus. The diagram illustrates multiple levels of chromatin folding from the primary structural unit consisting of genomic (more ...)
Physical contacts between DNA segments can be measured with the 'chromosome conformation capture' (3C) technologies [11
]. The 3C approach (Figure ) uses formaldehyde to covalently link chromatin segments in vivo
. Cross-linked chromatin is then digested with a restriction enzyme and ligated under conditions promoting intermolecular ligation of cross-linked segments. Cross-links are finally reversed by proteinase K digestion and DNA extraction to generate a '3C library'. 3C libraries contain pair-wise ligation products, where the amount of each product is inversely proportional to the original three-dimensional distance separating these regions. These libraries are conventionally analyzed by semi-quantitative PCR amplification of individual 'head-to-head' ligation junctions and agarose gel detection (for details, see [12
]). 3C was first used to show that long-range interactions are essential for gene expression in several important mammalian genomic domains. For example, it was demonstrated that the locus control region of the beta-globin locus specifically interacts with actively transcribed genes but not with silent genes [13
]. These contacts were required for gene expression and mediated by the hematopoietic transcription factors GATA-1 and co-factor FOG-1 [15
3C technology has been widely adopted for small-scale analysis of chromatin organization at high-resolution [17
]. However, this approach is technically tedious and not convenient for large-scale studies. Genome-scale conformation studies can be performed quantitatively using the 3C-carbon copy (5C) technology (Figure ) [16
]. The 5C approach combines 3C with the highly multiplexed ligation-mediated-amplification technique to simultaneously detect up to millions of 3C ligation junctions. During 5C, multiple 5C primers corresponding to predicted 'head-to-head' 3C junctions are first annealed in a multiplex setting to a 3C library. Annealed primers are then ligated onto 3C contacts to generate a '5C library'. Resulting libraries contain 5C products corresponding to 3C junctions where the amount of each product is proportional to their original abundance in 3C libraries. 5C libraries are finally amplified by PCR in a single step with universal primers corresponding to common 5C primer tails. These libraries can be analyzed on custom microarrays or by high-throughput DNA sequencing [16
]. Although 5C technology is an ideal discovery tool and particularly well suited to map functional interaction networks, this approach is not yet widely adopted partly due to the lack of available resources.
In this study, we used the THP-1 leukemia differentiation system characterized by Suzuki et al
] to identify chromatin conformation signatures (CCSs) associated with the transcription network of cellular differentiation. To this end, we mapped physical interaction networks with the 3C/5C technologies in the transcriptionally regulated HoxA
cluster and in a silent gene desert region. The HoxA
genes were selected for their pivotal roles in human biology and health. Importantly, the HoxA
cluster encodes 2 oncogenes, HoxA9
, which are over expressed in THP-1 cells. This genomic region plays an important role in promoting cellular proliferation of leukemia cells and HoxA
CCS identification should, therefore, help understand the mechanisms involved in regulating these genes.
Using 3C, we found that repression of HoxA9, 10, 11 and 13 expression is associated with formation of distinct contacts between the genes and with an overall increase in chromatin packaging. Chromatin remodeling was specific to transcriptionally regulated domains since no changes were observed in the gene desert region. We developed a suite of computer programs to assist in 5C experimental design and data analysis and for spatial modeling of 5C results. We used these tools to generate large-scale, high-resolution maps of both genomic regions during differentiation. 5C analysis recapitulated 3C results and identified new chromatin interactions involving the transcriptionally regulated HoxA region. Three-dimensional modeling provided the first predicted conformations of a transcriptionally active and repressed HoxA gene cluster based on 5C data. Importantly, these models identify CCSs of human leukemia, which may represent an entirely novel class of human disease biomarker. 5C research tools are now publicly available on our 5C resource website (see Materials and methods).