Active Enhancers : Recent Research Advances and Insights into Disease

During development and throughout an organism’s life, precise spatial and temporal regulation of cellular functions is ensured by the carefully regulated process of gene expression. By adjusting target gene transcription across vast genomic lengths, enhancers—important regulatory regions in the genome—manage this complexity. Since their discovery more than thirty years ago, enhancers have become important factors in determining the transcriptional landscape, allowing for dynamic and cell-type-specific patterns of gene expression.

Enhancers use chromatin remodeling, physical contacts with target promoters, and epigenetic changes to carry out their regulatory effects. A wide range of transcription factors, cofactors, and chromatin-binding proteins control this complex process, which together promotes enhancer activation and specificity. Crucially, enhancer malfunction has been linked to a wide range of human illnesses, including as neurological conditions, developmental abnormalities, and cancer, highlighting their importance as possible targets for treatment.

Our knowledge of enhancers has been completely transformed in recent years by developments in functional genomics and high-throughput sequencing methods. Methods like chromatin accessibility assays (like ATAC-seq), histone modification profiling (like ChIP-seq), and chromatin interaction mapping (like Hi-C) have made it possible to identify enhancers across the entire genome and have clarified how they work. Our understanding of enhancer biology has also been further broadened by the identification of enhancer RNAs (eRNAs) and the creation of CRISPR-based technologies, which present fresh chances for functional characterisation and therapeutic investigation.

The goal of this review Is to present a thorough analysis of enhancer biology, covering its structural and functional properties, research approaches, and functions in both health and sickness. We also talk about future approaches in this quickly developing science and examine the therapeutic potential of focusing on enhancers. This article aims to close the gap by emphasizing new trends and synthesizing existing knowledge.

Characteristics of Enhancers

Enhancers are non-coding DNA segments that, frequently from great genomic distances, increase the transcription of target genes to control gene expression. Enhancers and their roles are defined by a number of common traits, notwithstanding their diversity and complexity.

1. Epigenetic Modifications of Enhancers are often identified by particular epigenetic characteristics that set them apart from other regulatory components: Chromatin accessibility: ATAC-seq, FAIRE-seq, or DNase I hypersensitivity assays can be used to identify enhancers, which are found in open chromatin areas. Enhancer sequences can be bound by transcription factors and coactivators because of this accessibility. Changes to the Histones: Certain histone marks are indicative of poised and active enhancers:

Active Enhancers: Linked to H3 lysine 4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac). Poised Enhancers: Show H3K4me1 and H3 lysine 27 trimethylation (H3K27me3), which are frequently present in a repressed form but are prepared for activation. DNA Methylation While the relationship between DNA methylation and enhancer activity is still being studied, enhancers frequently show modest levels of DNA methylation when they are active.

2. Enhancer RNAs (eRNAs): Upon activation, enhancers are translated into non-coding RNAs called enhancer RNAs (eRNAs), which are frequently unstable and non-polyadenylated. It is believed that their transcription contributes to chromatin remodeling, enhancer activation, and enhancer-promoter communication. Although their exact function is unknown, eRNAs may aid in the recruitment of transcriptional machinery to specific promoters or the stabilization of enhancer-promoter loops.
3. Effects of Enhancers on Promoters Enhancers interact physically with target gene promoters to carry out their regulatory actions Cytokine Looping
4. Through the use of architectural proteins such as CTCF and the cohesin complex, chromatin looping facilitates transcriptional activation by bringing enhancers and promoters closer together.

Specificity of Contact: Highly specialized enhancer-promoter interactions are frequently limited in topologically associating domains (TADs). Disease and ectopic gene expression may result from disruption of these connections. The mapping of these interactions has been made possible by tools like Hi-C, Capture-C, and 3C.

