Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failure in pulmonary arterial hypertension (PAH). None of the established vasodilator drug therapies for PAH prevents or reverse established arterial wall thickening, stiffening, and hypercontractility. Therefore, new approaches are needed to achieve long-acting prevention and reversal of occlusive pulmonary vascular remodeling. Several promising new drug classes are emerging from a better understanding of pulmonary vascular gene expression programs. In this review, potential epigenetic targets for small molecules and oligonucleotides will be described. Most are in preclinical studies aimed at modifying the growth of vascular wall cells in vitro or normalizing vascular remodeling in PAH animal models. Initial success with lung-directed delivery of oligonucleotides targeting microRNAs suggests other epigenetic mechanisms might also be suitable drug targets. Those targets include DNA methylation, proteins of the chromatin remodeling machinery, and long noncoding RNAs, all of which act as epigenetic regulators of vascular wall structure and function. The progress in testing small molecules and oligonucleotide-based drugs in PAH models is summarized. The set of proteins expressed and the abundance of proteins in cells of the vascular wall is a complex function of transcription, translation, and regulation of protein lifespan. In addition to the heritable protein-coding sequences in DNA, there are epigenetic processes in somatic cells that modify gene expression within a generation to define the somatic epitype  1]. In . In 1]. Epigenetic therapies could be developed that are effective with tolerable off-target effects. To establish appropriate drug targets, the timing and the necessity for particular epigenetic adaptations in developing pulmonary hypertension need to be defined. Many new drug targets have been described in preclinical studies which are summarized in this review and in several recent reviews of the same topic ,3,4,5,6.Targeting epigenetic mechanisms is appealing because enzymatically catalyzed reactions are often reversible, and the enzymes are targets for small molecules and oligonucleotide drugs. This is in sharp contrast to mutations in genomic and mitochondrial DNA, which are not routinely correctable in a clinical setting, although remarkable advances in gene editing technology have created new opportunities that are advancing rapidly . This rePulmonary arterial hypertension (WHO Group I) is a rare, progressive pulmonary vascular disease characterized by elevated pulmonary artery pressure (>25 mm Hg), dyspnea, exercise intolerance, and ultimately right heart failure. In the USA, it is more common in women than men (3–5:1) and in Caucasians (85%) compared to African-Americans 12%) and Hispanics (<3%) % and His,14,15. IBMPR2) expression and function in subjects with familial PAH? In pulmonary vascular remodeling and repair, the druggable targets include enzymes that catalyze DNA methylation and demethylation, enzymes of DNA repair pathways, enzymes that catalyze histone posttranslational modifications, and noncoding RNAs. Most studies described below are early-stage preclinical trials arranged by biochemical and molecular classes.There are several issues to address when developing therapies to reprogram the somatic epitype in order to prevent or reverse vascular remodeling. Which processes in which cells should be targeted ? What arGLYN) occurs in peripheral blood monocytes of human subjects with pulmonary veno-occlusive disease, but not in patients with PAH [GLYN genes in several T-cell populations  were not altered in PAH. This suggests some disease-restricted effects on immune cell gene silencing that offer selective targets for drugs altering DNA methylation. Further work in PAH samples is required to find analogous PAH-restricted methylation patterns.One of the major determinants of whether a gene is transcribed is methylation of DNA, typically but not exclusively in CpG islands in promoter regions . DNA metwith PAH . The autBMPR2 promoter in scleroderma patients is another relevant observation [BMPR2 promoter methylation and reduced BMPR2 expression. Later studies of BMPR2 silencing by DNA methylation in PAH patients are mixed. One study of peripheral blood cell DNA reported no methylation of the BMPR2 promoter [TET2) gene coding for a DNA demethylase have been reported in humans with PAH. Furthermore, TET2 knockout mice develop a mild form of PAH [Methylation of the ervation . Patientpromoter , but a mpromoter . Severalm of PAH . These lm of PAH . SimilarThe timing of a therapeutic intervention that reduces DNA methylation will be important to establish. If changes in DNA methylation occur prior to diagnosis (drivers) the damage may be difficult to reverse versus ongoing DNA methylation during the progression of the disease (adaptive responses). It is not clear whether DNA methylation can be selectively modulated with drugs, but there is some reason for optimism. De novo DNA methylation is dynamic and reversible by the action of demethylases. Blocking DNMT activity might be effective in allowing vascular repair, as shown by Archer and coworkers using 5-azacytidine in a rat model of PAH . This stPost-translational modifications of histones control chromatin structure by charge effects and by recruiting additional chromatin remodeling enzymes . In geneA growing set of pharmacological studies implicate histone acetylation in PAH by virtue of the effects of HDAC inhibitors on vascular remodeling and cardiac function. Detailed progress at the molecular level is somewhat limited by the modest understanding of exactly which genes to focus on. Some of the earliest evidence supporting HDACs in pulmonary hypertension is from a study of the bovine model of hypoxic pulmonary hypertension . ApicidiNOX gene promoters [Inhibiting HDACs in PAH might be beneficial in part because it alters ROS production. In macrophages and THP-1 cells, HDAC inhibitors reduced NOX 1, 2, and 4 expression of macrophages and THP-1 cells by reducing Pol II and p300 HAT loading on romoters . H3K4me3romoters . The claromoters ,37. Someromoters . StudiesThere are only a few studies of histone acetyltransferases in PAH. It is known that histone acetyltransferase activity is higher in lung tissue of PAH patients , but it Methylation of histones has a cooperative effect with DNA methylation to favor heterochromatin formation and gene repression . During ClinicalTrials.gov Identifier: NCT03655704). Recent reports of cardiac toxicity temper enthusiasm for broad-spectrum BET inhibitors [Bromodomain-containing proteins bind acetylated lysine residues on histones and function as transcriptional coactivators in the histone code “reader” machinery ,46. Vaschibitors . Isoformhibitors .In contrast to the significant literature on small molecule inhibitors of histone remodeling in PAH animal models, there is a lack of studies of oligonucleotides targeting HDACs, HATs, or BET proteins in vascular tissues. Although there are numerous antisense oligonucleotides available to modify chromatin remodeling machinery in vitro, there are few or no preclinical drug development studies of PAH models. Drug delivery and tissue specificity are two important problems slowing the development of new oligonucleotide drugs. These two issues have been addressed to some extent by recent studies of oligonucleotides targeting microRNAs in animal models of PAH.Several classes of noncoding RNAs regulate gene expression and protein abundance to fine-tune cell phenotypes in conditions of health and disease . MicroRNNumerous correlative studies of miRNA expression in vascular tissue from humans have defined altered miRNA expression patterns in PAH. The miR-17~92 cluster, MiR-21, miR-145, and miR-204 were the first miRNAs associated with PAH ,52,53,54One limitation of initial studies of miRNA mimics and antagonists is that none used intravenous therapy to achieve lung-directed delivery. They also used prevention protocols rather than a rescue protocol to reverse severe occlusive vascular remodeling. This limitation is relevant to drug therapy because humans are diagnosed with PAH after it is well established, not prior to the development of arteriopathy and cardiac dysfunction. A later reversal study  employinOne question that remains unanswered is which cell types should be targeted in treating PAH with oligonucleotide drugs? Many mechanistic studies of miRNAs in pulmonary artery remodeling have focused on endothelial cells and vascular smooth muscle cells. However, some miRNAs associated with PAH in humans  also regulate inflammation and immune cell biology. For example, IL-6 upregulates expression of the miR-17~92 cluster, which targets BMPR2, suggesting that inflammation exacerbates PAH by inhibiting BMPR2 signaling. MiR-145 is known to regulate cell fate during early embryogenesis and is a master regulator of smooth muscle contractile phenotype. It also regulates the immune response by promoting M1 to M2 macrophage polarization  and Th2 Long RNAs (>200nt) not translated to proteins (lncRNA) are transcribed from a variety of sites in the nuclear genome. There are lncRNAs from enhancer sequences of protein-coding genes (eRNAs) , RNAs frMALAT1 gene have been described in humans with essential hypertension [Although recent observational studies reveal the association between lncRNAs and PAH, it will be important to determine what strategies are used to modify lncRNA expression and function in the pulmonary circulation. Antisense oligonucleotides (“antagoNATs”) that inactivate lncRNAs in cells and in animal models of disease have been proposed  and usedrtension . MALAT1 rtension , antiprortension .The prospect of modifying epigenetic processes to alter the interaction of genes and the environment in pulmonary vascular cells is exciting and important. The premise is that cell phenotype and vessel architecture are determined by reversible biochemical processes acting on the genetic substrate. This is the mechanistic basis for several anticancer drugs that modify the epigenetic processes described in this review. In cancer chemotherapy, single epigenetic modifying drugs and combination therapies that include epigenetic modifiers are effective in some cases, but ideal drug regimens are still being defined. Successful epigenetic reprogramming in lung cancer chemotherapy is very encouraging and highly relevant to epigenetic therapy for PAH ,94. If nThere are still significant gaps in knowledge that limit the development of epigenetic modifying oligonucleotide drugs. We need better descriptions of which miRNAs, lncRNAs, circRNAs, histone modifications, and DNA methylation sites are present at the single-cell level in PAH. We also need important longitudinal studies of epigenetic features in individual pulmonary vascular wall cells and in key genes controlling cell phenotype . Serial


