Hierarchical co-regulation

Numerous studies have shown that long-range chromosomal contacts coincide with changes in gene expression. To analyze whether such contacts are required for cotranscription in multigene complexes that are activated by the same transcription factor, Mhlanga and colleagues studied a well-characterized tumor necrosis factor-α (TNF-α)-induced multigene complex, in which three interacting genes (SAMD4A, TNFAIP2 and SLC6A5) are activated by transcription factor NF-κB. TNF-α–induced formation of chromosomal contacts between these genes was accompanied by cotranscription of all three genes in a relatively small fraction of cells, thus suggesting that only certain cells in a population have the correct spatial arrangement of their chromosomes to permit co-regulation. The authors therefore devised a single-cell–based assay, in which TALE nuclease (TALEN)-mediated disruption of the SAMD4A gene loop in single cells was visualized by DNA fluorescence in situ hybridization (FISH), and abrogated SAMD4A RNA and protein expression was analyzed by RNA FISH and immunofluorescence. Parallel monitoring of the other two interacting genes showed that their transcription was abolished. TALENs were also targeted to sites of chromosomal contacts in TNFAIP2 and SLC6A5 while transcription of the other two interacting genes was monitored. Interestingly, disruption of the TNFAIP2 loop did not have a significant effect on SAMD4A transcription, yet transcription of both TNFAIP2 and SLC6A5 was reduced, thus suggesting a hierarchical mode of regulation. Disruption of the SLC6A5 loop had an even greater hierarchical effect, as transcription of both interacting genes was unaffected. Disrupted gene loops could be successfully repaired in a sequence-independent manner to restore transcription of the interacting genes. The single-cell TALEN assay seems a powerful approach to interrogate transcriptional regulation in three dimensions, and it will be interesting to explore the molecular basis for the hierarchical mode of transcription in a multigene complex. (Cell 155, 606–620, 2013)