An oligodendrocyte silencer element underlies the pathogenic impact of lamin B1 structural variants.

The role of non-coding regulatory elements and how they might contribute to tissue type specificity of disease phenotypes is poorly understood. Autosomal Dominant Leukodystrophy (ADLD) is a fatal, adult-onset, neurological disorder that is characterized by extensive CNS demyelination. Most cases of ADLD are caused by tandem genomic duplications involving the lamin B1 gene ( LMNB1 ) while a small subset are caused by genomic deletions upstream of the gene. Utilizing data from recently identified families that carry LMNB1 gene duplications but do not exhibit demyelination, ADLD patient tissues, CRISPR modified cell lines and mouse models, we have identified a novel silencer element that is lost in ADLD patients and that specifically targets overexpression to oligodendrocytes. This element consists of CTCF binding sites that mediate three-dimensional chromatin looping involving the LMNB1 and the recruitment of the PRC2 repressor complex. Loss of the silencer element in ADLD identifies a previously unknown role for silencer elements in tissue specificity and disease causation.

Uncovering oligodendrocyte enhancers that control Cnp expression.

Oligodendrocytes (OLs) produce myelin sheaths around axons in the central nervous system (CNS). Myelin accelerates the propagation of action potentials along axons and supports the integrity of axons. Impaired myelination has been linked to neurological and neuropsychiatric disorders. As a major component of CNS myelin, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) plays an indispensable role in the axon-supportive function of myelin. Notably, this function requires a high-level expression of CNP in OLs, as evidenced by downregulated expression of CNP in mental disorders and animal models. Little is known about how CNP expression is regulated in OLs. Especially, OL enhancers that govern CNP remain elusive. We have recently developed a powerful method that links OL enhancers to target genes in a principled manner. Here, we applied it to Cnp, uncovering two OL enhancers for it (termed Cnp-E1 and Cnp-E2). Epigenome editing analysis revealed that Cnp-E1 and Cnp-E2 are dedicated to Cnp. ATAC-seq and ChIP-seq data show that Cnp-E1 and Cnp-E2 are conserved OL-specific enhancers. Single cell multi-omics data that jointly profile gene expression and chromatin accessibility suggest that Cnp-E2 plays an important role in Cnp expression in the early stage of OL differentiation while Cnp-E1 sustains it in mature OLs.

Identifying an oligodendrocyte enhancer that regulates Olig2 expression.

Olig2 is a basic helix-loop-helix transcription factor that plays a critical role in the central nervous system. It directs the specification of motor neurons and oligodendrocyte precursor cells (OPCs) from neural progenitors and the subsequent maturation of OPCs into myelin-forming oligodendrocytes (OLs). It is also required for the development of astrocytes. Despite a decade-long search, enhancers that regulate the expression of Olig2 remain elusive. We have recently developed an innovative method that maps promoter-distal enhancers to genes in a principled manner. Here, we applied it to Olig2 in the context of OL lineage cells, uncovering an OL enhancer for it (termed Olig2-E1). Silencing Olig2-E1 by CRISPRi epigenome editing significantly downregulated Olig2 expression. Luciferase assay and ATAC-seq and ChIP-seq data show that Olig2-E1 is an OL-specific enhancer that is conserved across human, mouse and rat. Hi-C data reveal that Olig2-E1 physically interacts with OLIG2 and suggest that this interaction is specific to OL lineage cells. In sum, Olig2-E1 is an evolutionarily conserved OL-specific enhancer that drives the expression of Olig2.

Exosomes/microvesicles target SARS-CoV-2 via innate and RNA-induced immunity with PIWI-piRNA system.

Murine neural stem cells (NSCs) were recently shown to release piRNA-containing exosomes/microvesicles (Ex/Mv) for exerting antiviral immunity, but it remains unknown if these Ex/Mv could target SARS-CoV-2 and whether the PIWI-piRNA system is important for these antiviral actions. Here, using in vitro infection models, we show that hypothalamic NSCs (htNSCs) Ex/Mv provided an innate immunity protection against SARS-CoV-2. Importantly, enhanced antiviral actions were achieved by using induced Ex/Mv that were derived from induced htNSCs through twice being exposed to several RNA fragments of SARS-CoV-2 genome, a process that was designed not to involve protein translation of these RNA fragments. The increased antiviral effects of these induced Ex/Mv were associated with increased expression of piRNA species some of which could predictably target SARS-CoV-2 genome. Knockout of piRNA-interacting protein PIWIL2 in htNSCs led to reductions in both innate and induced antiviral effects of Ex/Mv in targeting SARS-CoV-2. Taken together, this study demonstrates a case suggesting Ex/Mv from certain cell types have innate and adaptive immunity against SARS-CoV-2, and the PIWI-piRNA system is important for these antiviral actions.

