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.

SUMMARY 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.

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, 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, 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. ATAC-seq and 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.

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.

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.Myrf (myelin regulatory factor, previously known as C11orf9 [the human gene] and Mrf [the mouse gene]) is a pleiotropic membrane-bound transcription factor 1-7 . Throughout this paper, MYRF and Myrf refer to the human and mouse genes, respectively. In the murine central nervous system (CNS), Myrf is specifically expressed by oligodendrocytes (OLs) 1,8 . Conditional knockout of Myrf in OL lineage cells led to widespread dysmyelination and severe neurological deficits 1 . Myrf is also indispensable for the life-long maintenance, plasticity, and regeneration of CNS myelin 9-11 . These studies left the impression that Myrf is a "myelin" transcription factor, and hence the name Myrf. We now know that MYRF is also expressed in other tissues such as stomach, lung, heart, ovary, eye, and developing gonads [12][13][14][15] . Consistently, MYRF coding variants have been implicated in both myelin and non-myelin diseases [13][14][15][16][17][18][19] , and whole-body Myrf knockout led to embryonic lethality in mouse independent of OL development 1 . In keeping with myelin-independent functions of Myrf, Myrf orthologs are found and play an important role in organisms without myelin 2,3,6,7 .Myrf is generated as a type-II membrane protein in the endoplasmic reticulum (ER) 4-6 . The Intramolecular Chaperone Auto-processing (ICA) domain is pivotal to the auto-cleavage of Myrf 4,5,20 . The ICA domain forms a homo-trimer, which as an intramolecular chaperone helps Myrf to form a homo-trimer in the ER membrane by chaperoning the formation of a triple β-helix (the intramolecular chaperone function, Fig. 1) 4,5,20-23 . As soon as the triple β-helix is completed, the ICA homo-trimer performs the auto-cleavage reactions that cleave the three P586-S587 peptide bon...