5. Context – Dependent activity: Activity That Depends on Context Environmental factors, developmental stages, and cell types all affect enhancer activity, which is very context-specific:

Environmental factors, developmental stages, and cell types all affect enhancer activity, which is very context-specific: Enhancers attract transcription factors and cofactors relevant to cell types in order to attain specificity. Genes essential for determining cell identity and function are expressed by super-enhancers, which are collections of extremely active enhancers.

6. Conservation and Evolution: Different species may exhibit different levels of conservation in enhancer sequences. Because of their functions in evolutionary adaptation, certain enhancers are species-specific, whereas others are extremely conserved. The dynamic character of enhancer evolution is emphasized b enhancer turnover, which occurs when one enhancer is swapped out for another with a comparable function.
7. Booster Robustness and Redundancy  Multiple enhancers control a large number of genes, guaranteeing robust gene expression: Redundant enhancers can make up for each other’s loss, making them resilient to environmental changes or mutations. This overlap in functionality makes it more difficult to study

Technologies for High-Throughput Sequencing in Enhancer Research

High-throughput sequencing technology breakthroughs have completely changed our capacity to find and describe enhancers across the whole genome. These methods offer vital information about the spatial, transcriptional, and epigenetic properties of enhancers as well as how they interact with target promoters. The main high-throughput sequencing techniques used in enhancer research are described here.

1. Assays for Chromatin Accessibility  

One characteristic of active enhancers is chromatin accessibility. Among the methods for mapping accessible chromatin are: DNase-Seq: Marks open chromatin regions, including enhancers, and identifies DNase I hypersensitive locations. Tn5 transposase is used in ATAC-Seq (Assay for Transposase-Accessible Chromatin), a quicker and more sensitive technique for mapping accessible chromatin. By using formaldehyde crosslinking and phenol-chloroform extraction, FAIRE-Seq (Formaldehyde-Assisted Isolation of Regulatory Elements) enriches open chromatin regions.

2. Profiling of Histone Modification  

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is the gold standard for mapping histone modifications, which are important markers of enhancer activity. Active enhancers are linked to H3K4me1 and H3K27ac. H3K27me3: Identifies repressed or poised enhancers. In contrast to conventional ChIP-Seq, emerging options like CUT&RUN (Cleavage Under Targets and Release Using Nuclease) and CUT&Tag (Cleavage Under Targets and Tagmentation) provide higher resolution, lower input needs, and less background noise.

3. Enhancer Transcriptomics.

Enhancer activation frequently results in the production of enhancer RNAs (eRNAs), which are non-coding RNAs: RNA-Seq: Provides information on enhancer activity by detecting eRNA transcripts. RNA Profiling of Nascents: By capturing early eRNA transcription, methods such as Global Run-On Sequencing (GRO-Seq) and Precision Run-On Sequencing (PRO-Seq) offer a dynamic picture of enhancer activity.

4. Mapping Chromatin Interactions   

Enhancer function depends on enhancer-promoter interactions, which can be mapped using a variety of sequencing-based techniques: Hi-C: A genome-wide method that uses the 3D genome structure to identify chromatin connections, such as those between enhancers and promoters. By concentrating on particular genomic regions of interest, including enhancer-promoter pairs, Capture-C is a focused method that improves resolution. To find protein-mediated chromatin connections, ChIA-PET (Chromatin Interaction Analysis by Paired-End Tagging) combines chromatin immunoprecipitation and interaction mapping. These instruments show how enhancers interact with target genes and are arranged spatially throughout the nucleus.

5. Spatial and Single-Cell Transcriptomics.

Single-cell resolution enhancer research are now possible thanks to emerging technologies: Single-Cell ATAC-Seq: Indicates enhancer activity specific to a cell type by profiling chromatin accessibility in individual cells. Transcriptome data and spatial information are used in spatial transcriptomics to reveal enhancer activity in tissue architecture. These methods are especially useful for researching enhancers during development and in diverse tissues.

