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.

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.

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.

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.

Age associated high level of major vault protein is p53 dependent.

Major vault protein (MVP) represents the main component of vaults and has been linked to multi-drug resistance (MDR) in cancer cells. We previously reported that MVP plays an important role in the resistance of senescent human diploid fibroblasts (HDFs) to apoptosis and also that MVP expression is markedly reduced in young HDFs but not in senescent HDFs. In this study, designed to elucidate the regulation of MVP in young and senescent HDFs, we examined the levels of transcriptional factors for the MVP gene, which revealed that among the putative transcriptional factors, p53 decreased only in young HDFs, but not in senescent HDFs in response to H(2)O(2) treatment in the same mode as the expression of MVP. Moreover, the phosphorylation status of p53 increased only in senescent HDFs but not in young HDFs in response to H(2)O(2) treatment. Therefore, we tested the possibility of MVP regulation by p53 status. MVP is upregulated in p53 over-expressing young HDFs, while MVP is downregulated in p53-specific small interfering RNA (siRNA)-transfected senescent HDFs, which suggests that the expression of MVP would be p53 dependent. Furthermore, using chromatin immunoprecipitation (ChIP) assay, we observed that p53 binds directly to the MVP promoter. Taken together, these results suggest that p53 would be a major transcriptional factor for MVP gene expression.