The review describes the role of adiponectin in liver diseases in the presence and absence of surgery reported in the literature in the last ten years. The most updated therapeutic strategies based on the regulation of adiponectin including pharmacological and surgical interventions and adiponectin knockout rodents, as well as some of the scientific controversies in this field, are described. Whether adiponectin could be a potential therapeutic target for the treatment of liver diseases and patients submitted to hepatic resection or liver transplantation are discussed. Furthermore, preclinical and clinical data on the mechanism of action of adiponectin in different liver diseases  in the absence or presence of surgery are evaluated in order to establish potential targets that might be useful for the treatment of liver disease as well as in the practice of liver surgery associated with the hepatic resections of tumors and liver transplantation. Herein, we review the preclinical and clinical studies reported over the last ten years that investigate the mechanisms of action of adiponectin in liver diseases, in both the presence and absence of surgery. Potential targets and pharmacological strategies aimed at regulating adiponectin in liver diseases are also discussed. Our reasons for this research are threefold. Firstly, there is a need for useful pharmacological strategies to treat nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) and avoid progression to cirrhosis and cancer. Secondly, given the prevalence of NAFLD in both society at large and liver transplantation (LT) donation (30% in cadaveric and 20% in living donors), NAFLD pathology has become a major focus of scientific and clinical research ,2. SpeciAdiponectin is a 28 kDa protein hormone including 274 amino acids encoded by the AdipoQ gene. Adiponectin exists in plasma as three distinct oligomeric complexes: the homotrimer , the hexamer  and 12–18 protomer  .The total and distribution of all three adiponectin isoforms  are decreased in NAFLD patients without any surgical intervention . HMW adiDifferent studies of liver diseases related to NAFLD/NASH without any surgical intervention have suggested that adipose tissue is a major site of endogenous adiponectin production ,13,14,15Adiponectin enhances the binding of APPL1  to the two adiponectin receptors (adipoR1 and adipoR2). These interactions are essential for subsequent adiponectin actions ,19. HoweAs mentioned above, the data suggest that the role of adiponectin depends on the surgical conditions and type of liver. Thus, adiponectin is injurious to steatotic livers submitted to 60 min of normothermic ischemia . HoweverIn obesity, it has been reported that adiponectin protects liver from steatosis and inflammation: it increases the capacity of insulin to suppress glucose production . A diet It should be also noted that adiponectin reduces chronic inflammation in target organs including vasculature, lung and heart, thereby leading to protection against various obesity-related disorders . HoweverThe effects of adiponectin in steatotic livers undergoing surgery and on various obesity-related disorders should not be ignored. Indeed, the different signaling pathways such as AMPK and PPARα involved in the anti-steatotic effects of adiponectin also play a role in protecting steatotic livers undergoing I/R against damage .. Indeed, in vitro studies based mainly on culture-activated HSCs suggest that hepatic fibrosis and its resolution are controlled by adiponectin and leptin.Hepatic fibrosis is the last prevalent pathway prior to cirrhosis and eventually requires LT. Among the different adipocytokines (retinol binding protein 4 (RBP4), resistin, apelin, chemerin, vaspin, etc.), adiponectin is one chiefly involved in hepatic fibrosis . In patiAdiponectin has strong-antifibrotic features whereas leptin acts as a profibrogenic molecule. This is because adiponectin prevents leptin signal transduction . In factIn a choline-deficient mouse NASH model, low levels of adiponectin have been reported , althougIn an experimental model of LT, the administration of adiponectin reduced hepatic injury and increased regeneration in both steatotic and non-steatotic grafts submitted to 6 h of cold ischemia . AdiponePharmacological strategies aimed at modulating the actions of adiponectin are ineffective in non-steatotic livers from Zucker rats undergoing 60 min of warm ischemia, as well as in surgical procedures requiring liver regeneration such as partial hepatectomy with I/R . HoweverRegarding the reported effects of adiponectin on liver regeneration, treatment with the anti-diabetic drug rosiglitazone, which is thought to act by elevating serum adiponectin, inhibits liver mass recovery . NonetheIn a study using rats fed ethanol, the anti-inflammatory effects of adiponectin were mediated by regulation of toll-like receptor 4 (TLR4) signaling via the myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) pathways  Table 2Table 2. It has also been suggested that one explanation for the absence of a NAFLD phenotype might be upregulation of adiponectin, which activates the AMPK–forkhead box protein O (FOXO) signaling axis and probably overrides detrimental oxidative stress and JNK signaling  Figure .Decreased serum levels of adiponectin and decreased gene expression of ileum fibroblast growth factor 15 (FGF15) have been reported in chronic ethanol fed mice, whereas elevated levels of circulating adiponectin and FGF15 protected against inflammation and liver damage ,70. ThesLactobacillus gasseri (SBT 2055) reduced abdominal visceral fat and increased serum adiponectin in obese people [Pharmacological intervention aimed at elevating adiponectin levels have been reported as promising for the treatment and progression of NAFLD . Thus, oe people . Thus, te people ,82,83,84e people ,86  require a certain pretreatment time before they exert their effects, and this is a major problem in LT from brain-dead or cardiac-arrest donors. The potential role of adiponectin in connecting the intestine and liver is scientifically and clinically interesting, and so is its role in regulating FGF15/19 signaling, which is of potential interest due to the benefits on bile acid homeostasis, a critical problem in liver diseases including NASH, cirrhosis and LT. However, appropriate experimental models are required to elucidate the relevance of adiponectin-FGF15/19 in liver diseases and surgery. This is also the case with the effects of adiponectin in fibrosis, since the results are mainly based on in vitro studies, far removed from clinical practice.Different clinical results have been reported in relation with the levels of adiponectin and the pathological characteristics of NAFLD/NASH and its progression. In the context of hepatic resections and LT, the research into prognostic factors are of clinical and scientific relevance for the treatment of patients but the reported results are very limited and inconclusive. In our view, whether the levels of adiponectin reported in liver diseases might be the result of or the reason for the pathology, itself remains to be elucidated. Potential differences in the levels of adiponectin in liver diseases depending on gender should be considered . In addition, although adiponectin might be elevated in liver diseases, it might not exert protection if its receptor is downregulated or the adiponectin signaling pathway is dysfunctional. In addition, in line with other authors, in our view, adiponectin expression may initially be elevated to compensate disease progression, but then higher adiponectin levels could turn out to be ineffective because of an overall deterioration of the patient’s condition . All of


This Review depicts the evolving role of MRI in the diagnosis and prognostication of anomalies of the fetal body, here including head and neck, thorax, abdomen and spine. A review of the current literature on the latest developments in antenatal imaging for diagnosis and prognostication of congenital anomalies is coupled with illustrative cases in true radiological planes with viewable three-dimensional video models that show the potential of post-acquisition reconstruction protocols. We discuss the benefits and limitations of fetal MRI, from anomaly detection, to classification and prognostication, and defines the role of imaging in the decision to proceed to fetal intervention, across the breadth of included conditions. We also consider the current capabilities of ultrasound and explore how MRI and ultrasound can complement each other in the future of fetal imaging. Fetal movements can cause artifacts in MRI and various approaches are used to reduce their negative effect on images, including adjustment of acquisition parameters. However, such adjustment limits the detail derived from the scan (panel). Modern post-acquisition reconstruction techniques allow for motion-correction, three-dimensional (3D) segmentation, and volumetry. The refinement of these techniques has given fetal MRI a well defined role in the assessment of fetal CNS anomalies,Fetal body MRI is now complementary to ultrasound for the assessment of head and neck masses, anterior abdominal wall lesions, and thoracic or abdominal pathology. Many of these body regions have been particularly challenging to image, owing to fetal movement or the deformable nature of the cavity wall. This problem has limited the role of MRI to the assessment of contiguous but misaligned two-dimensional (2D) slices, which has clear disadvantages in the assessment of subtle abnormalities and undersells the potential of MRI for providing 3D data. This illustrative Review summarises current advances in MRI for malformations of the fetal body that might be amenable to fetal or early postnatal intervention. The widely recognised gold standard of fetal ultrasound is used as a comparator, and the circumstances are shown in which the two methods can complement each other in the assessment of challenging cases.5This Review covers lesions of the head and neck, thorax, abdomen and pelvis, and spine, for which MRI has a less established role than for lesions of the fetal brain or heart. Each section contains several illustrative case reports with relevant antenatal history and information derived from images. New image reconstruction techniques are shown that could further increase the benefits of MRI; specifically illustrative examples of deformable slice-to-volume registration, which allows reconstruction of high-resolution 3D images in modifiable planes and 3D segmentation to improve accuracy of volume measurement .56 The predominant concern is that of neonatal airway compromise, and the advent of ex-utero intrapartum treatment has enabled life-saving measures for the fetus with a critical airway. These measures involve doing a carefully planned airway intervention while maintaining feto–placental circulation until the fetus can be separated from the umbilical cord to complete the caesarean section delivery. Identifying which fetuses might need ex-utero intrapartum treatment remains a challenge, and requires referral to specialised centres that can coordinate the necessary investigation and intervention.7 Evaluation by fetoscopy is an alternative means of imaging to assess the fetal airway; however, the associated procedural risks should be recognised.8 However, although ex-utero intrapartum treatment in specialist centres has proved to be successful, it is an invasive procedure with substantial risks to both mother and fetus. Maternal and fetal factors, and amount of institutional expertise, should be considered, with the availability of accurate imaging clearly being important.Most antenatally diagnosed neck masses are either lymphatic malformations  or terat9 and this is important if considering the need for a tracheostomy, given that passage of the tumour anterior to the trachea might render this procedure infeasible. A large retrospective series showed that fetuses requiring ex-utero intrapartum treatment could be predicted using MRI-measured dimensions of polyhydramnios and evidence of mass effect on the trachea, with a sensitivity of 95% and a specificity of 80%, although tumour size itself was not a predictive factor.10For fetal neck masses, MRI provides a clear advantage over conventional ultrasound for assessing tumour extension and giving a 3D visualisation of the tumour’s relation to the airway . MRI can11 because it can be more sensitive than 3D ultrasound in detecting cases of isolated cleft palate,12 for which the absence of an associated labial defect could render diagnosis by ultrasound difficult.For assessing bony defects such as orofacial clefts, the value of MRI is generally surpassed by that of 3D ultrasound, because the latter gives a detailed depiction of the fetal face. However, MRI is still of use in suspected cases,3. Lateral displacement of the tongue and effacement of the oropharyngeal cavity were appreciable on the fetal ultrasound scan. MRI was able to establish the margins of the fetal airway and supported the decision to give ex-utero intrapartum treatment, in which video-assisted oral intubation was done before neonatal tracheostomy. Detailed