Genome‐Wide Approaches to Identify the Interplay of Transcription Factors, Regulatory Elements and Target Genes


Tightly controlled gene regulation is essential for embryogenic development and homeostasis. One of the key questions in gene regulation is the interplay between transcription factors, regulatory elements and target genes. Recent development of high‐throughput technologies has made analyses of the complex interplay possible at the genome‐wide scale, and provides insights into the molecular mechanism of gene regulation. These state‐of‐the‐art genome‐wide approaches include RNA (ribonucleic acid)‐seq for gene expression, ChIP‐seq (chromatin immunoprecipitation followed by sequencing) for transcription factor binding and histone modifications, and methods for analysing chromatin accessibility and three‐dimensional chromatin conformation. For each of these approaches, we discuss the background, the current working pipeline and ongoing development. As computational analysis packages and methods are essential for interpreting genome‐wide large data sets, the commonly used ones are summarised with the corresponding technologies.

Key Concepts

  • The complex interplay of transcription factors, cis‐regulatory elements and target genes is essential for proper gene regulation.
  • RNA‐seq analyses have revolutionised gene expression analyses.
  • ChIP‐seq analyses assess the binding of transcription factor and the occupancy of histone modifications and provide potential cis‐regulatory elements and gene regulation in the genome.
  • Analyses of chromatin accessibility reveal cis‐regulatory elements and complement ChIP‐seq analyses.
  • Analyses of three‐dimensional chromatin conformation provide information on the chromatin domains that facilitate or restrict gene regulation.

Keywords: genome‐wide; transcriptional factors; cis‐regulatory elements; target genes; RNA‐seq; ChIP‐seq; chromatin accessibility; three‐dimensional chromatin conformation

Figure 1. An overview of transcriptional regulation.
Figure 2. RNA‐seq pipeline. After total RNA (ribonucleic acid) extraction, mRNA is captured by Oligo‐dT beads hybridising polyA tails or by rRNA depletion with a hybridisation‐capture process. Afterwards, mRNAs are fragmented and reverse transcribed to double‐stranded cDNA (complementary deoxyribonucleic acid) fragments. Sequencing adaptors are then added to each cDNA fragment followed by standard sample preparation. After squencing, the reads that contain short sequences obtained from each cDNA are aligned to the reference genome or transcriptome.
Figure 3. ChIP‐seq pipeline. First, proteins are cross‐linked to DNA (dexyribonucleic acid) with formaldehyde. Subsequently, the chromatin is sheared into short fragments. Then specific chromatin fractions that interact with the SS‐TF (sequence‐specific transcription factor)/histone modification are isolated using antibodies specifically recognising the SS‐TF/ the histone modification by immunoprecipitation. Afterwards, the immunoprecipitated DNA is decross‐linked, purified and sequenced. By aligning sequenced DNA reads to the reference genome and searching for enriched regions, we can determine where SS‐TF/ histone modification is located along the genome.
Figure 4. 3C and its derivative genome‐wide methods. In 3C, DNA–DNA interactions in close proximity are cross‐linked with formaldehyde. Afterwards, the cross‐linked DNA is digested with an excess of restriction enzyme followed by a ligation step between DNA fragments in proximity. After decross‐linking and DNA purification, PCR (polymerase chain reaction) anayses with primers specific for the loci of interest is performed. 4C includes another step of restriction enzyme digestion and ligation followed by inverse PCR while 5C uses multiplexed ligation‐mediated amplification. In Hi‐C, after restriction enzyme digestion, the 5′ DNA overhang is filled and marked with biotin, which enables streptavidin beads binding after the ligation step. The streptavidin beads selected fragments are then sequenced, which can give a genome‐wide view of interactions.


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Further Reading

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Qu, Jieqiong, and Zhou, Huiqing(Feb 2018) Genome‐Wide Approaches to Identify the Interplay of Transcription Factors, Regulatory Elements and Target Genes. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0027370]