Folding Of Domain Research Articles

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx)

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

The Influence of Calcium toward Order/Disorder Conformation of Repeat-in-Toxin (RTX) Structure of Family I.3 Lipase from Pseudomonas fluorescens AMS8.

Calcium-binding plays a decisive role in the folding and stabilization of many RTX proteins, especially for the RTX domain. Although many studies have been conducted to prove the contribution of Ca2+ ion toward the folding and stabilization of RTX proteins, its functional dynamics and conformational structural changes remain elusive. Here, molecular docking and molecular dynamics (MD) simulations were performed to analyze the contribution of Ca2+ ion toward the folding and stabilization of the RTX lipase (AMS8 lipase) structure. AMS8 lipase contains six Ca2+ ions (Ca1–Ca6). Three Ca2+ ions (Ca3, Ca4, and Ca5) were bound to the RTX parallel β-roll motif repeat structure (RTX domain). The metal ion (Ca2+) docking analysis gives a high binding energy, especially for Ca4 and Ca5 which are tightly bound to the RTX domain. The function of each Ca2+ ion is further analyzed using the MD simulation. The removal of Ca3, Ca4, and Ca5 caused the AMS8 lipase structure to become unstable and unfolded. The results suggested that Ca3, Ca4, and Ca5 stabilized the RTX domain. In conclusion, Ca3, Ca4, and Ca5 play a crucial role in the folding and stabilization of the RTX domain, which sustain the integrity of the overall AMS8 lipase structure.

Protein structural and mechanistic basis of progeroid laminopathies.

Progeroid laminopathies are characterized by the premature appearance of certain signs of physiological aging in a subset of tissues. They are caused by mutations in genes coding for A-type lamins or lamin-binding proteins. Here, we review how different mutations causing progeroid laminopathies alter protein structure or protein-protein interactions and how these impact on mechanisms that protect cell viability and function. One group of progeroid laminopathies, which includes Hutchinson-Gilford progeria syndrome, is characterized by accumulation of unprocessed prelamin A or variants. These are caused by mutations in the A-type lamin gene (LMNA), altering prelamin A itself, or in ZMPSTE24, encoding an endoprotease involved in its processing. The abnormally expressed farnesylated proteins impact on various cellular processes that may contribute to progeroid phenotypes. Other LMNA mutations lead to the production of nonfarnesylated A-type lamin variants with amino acid substitutions in solvent-exposed hot spots located mainly in coil 1B and the immunoglobulin fold domain. Dominant missense mutations might reinforce interactions between lamin domains, thus giving rise to excessively stabilized filament networks. Recessive missense mutations in A-type lamins and barrier-to-autointegration factor (BAF) causing progeroid disorders are found at the interface between these interacting proteins. The amino acid changes decrease the binding affinity of A-type lamins for BAF, which may contribute to lamina disorganization, as well as defective repair of mechanically induced nuclear envelope rupture. Targeting these molecular alterations in A-type lamins and associated proteins identified through structural biology studies could facilitate the design of therapeutic strategies to treat patients with rare but severe progeroid laminopathies.

Insight into the Folding and Dimerization Mechanisms of the N-Terminal Domain from Human TDP-43.

TAR DNA-binding protein 43 (TDP-43) is a 414-residue long nuclear protein whose deposition into intraneuronal insoluble inclusions has been associated with the onset of amyotrophic lateral sclerosis (ALS) and other diseases. This protein is physiologically a homodimer, and dimerization occurs through the N-terminal domain (NTD), with a mechanism on which a full consensus has not yet been reached. Furthermore, it has been proposed that this domain is able to affect the formation of higher molecular weight assemblies. Here, we purified this domain and carried out an unprecedented characterization of its folding/dimerization processes in solution. Exploiting a battery of biophysical approaches, ranging from FRET to folding kinetics, we identified a head-to-tail arrangement of the monomers within the dimer. We found that folding of NTD proceeds through the formation of a number of conformational states and two parallel pathways, while a subset of molecules refold slower, due to proline isomerism. The folded state appears to be inherently prone to form high molecular weight assemblies. Taken together, our results indicate that NTD is inherently plastic and prone to populate different conformations and dimeric/multimeric states, a structural feature that may enable this domain to control the assembly state of TDP-43.

Non-native fold of the putative VPS39 zinc finger domain.

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Activation of viral transcription by stepwise largescale folding of an RNA virus genome.