illustration of the 3D reconstruction of these two lesions is provided in 13 Congenital pulmonary airway malformations can be solid or cystic; fetal ultrasound can reasonably reliably define these lesions and differentiate them from broncho-pulmonary sequestrations on the basis of a characteristic systemic arterial blood supply identified using colour doppler  can reliably produce volumetric measurement, even from motion corrupted sequences  along with the remaining total lung volume (12·82 cm3). There was displacement of the mediastinum, including the heart, into the right hemithorax; however, notably there was no hydrops. Detailed illustrations of the 3D reconstructed image and segmentations are in 3). Detailed illustrations of the 3D reconstructed image and segmentations can be seen in 3. The patient had postnatal repair on day 6 of life and is currently being followed-up at age 3 years. Detailed illustrations of the 3D reconstructed image and segmentations can be seen in 30 A systematic review found that MRI used in conjunction with ultrasound provided sensitivity greater than 94% and specificity of 89%.31 Findings of dilated upper oesophagus with bowing of the trachea can be readily seen on MRI  is a spectrum of disorders that has an overall prevalence of about one in 3000. There is usually an associated fistula, ligation of which is necessary in the first days of life, ideally with simultaneous repair of the atresia. Currently, fetal ultrasound manages to identify oesophageal atresia in less than a third of cases. Findings of a small stomach and polyhydramnios elevate the index of suspicion, but confirmation of a oesophageal atresia diagnosis is rarely possible because of the low specificity of these findings, which can be related to oropharyngeal or mediastinal masses, poor fetal swallowing, or simply a normal variant.n on MRI  and have34 The Review authors anticipate that reliable and accurate diagnosis of such cases could create a role for tissue-engineered constructs, similar to what has been achieved with congenital diaphragmatic hernias.29When the authors of this Review used post-acquisition reconstruction, we were unable to improve upon the diagnostic accuracy of 2D MRI images because the averaging process of reconstruction algorithms can potentially reduce any appearance of distension in an organ with peristalsis. The Review authors found that with improved image acquisition , the dis35 However, a small proportion enlarge or cause problems owing to mass effect,36 and non-ovarian cysts are still challenging to diagnose on ultrasound scans.37 These rarer diagnoses might require early specialist referral—eg, in cases of choledochal malformation, lymphatic malformation, or fetal tumour. Antenatal counselling requires reliable delineation of the cyst and its spatial and anatomical relations. A small series suggested that the spatial resolution of MRI in 3D allows for more accurate anatomical delineation and improved diagnostic accuracy compared with ultrasound.38 However, caution should be used regarding absolute reliance on MRI, because two cases in the series were incorrectly reclassified after MRI, including a case of adrenal haemorrhage. It is worth noting that modern T2-weighted MRI sequences are haemorrhage-sensitive, but although widely used in placental MRI research,39 these sequences have not been optimised for fetal clinical application.Abdominopelvic cysts are a common finding on fetal ultrasound scans, documented in approximately one per 1000 pregnancies, and are typically ovarian in origin. The clinical postnatal outcomes of antenatally diagnosed cysts have been well described; many will involute during antenatal or early postnatal life, and they are generally managed expectantly.40 Accurate sizing of the defect is another advantage of MRI, because defects larger than 5 cm can be associated with prolonged respiratory morbidity, and to morbidity related to feeding associated with the process of restoring abdominal organs and achieving abdominal closure.Fetal ultrasound is usually able to diagnose, and differentiate between, common abdominal wall defects, exomphalos, and gastroschisis. This distinction is important, because the antenatal counselling and post-natal outcomes of each of these conditions is markedly different. Exomphalos, with a prevalence of one per 13 000, is commonly associated with other midline structural defects that can be shown on ultrasound  an absence of antenatal diagnostic features, such as rectal dilatation. However, the spatial relationship of urogenital and intestinal structures in the fetal pelvis have enabled cloacal anomalies to be diagnosed with reasonable reliability using MRI.The 53 Defining the consistency of the overall tumour regarding its solid or cystic proportion also carries important prognostic relevance in terms of tumour grade; solid, hypervascularised masses can lead to fetal cardiac failure, and are candidates for fetal intervention involving debulking or expedited delivery.56 MRI is useful for showing the internal characteristics of the tumour, its intrapelvic extent, and involvement of the spinal canal.57 Also, MRI is particularly useful for distinguishing the fat tissue of the mass from the surrounding pelvic side wall and the meconium content of the fetal bowel  leads to a typical banana-shaped cerebellum with a lemon-shaped sculpting of the skull, both commonly seen on mid-gestation ultrasound. The vertebral defect and herniated content are usually also seen, and sonographic assessment of lower limb and bladder function can also be made. First trimester ultrasound diagnosis is also feasible using the so-called crash sign, which describes posterior displacement and deformation of the mesencephalon against the occipital bone in axial view.60 Here fetal MRI allows the spinal defect extent to be assessed with similar accuracy to ultrasound . MRI can be used to predict long-term lower limb, bladder, and bowel function. MRI is also a useful adjunct to a fetal ultrasound that has detected closed neural tube defects, in providing further characterisation of spinal cord anomalies, such as tethering.64 Lower urinary tract obstruction can result in a reduced amniotic fluid volume, which can make ultrasound examination of the underlying lesion difficult. However, the paucity of amniotic fluid is of little consequence in MRI acquisition and reconstruction. Management is complex, because a distended bladder in the first trimester often either empties spontaneously or resolves after a single drainage guided by ultrasound65 and the benefit of more invasive treatments  is unclear, as shown by the PLUTO trial.66 There is recognition among fetal interventionalists that better patient selection for bladder shunting is needed. This selection requires antenatal assessment of both renal parenchyma and amniotic fluid volume, which can be done with modern MRI acquisition and processing.68 Although fetal MRI is not a gold standard for diagnosis, it is a recognised useful adjunct to fetal ultrasound in the evaluation of identified pathology.70Hydronephrosis is a common finding on fetal imaging, with a prevalence of 1% of all pregnancies. The condition might be self-limiting and idiopathic; however, it can also be a consequence of several different abnormalities of the renal collecting system and ureter, or of lower urinary tract obstruction. Posterior urethral valves can be shown on fetal MRI, with the characteristic appearance of a dilated posterior urethra. A finding of a dilated bladder, or bilateral antenatal hydronephrosis, or both, requires careful investigation, because fetal megacystis can present in several chromosomal abnormalities.71 Diagnosis of bilateral renal agenesis is a further condition in which MRI might be useful, because on ultrasound, in the presence of anhydramnios, the adrenal glands can be mistaken for renal tissue, whereas MRI is able to better indicate that the kidneys are absent.72Fetal MRI has further prognostic value, in assessing the renal parenchyma in detail for the presence of cysts and parenchymal dysplasia, detailed in the illustrative cases that follow. Furthermore, MRI can help to measure lung volumes in the context of severe oligohydramnios, in which lung hypoplasia presents an extreme risk to a neonate’s survival.Two examples of hydronephrosis and one of unilateral multicystic dysplastic kidney are used. The Fetal MRI has been shown to complement fetal ultrasound in many conditions, and to be superior to ultrasound in others because of its precise delineation of body anatomy. Wider adoption of fetal MRI could accelerate its validated use for the diagnosis and investigation of many fetal anomalies, such as those discussed in this Review, and facilitate routine referrals through established pathways. MRI also overcomes several technical limitations of ultrasound, such as atypical fetal position, reduced amniotic fluid volume, or high maternal body-mass index.Our unit has experience in doing postacquisition reconstruction on externally acquired MRI images; however, these outputs depend on the quality of the original images and acquisition of sufficient image stacks in all three planes. Although the deformable slice-to-volume registration reconstruction tool is already available for download, further technical developments are needed to support its full clinical translation, including robustness to fetal motion, and evaluation of the effects on volumetric measurements. It is important to reiterate that reconstructed volumes should be used in conjunction with 2D images for diagnosis, because the averaging process might occlude visualisation of dynamic physiological processes such as peristalsis or cause blurring owing to peristaltic motion.A benefit common to all conditions discussed in this Review is the communicability of the images gained with MRI; 3D ultrasound gives excellent spatial resolution of craniofacial and skeletal anomalies but (for non-expert sonographers) involves greater difficulty in interpreting soft tissue lesions within the thorax and abdomen. The cross-sectional images obtained on conventional MRI are readily interpretable for a surgeon, and with progress made in 3D reconstruction, are likely to guide treatment with increased precision.73 We believe that fetal MRI has the potential to aid fetal medicine, and assist neonatal and paediatric surgical specialists in diagnosis, prognostication, parental counselling, and planning prenatal and postnatal care of babies with congenital anomalies.The superiority of MRI to ultrasound in prognostication is supported by data in only a few conditions at present, reflecting the current early stage of development of fetal body MRI.Supplementary AppendixSupplementary video 1. Cervical lymphatic malformation (from figure 1A)Supplementary video 10. Sacrococcygeal teratoma (from appendix p 3)Supplementary video 11. Open spina bifida (from figure 5A)Supplementary video 12. Open spina bifida (from figure 5A)Supplementary video 13. Hydronephrosis Supplementary video 14. Multicystic dysplastic kidney Supplementary video 3. Congenital pulmonary airway malformation (from figure 2A)Supplementary video 4. Congenital pulmonary airway malformation (from figure 2A)Supplementary video 5. Bronchopulmonary pulmonary sequestration and (from figure 2B)Supplementary video 6. Left congenital diaphragmatic hernia (from figure 2C)Supplementary video 7. Left congenital diaphragmatic hernia (from figure 2C)Supplementary video 8. Exomphalos and left congenital diaphragmatic hernia (from figure 4A)Supplementary video 9. Exomphalos and left congenital diaphragmatic hernia (from figure 4A)upplementary video 2. Oral teratoma (from figure 1B)