6. Tools for Functional Genomics.

 Functional genomics techniques are used in conjunction with high-throughput sequencing to confirm enhancer activity: Enhancer regions can be perturbed using CRISPR/Cas9 screens to examine how they affect gene expression. Self-Transcribing Active Regulatory Region Sequencing, or STARR-Seq, is a massively parallel reporter experiment that uses transcription of the enhancer sequence itself to evaluate enhance activity directly. High-Throughput Sequencing Applications in Disease Studies and Enhancer Research: detection of mutations in enhancer areas linked to illness.

Table: 1 ; Technologies for high – throughput  sequencing in enhancer research

 Understanding the biological functions of enhancers and their modes of activity requires the use of functional genomics techniques. These techniques evaluate enhancer regions’ activity, specificity, and contributions to gene regulation in several settings, going beyond simply mapping them. We list the main functional genomics methods below.

1. Technologies Based – on CIRSPR

Functional enhancer studies have been revolutionized by the development of CRISPR/Cas systems, which allow for precise genomic changes. CRISPR Knockout (CRISPR-KO): Enhancer areas are deleted to examine how they affect the expression of the target gene. Helpful for evaluating the robustness and redundancy of enhancers. Repression of enhancer activity without changing the DNA sequence is known as CRISPR Interference (CRISPRi). Accomplished by combining transcriptional repressors such as KRAB with catalytically inactive Cas9 (dCas9). CRISPR Activation (CRISPRa): Enhancer activation with dCas9 linked to activators such as p300 or VP64. Makes it possible to evaluate whether an enhancer is sufficient to activate a target gene. CRISPR Screens: High-throughput enhancement area perturbation to find those essential for particular gene expression programs or phenotypes.

2. MPRAs, or massively parallel reporter assays MPRAs

Evaluate thousands of potential enhancers’ activity at once: One type of MPRA that positions putative enhancers downstream of a minimum promoter within the reporter construct is called STARR-Seq (Self-Transcribing Active Regulatory Region Sequencing). A direct indicator of enhancer activity, active enhancers promote the transcription of their own sequences. Plasmid-Based MPRAs: Upstream of a reporter gene, candidate enhancers are cloned and tested in cell lines. provide numerical information about enhancer strength. In Vivo MPRAs: Used to evaluate enhancer activity in a physiological setting using animal models.

3. Epigenetic editing and chromatin  

The regulatory mechanisms of enhancer regions can be revealed by directly manipulating their chromatin states: Editing the epigenome:uses histone modifiers coupled to dCas9 to change enhancer chromatin states. Histone acetylation (enhancer activation) and histone deacetylation (enhancer suppression) can be achieved with dCas9-p300 and dCas9-LSD1, respectively. Artificial Enhancers: artificially created enhancers that are inserted into cells to examine particular interactions or sequence motifs.

4. Perturb-seqencing :

 A mix of single-cell RNA sequencing (scRNA-seq) with CRISPR screens: connects particular alterations to subsequent transcriptional changes in individual cells, allowing for high-throughput functional evaluation of enhancers. Beneficial for researching enhancer functions particular to cell types.

5. Functional Genomics of Single Cells 

At the individual cell level, single-cell technologies offer insights on enhancer function: CRISPR Screens for Single Cells: Examine how enhancers affect the regulation of genes in diverse cell groups. Individual Cell ATAC-Seq In conjunction with CRISPR: monitors changes in chromatin accessibility following enhancer perturbation.

6.  Models of Enhancer Deletion Mouse Models 

CRISPR or homologous recombination is used to target enhancers in mice for the purpose of studying their physiological and developmental functions. Human Organoids: Enhancer function in organ development or disease is modeled by perturbing enhancers in organoids made from human pluripotent stem cells.

7.  Functional Validation with eRNAs Knockdown/Overexpression.

Using CRISPR tools or antisense oligonucleotides, enhancer RNAs (eRNAs) can be overexpressed or silenced to examine their functional role. Finding eRNAs’ protein partners in order to clarify their modes of action is known as eRNA binding studies.