The genomes of RNA viruses contain regulatory elements of varying complexity. Many plus-strand RNA viruses employ largescale intra-genomic RNA-RNA interactions as a means to control viral processes. Here, we describe an elaborate RNA structure formed by multiple distant regions in a tombusvirus genome that activates transcription of a viral subgenomic mRNA. The initial step in assembly of this intramolecular RNA complex involves the folding of a large viral RNA domain, which generates a discontinuous binding pocket. Next, a distally-located protracted stem-loop RNA structure docks, via base-pairing, into the binding site and acts as a linchpin that stabilizes the RNA complex and activates transcription. A multi-step RNA folding pathway is proposed in which rate-limiting steps contribute to a delay in transcription of the capsid protein-encoding viral subgenomic mRNA. This study provides an exceptional example of the complexity of genome-scale viral regulation and offers new insights into the assembly schemes utilized by large intra-genomic RNA structures.

A Systems View of the Genome Guardians: Mapping the Signaling Circuitry Underlying Oligonucleotide/Oligosaccharide-Binding Fold Proteins.

The oligonucleotide/oligosaccharide-binding (OB)-fold domain proteins are considered as genome guardians, whose functions are extending beyond genomic stability. The broad functional diversity of the OB-fold proteins is attributed to their protein-DNA, protein-RNA, and protein-protein interactions (PPI). To understand the connectivity of the human OB-fold proteins, we report here a systems-level approach. Specifically, we mapped all human OB-fold PPI networks and evaluated topological features such as network robustness and network hub, among others. We found that the OB-fold network comprised of 227 nodes forming 5523 interactions, and has a scale-free topology having UBA52, ATR, and TP53 as leading hub proteins that control efficient communication within the network. Furthermore, four different clusters and subclusters have been identified, which are implicated in diverse cellular processes, including DNA replication, repair, maintenance of genomic stability, RNA processing, spermatogenesis, complement system, and telomere maintenance. The importance of these clusters is further strengthened by knockout studies, which showed a significant decrease in topological properties. In summary, this study provides new insights on the role of OB-fold protein as genome guardians in regard to the underlying mechanism of signaling pathways, the roles of key regulators, and thus, offers new prospects as potential targets for diagnostics and therapeutics purposes.

Folding of Truncated Granulin Peptides.

Granulins are a family of unique protein growth factors which are found in a range of species and have several bioactivities that include cell proliferation and wound healing. They typically contain six disulfide bonds, but the sequences, structures and bioactivities vary significantly. We have previously shown that an N-terminally truncated version of a granulin from the human liver fluke, Opisthorchis viverrini, can fold independently into a “mini-granulin” structure and has potent wound healing properties in vivo. The incorporation of a non-native third disulfide bond, with respect to the full-length granulin module, was critical for the formation of regular secondary structure in the liver fluke derived peptide. By contrast, this third disulfide bond is not required for a carp granulin-1 truncated peptide to fold independently. This distinction led us to explore granulins from the zebrafish model organism. Here we show that the mini-granulin fold occurs in a naturally occurring paragranulin (half-domain) from zebrafish, and is also present in a truncated form of a full-length zebrafish granulin, suggesting this structure might be a common property in either naturally occurring or engineered N-terminally truncated granulins and the carp granulin-1 folding is an anomaly. The in vitro folding yield is significantly higher in the naturally occurring paragranulin, but only the truncated zebrafish granulin peptide promoted the proliferation of fibroblasts consistent with a growth factor function, and therefore the function of the paragranulin remains unknown. These findings provide insight into the folding and evolution of granulin domains and might be useful in the elucidation of the structural features important for bioactivity to aid the design of more potent and stable analogues for the development of novel wound healing agents.

Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Contriving Multi-Epitope Subunit of Vaccine for COVID-19: Immunoinformatics Approaches.