One such example is the C2H2-type zinc finger, AtSTOP1, which is a transcriptional regulator of a number of Arabidopsis Al tolerance genes, including AtMATE and AtALMT1, and has been shown to activate AtALMT1, not only in response to Al but also low soil P. The large WRKY family of transcription factors are also known to affect a broad spectrum of phenotypes, some of which are related to acidic soil abiotic stress responses. Hence, we focus here on signaling proteins such as TFs and protein kinases to identify, from the literature, evidence for unifying regulatory networks controlling Al tolerance, P efficiency and, also possibly drought tolerance. Particular emphasis will be given to modification of root system morphology and architecture, which could be an important physiological “hub” leading to crop adaptation to multiple soil-based abiotic stress factors.Crop tolerance to multiple abiotic stresses has long been pursued as a Holy Grail in plant breeding efforts that target crop adaptation to tropical soils. On tropical, acidic soils, aluminum (Al) toxicity, low phosphorus (P) availability and drought stress are the major limitations to yield stability. Molecular breeding based on a small suite of pleiotropic genes, particularly those with moderate to major phenotypic effects, could help circumvent the need for complex breeding designs and large population sizes aimed at selecting transgressive progeny accumulating favorable alleles controlling polygenic traits. The underlying question is twofold: do common tolerance mechanisms to Al toxicity, P deficiency and drought exist? And if they do, will they be useful in a plant breeding program that targets stress-prone environments. The selective environments in tropical regions are such that multiple, co-existing regulatory networks may drive the fixation of either distinctly different or a smaller number of pleiotropic abiotic stress tolerance genes. Recent studies suggest that genes contributing to crop adaptation to acidic soils, such as the major Arabidopsis Al tolerance protein, AtALMT1, which encodes an aluminum-activated root malate transporter, may influence both Al tolerance and P acquisition via changes in root system morphology and architecture. However,  Al3+ is quite toxic to roots, inhibiting both root elongation and root meristem cell division  are quite extensive worldwide, comprising up to 50% of the world’s potentially arable lands . As the cis elements in the promoter region. Depending also on more complex aspects such as chromatin structure, this gives TFs potential for promiscuous binding to many targets, giving rise to complex regulatory circuits. A good example of how the promiscuity of TF binding sites can impact evolutionary adaptation is presented in In searching for classes of genes involved in mediating resistance concurrently to these three stresses, it is more likely that “upstream” genes that control regulatory and signaling networks such as transcription factors (TFs), kinases and phosphatases would be more likely candidates than structural genes such as root plasma membrane transporters that mediate efflux of Al-binding organic acid anions that have been shown to be involved in crop Al tolerance , 2015. RIn this review, we have focused on signaling/regulatory proteins such as TFs and protein kinases to identify, from the literature, evidence for unifying regulatory networks controlling Al tolerance, P efficiency and also possibly drought tolerance. Particular emphasis will be given to modification of root system morphology and architecture, which could be an important physiological “hub” leading to crop adaptation to multiple soil-based abiotic stress factors.Aluminum (Al) on acidic soils intoxicates root regions involved in root growth (meristem and elongation zone). Cells in these regions are subject to rapid alterations in Al-induced transcription, resulting in the induction of expression of several Al tolerance genes associated with root tip Al exclusion and detoxification mechanisms .STOP1 in Arabidopsis and ART1 in rice are the best characterized TFs regulating Al tolerance. STOP1, a C2H2-type zinc finger transcription factor, was identified via positional cloning of a low-pH-sensitive Arabidopsis mutant. Although AtSTOP1 expression is not induced by Al, the stop1-mutant is also Al hypersensitive  and AtALS3 (ABC transporter-like protein)  have been reported to be involved in crop Al tolerance, and the majority of these TFs belong to zinc finger and WRKY transcription factor families. Among them, ensitive . AtSTOP1nce gene , closely(TaALMT1 . These tbidopsis . Furtherprotein) .AtSTOP1 is ubiquitously expressed in the root with higher expression in the root tip, and its expression is not affected by Al stress. In turn, AtSTOP1 downstream genes  are induced by Al  Al-induced early and late expression; (2) root tip-specific expression; and (3) repression of ALMT1 expression. In addition, it was reported that the transcription factor, CAMTA2 , binds to the AtALMT1 promoter at a cis-element in a different binding region than STOP1, and appears to be involved in induction of AtALMT1 expression only in late Al stress. Previously, it was also demonstrated that AtWRKY46 binds to W-box sequences in the AtALMT1 promoter, repressing its expression in the absence of Al , Holcus lanatus (HlALMT1) and rice (OsFRDL4) carry more STOP1/ART1 binding sites in their promoters and exhibit higher expression levels than the same genes in the respective sensitive accessions  are involved in Al tolerance in rice  and XYLOGLUCAN ENDOTRANSGLUCOSYLASE-HYDROLASES17 (XTH17), that are involved in cell wall modification. Within those, XTH17 works in modifying hemicellulosic polymers during cell expansion , a rice bean NAC transcription factor, is up-regulated by Al in the root apex  and VuWAKL1 (Vigna umbellata WAK1-like) promoters, positively regulating their expression , have been implicated as sensing/signaling molecules in Al signaling  of −1.6 MPa) might play a key role in the prevention of root growth elongation inhibition under drought stress  family of peptides are small peptides that function as plant hormones via release from cells into the extracellular space, where they function as intercellular signaling molecules. They have been shown to bind to receptor-like proteins at the outer surface of the plant cell plasma membrane to help mediate signal transmission. CLE peptides have been shown to regulate a range of physiological and developmental processes, including drought responses. CLE proteins have recently been shown to be a mobile signal transmitted from roots to shoots and involved in increased ABA biosynthesis after dehydration stress . In thisPlant cellular and apoplastic pH has been proposed to be another signaling factor that could play a role in stomatal aperture regulation . Droughtin silico target analysis led to the identification of two to three hundred novel target genes for the drought stress response of the entire root system, along with responses of the divided root system to drought and well-watered conditions. From the target analysis, the authors proposed these miRNAs could be involved in a number of drought response pathways, including ABA and calcium signaling, detoxification of free radicals induced by drought, and stimulation of lateral root initiation and growth, which could lead to bigger and deeper root systems that could more effectively acquire water located deeper in the soil profile under drought.Recent studies have identified specific microRNAs that are responsive to drought stress  and thisIt is well known that transcription factor (TF) proteins can play major roles in regulatory and signaling networks, and plant drought response is no exception. A number of recent studies have been conducted to identify TFs responsive to drought and in some these studies, the function of the identified TFs has been elucidated . TFs resAtAREB1 exhibited enhanced performance under drought without any penalty on yield. From changes in expression profiles for phosphatases (PP2C) and kinases (SnRK2) in the AtAREB1-overexpressing transgenic plants, the authors noted that the observed lower expression of phosphatases and higher expression of kinases are known to be linked to ABA-dependent stomatal closure, and the resulting reduced stomatal conductance to water in the OE lines could explain the observed drought resistance. This overexpression line also had a higher leaf area index and elevated intrinsic water use in subsequent research by OsERF71 altered expression of genes that regulate lignin biosynthesis and cell wall loosening enzymes, leading to increased root radial growth, more cell layers in the vasculature, and increased root aerenchyma, and these root structural changes were associated with reduced water transpiration and increased drought tolerance , through its interaction with ABSCISIC ACID RESPONSE ELEMENT (ABRE) motifs in target gene promoters. Hence, ABI5 has been shown or proposed to be involved in many ABA-related activities, including seed germination, seedling stress tolerance, integration of hormone interactions, and ABA biosynthesis . Hence, leaf isotope C discrimination can be used as a proxy phenotype for transpirational efficiency. Using this approach, leaf isotope C discrimination was used to phenotype an Arabidopsis Col-4 x Ler RIL population. Genetic analysis of the data yielded a significant QTL for transpirational efficiency for leaf isotope C discrimination that was then fine mapped on chromosome 2 (48.96–51.02 cM) and explained up to 64% of the phenotypic variation for this trait in the RIL population. The population parents, Col and Ler, contain ERECTA (ER) and er1 alleles, respectively. Upon screening of candidate genes residing in that region, they found that the ERECTA gene was located in the center of the QTL interval. They also observed contrasting values of leaf isotope C discrimination for individuals with the ER or er1 alleles. For functional validation of the candidate gene for transpirational efficiency, multiple ERECTA mutants were compared with near-isogenic lines containing ERECTA allele homozygotes. They observed that all of the er mutants exhibited higher leaf isotope C discrimination and therefore lower transpirational efficiency than lines homozygous for ERECTA allele. Further, in transgenic lines which complemented the mutation with the ERECTA allele, they confirmed the identity of ERECTA as a transpirational efficiency gene. By dissecting leaf anatomical features, lower stomatal conductance because of lower stomatal density caused by epidermal cell expansion, was observed as the anatomical effect of the ERECTA gene. Loosely packed fewer and smaller mesophyll cells were also observed, and it was concluded that all of these phenotypes collectively are affecting transpirational efficiency. These anatomical phenotypic traits were maintained under drought stress which suggests that the ERECTA gene could be an important genetic tool to increase transpirational efficiency in crops in drought stress environments.The  anatomy . Leaf caTaER genes were positively correlated with transpiration efficiency and yield traits. Gene sequences for ERECTA orthologs in wheat were identified by in silico approaches to compare TaER cDNA sequences to a wheat genome sequence database. The authors also observed significant variation in expression of these genes among 48 wheat varieties in the flag leaves at grain filling and at the heading growth stages. There was a significant positive correlation in TaER expression with water use efficiency, flag leaf area and yield traits (biomass and gain yield), whereas the rate of transpiration, stomatal density and rate of photosynthesis were negatively correlated. These results were consistent with ER (SbER2-1) gene and the transgenic overexpression lines exhibited increased rates of photosynthesis and water use efficiency.Water deficit may disrupt the lipid bilayer in cell membranes, triggering protein denaturation and accumulation of cellular electrolytes, which may lead to osmotic imbalance in plant cells . Hence, stop1 knockout lines showed enhanced drought tolerance in Arabidopsis (stop1 mutant was the CBL-interacting protein kinase 23 (CIPK23), which may be involved in K+/Na+ homeostasis via regulation of K+ transporters. In agreement with K+ involvement in stomatal