Identifying oligodendrocyte enhancers governing Plp1 expression.

Oligodendrocytes (OLs) produce myelin in the central nervous system (CNS), which accelerates the propagation of action potentials and supports axonal integrity. As a major component of CNS myelin, proteolipid protein 1 (Plp1) is indispensable for the axon-supportive function of myelin. Notably, this function requires the continuous high-level expression of Plp1 in OLs. Equally important is the controlled expression of Plp1, as illustrated by Pelizaeus-Merzbacher disease for which the most common cause is PLP1 overexpression. Despite a decade-long search, promoter-distal OL enhancers that govern Plp1 remain elusive. We have recently developed an innovative method that maps promoter-distal enhancers to genes in a principled manner. Here, we applied it to Plp1, uncovering two OL enhancers for it (termed Plp1-E1 and Plp1-E2). Remarkably, clustered regularly interspaced short palindromic repeats (CRISPR) interference epigenome editing showed that Plp1-E1 and Plp1-E2 do not regulate two genes in their vicinity, highlighting their exquisite specificity to Plp1. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) data show that Plp1-E1 and Plp1-E2 are OL-specific enhancers that are conserved among human, mouse and rat. Hi-C data reveal that the physical interactions between Plp1-E1/2 and PLP1 are among the strongest in OLs and specific to OLs. We also show that Myrf, a master regulator of OL development, acts on Plp1-E1 and Plp1-E2 to promote Plp1 expression.

Functional mechanisms of MYRF DNA-binding domain mutations implicated in birth defects.

Myrf is a pleiotropic membrane-bound transcription factor that plays critical roles in diverse organisms, including in oligodendrocyte differentiation, embryonic development, molting, and synaptic plasticity. Upon autolytic cleavage, the Myrf N-terminal fragment enters the nucleus as a homo-trimer and functions as a transcription factor. Homo-trimerization is essential for this function because it imparts DNA-binding specificity and affinity. Recent exome sequencing studies have implicated four de novo MYRF DNA-binding domain (DBD) mutations (F387S, Q403H, G435R, and L479V) in novel syndromic birth defects involving the diaphragm, heart, and the urogenital tract. It remains unknown whether and how these four mutations alter the transcription factor function of MYRF. Here, we studied them by introducing homologous mutations to the mouse Myrf protein. We found that the four DBD mutations abolish the transcriptional activity of the Myrf N-terminal fragment by interfering with its homo-trimerization ability by perturbing the DBD structure. Since the Myrf N-terminal fragment strictly functions as a homo-trimer, any loss-of-function mutation has the potential to act as a dominant negative. We observed that one copy of Myrf-F387S, Myrf-Q403H, or Myrf-L479V, but not Myrf-G435R, was tolerated by the Myrf N-terminal homo-trimer for structural and functional integrity. These data suggest that F387S, Q403H, and L479V cause birth defects by haploinsufficiency, while G435R does so via dominant negative functionality.

PRC2 Acts as a Critical Timer That Drives Oligodendrocyte Fate over Astrocyte Identity by Repressing the Notch Pathway.

PRC2 creates the repressive mark histone H3 Lys27 trimethylation. Although PRC2 is involved in various biological processes, its role in glial development remains ambiguous. Here, we show that PRC2 is required for oligodendrocyte (OL) differentiation and myelination, but not for OL precursor formation. PRC2-deficient OL lineage cells differentiate into OL precursors, but they fail to trigger the molecular program for myelination, highlighting that PRC2 is essential for directing the differentiation timing of OL precursors. PRC2 null OL lineage cells aberrantly induce Notch pathway genes and acquire astrocytic features. The repression of the Notch pathway restores the myelination program and inhibits abnormal astrocytic differentiation in the PRC2-deficient OL lineage, indicating that Notch is a major target of PRC2. Altogether, our studies propose a specific action of PRC2 as a novel gatekeeper that determines the glial fate choice and the timing of OL lineage progression and myelination by impinging on the Notch pathway.

Functional mechanism and pathogenic potential of MYRF ICA domain mutations implicated in birth defects.

