STARR-seq (short for self-transcribing active regulatory region sequencing) is a method to assay enhancer activity for millions of candidates from arbitrary sources of DNA. It is used to identify the sequences that act as transcriptional enhancers in a direct, quantitative, and genome-wide manner. [1]
In eukaryotes, transcription is regulated by sequence-specific DNA-binding proteins (transcription factors) associated with a gene’s promoter and also by distant control sequences including enhancers. Enhancers are non-coding DNA sequences, containing several binding sites for a variety of transcription factors. [2] They typically recruit transcriptional factors that modulate chromatin structure and directly interact with the transcription machinery placed at the promoter of a gene. Enhancers are able to regulate transcription of target genes in a cell type-specific manner, [1] independent of their location or distance from the promoter of genes. In certain contexts (see Transvection (genetics)), they can even regulate transcription of genes located in a different chromosome. [3] However, the knowledge about enhancers so far has been limited to studies of a small number of enhancers, as they have been difficult to identify accurately at a genome-wide scale. [2] Moreover, many regulatory elements function only in certain cell types and specific conditions. [4]
Enhancer detection in Drosophila is an original methodology using random insertion of transposon-derived vector that encodes a reporter protein downstream of a minimal promoter. This approach allows to observe the expression of reporter in transgenic animals and provides information about nearby genes that are regulated by these sequences. The discovery and characterization of cell types along with genes involved in their determination have been significantly improved by the discovery of this technique. [5] [6] [7] [8]
During the past few years, post-genomic technologies, have displayed specific features of poised and active enhancers that have improved enhancer discovery. [2] Development of new methods such as deep sequencing of DNase I hypersensitive sites (DNase-Seq), formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-Seq), chromatin immunoprecipitation followed by deep sequencing (ChIP-sequencing), and MNase-defined cistrome-Occupancy Analysis (MOA-seq [9] ), provide genome-wide enhancer predictions by enhancer-associated chromatin features. [1]
However, DNase-seq and FAIRE-seq alone fail to provide a direct functional or quantitative readout of enhancer activity, so reporter assays that can deduce enhancer strength from the quantitative enrichment of reporter transcripts are needed to assess enhancer activity quantitatively. Yet, these assays are not high-throughput (High throughput biology), as it is impossible to conduct millions of tests required for identification of enhancers in a genome-wide manner. [1] The development of STARR-seq attempts to circumvent this analytical barrier. Taking advantage of the knowledge that enhancers can work independently of their relative locations, candidate sequences are placed downstream of a minimal promoter, allowing the active enhancers to transcribe themselves. The strength of each enhancer is then reflected by its relative enrichment among cellular RNAs. Such a direct coupling of candidate sequences to enhancer activity enables the parallel evaluation of millions of DNA fragments from arbitrary sources. [1]
Genomic DNA is randomly sheared and broken down to small fragments. Adaptors are ligated to size-selected DNA fragments. Next, adaptor-linked fragments are amplified and the PCR products are purified followed by placing candidate sequences downstream of a minimal promoter of screening vectors, giving them an opportunity to transcribe themselves. Candidate cells are then transfected with reporter library and cultured. Thereafter, total RNAs are extracted and poly-A RNAs isolated. Using reverse transcription method, cDNAs are produced, amplified and then candidate fragments are used for high-throughput paired end sequencing. Sequence reads are mapped to the reference genome and computational processing of data is carried out. [1]
Applying this technology to Drosophila genome, Arnold et al. [1] found 96% of the non-repetitive genome with at least 10-fold coverage. Authors discovered that most identified enhancers (55.6%) were placed within introns, particularly in the first intron and intergenic regions. 4.5% of enhancers were located at transcription start sites (TSS), suggesting that these enhancers can start transcription and also improve transcription from a distant TSS. [1] The strongest enhancers were near housekeeping genes such as enzymes or component of the cytoskeleton and developmental regulators such as the transcription factors. The strongest enhancer was located within the intron of the transcription factor zfh1. This transcription factor regulates neuropeptide expression and growth of larval neuromuscular junctions in Drosophila. [10] The ribosomal protein genes were the only class of genes with poor enhancers ranking. Moreover, authors demonstrated that many genes are regulated by several independent active enhancers even in a single cell type. Furthermore, gene expression levels on average were correlated with the sum of the enhancer strengths per gene, supporting direct link between gene expression and enhancer activity. [1]
Applying this technology to the characterization and discovery of regulatory variant alleles, Vockley et al. [11] characterized the effects of human genetic variation on non-coding regulatory element function, measuring the activity of 100 putative enhancers captured directly from the genomes of 95 members of a study cohort. This approach enables the functional fine-mapping of causal regulatory variants in regions of high linkage disequilibrium identified by eQTL analyses. This approach provides a general path forward to identify perturbations in gene regulatory elements that contribute to complex phenotypes.
STARR-seq has been used to measure the regulatory activity of DNA fragments that have been enriched for sites occupied by specific transcription factors. Cloning ChIP DNA libraries generated from chromatin immunoprecipitation of the glucocorticoid receptor into STARR-seq enabled genome-scale quantification of glucocorticoid-induced enhancer activity. [12] This approach is useful for measuring the differences in enhancer activity between sites that are bound by the same transcription factor.