opening , inhibits primary root growth and enhances lateral root proliferation under P deficiency, possibly favoring P acquisition via Fe-mediated RAM exhaustion (see section “Major Al Tolerance Genes That Are Also Involved in P Deficiency Stress Pathways”). In addition, STOP1 may also influence both salt and drought tolerance . This emerging pleiotropic role of STOP1 in abiotic stress tolerance has been further supported by the recent finding that bidopsis . Among t opening , furtherolerance . Howeverolerance , which mRD26, indicating that the expression of stress-inducible NACs is under control of the central ABA perception and signaling module , and many stress-related genes were up-regulated in the SNAC1-overexpressing rice plants. The transgenic lines were also more sensitive to ABA and showed reduced water loss due to enhanced stomatal closing (with apparent no effect in photosynthesis), possibly by drought induction of SNAC1 in guard cells  TFs have been implicated in drought regulation of stress-related genes via a wide-range of possible tolerance mechanisms, including stomatal development, ABA signaling, trichome and root hair development, osmoregulation, photosynthesis and growth regulation, in addition to ROS scavenging . In maize, ZmPTF1 was also shown to enhance lateral root development and confer drought tolerance . Enhancese genes . Thus, aVuNAR1 in the Vigna umbellata root apex , were more tolerant to bacterial infection, heat, and osmotic stress , which may both be a toxic product and also can be a signal. erf74 mutant lacked the reactive oxygen species burst often seen in the early stages of various stresses, which was due to lower expression level of RESPIRATORY BURST OXIDASE HOMOLOG D (RbohD) in the erf74 mutant. Possibly this is part of a ROS signaling pathway conditioning tolerance to stresses such as Al toxicity, drought and temperature extremes, which may be related to ROS signals (AtALMT1 and AtMATE expression (see Section 2.2b), other, yet unidentified factors may connect drought and Al tolerance via ABA-dependent pathways.Ethylene response factors are TFs in the AP2/ERF superfamily, which are involved in tolerance to multiple abiotic stresses . Well-kn signals . While tIn this review, we examined the literature for common elements shared between the three major stresses that often co-occur on acidic soils: Al toxicity, low P availability and drought, with a focus on genes/proteins involved in signaling and/or regulatory pathways and networks controlling plant responses and adaptations to these three abiotic stresses. In general, research on crop adaptation to acidic soils has focused on one of the two primary stresses resulting from the unique chemistry of acidic soils, Al toxicity or P deficiency. But as this field has advanced and matured, we showed here that quite recently, a number of genes have been identified that are involved both in Al resistance and adaptation to P deficiency. The very broad field of research on crop adaptations to drought, on the other hand, has not focused much on the possible role of drought resistance mechanisms in adaptations to acidic soils. This is primarily because drought, by its very nature, occurs on all types of soils in many different eco-agricultural systems. Nonetheless, in this review we did identify several genes and the proteins they encode that could play a role in adaptation to all three of these abiotic stresses. At the beginning of this review, we speculated that genes involved in signaling/regulatory networks might be the best source for candidates involved in crop adaptation to all three stresses. This turned out to be the case as we identified transcription factors from several TF families that could play this pleiotropic role. These TF’s are summarized here and a model of the function of the best candidate TF’s in the three abiotic stresses is presented in (1)ALMT1 . Furthermore, in a heterologous system, Xenopus oocytes, CIPK23 can activate via phosphorylation the guard cell PM anion channel, SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1)  , the dir(2)ALMT1 expression in the absence of Al miR399: There is good evidence for this MYB transcription factor co-regulating P efficiency and drought tolerance via regulation of miR399 , and to salt and drought  involved in Al toxicity, drought stress and P deficiency. However more research is needed to say with certainty that the same factors can regulate tolerance to all three stresses. If that is the case, it will be quite intriguing to determine if these genes would be useful in a plant breeding program for multiple environmental stresses. Within a scenario where the same genes in some way influence tolerance to multiple stresses, instances of conflicting effects may be foreseen, as previously discussed. If the same gene acts simultaneously as positive and negative regulators of tolerance to different stresses, this may cancel its final effect in phenotypic expression or even be detrimental to crop performance on acidic soils. Within the context of molecular breeding strategies targeting quantitative traits such as genomic selection, these and other negative effects may be filtered out along the selection process via genomic estimation of breeding values. However, transgenic approaches may be more sensitive to this problem. In that regard, gene editing has emerged as a powerful approach to fine tune gene expression, which could help circumvent negative effects coming from pleiotropy or epistasis. This approach has proven useful in bypassing negative epistasis effects on yield in tomato (In conclusion, there are tantalizing links in the literature between the regulation of plant adaptation and responses to Al toxicity, P deficiency and drought stress. To provide the readers with a summary of the work reviewed in this paper, we have included n tomato  and to tn tomato . ParticuJM and LK also handled the revising and editing of the full version of the manuscript. All authors contributed by conducting literature research and writing of different parts of the text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


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.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.Enhancers are 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. Common human diseases such as cancer, cardiovascular disorders, diabetes, and mental illness represent the major contributors to morbidity and mortality in both the developed and developing world. Although our understanding for the pathogenic mechanisms underlying most common diseases has significantly improved over the last decades, the prevention and treatment of these disorders are still largely ineffective.1 Initially scientists focused on association studies between variation in candidate genes and common diseases. With the development of international efforts such as the HapMap project, the scientific community rapidly moved from probing variation present in a few candidate genes to testing millions of single-nucleotide polymorphisms (SNPs) located throughout the genome.2Most common diseases are highly heterogeneous, with few cases characterized by simple etiology dominated by a single genetic mutation, while the most cases are caused by the interplay of multiple genetic and environmental factors. Before the completion of the Human Genome Project, efforts to map genetic variants were mostly based on linkage studies that searched for shared haplotypes among related affected individuals. However, linkage studies do not have enough power to detect common variants with modest effect, and they have been slowly replaced by association studies.4 Large-scale GWAS have discovered hundreds of SNPs that are associated with the risk of common disorders. However, the majority of disease-risk variants are located outside protein-coding genes, suggesting a role for noncoding variation in the human genome.4Genome-wide association studies (GWAS) provide a genetic approach to identifying molecular pathways involved in complex traits and diseases by defining associations between genetic variants and phenotypes of interest.The emergence of next-generation sequencing technologies combined with genome-wide approaches has led to the identification of key biological regulators located in noncoding regions of the human genome. Advances in the field of genomics and transcriptomics have uncovered the role of DNA and RNA elements, such as enhancer regions and long noncoding RNAs (lncRNAs), in the fine-tuning of crucial physiological processes. Specifically, these studies have shown that genetic polymorphisms located in such regions have a regulatory role and, hence, a subtler impact on the pathogenesis of human diseases when compared with mutations underlying mendelian disorders. Altogether, these recent findings highlight the need for a new approach to systematically study the functional outcome of noncoding polymorphisms present in the genome.In this perspective, we describe the latest findings about emerging regulatory roles of noncoding elements in the human genome and their association with the predisposition to common diseases. We mention the latest technological advances that allow researchers to unveil the functional role of genetic variants located in different genomic regions. Finally, we propose a new framework to investigate the functional role of noncoding variation in the genome and, in turn, to identify new pharmacological and therapeutic targets.5 A key feature of most TAD boundaries is the presence of the CCCTC binding factor (CTCF) together with the structural maintenance of the chromosome cohesion complex.5 The binding of CTCF at TAD borders is crucial, as removal one of its binding sites is sufficient to abrogate a TAD boundary.6 Notably, intra-TAD interactions between regulatory elements are cell type specific and investigation of DNA-DNA contacts with high-throughput sequencing technologies can reveal multiple layers of chromatin organization and regulation.Chromosomal organization is an evolutionarily conserved feature that requires hierarchical folding of the chromatin into large compartments composed of smaller domains called topologically associating domains (TADs). TADs are stable across cell divisions, invariant across different cell types, and evolutionarily conserved, indicating their fundamental importance in chromatin organization.cis-acting elements: the promoter, a genomic region defining the initiation of transcription, and more distal regulatory elements called enhancers. While promoters provide the essential sites of transcriptional initiation of RNAs, they are frequently not sufficient to direct appropriate developmental and signal-dependent levels of gene expression.8 This additional information is provided by enhancers, short regions of DNA that, when bound by transcription factors (TFs), enhance RNA expression from target promoters. Enhancers can reside hundreds of thousands of base pairs away from their target gene and are typically well conserved across genomes, and their function is generally considered to depend on three-dimensional (3D) enhancer–promoter interactions.9 Within TADs, DNA folding allows fine-tuning of gene expression by facilitating contacts between regulatory sequences such as promoters and enhancers. Enhancer selection is driven by cell-type-specific combinations of lineage-determining TFs that, in turn, specify the binding of signal-dependent TFs. As a consequence, each cell has a unique enhancer repertoire that underlies its particular pattern of gene expression and enables cell-type-specific responses to intra- and extracellular signals.10 Furthermore, active enhancers are characterized by bidirectional transcription, which results in the production of enhancer RNAs (eRNAs) believed to facilitate long-range enhancer–promoter looping. Accordingly, genetic variation affecting enhancer selection and function is considered to be a major determinant of differences in cell-type-specific gene expression between individuals.Regulation of gene