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Derailed regulatory network in childhood acute leukaemia

The regulation of gene expression is controlled by multiple mechanisms. The primary level of control is embedded within the sequence itself, as the transcription factors bind specific deoxyribonucleic acid (DNA) sequences at the gene regulatory regions and either activate or silence the genes. A secondary level of control includes enhancer sequences, which may lie megabases away from the gene and are necessary for the induction of gene expression in the human genome (1). A third level of control is the three-dimensional structure of chromatin, which can bring enhancers and promoters into contact with one another and can be restricted by the CCCTC-binding factor (CTCF) that insulates chromatin segments into topologically associated domains. A number of novel methodological approaches have recently hastened characterisation of the regulatory elements in the genome (2). One commonly used approach is to couple the chromatin immunoprecipitation sequencing of the transcription factors with gene expression profiling to study the transcription factor fusion targets in leukaemia (3,4). In comparison, the assay named global nuclear runon sequencing allows simultaneous exploration of the dynamics of gene expression and regulatory element activity. In a cell model with inducible ETS variant 6-runtrelated transcription factor 1 (ETV6-RUNX1) fusion, we were able to follow up the interplay of the enhancer elements and their host genes that occur downstream of this structural variation, which is the most common one found in childhood leukaemias (5). The ETV6-RUNX1 fusion bound the predominantly RUNX1 motif containing DNA sequences and caused repression of the enhancer activity as measured by the enhancer ribonucleic acid (RNA) levels. Interestingly, in most cases, the repression at the enhancers led to lower transcription at the nearby gene loci. In particular, the ETV6-RUNX1 aberration predominantly altered the expression of genes regulating B-cell adhesion and signalling. Furthermore, we noted a significantly higher repression of cluster of differentiation (CD)19/20 associated super-enhancers, in comparison with stem cell-linked CD34 super-enhancers (5). Super-enhancers are regions of the genome with clusterings of multiple enhancer sites and are deemed essential for lineage specification (6). They could provide a possible explanation for the disruption of B-cell differentiation, which is a hallmark of precursor leukaemias. A significant number of somatic mutations in leukaemia are located outside the coding regions of the genome. Mansour et al. (7) identified a small insertional mutation in a regulatory element at the T-cell acute lymphocytic leukaemia protein 1 (TAL1) gene locus in patients with T-cell acute lymphoblastic leukaemia. The mutation introduced a novel transcription factor binding site, for transcription factor myeloblastosis (MYB), thereby creating an aberrant super-enhancer that led to gross overexpression of the TAL1 gene. Such changes at the binding sites may also be inherited, as exemplified by the risk of neuroblastomas associated with a common single nucleotide polymorphism that alters a site where GATA transcription factors bind with DNA in a super-enhancer region of the LIM domain-only 1 (LMO1) gene (8). Moreover, the regulatory boundaries at the oncogene loci may be lost during disruption of the CTCF binding sites by mutations, as shown in T-cell acute lymphoblastic leukaemia (9), which leads to aberrant transcriptional activity. In addition to explaining the aberrant activation of oncogenes in leukaemic cells, highly active enhancers may create genetic instability. We explored the transcriptional features associated with structural variation sites in precursor B-cell leukaemias, which are represented by deletions, insertions, translocations and inversions. Structural variation sites tended to accumulate at certain loci in the leukaemia genomes and were more prevalent at actively transcribed regions. We discovered that simultaneous transcription from both DNA strands was often associated with these recurring structural variation sites (10). Enhancers that overlapped gene regions displayed this feature, which was accompanied by polymerase II stalling, access to DNA cleavage motifs and R-loop formation, a DNA:RNA hybrid formed during the transcription, which could lead to double-stranded DNA breaks that promote off-target cleavage by recombination-activating genes and activation-induced cytidine deaminase complexes. Stalled polymerases were found within the first intron of many key regulators of cell differentiation and tumour suppressor genes, including paired box protein 5 (PAX5) and B-cell translocation gene 1 (BTG1) protein (10,11). The preleukaemic cells arrested at the pre-B stage are thereby vulnerable to secondary lesions at such genomic regions. Taken together, these observations highlighted that altered activity or mutations in both coding and noncoding