8. Super-Enhancer Research

Clusters of enhancers known as super-enhancers control important genes for both illness and cell identity: Super-enhancers are frequently perturbed in functional genomics techniques to examine their cooperative effects. Their structure-function links are revealed by methods such as CRISPR tiling screens, in which super-enhancer regions are targeted by overlapping guide RNAs.

9. In Vivo Approaches

Transgenic Reporter Mice: To examine their activity in vivo, introduce fluorescent or luminous reporter structures under enhancer control. Viral Vector Delivery: Introduce enhancer-reporter structures into particular cell types or tissues using viral vectors. Functional Genomics Applications in Enhancer Research

1. 1. Finding Disease-Associated Enhancers: Non-coding variations associated with human disorders are functionally validated.
   2. Comprehending Developmental Dynamics: Determining the functions of enhancers in organogenesis and tissue differentiation.
   3. Therapeutic Targeting: Investigating enhancer modulation as a possible treatment for neurological conditions, cancer, and other conditions. Our knowledge of enhancer biology has significantly increased because to functional genomics techniques, which provide strong instruments for analyzing their intricate functions in gene regulation and their consequences for both health and illness.

Enhancers in Human Illness

Numerous human disorders can result from the malfunction of enhancers, which are essential for controlling gene expression. Enhancers are frequently implicated in the pathophysiology of disease due to mutations or dysregulation of enhancer activity, which can result in abnormal gene expression, disturbed cellular functions, and the onset of disease. The different ways that enhancers contribute to human disease are examined below.

1. Enhancer Mutations Associated with Disease

Disease can result from mutations that alter normal gene regulation in enhancer regions. These mutations may consist of: SNPs, or single nucleotide polymorphisms: By altering the chromatin environment or the binding affinity of transcription factors, disease-associated SNPs, which frequently sit in enhancer regions, can change the function of enhancers. For instance, since they interfere with immune-related gene regulation, SNPs in enhancers have been connected to autoimmune disorders like Crohn’s disease and rheumatoid arthritis

2. Additions and Removals (Indels): 

Gene expression can be disrupted by small insertions or deletions in enhancer regions, which can either produce or remove enhancer motifs. Congenital diseases, for example, can result from mutations in the enhancers of genes involved in developmental processes.

3. Structural Variants:

 Genes may be misregulated as a result of large chromosomal rearrangements, including deletions, duplications, or translocations, which may affect enhancer regions. The translocation of enhancer regions in some malignancies, including Burkitt lymphoma, is a well-known example, where the MYC oncogene is overexpressed due to the enhancer region.

2. Cancer’s Enhancer Dysregulation

 One of the most researched diseases where enhancer dysregulation has a role is cancer: The activation of oncogenes: Enhancers that are close to oncogenes are improperly active in many malignancies, which results in the overexpression of genes that promote cell survival and proliferation. For instance, oncogenes like MYB or TAL1 may become active in specific forms of leukemia due to alterations in enhancer regions. 

Deregulation of Super-Enhancers:

It has been demonstrated that super-enhancers, which are groups of enhancers that drive the expression of important genes, control genes essential for the identity of cancer cells. Super enhancers are reprogrammed to induce the expression of genes that promote carcinogenesis in some types of cancer. Research on cancer treatments that target super-enhancers shows promise. Silencing of Tumor Suppressor Genes:

Conversely, improper enhancer silencing may inhibit tumor suppressor gene expression, which could accelerate the development of cancer. Epigenetic alterations like DNA methylation or histone modifications can cause enhancer repression, which lowers the expression of genes that would normally prevent tumor growth.

3. Neurological Disorder Enhancers 

Enhancers are essential for the accurate control of genes related to neuronal function and brain development. Numerous neurological conditions can result from enhancer dysregulation, including: Disorders of Neurodevelopment: A congenital neurodevelopmental condition may result from mutations in enhancer areas involved in brain development. For example, a neurodevelopmental disease marked by intellectual disability and speech difficulties has been associated with mutations in enhancers controlling the expression of the MEF2C gene.