Myrf is a membrane-bound transcription factor that plays a key role in various biological processes. The Intramolecular Chaperone Auto-processing (ICA) domain of Myrf forms a homo-trimer, which carries out the auto-cleavage of Myrf. The ICA homo-trimer-mediated auto-cleavage of Myrf is a prerequisite for its transcription factor function in the nucleus. Recent exome sequencing studies have implicated two MYRF ICA domain mutations (V679A and R695H) in a novel syndromic form of birth defects. It remains unknown whether and how the two mutations impact the transcription factor function of Myrf and, more importantly, how they are pathogenic for congenital anomalies. Here, we show that V679A and R695H cripple the ICA domain, blocking the auto-cleavage of Myrf. Consequently, Myrf-V679A and Myrf-R695H do not exhibit any transcriptional activity. Molecular modeling suggests that V679A and R695H abrogate the auto-cleavage function of the ICA homo-trimer by destabilizing its homo-trimeric assembly. We also found that the ICA homo-trimer can tolerate one copy of Myrf-V679A or Myrf-R695H for its auto-cleavage function, indicating that V679A and R695H are not dominant negatives. Thus, if V679A and R695H in a heterozygous state caused birth defects, it would be via haploinsufficiency of MYRF.

Molecular mechanism for the multiple sclerosis risk variant rs17594362.

Multiple sclerosis (MS) is known as an autoimmune demyelinating disease of the central nervous system. However, its cause remains elusive. Given previous studies suggesting that dysfunctional oligodendrocytes (OLs) may trigger MS, we tested whether single nucleotide polymorphisms (SNPs) associated with MS affect OL enhancers, potentially increasing MS risk by dysregulating gene expression of OL lineage cells. We found that two closely spaced OL enhancers, which are 3 Kb apart on chromosome 13, overlap two MS SNPs in linkage disequilibrium-rs17594362 and rs12429256. Our data revealed that the two MS SNPs significantly up-regulate the associated OL enhancers, which we have named as Rgcc-E1 and Rgcc-E2. Analysis of Hi-C data and epigenome editing experiments shows that Rgcc is the primary target of Rgcc-E1 and Rgcc-E2. Collectively, these data indicate that the molecular mechanism of rs17594362 and rs12429256 is to induce Rgcc overexpression by potentiating the enhancer activity of Rgcc-E1 and Rgcc-E2. Importantly, the dosage of the rs17594362/rs12429256 risk allele is positively correlated with the expression level of Rgcc in the human population, confirming our molecular mechanism. Our study also suggests that Rgcc overexpression in OL lineage cells may be a key cellular mechanism of rs17594362 and rs12429256 for MS.

A principled strategy for mapping enhancers to genes.

Mapping enhancers to genes is a fundamental goal of modern biology. We have developed an innovative strategy that maps enhancers to genes in a principled manner. We illustrate its power by applying it to Myrf. Despite being a master regulator of oligodendrocytes, oligodendrocyte enhancers governing Myrf expression remain elusive. Since chromatin conformation capture studies have shown that a gene and its enhancer tend to be found in the same topologically associating domain (TAD), we started with the delineation of the Myrf TAD. A genome-wide map of putative oligodendrocyte enhancers uncovered 6 putative oligodendrocyte enhancers in the Myrf TAD, narrowing down the search space for Myrf enhancers from the entire genome to 6 loci in a principled manner. Epigenome editing experiments revealed that two of them govern Myrf expression for oligodendrocyte development. Our new method is simple, principled, and powerful, providing a systematic way to find enhancers that regulate the expression of a gene of interest. Since it can be applied to most cell types, it would greatly facilitate our effort to unravel transcriptional regulatory networks of diverse cell types.

Elucidating the transactivation domain of the pleiotropic transcription factor Myrf.

Myrf is a newly discovered membrane-bound transcription factor that plays an essential role in as diverse organisms as human, worm, and slime mold. Myrf is generated as a type-II membrane protein in the endoplasmic reticulum (ER). It forms homo-oligomers to undergo auto-cleavage that releases Myrf N-terminal fragment from the ER membrane as a homo-trimer. The homo-trimer of Myrf N-terminal fragments enters the nucleus and binds the Myrf motif to activate transcription. Despite its prominent role as a transcriptional activator, little is known about the transactivation domain of Myrf. Here, we report that the N-terminal-most (NTM) domain of Myrf is required for transcriptional activity and, when fused to a Gal4 fragment, enables it to activate transcription. The transactivation function of the NTM domain did not require homo-trimerization. We also discovered that the NTM domain can be sumoylated at three lysine residues (K123, K208, and K276), with K276 serving as the main acceptor. K276 sumoylation repressed the transactivation function of the NTM domain without affecting the stability or nuclear localization of Myrf N-terminal fragment. In sum, this study identifies the NTM domain as the transactivation domain of Myrf and the potential regulatory impact of its K276 sumoylation.