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By combining traditional approach with high-throughput sequencing technology and highly specialized bio-computing methods, STARR-seq is able to detect enhancers in a quantitative and genome-wide manner. The study of gene regulation and their responsible pathways in the genome during normal development and also in disease can be very demanding. Therefore, applying STARR-seq to many cell types across organisms supports identifying cell type-specific gene regulatory elements and practically assesses non-coding mutations causing disease. Recently, a related approach coupling capture of regions of interest to STARR-seq technique have been developed and extensively validated in mammalian cell lines. [13]
Functional genomics is a field of molecular biology that attempts to describe gene functions and interactions. Functional genomics make use of the vast data generated by genomic and transcriptomic projects. Functional genomics focuses on the dynamic aspects such as gene transcription, translation, regulation of gene expression and protein–protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures. A key characteristic of functional genomics studies is their genome-wide approach to these questions, generally involving high-throughput methods rather than a more traditional "candidate-gene" approach.
DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins in vitro. This technique can be used to study protein-DNA interactions both outside and within cells.
ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized to study these protein–DNA relations.
Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.
Chromatin Interaction Analysis by Paired-End Tag Sequencing is a technique that incorporates chromatin immunoprecipitation (ChIP)-based enrichment, chromatin proximity ligation, Paired-End Tags, and High-throughput sequencing to determine de novo long-range chromatin interactions genome-wide.
Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly define cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of epigenomics and learning more about epigenetic phenomena.
In genetics, DNase I hypersensitive sites (DHSs) are regions of chromatin that are sensitive to cleavage by the DNase I enzyme. In these specific regions of the genome, chromatin has lost its condensed structure, exposing the DNA and making it accessible. This raises the availability of DNA to degradation by enzymes, such as DNase I. These accessible chromatin zones are functionally related to transcriptional activity, since this remodeled state is necessary for the binding of proteins such as transcription factors.
Enhancer-FACS-seq (eFS), developed by the Bulyk lab at Brigham and Women’s Hospital and Harvard Medical School, is a highly parallel enhancer assay that aims for the identification of active, tissue-specific transcriptional enhancers, in the context of whole Drosophila melanogaster embryos. This technology replaces the use of microscopy to screen for tissue-specific enhancers with fluorescence activated cell sorting (FACS) of dissociated cells from whole embryos, combined with identification by high-throughput Illumina sequencing.
H3K4me3 is an epigenetic modification to the DNA packaging protein Histone H3 that indicates tri-methylation at the 4th lysine residue of the histone H3 protein and is often involved in the regulation of gene expression. The name denotes the addition of three methyl groups (trimethylation) to the lysine 4 on the histone H3 protein.
H3K27ac is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates acetylation of the lysine residue at N-terminal position 27 of the histone H3 protein.
CUT&RUN sequencing, also known as cleavage under targets and release using nuclease, is a method used to analyze protein interactions with DNA. CUT&RUN sequencing combines antibody-targeted controlled cleavage by micrococcal nuclease with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN sequencing does not.
H3K9me3 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the tri-methylation at the 9th lysine residue of the histone H3 protein and is often associated with heterochromatin.
H3K4me1 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the mono-methylation at the 4th lysine residue of the histone H3 protein and often associated with gene enhancers.
H4K20me is an epigenetic modification to the DNA packaging protein Histone H4. It is a mark that indicates the mono-methylation at the 20th lysine residue of the histone H4 protein. This mark can be di- and tri-methylated. It is critical for genome integrity including DNA damage repair, DNA replication and chromatin compaction.
H4K8ac, representing an epigenetic modification to the DNA packaging protein histone H4, is a mark indicating the acetylation at the 8th lysine residue of the histone H4 protein. It has been implicated in the prevalence of malaria.
H4K12ac is an epigenetic modification to the DNA packaging protein histone H4. It is a mark that indicates the acetylation at the 12th lysine residue of the histone H4 protein. H4K12ac is involved in learning and memory. It is possible that restoring this modification could reduce age-related decline in memory.
H3K14ac is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the acetylation at the 14th lysine residue of the histone H3 protein.
H3K9ac is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the acetylation at the 9th lysine residue of the histone H3 protein.
H3K36ac is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the acetylation at the 36th lysine residue of the histone H3 protein.
MNase-seq, short for micrococcal nuclease digestion with deep sequencing, is a molecular biological technique that was first pioneered in 2006 to measure nucleosome occupancy in the C. elegans genome, and was subsequently applied to the human genome in 2008. Though, the term ‘MNase-seq’ had not been coined until a year later, in 2009. Briefly, this technique relies on the use of the non-specific endo-exonuclease micrococcal nuclease, an enzyme derived from the bacteria Staphylococcus aureus, to bind and cleave protein-unbound regions of DNA on chromatin. DNA bound to histones or other chromatin-bound proteins may remain undigested. The uncut DNA is then purified from the proteins and sequenced through one or more of the various Next-Generation sequencing methods.