Autism Spectrum Disorders (ASD):

Autism has been linked to variations in enhancer regions that control genes related to synapse function, neuronal signaling, and neuroplasticity. The normal expression of these genes may be disrupted by enhancer mutations, which may contribute to the behavioral and neurological characteristics of ASD.

Neurodegenerative illnesses: Parkinson’s and Alzheimer’s illnesses are two examples of neurodegenerative diseases that can be exacerbated by enhancer dysfunction. For instance, these illnesses have been linked to mutations in enhancers that impact the expression of genes involved in neuronal survival or cellular stress responses.

4. Heart related condition

Genes necessary for cardiovascular development and function are likewise regulated by enhancer regions. Heart disease may be exacerbated by the dysregulation of these enhancers

Congenital Heart Defects: Congenital heart abnormalities can result from mutations in enhancers that regulate genes involved in heart development. Mutations in enhancers that control genes crucial for heart development, such as NKX2-5 and TBX5, are one example.

Atherosclerosis: The accumulation of plaque in blood vessels is a symptom of atherosclerosis, which can be exacerbated by immune cells’ dysregulated enhancer activity. This condition can be made worse by enhancer mutations that impact genes related to inflammation or lipid metabolism.

5. Metabolic Conditions

Metabolic problems can result from the disruption of enhancers, which are essential for the regulation of genes involved in metabolism: Diabetes type 2: It has been demonstrated that a number of genetic loci linked to Type 2 diabetes include mutations in enhancer regions that control genes involved in glucose homeostasis and insulin synthesis. These enhancer mutations may contribute to the disease by causing pancreatic β-cells to express genes improperly. Obesity: Obesity can cause dysregulation of enhancers that regulate genes related to energy balance and fat storage. For instance, a higher risk of obesity has been associated with enhancer mutations that impact the FTO gene, which is involved in fat metabolism.

6. Immune system disorders and infectious diseases 

Numerous immune-related disorders can result from mutations or dysregulation of enhancers, which are also essential for the control of immune response genes:

Autoimmune Conditions:

Autoimmune conditions like type 1 diabetes and systemic lupus erythematosus (SLE) have been connected to enhancer mutations in immune-related genes. Uncontrolled immune responses may arise from variations in enhancers that affect immune cell development or cytokine genes.

Viral Infections:

The host’s reaction to viral infections may also be influenced by enhancers. For example, the expression of antiviral genes may be impacted by immune cell enhancer activation. Chronic viral illnesses can result from dysregulation of these enhancers, which can impair the immune system’s capacity to combat infections.

Targeting Enhancers Therapeutically in Disease Enhancers are appealing targets for therapeutic interventions because of their function in gene control. Among the methods for adjusting enhancer activity are:

Little Molecules:

In disorders brought on by enhancer dysfunction, the development of drugs that target certain epigenetic marks at enhancers, such as acetylation or methylation inhibitors, can aid in the restoration of normal gene expression. Gene editing: Enhancer mutations may be fixed or enhancer activity may be reprogrammed using CRISPR/Cas9 and other genome-editing methods. This strategy shows great promise for diseases like some forms of cancer or genetic abnormalities that are brought on by unique enhancer mutations. Treatments Based on RNA: A potential strategy to modify enhancer activity and restore normal gene expression may be to target enhancer RNAs (Erna’s) with antisense oligonucleotides or other RNA-based therapeutics.

In conclusion Numerous human diseases, including cancer, neurological conditions, metabolic and cardiovascular ailments, are linked to enhancer dysregulation. Developing tailored therapeutic options requires an understanding of the mechanisms via which enhancer mutations and changes cause disease. Enhancers have great potential as biomarkers and therapeutic targets as functional genomics and gene-editing technologies continue to progress.

Fig : 3  Proportional involvements of enhancers in human diseases