COVID-19 has recently become the most serious threat to public health, and its prevalence has been increasing at an alarming rate. The incubation period for the virus is ~1–14 days and all age groups may be susceptible to a fatality rate of about 5.9%. COVID-19 is caused by a novel single-stranded, positive (+) sense RNA beta coronavirus. The development of a vaccine for SARS-CoV-2 is an urgent need worldwide. Immunoinformatics approaches are both cost-effective and convenient, as in silico predictions can reduce the number of experiments needed. In this study, with the aid of immunoinformatics tools, we tried to design a multi-epitope vaccine that can be used for the prevention and treatment of COVID-19. The epitopes were computed by using B cells, cytotoxic T lymphocytes (CTL), and helper T lymphocytes (HTL) base on the proteins of SARS-CoV-2. A vaccine was devised by fusing together the B cell, HTL, and CTL epitopes with linkers. To enhance the immunogenicity, the β-defensin (45 mer) amino acid sequence, and pan-HLA DR binding epitopes (13aa) were adjoined to the N-terminal of the vaccine with the help of the EAAAK linker. To enable the intracellular delivery of the modeled vaccine, a TAT sequence (11aa) was appended to C-terminal. Linkers play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable. The secondary and three-dimensional (3D) structure of the final vaccine was then predicted. Furthermore, the complex between the final vaccine and immune receptors (toll-like receptor-3 (TLR-3), major histocompatibility complex (MHC-I), and MHC-II) were evaluated by molecular docking. Lastly, to confirm the expression of the designed vaccine, the mRNA of the vaccine was enhanced with the aid of the Java Codon Adaptation Tool, and the secondary structure was generated from Mfold. Then we performed in silico cloning. The final vaccine requires experimental validation to determine its safety and efficacy in controlling SARS-CoV-2 infections.

Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking.

Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides’ ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.

Amyloid aggregation of spin-labeled β-lactoglobulin. Part II: Identification of spin-labeled protein and peptide sequences after amyloid aggregation

Site-directed spin labeling (SDSL) of natural β-lactoglobulin (β-lg) was established. Combined electron paramagnetic resonance (EPR) and mass spectrometric analysis following tryptic digestion demonstrated that spin labels bind site-specifically but are not directed to all five cysteine residues to various preferred and reproducible extents. MTSSL and iodoacetamido-proxyl spin label (IPSL) were 80 and 60% reliably bound to the H strand, respectively, and combined in one spectral component and buried in the protein core. After heat incubation at pH 2 and fractionation, all labeled side chains (peptides) were part of the amyloid and non-amyloid fractions, even if they could not detect amyloid structures. It was assumed that the IPSL-labeled side chains of peptides with Cys160 from random coil were incorporated into small non-amyloid aggregates in non-polar environments. After heating at pH 3.5, a rearrangement of the previous α-helix was assumed to shift from the autonomous folding domain during partial unfolding, which improved the accessibility of β-sheets to the water/DMSO-environment. β-sheets were likely densely packed by the accumulation of intermolecular β-sheets, which suggests that amyloid-like structures can be formed from building blocks of the entire primary β-lg structure. Double electron-electron resonance (DEER) confirmed that the spatial distribution of labels within the amyloid-like fraction in a one-dimensional arrangement of the entire protein aggregates was similar to a string of pearls. Thus, SDSL of proteins containing several cysteine residues can be used to gain deep insights into the aggregation mechanism of proteins under food processing conditions.

NC2 complex is a key factor for the activation of catalase-3 transcription by regulating H2A.Z deposition

Negative cofactor 2 (NC2), including two subunits NC2α and NC2β, is a conserved positive/negative regulator of class II gene transcription in eukaryotes. It is known that NC2 functions by regulating the assembly of the transcription preinitiation complex. However, the exact role of NC2 in transcriptional regulation is still unclear. Here, we reveal that, in Neurospora crassa, NC2 activates catalase-3 (cat-3) gene transcription in the form of heterodimer mediated by histone fold (HF) domains of two subunits. Deletion of HF domain in either of two subunits disrupts the NC2α–NC2β interaction and the binding of intact NC2 heterodimer to cat-3 locus. Loss of NC2 dramatically increases histone variant H2A.Z deposition at cat-3 locus. Further studies show that NC2 recruits chromatin remodeling complex INO80C to remove H2A.Z from the nucleosomes around cat-3 locus, resulting in transcriptional activation of cat-3. Besides HF domains of two subunits, interestingly, C-terminal repression domain of NC2β is required not only for NC2 binding to cat-3 locus, but also for the recruitment of INO80C to cat-3 locus and removal of H2A.Z from the nucleosomes. Collectively, our findings reveal a novel mechanism of NC2 in transcription activation through recruiting INO80C to remove H2A.Z from special H2A.Z-containing nucleosomes.