expression generally involves two different types of 12 In particular, lncRNAs (lncRNAs) have emerged as important regulators of various biological processes.13 LncRNAs are characterized by a length longer than 200 nucleotides and the absence of open reading frames. As such, the definition is very general, and under the name lncRNAs we include different transcripts, such as long intergenic noncoding RNAs (lincRNAs), eRNAs, and sense and antisense RNAs.14 Like mRNAs, lncRNAs are transcribed by RNA polymerase II and are capped and polyadenylated. However, compared with protein-coding transcripts, lncRNAs have strict spatiotemporal expression patterns, exhibiting tissue- and cell-type specificity.15 Also, unlike coding transcripts that are predominantly cytosolic,16 approximately 60% of noncoding transcripts are enriched in the nucleus, specifically in the chromatin fraction,18 suggesting that their action might depend on their interactions with chromatin and chromatin binding factors.18 As the cytoplasmic functions of lncRNAs have been already reviewed elsewhere,19 we focus on the nuclear functions of lncRNAs in this perspective.Recent international scientific efforts have discovered that the vast majority of mammalian genomes are pervasively transcribed, accounting for a previously unappreciated complexity of the noncoding RNA (ncRNA) fraction.Fig. 1a), organization of the chromatin structure , and transcriptional regulation , with some lncRNAs, such as XIST, being able to perform a combination of functions (scaffolding and guiding). In the next sections, we discuss the different mechanisms of action for nuclear lncRNAs in further detail.During recent years, functional classification of lncRNAs has proven to be a challenging task, as we are only now starting to appreciate the multiple mechanisms of action for these transcripts. Nuclear lncRNAs are able to perform diverse functions, such as involvement in nuclear architecture  and MALAT1 , have been demonstrated to play important scaffolding roles in the assembly of nuclear paraspeckles and speckles, respectively, by interacting with protein components.15MALAT1 is a highly abundant nuclear lncRNA that localizes in nuclear speckles by physically interacting with splicing factors such as SRSF1 and SC-35 (aka SRSF2).20 Although MALAT1 has been shown to be dispensable for nuclear speckle formation, it is thought to facilitate the localization of nuclear speckles at transcriptionally active gene loci.14Two of the most well-studied lncRNAs, NEAT1 provides another example of an architectural lncRNA, involved in the formation of nuclear paraspeckles. Paraspeckles are nuclear bodies formed by interacting protein and RNA components, believed to be crucial for mRNA retention, mRNA cleavage, and protein sequestration.22 In humans, the NEAT1 transcript is processed to produce two distinct transcripts, a 3.7 kb NEAT1_1 and a 22.7 kb NEAT1_2, often referred to as the short and long NEAT1 isoforms.14 Interestingly, only the longer isoform NEAT1_2 is essential for paraspeckle assembly,23 by interacting with essential paraspeckle protein components such as NONO/P54NRB and SFPQ.24FIRRE  is a representative example of the nuclear organization properties of some lncRNAs. The FIRRE locus is localized on the X chromosome and has been observed to escape X chromosome inactivation, localizing to a 5 Mb domain in the vicinity of its site of transcription.25FIRRE RNA contains multiple repeats of a 156 nt long sequence that enables binding to the nuclear matrix organizing protein HNRNPU. This lncRNA can establish trans-chromosomal interactions across five distinct loci, bringing in spatial proximity to its site of transcription and contributing to higher-order chromatin organization. Additionally, FIRRE RNA has been demonstrated to bind the architectural protein CTCF, targeting the inactive X chromosome in the perinucleolar region.26The evolutionarily conserved, nuclear, lincRNA 15 As such, these RNAs can deliver proteins to specific gene locations and coordinate genetic programs. Multiple studies have shown that lncRNAs can regulate expression of target genes in cis (in proximity to their site of transcription or more distantly but on the same chromosome of origin) or in trans (on chromosomes different from the chromosome of origin).Most lncRNAs exhibit nuclear localization with enrichment for the chromatin fraction; furthermore, they have the ability to fold into space and associate with specific proteins such as TFs to fine-tune gene expression.cis-acting lncRNA is XIST (X-inactive specific transcript), a crucial transcript for the process of dosage compensation in female mammal cells. Dosage compensation is an essential process to ensure that gene expression levels from two X chromosomes in female cells are equivalent to gene expression levels from the single X chromosome found in male cells. XIST is transcribed from one of the two X chromosomes and physically associates with the Polycomb repressive complex 2 (PRC2) to initiate epigenetic repression of the entire X chromosome through the deposition of H3K27me3 histone marks by EZH2.27 Following the epigenetic silencing, the inactive X chromosome localizes to the nuclear periphery.Probably the most well-studied example of a HOTAIR) is a well-known trans-acting lncRNA. HOTAIR is transcribed in the antisense direction to the HOXC locus and located on chromosome 12 in humans. Despite being initially thought to be a cis-acting, negative regulator of HOXC locus expression, its inhibition does not affect HOXC expression. In contrast, HOTAIR inactivation leads to a significant upregulation of the HOXD locus, located on a different chromosome (chromosome 2 in humans). Similar to XIST, HOTAIR interacts with PRC2 to guide the repressive machinery to the HOXD locus and initiate epigenetic silencing through H3K27 trimethylation (H3K27me3).28The HOX antisense intergenic RNA , the genetic variant results in impaired TF binding, leading to unsuccessful chromatin looping and lack of enhancer–promoter interactions. In turn, the target gene’s transcriptional activation is inefficient, resulting in basal transcription levels.Enhancers represent genomic regulatory elements (GREs) that are crucial for the regulation of distinct spatiotemporal transcriptional programs. Consequently, disease-associated variations within enhancer elements are likely to result in transcriptional dysregulation of cell-type-specific gene expression. Indeed, genetic risk variants are enriched in enhancer-like elements, featuring increased DNAse hypersensitivity sites and the aforementioned histone marks.30 In the case of Parkinson’s disease (PD), more than 90% of reported cases present non-Mendelian inheritance. A study by Soldner and colleagues found a major risk allele for PD within an enhancer of the α-synuclein gene, which results in altered TF binding and upregulation of the protein.31 Similarly, 30% of noncoding variants associated with Alzheimer’s disease (AD) were found to be located in enhancer elements, with their target genes involved with amyloid-beta plaque clearance, synaptic transmission, and immune responses.32 Furthermore, these variants were observed to affect TF and CTCF binding sites, suggesting that their presence might influence AD risk by affecting TF binding and enhancer–promoter chromatin looping mechanisms.32 A large class of complex disorders includes autoimmune disorders such as systemic lupus erythematosus (SLE), multiple sclerosis, and type 1 diabetes. In a large study investigating noncoding variants in 21 autoimmune diseases, it was determined that approximately 60% of likely causal variants map to enhancer-like elements that are often stimulus-dependent CD4+ T-cell-specific enhancers.33 Additionally, in the case of SLE, two risk variants were found to locate in a distal enhancer, regulating the expression of the NFκB inhibitor A20, encoded by the TNFAIP gene. The nonrisk enhancer was found to interact with the promoter of TNFAIP through long-range chromatin looping and enhance its expression upon direct binding of NFκB. However, the presence of the risk variants impairs NFκB binding, disrupts the long-range enhancer–promoter interactions, and results in low expression of TNFAIP.34Disease-associated enhancer variants are more likely to be found in enhancer sequences that are active in cells and tissues relevant to the disease of interest. For example, pancreatic islet enhancers are specifically enriched in risk variants associated with type 2 diabetes.cis-regulatory element, considered to act as a transcriptional enhancer for the MYC proto-oncogene.35 The variant is associated with enhancer activation through differential binding of the beta-catenin coactivator and Wnt signaling effector TCF7L2.35 Furthermore, a risk SNP was identified as one of the strongest predisposing variants to neuroblastoma. The risk SNP is located within a superenhancer element, regulating the expression of LIM domain only 1 (LMO1) through the binding of the TF GATA3, which in turn facilitates long-distance enhancer–promoter looping and leads to the upregulation of LMO1.36Enhancer variants have also been identified in various types of cancers. In the case of colorectal cancer, a variant mapping to the 8q24 locus, known as rs6983267, was observed to map to a distal PCAT19 locus, regulating the expression of the PCa-associated genes PCAT19 and CEACAM21.38 The variant is characterized by stronger binding affinity for the TF HOXA2, which leads to the transcriptional activation of the aforementioned genes, resulting in increased proliferation and metastasis.37A major noncoding risk allele for prostate cancer (PCa) susceptibility and aggressiveness, rs11672691, was found to reside within an enhancer element, located in intron 2 of the lncRNA HOTAIR (rs920778) has been observed to contribute to increased susceptibility for the development of esophageal squamous cell carcinoma and gastric cancer through a putative, de novo intronic enhancer element, leading to HOTAIR upregulation and further target gene expression dysregulation.40A genetic variant located in the intron of the lncRNA FTO (fat mass and obesity-associated protein). SNP variants located in a 47 kb region, spanning introns 1 and 2 of the FTO gene, have been strongly associated with adult and childhood obesity as well as an increased risk for type 2 diabetes.42 Although SNPs in FTO were determined to have no effect on its expression, the 47 kb region has been demonstrated to contain multiple TF binding sites, enhancer-associated chromatin modifications, and DNase hypersensitivity sites, suggestive of the putative enhancer potential for this region. Additionally, using chromatin conformation capture technologies, the region has been observed to establish long-range interactions with the obesity-related gene IRX3 and FTO risk alleles correlating with increased IRX3 expression and body mass index in humans, mice, and zebrafish.43Another intriguing example of functional enhancer variation has been reported for the obesity-associated gene LMNB) gene, chromosomal rearrangements affecting TAD boundaries have been demonstrated to lead to enhancer adoption and eventual LMNB overexpression.44 Furthermore, TAD boundary disruptions and mutations in CTCF binding sites have been implicated in enhanced oncogene expression, through the formation of abnormal enhancer–promoter interactions.4547Aberrant enhancer adoption could result from genetic variations affecting TADs or TAD boundaries. An example of this can be seen in the case of autosomal dominant adult-onset demyelinating leukodystrophy (ADLD). Although ADLD has been known to be the result of genomic duplication of the lamin B . In physiological conditions, a locus X results in the production of the lncRNA X, which mediates the recruitment of histone modifier proteins to the target genes, leading to the deposition of repressive chromatin marks and transcriptional inhibition . However, in the case of genetic variation, the locus can result in the production