Estrogen receptor action in three dimensions - looping the loop

Due to advances in genomic technologies, our understanding of estrogen receptor (ER)-mediated transcription in breast cancer cells has evolved significantly in recent years. Genome-wide mapping experiments revealed thousands of ER-binding events, but linking them to the target genes has been an ongoing struggle. A recent paper describes a new technique, called ChIA-PET (chromatin interaction analysis using paired-end tag sequencing), that can directly address these questions. ChIA-PET is an unbiased approach for simultaneously identifying all genome-wide binding events of a transcription factor and those involved in long-range chromatin loops.


Background
Estrogen receptor (ER) has been one of the most intensively studied transcription factors, primarily due to its importance in breast cancer. Much eff ort has been invested in identifying the target genes of ER, mainly by expression arrays in breast cancer cell lines and in human tumour samples [1,2]. In the past few years, the evolution of genomic technologies has permitted mapping of ER-binding events in an unbiased manner [3,4]. Th is involves combining chromatin immunoprecipitation with micro arrays or highthroughput sequencing in order to identify ER contact points within the genome. Th ese genome-wide experiments revealed a number of surprising features about ER biology, the most unexpected one being that ER regulates transcription mostly from distal enhancers. Th is fi nding raised the subsequent question: which of the thousands of ER-binding events are the functional ones that regulate expression of the few hundred ER target genes? Fullwood and colleagues [5] have now established a method for addressing this question, allowing researchers to assign ERbinding events to transcriptional hubs and target genes.

The article
Researchers at the Genome Institute of Singapore have developed a technique called ChIA-PET (chromatin interaction analysis using paired-end tag sequencing) [5], which couples chromosome conformation capture [6], a method for identifying interacting chromatin regions, with high-throughput sequencing. Th is permits the global identifi cation of chromatin loops formed at a specifi c time point. Th e authors used a well-established system whereby MCF-7 breast cancer cells are cultured in hormone-deprived media and subsequently treated with estrogen for a short time period, resulting in synchronisation of transcriptional events [7]. Using this system, the authors could capture the high-confi dence chromatin interactions that form during the fi rst wave of estrogen-ER-mediated transcription. Th ese interactions were extensively validated, supporting their conclusions. Th e implications of this investigation are profound. It is revealed that chromatin interactions can occur in clusters of contact points and that the actual anchor points, in a linear two-dimensional represen ta tion, can be found separ ated by hundreds of kilobases and in some cases megabases. Contrary to previous sugges tions [8], interchromosomal interactions do not appear to be a common or experimentally verifi able pheno menon. Th is suggests that ER-binding events regulate genes within mostly a spatially confi ned environ ment, which in many cases involves the folding of multiple regulatory regions into one transcriptional hub.
In summary, the authors fi nd 689 ER-associated chromatin interaction complexes made up of duplexes and more complex interactions. Th ese tend to involve the stronger ER-binding events, which are biased toward specifi c histone marks and other transcriptional regulators important for ER function, such as FoxA1 [9]. By combining the ChIA-PET data with gene expression data, they could discover individual transcriptional hubs and the target genes localised in the vicinity.

The viewpoint
Until now, researchers have been linking enhancers and cis-regulatory elements with the target genes one at a time [10,11]. ChIA-PET circumvents this limitation and Abstract Due to advances in genomic technologies, our understanding of estrogen receptor (ER)-mediated transcription in breast cancer cells has evolved signifi cantly in recent years. Genome-wide mapping experiments revealed thousands of ER-binding events, but linking them to the target genes has been an ongoing struggle. A recent paper describes a new technique, called ChIA-PET (chromatin interaction analysis using paired-end tag sequencing), that can directly address these questions. ChIA-PET is an unbiased approach for simultaneously identifying all genome-wide binding events of a transcription factor and those involved in long-range chromatin loops.
can identify all active loops at a given time point, allowing us to redefi ne the functional ER-binding events. While this constitutes the fi rst snapshot of ER-mediated chroma tin loops, it is a quantum leap in our understanding of transcription dynamics. However, this pioneer ing work leads to a series of new questions. Th e authors associate the DNA loops that they captured at 45 minutes of estrogen treatment with a time course expression profi le of cells treated with estrogen from 0 to 48 hours of treatment. A challenging concept that needs further testing is whether the chromatin loops (captured at 45 minutes) are stable over time or whether they change, leading to the formation of new loops at later time points to regulate transcription of the 'late' target genes. Moreover, what is the role of co-operating transcription factors in loop formation? Also, since many of the ER functional binding events do not directly involve promoter regions, one can speculate that some DNA loops might circum vent the need for a transcription factor to be present at the promoter by concentrating the transcription machinery in the three-dimensional hubs.
Furthermore, this technology can be used to explore ER chromatin loops and transcriptional hubs in diff erent breast cancer cell lines and treatment conditions. Th e identifi cation of transcriptionally active binding events in the diff erent contexts will illuminate the factors that dictate diff erential binding and transcriptional activity. However, the major limitations of the ChIA-PET technology are the complex technical aspects of the procedure, the heavy bioinformatics demand and the requirement for high-throughput sequencers. Neverthe less, these genomic tools are becoming more readily available, and as such, ChIA-PET will prove to be a powerful tool for delineating mechanisms of transcription in a spatial and temporal manner, applicable to any transcription factor of interest.

Abbreviations
ChIA-PET = chromatin interaction analysis using paired-end tag sequencing; ER = estrogen receptor.