Enhancers and Genetics

By | July 10, 2021
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Enhancers

They are short DNA fragments with several prominent features such as:

  • driving the expression of the target gene,
  • orientation relative to a target gene promoter,
  • functionally independent of genomic distance,
  • sensitivity towards DNase treatment (since its neucleosome free region),
  • functional influence over chromatin architecture by allowing binding of transcription co-activators and histone acetylation,
  • and contains specific DNA sequences for the binding of transcription factors, undertaking transcriptional regulation.

History

The enhancer was first discovered in simian virus SV40 where it was a 72 bp long DNA fragment that increased the expression of reporter gene promoter by 200 fold. Further studies were performed to find cellular enhancers in vivo. Eventually, realizing the potential of enhancers, they were widely used in genomics and molecular biology studies where they regulated important functions in various developmental systems and cell-type specificity studies.

Recent research studies suggest that functional enhancers encode non-coding RNAs, and also act as transcription units. spatiotemporal pattern of gene expression is influenced by a network of regulatory enhancers dictating cellular identities and cellular functions.

Molecular biology techniques to study and detect enhancers

  • Reverse transcription-PCR (RT-PCR): A PCR-based method where enzyme reverse transcriptase is used to convert RNAs to complementary DNA (cDNA). The amount of the target enhancer can be determined using qPCR or real-time PCR. It is time and cost-effective for single locus experiments. It might result in low throughput due to ongoing transcriptional activity with the target enhancer.
  • RNA fluorescence in situ hybridization (RNA FISH): Pre-designed fluorescent labelled oligonucleotides or primers are used as probes to hybridize with the target RNAs when gragmented genome is used as template. This can be detected by the fluorescent signal emitted by the primer. This is suitable for studying single cell and spatiotemporal relationship between molecules involved in a particular process. It might result in low throughput because of the dicey nature of in situ hybridization technique.
  • RNA polymerase II immunoprecipitation along with high-throughput sequencing (RNAPII ChIP-seq): This method involves study of chromatin regions that are associated with RNA polymerase II. It involves immunoprecipitation i.e., crosslinking of RNAPII subunit using specific antibodies. In this way, potential transcription site can be determined that can be further subjected to high-throughput sequencing to identify the enhancer regions. Lower specificity and quality of antibodies is a disadvantage in this method. This only gives us an indirect evidence of transcription site because of multiple forms of modified RNAPII.
  • Global run-on sequencing (GRO-seq): Nascent transcriptome (primary transcripts that needs to be transpoted to the cytoplasm and processed for translation) of a cell population by re-initiating the transcription of RNAPII in vitro. Nascent transcriptome is labelled using radiolabeled mononucleotides. Along with sequencing, this method is high throughput due to its high efficiency in detecting dynamic transcriptional activity including non-coding RNAs or enhancer RNAs.
  • 5′ GRO-seq: An alternate version of GRO-seq where m7G-capped nascent RNAs are targeted. By this method, precise start site of the transcription process could be identified.
  • BruUV-seq: This technique involves inducing DNA lesions through UV light and blocking transcription randomly in the genome. This is followed by BrUTP incorporation and RNA seq. This is more effective in identifying the transcription start site due to the BrUTP incorporation that preserves the RNAPII position in vivo.
  • RNA seq: RNA composition of a cell or group of cells at a moment is sequenced and studied. This method is high throughput, precise, and easy to perform.
  • RNA seq (poly (A)): Oligo-dT primers are used to detect the amount of RNAs and converted to cDNAs using reverse transcription. These cDNAs are used for sequencing. This is efficient in identifying RNAs with poly A tails.
  • Cap analysis of gene expression (CAGE): m7G capped RNAs are captured using genome wide tool in a transcriptome.
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The aim of these methods is to find epigenomic marks. Epigenomic marks mean active enhancers. A group of clustered enhancers is a high binding density of transcription factors and other chromatin regulators. Enhancers also exhibit robust eRNA transcription. The eRNA-producing enhancers have high enrichment of active histone marks as well as protection from repressive marks such as DNA methylation.

The enhancer-promoter looping: How are promoters regulated by enahncers?

The tracking model:The RNAPII and its transcription machinery track through the intervening DNA in-between promoters and enhancers.

The looping model: The transcription machinery is loaded at enhancers and then reaches the promoter leading to a physical interaction through looping.

In both the models, the enhancers activate the gene promoter transcriptionally. The looping model is the most favored one since that allows exchange of transcriptional machinery in both the directions. But this possibility has been less explored.

Credit: https://en.wikipedia.org/wiki/Enhancer_(genetics)

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