of an lncRNA X that is unable to bind the respective chromatin modifier, leading to absence of repressive histone modification deposition and aberrant transcription of the target genes, represented in Fig. 3c).During recent years, genetic variation in loci encoding for lncRNAs has been associated with the predisposition to several complex disorders. A recent study has shown that variation in genes encoding lncRNAs is more likely to include polymorphisms associated with common disorders compared with protein-coding genes.ANRIL (antisense noncoding RNA in the INK4 locus). ANRIL, also known as the CDKN2B-AS1 transcript, is one of at least five genes included in the 9p21 region. This chromosomal region has been identified as the strongest susceptibility region for cardiovascular disease and a hotspot for various disease-associated mutations.49ANRIL expression and its splicing isoforms have been associated with several diseases, such as atherosclerosis, calcific aortic stenosis, myocardial infarction, type 2 diabetes, AD, glaucoma, and endometriosis.4954ANRIL is known to act primarily in cis, mediating the epigenetic silencing of CDKN2A and CDKN2B loci. Specifically, ANRIL has been shown to interact with PRC1 and PRC2, leading to the deposition of H3K27me3 marks on target genes.56 Because of its roles in regulating expression from the P16/INK4 locus, ANRIL is considered a major regulator of mechanisms involving cell growth, proliferation, and senescence.58 Also, some reports demonstrate the ability of ANRIL to act in trans, regulating the transcription of genes on different chromosomes, potentially due to the presence of Alu motifs in ANRIL lncRNA, essential for transcriptional regulation in trans.60One of the most well-known disease-associated lncRNAs is ANRIL, an SNP affecting the lncRNA MIAT  was found to be significantly associated with the development of myocardial infarction.62 However, unlike other lncRNAs, MIAT does not appear to be associated with chromatin, but is considered to be a competitive endogenous RNA (ceRNA), often termed “sponge” lncRNAs. MIAT has been shown to participate in negative feedback loops of the VEGF and TGFb1 pathways, by inhibiting microRNAs miR-150-5p and miR-24, respectively, resulting in the regulation of endothelial cell proliferation, migration, and apoptosis.6163In addition to LNC13 is a recently characterized lncRNA that is involved in the inhibition of pro-inflammatory gene expression in macrophages and associated with susceptibility to celiac disease (CeD).64 The LNC13 locus is located on chromosome 2 in proximity to the pro-inflammatory gene IL18RAP and its RNA forms a complex with HNRNPD, a ubiquitously expressed heterogeneous nuclear ribonucleoprotein and the histone deacetylase enzyme HDAC1. Upon pro-inflammatory stimulation, LNC13 is degraded, allowing the expression of IL18RAP. However, in biopsies of intestinal tissue from CeD patients, LNC13 expression is significantly reduced and is associated with the rs917997 variant characterized by inefficient binding of hnRNPD, leading to impaired transcriptional repression of IL18RAP and continuous pro-inflammatory gene expression.65LINC00513, was recently associated with increased susceptibility to SLE.66 Two variants located in the promoter region of the lncRNA, rs205764 and rs547311, were demonstrated to upregulate its expression and increase STAT1 and STAT2 phosphorylation with further activation of the IFN pathway. As its expression is significantly upregulated in patients where the disease is very active, LINC00513 is proposed to play a major role in SLE progression and pathogenesis.66A novel lncRNA, termed HOTAIR has been extensively investigated in cancer pathogenesis and progression. Gupta and colleagues reported that upregulation of HOTAIR in breast cancer patients is associated with higher mortality risk through PRC2-dependent silencing of proliferation and metastasis inhibitors.67 Additional studies have revealed its implication in various types of cancers, such as glioblastoma,68 castration-resistant prostate cancer,69 gastrointestinal tumors,70 and pancreatic cancer.71The lncRNA The emerging evidence for the role of noncoding variation in the predisposition to common diseases has highlighted the need for a new approach that combines the concepts discussed above with new technological advances.72 More recently, several technologies aimed at mapping genome-wide RNA–chromatin interactions have been developed, holding the great potential to identify the target genes of multiple transcripts at once.7376 LncRNAs function through interactions with other transcripts or proteins in order to perform their biological functions. During the last decade, the advent of technologies such as RIP77 and CLIP78 has enabled the identification of multiple transcripts associated with different classes of proteins. More recently, the development of sequencing-based technologies to investigate RNA-RNA interactions at the genome-wide level has unveiled an unexpected complexity for RNA-based regulation.79Recent advances in genome-wide technologies to study DNA-DNA and RNA-DNA interactions in intact eukaryotic nuclei have allowed researchers to assess chromatin topology and the role of transcription in maintenance of the genome architecture. Chromatin conformation capture methodology and its later adaptations have allowed the mapping of DNA regions that are observed to be in proximity more frequently than expected, leading to the identification of the functional interaction between regulatory elements and promoters. In the past, methodologies to dissect RNA–chromatin interactions mediated by specific transcripts have identified the target genes for several lncRNAs.Fig. 4). The first important step is to assess whether the variant of interest is located within an already annotated locus. Below we discuss the functional consequence of genetic variants present in different genomic regions.The establishment of novel technological approaches to probe the involvement of noncoding elements at both the DNA and RNA level is quickly becoming a key feature for the identification of novel regulatory mechanisms for the control of gene expression and, consequently, cell identity Table 2.43 Another possible functional outcome for noncoding variation in a GRE is disruption of the binding site for lncRNAs (from RNA–chromatin interactions technologies). Exonic variants can alter 3D contacts if encompassing a GRE. However, such variants can have a more pronounced effect by affecting either the aminoacidic sequence or the stability of the final protein product.Polymorphisms located in protein-coding genes can have different functional outcomes depending on the gene region they are embedded in. Variants in nonexonic regions  can directly affect expression levels or the stability of the transcript through various mechanisms. Furthermore, polymorphisms located in a GRE  encompassing a nonexonic region could result in disruption of the TF binding site and, in turn, affect the expression level of nearby genes. As illustrated in 65Polymorphisms located in noncoding genes can have different functional outcomes whether the variant acts on the DNA or RNA level. The GWAS-associated SNP can encompass a GRE and affect the binding of key TFs, disrupting possible interactions with promoter regions with functional consequences on the expression of target genes. Alternatively, the mutation can affect the sequence of the mature ncRNA, leading to structural alterations of the transcript. Technologies to identify RNA-DNA, RNA-RNA, and RNA–protein interaction can unveil novel targets for the physiological role of the RNA and its alterations in disease. Indeed, a polymorphism located in lnc13 and associated with CeD was shown to affect the interaction of the lncRNA and HNRPD protein by using RIP technology.44Functional characterization of GWAS-associated variants that map in nonannotated regions presents further challenges. As the genomic region of interest has not yet been characterized, all possible scenarios should be taken into consideration. The polymorphism could be located in a novel (coding or noncoding) gene that in turn is responsible for the observed phenotype. In this case, it is important to look into expression datasets to assess if the region harboring the variant is transcribed. Another possibility is that the variant lies within a GRE whose disruption is associated with the disorder. Indeed, characterization of a putative enhancer element revealed enhancer adoption for LMNB that in turn caused ADLD due to overexpression of the gene.Functional characterization of GWAS-associated noncoding variants opens new possibilities for therapeutic treatments, including the possibility for a new generation of pharmaceutical targets. Again, we need to distinguish between variants acting at the level of DNA from polymorphisms acting at the RNA level.81 One limitation for this approach is to have a delivery method that is at the same time cell type specific and does not elicit a strong immunological response.For genetic polymorphisms associated with GWAS and functioning as GREs, it is possible to focus on the interacting gene as revealed by chromatin conformation studies. An important distinction would be to identify whether the polymorphism leads to a loss or an increase of enhancer function. This would in turn determine the nature of the pharmacological agents and their mechanisms of action . Another possibility to correct genetic variants active at DNA level is the use of new genome-editing technologies.82 Although a delivery method to ensure cell specificity is still a limitation, the great potential of this approach has been shown in mice studies targeting the expression lncRNAs involved in human diseases.82 For pathological genetic variants that affect the lncRNA structure, pharmacological molecules can be envisioned to repair the 3D structure of lncRNAs. Aptamers represent a promising class of single-stranded nucleic acids that have been shown to efficiently recognize the secondary structure of specific lncRNAs and, in turn, to prevent interactions mediated by the transcript.83 Another possibility is to use small molecules to alter the lncRNA 3D structure and potential for interactions with DNA, other RNAs, or proteins. Albeit still in its infancy, this approach has yielded some encouraging results.84 Finally, the continuously expanding palette of genome-editing technologies allows researchers to edit the sequence of interest either directly in the genome or at the RNA level, with a recent approach using the CRISPR-Cas13 complex.85For GWAS-associated noncoding variants active at the RNA level, it is feasible to target the lncRNA by modulating its expression levels. Aberrant expression of the transcript can be reduced using gene silencing technologies such as antisense oligos (ASOs), short interfering RNAs (siRNAs), or the interference system of clustered regularly interspaced short palindromic repeats (CRISPRi).Most common disorders develop during adult life as opposed to single monogenic disorders that usually manifest at an earlier age. As most GWAS-associated genetic variants reside in regions of the genome that do not encode for proteins, the late onset of common diseases highlights the subtle regulatory role played by genetic noncoding elements.Both enhancers and lncRNAs have restricted spatiotemporal activity patterns, making them ideal regulatory elements for the control of cell fate and coordinated gene expression during development. Recent studies have started to reveal diverse functional consequences for genetic variation located within enhancers and lncRNAs, and the availability of greatly improved or novel technologies will further expand our current knowledge on the mechanisms by which noncoding elements play a key role in human physiology and pathology.Although still in its infancy, the development of novel pharmacological therapies targeted at noncoding elements holds great promise for the cure and treatment of common human disorders.