Structural study of the flagellar junction protein FlgL from Legionella pneumophila

Legionella pneumophila is a flagellated pathogenic bacterium that causes atypical pneumonia called Legionnaires’ disease. The flagellum plays a key role in the pathogenesis of L. pneumophila in the host. The protein FlgL forms a junction between the flagellar hook and filament and has been reported to elicit the host humoral immune response. To provide structural insights into FlgL-mediated junction assembly and FlgL-based vaccine design, we performed structural and serological studies on L. pneumophila FlgL (lpFlgL). The crystal structure of a truncated lpFlgL protein that consists of the D1 and D2 domains was determined at 3.06 Å resolution. The D1 domain of lpFlgL adopts a primarily helical, rod-shaped structure, and the D2 domain folds into a β-sandwich structure that is affixed to the upper region of the D1 domain. The D1 domain of lpFlgL exhibits structural similarity to the flagellar filament protein flagellin, allowing us to propose a structural model of the lpFlgL junction based on the polymeric structure of flagellin. Furthermore, the D1 domain of lpFlgL exhibited substantially higher protein stability than the D2 domain and was responsible for most of the antigenicity of lpFlgL, suggesting that the D1 domain of lpFlgL would be a suitable target for the development of an anti-L. pneumophila vaccine.

Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Structural and Functional Characterisation of the Domains of Ubiquitin-Activating Enzyme (E1) of Saccharomyces cerevisiae.

Ubiquitin-activating enzyme (E1) is the first enzyme of the ubiquitination pathway and is required to activate ubiquitin. E1 of Saccharomyces cerevisiae is a large multidomain monomeric protein. There are no studies available on the domains of yeast E1 as independent entities. Four domains of E1 namely, first catalytic cysteine half-domain (FCCH), four-helix bundle (4HB), second catalytic cysteine half-domain (SCCH) and ubiquitin fold domain (UFD) were characterised to understand their structural and functional independence vis-a-vis full length E1. Spectroscopic characterisation using circular dichroism and fluorescence suggested that these domains can act as independent folding units and attain native-like secondary structure. The structural features obtained with the peptides SCCH and FCCH of S. cerevisiae bear a high degree of structural similarity to the corresponding fragments of mouse from literature. Nearly 50% of the residues of the 4HB domain of the S. cerevisiae sample showed helical conformation. They displayed a high degree of conservation when compared with 4HB of mouse with respect to their identity and arrangement. The fragment UFD of yeast formed an α/β domain as in the whole protein and exhibited 45% homology with that of mouse, showing a similar arrangement of α and β elements in its secondary structure. Overexpression of the domains in vivo indicated that the SCCH domain and to some extent UFD apparently interfere with cellular functions such as survival under various stresses.

Crystallographic Structure Determination of Bacteriophage Endolysins.

Bacteriophages produce endolysins that target and cleave the hosts peptidoglycan to release their progeny at the end of the infection cycle. These proteins can be used for the eradication of pathogenic bacteria, but also for their detection. Endolysins may contain a single catalytic domain or several domains, including a cell wall binding domain. To understand their function in detail and design mutated or chimeric molecules with novel properties, knowledge of their structures and detailed mechanisms is necessary. X-ray protein crystallography is an excellent method to obtain high-resolution structures of biological macromolecules, and here we describe the method and the folds of known endolysin domains.

Non-cooperative 4E-BP2 folding with exchange between eIF4E-binding and binding-incompatible states tunes cap-dependent translation inhibition

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.

Cotranslational folding cooperativity of contiguous domains of α-spectrin

Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using force profile analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other's folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in piconewtons. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of ∼15 pN during cotranslational folding.

The New Frontier of Functional Genomics: From Chromatin Architecture and Noncoding RNAs to Therapeutic Targets.

Common diseases are complex, multifactorial disorders whose pathogenesis is influenced by the interplay of genetic predisposition and environmental factors. Genome-wide association studies have interrogated genetic polymorphisms across genomes of individuals to test associations between genotype and susceptibility to specific disorders, providing insights into the genetic architecture of several complex disorders. However, genetic variants associated with the susceptibility to common diseases are often located in noncoding regions of the genome, such as tissue-specific enhancers or long noncoding RNAs, suggesting that regulatory elements might play a relevant role in human diseases.Enhancers are cis-regulatory genomic sequences that act in concert with promoters to regulate gene expression in a precise spatiotemporal manner. They can be located at a considerable distance from their cognate target promoters, increasing the difficulty of their identification. Genomes are organized in domains of chromatin folding, namely topologically associating domains (TADs). Identification of enhancer–promoter interactions within TADs has revealed principles of cell-type specificity across several organisms and tissues.The vast majority of mammalian genomes are pervasively transcribed, accounting for a previously unappreciated complexity of the noncoding RNA fraction. Particularly, long noncoding RNAs have emerged as key players for the establishment of chromatin architecture and regulation of gene expression.In this perspective, we describe the new advances in the fields of transcriptomics and genome organization, focusing on the role of noncoding genomic variants in the predisposition of common diseases. Finally, we propose a new framework for the identification of the next generation of pharmacological targets for common human diseases.