Homo-trimerization is essential for the transcription factor function of Myrf for oligodendrocyte differentiation.

Myrf is a key transcription factor for oligodendrocyte differentiation and central nervous system myelination. We and others have previously shown that Myrf is generated as a membrane protein in the endoplasmic reticulum (ER), and that it undergoes auto-processing to release its N-terminal fragment from the ER, which enters the nucleus to work as a transcription factor. These previous studies allow a glimpse into the unusual complexity behind the biogenesis and function of the transcription factor domain of Myrf. Here, we report that Myrf N-terminal fragments assemble into stable homo-trimers before ER release. Consequently, Myrf N-terminal fragments are released from the ER only as homo-trimers. Our re-analysis of a previous genetic screening result in Caenorhabditis elegans shows that homo-trimerization is essential for the biological functions of Myrf N-terminal fragment, and that the region adjacent to the DNA-binding domain is pivotal to its homo-trimerization. Further, our computational analysis uncovered a novel homo-trimeric DNA motif that mediates the homo-trimeric DNA binding of Myrf N-terminal fragments. Importantly, we found that homo-trimerization defines the DNA binding specificity of Myrf N-terminal fragments. In sum, our study elucidates the molecular mechanism governing the biogenesis and function of Myrf N-terminal fragments and its physiological significance.

Mercury-induced amyloid-beta (Aß) accumulation in the brain is mediated by disruption of Aß transport.

According to a recent study, mercury (Hg) exposure contributes to Alzheimer's disease (AD). However, the underlying mechanisms are not understood. This study investigated the effect of methylmercury (MeHg) treatment on the generation, degradation, and transport of amyloid ß-protein (Aß) in the brain. Wistar rats were administered MeHg by gavage (0, 20, 200, and 2,000 µg Hg/kg/day) for 4 weeks. The total Hg in the blood and brain regions was measured, and the levels of Aß42 in plasma, cerebrospinal fluid (CSF), and brain regions were estimated. The expression of amyloid precursor protein (APP), beta-site APP-cleaving enzyme 1 (BACE1), and neprilysin (NEP) in the brain regions was determined, in addition to the expression of low-density lipoprotein receptor-related protein 1 (LRP1) and the receptor for advanced glycation end products (RAGE) in the brain capillary endothelium (BCE). Finally, the amount of soluble low-density lipoprotein receptor-related protein (sLRP) in the plasma was determined. Aß42 levels were decreased in the CSF of the 2,000 µg Hg/kg/day group compared with controls, and Aß42 levels increased in the hippocampus (HC) in a dose-dependent manner. MeHg decreased LRP1 expression but increased RAGE levels in BCE. sLRP levels were decreased in the plasma of the MeHg-treated rats. They were positively correlated with CSF Aß42 and negatively correlated with Aß42 and Hg levels in HC. These results imply that MeHg reduces the transportation of Aß, thereby resulting in the accumulation of the protein in the HC. Plasma sLRP levels may be an early biomarker of Hg-induced Aß accumulation in the brain.

Reference levels of blood mercury and association with metabolic syndrome in Korean adults.

PURPOSE: Mercury (Hg) is a nonessential and toxic metal that is widely distributed in the environment. This study was performed to estimate the representative blood Hg level, to determine the contributing factors to Hg exposure, and to analyze the association of blood Hg with metabolic syndrome in Korean adults. METHODS: Mercury exposure is assessed by total Hg concentration in blood. A total of 2,114 healthy adults who have not been exposed to Hg occupationally were sampled by the multistaged, sex-, and age-stratified probability method. Information was collected regarding the subjects' demographic characteristics, lifestyles, and past medical history. The participants then underwent physical examination and blood sampling. RESULTS: The geometric mean concentration of Hg in whole blood was 3.90 µg/L, which was significantly influenced by sex, age, smoking, alcoholic consumption, residence area, and seafood intake after adjustment for confounders. Significant increases in body mass index, waist circumference, diastolic blood pressure, total cholesterol, and triglyceride were observed according to the blood Hg levels after adjustment for covariates. Also, Hg exposure was significantly associated with metabolic syndrome and their components such as obesity and increased fasting glucose. CONCLUSION: The blood Hg level in Korean adults is higher than that in USA and other Western countries, while it is similar to or lower than that in other Asian countries. The blood Hg level is influenced by sociodemographic factors and individual lifestyles including dietary habits. Furthermore, blood Hg is associated with metabolic syndrome, in which Hg exposure may play a role as a possible risk factor for cardiovascular diseases.