Ambler’s laboratory notebook marked the discovery of protein methylation and a segue into a new field of scientific research Salmonella typhimurium flagellin, this “kink” was interpreted as a “new amino acid”, the N-methyl-lysine (NML). This discovery, bolstered by a subsequent examination of purified NML, was the first glimpse of protein methylation in living cells Although this exciting new discovery led to an initial surge in interest, focus on protein methylation quickly waned for a number of decades. By the time protein methylation emerged as a field of interest, research into other post-translational modifications (PTMs) was firmly underway. For example, the discovery of lysine (Lys) methylation predated tyrosine (Tyr) phosphorylation by two decades, following a fortuitous discovery of this new type of protein modification in v-Src associated kinase activity Although it was first discovered in 1959, protein methylation has only become a prolific area of discovery in recent decades. What we currently know of its properties and significance in biological function leaves many unanswered questions, making it all the more intriguing as research efforts continue.i.e., H3K4me) is a well-established marker of gene activation PTM is well-known to regulate a wide range of biological functions, including the regulation of numerous protein interactions, protein localization, protein stability, and enzyme function Following the extensive characterization of histone methylation, Lys methylation of non-histone proteins has been recently found to regulate many cellular processes Salmonella typhimurium provided the scientific community with proof of protein methylation in living cells Ambler and Rees’ observation of methyllysine in the flagellin of Although the physiological and regulatory roles of other PTMs such as phosphorylation had already been explored S-adenosylmethionine (SAM) to Lys, Arg, Asp, or Glu residues Following this initial curiosity, there was a precipitous drop in research in subsequent decades, in large part because no causal link could be established between protein methylation and regulation of biological processes. Kim and Paik diverted their focus to the enzymes involved in methylation throughout the 1960s and 70s. This was a fortunate detour, as they were able to establish the first methyltransferase activity: the enzymatic transfer of a methyl group from  residue  [18].FigIt was not until the 1990s that hints of a functional role for Lys methylation were finally beginning to be uncovered. The progress in genetics and molecular biology were propellers of these advancements, notably the expansion of genomic sequence information and techniques for studying chromatin biology In what is referred to as the “histone code”, PTMs of histones (such as methylation) were interpreted as a unique language which spelled out transcription and chromatin structure Shortly thereafter, large families of protein methyl-binding domains such as plant homeodomain (PHD) finger-containing domains, Tudor domains, malignant brain tumour (MBT) repeats, chromo domains were added to the list Zhang and his colleagues added Jumonji C-terminal (JmjC) domain-containing KDMs. JmjC-domain-containing proteins  were a class of alpha-ketoglutarate-dependent KDMs, which demonstrated a unique method of methylation. While LSD1 demethylates by oxidizing the While the role of Lys methylation in histones had been already being elucidated, the discovery of RNA-binding protein (RBP) methylation in 1998 expanded the scope of protein methylation. The methylation of RBPs was shown to have a regulatory role in RNP assembly, pre-mRNA splicing, and mRNA stability i.e., loss of p53K370me2), thereby disrupting the methyl-reader abilities of the Tudor domains of p53-binding protein 1 (53BP1) In 2004, Reinberg and his team observed an important function of Lys methylation in p53 tumor suppressor protein. In particular, they found that methylation of p53 by the KMT, SET domain-containing protein 7 (SETD7), resulted in enhanced transcriptional activity, nuclear stability, as well as apoptosis i.e., Rpl29K5), a ribosomal protein that is prevalent in all cell types. Lys methylation of Rpl29 dictates its regulation and impacts subcellular localization. It has been determined that methylation of Rpl29K5 is so frequent that the methylation itself may be used as a cellular biomarker for SETD7 activity. Consequently, it is possible that Rpl29 methylation can be used as a target for SETD7 inhibitors Further highlighting the broad reach of KMTs in non-histone Lys methylation, SETD7 has also been found to be the primary KMT for the methylation of ribosomal protein L29 (Rpl29) at K5 (Ubiquitin-like with PHD and RING finger domains1 (UHRF1) are also methylated by SETD7 and demethylated by LSD1. UHRF1 functions to regulate DNA methylation as well as heterochromatin formation. Methylation of UHRF1 has been shown to have unique functional response to DNA damage by regulating the enzymatic activity of repair proteins or the binding affinity of repair-associated transcription factors. Specifically, the methylation of UHRF1 induces the homologous recombination required for DNA repair, thus playing a critical role in the double-strand break repair mechanism Despite the rapid growth in our understanding of the function of Lys methylation, the field has experienced limited growth as a result of a lack of suitable identification technologies. Methylation exists as a relatively small uncharged protein modification. As a result, it is difficult to develop antibodies that do not suffer from low affinity or poor specificity, or that do not maintain specificity for the amino acid sequences surrounding the modified Lys. Although there have been several reports of successful immunoaffinity-enrichment of methyllysine peptides As we expand the breadth of protein Lys methylation events, there is a growing realization that Lys methylation plays a critical role in the development of many human diseases. Given the knowledge that Lys methylation plays a functional role in the regulation of an ever-expanding list of cellular processes , perhapse.g., the KMT2 (MLL) family proteins, are collectively among the most frequently dysregulated genes in many types of human cancers, and there is now a strong interest in developing targeted therapies against these modifying enzymes.Targeted therapies are not available for a number of cancers. For example, systemic chemotherapy is the only treatment option for triple negative breast cancer after surgery. However, chemotherapy is highly toxic and cancer cells can eventually become resistant to the treatment. New drug targets and innovative research strategies are key for the cancer therapeutics. Recently, research in cancer biology has discovered that genes encoding KMT and KDM enzymes, in vitro and robust efficacy in vivo, suggesting that targeting Lys methylation pathways and their regulating enzymes may be a relevant, emerging cancer therapeutic strategy.Given the extensive regulatory importance realized for Lys methylation, any mutations or dysfunction in KMT or KDM enzymes can lead to deregulated cell function, tumorigenesis, and chemotherapy resistance in vitro and in vivo on various cancer cells.To date, a handful of KMT and KDM inhibitors have been discovered or developed, still many inhibitors are at the preclinical stages of development  [47]. InAs the delicate balance in the activity of KMT and KDMs serves to tightly regulate Lys methylation and maintain healthy homeostatic conditions, it is not surprising that a disruption can lead to various pathologies. Although there has been research connecting non-histone Lys methylation to tumorigenesis, less has been explored related to other pathological conditions, which presents a unique opportunity for future research. The Lys methylation of heat shock protein 70 (HSP70) plays a role in homeostasis and an over-abundance of HSP70 has been found to be implicated in autoimmune diseases To date, the focus of non-histone Lys methylation has been toward its role in human pathology, however, recent research has suggested that non-histone Lys methylation may also be functionally significant in plant cells Decades of research into PTMs has characterized the functional importance of processes such as phosphorylation, acetylation, ubiquitination, and methylation of histones. Among these PTMs is a long-neglected and now rapidly expanding field of non-histone protein Lys methylation. Thanks to the advent of technologies, now we are able to perform molecular dynamics studies on KMT-catalyzed methylation of histone peptides that contain Lys and its sterically demanding analogs with mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy The authors have declared no competing interests.