key: cord-0002898-8iysset3 authors: Davidson, Nathan L.; Yu, Fengshan; Kijpaisalratana, Naruchorn; Le, Tuan Q.; Beer, Laurel A.; Radomski, Kryslaine L.; Armstrong, Regina C. title: Leukemia/lymphoma‐related factor (LRF) exhibits stage‐ and context‐dependent transcriptional controls in the oligodendrocyte lineage and modulates remyelination date: 2017-05-30 journal: J Neurosci Res DOI: 10.1002/jnr.24083 sha: a9ed744baa5ce70adff087cb1ce4e77afc969822 doc_id: 2898 cord_uid: 8iysset3 Leukemia/lymphoma‐related factor (LRF), a zinc‐finger transcription factor encoded by Zbtb7a, is a protooncogene that regulates differentiation in diverse cell lineages, and in the CNS, its function is relatively unexplored. This study is the first to examine the role of LRF in CNS pathology. We first examined LRF expression in a murine viral model of spinal cord demyelination with clinically relevant lesion characteristics. LRF was rarely expressed in oligodendrocyte progenitors (OP) yet, was detected in nuclei of the majority of oligodendrocytes in healthy adult CNS and during remyelination. Plp/CreER (T) :Zbtb7a (fl/fl) mice were then used with cuprizone demyelination to determine the effect of LRF knockdown on oligodendrocyte repopulation and remyelination. Cuprizone was given for 6 weeks to demyelinate the corpus callosum. Tamoxifen was administered at 4, 5, or 6 weeks after the start of cuprizone. Tamoxifen‐induced knockdown of LRF impaired remyelination during 3 or 6‐week recovery periods after cuprizone. LRF knockdown earlier within the oligodendrocyte lineage using NG2CreER (T) :Zbtb7a (fl/fl) mice reduced myelination after 6 weeks of cuprizone. LRF knockdown from either the Plp/CreER (T) line or the NG2CreER (T) line did not significantly change OP or oligodendrocyte populations. In vitro promoter assays demonstrated the potential for LRF to regulate transcription of myelin‐related genes and the notch target Hes5, which has been implicated in control of myelin formation and repair. In summary, in the oligodendrocyte lineage, LRF is expressed mainly in oligodendrocytes but is not required for oligodendrocyte repopulation of demyelinated lesions. Furthermore, LRF can modulate the extent of remyelination, potentially by contributing to interactions regulating transcription. Differentiation within the oligodendrocyte lineage is regulated by complex mechanisms that work together to accomplish derepression of myelin genes (Liu & Casaccia 2010) . Many components involved in these processes have been identified but the molecular interactions are not yet fully understood. Moreover, mechanisms may be modulated in different contexts, such as developmental myelination, myelin remodeling in normal adults, or remyelination in adult pathology. In the environment of demyelinated lesions, reactive astrocytes and activated microglia/macrophages express molecular signals that act, through transcriptional controls, to regulate OP differentiation (Gallo & Deneen, 2014) . Multiple transcription factors that regulate OP differentiation can interact with histone deacetylase 1 (HDAC1), including leukemia/lymphoma-related factor (LRF), myelin transcription factor 1, and Yin-Yang1 (Armstrong, Kim, & Hudson, 1995; Dobson, Moore, Tobin, & Armstrong, 2012; He, Sandoval, & Casaccia-Bonnefil, 2007; Liu et al., 2004; Nielsen, Berndt, Hudson, & Armstrong, 2004; Romm, Nielsen, Kim, & Hudson, 2005) . Each of these potential transcriptional repressors exhibits stage-specific expression within the oligodendrocyte lineage. In addition, HDAC1 represses the transcription factor Hes5 to promote OP differentiation and mediate derepression of myelin genes (Liu et al., 2006; Shen et al., 2008) . The notch-signaling pathway is one of the potent inhibitors of OP differentiation that limit remyelination (Hammond et al., 2014; Zhang et al., 2009 ). Jagged1, a notch ligand, is expressed in hypertrophic astrocytes in active multiple sclerosis plaques lacking remyelination (John et al., 2002) . Notch1 acts through Hes5 to inhibit OP differentiation (Wu, Liu, Levine, & Rao, 2003) . Hes5 is progressively down-regulated during OP differentiation yet elevated in demyelinated lesions in which remyelination is limited, such as in chronic lesions of multiple sclerosis (John et al., 2002; Kondo & Raff, 2000; Liu et al., 2006; Wang et al., 1998) . LRF warrants particular interest as a potential point of intersection between HDAC1 promoter regulation and notch signaling. LRF has been referred to as the "most exciting yet enigmatic" member of the POK/ZBTB family of transcription factors, which generally act as transcriptional repressors (Lunardi, Guarnerio, Wang, Maeda, & Pandolfi, 2013) . The 43 known members of this protein family contain a POK/ BTB domain at the N terminus, which mediates protein-protein interac-tions, while the C terminus contains multiple Kruppel-type zinc fingers that bind DNA. The gene zinc finger and BTB domain-containing protein 7A (Zbtb7a) encodes the protein referred to as LRF (mouse), OCZF (rat), or FBI-1 (human) and will be referred to as LRF here. LRF binds corepressors and recruits HDAC1 to gene targets with consensus LRF binding sites (Liu et al., 2004; Lunardi, Guarnerio, Wang, Maeda, & Pandolfi, 2013) . LRF plays a critical role in promoting differentiation of B cells by suppressing Notch1 signals that instruct differentiation along the T cell lineage (Lee et al., 2013; Maeda et al., 2007) . In multiple cell lines, LRF also interacts with sterol regulatory element-binding protein (SREBP) to synergistically activate transcription of fatty acid synthase (FASN) , which is essential for phospholipids in myelin and cell membranes . In OP cells, SREBPs are important regulators of oligodendrocyte maturation and FASN levels (Monnerie et al., 2017) . However, LRF activity can be stage-specific as shown for transcriptional regulation in the osteoclast lineage and also for fetal to adult type globin gene expression in erythroid cells (Masuda et al., 2016; Tsuji-Takechi et al., 2012) . Indeed, among hematopoietic lineages, LRF regulates differentiation by complexing with different key factors in a tissue-and context-dependent manner (Lunardi et al., 2013) . In the brain and spinal cord, LRF is strongly expressed in the nuclei of oligodendrocytes as well as in diverse neuronal populations (Dobson, Moore, Tobin, & Armstrong, 2012) . LRF expression in these postmitotic neural cells in the normal postnatal and adult CNS contrasts with the pro-mitotic role of LRF in cancer (Lee & Maeda, 2012) . LRF expression co-localized with NeuN in nuclei of both large motor neurons in the ventral horn and small sensory dorsal horn neurons, as well as in multiple neuronal populations distinguished by their size and laminar distribution in the cerebral cortex (Dobson et al., 2012) . In white matter of the postnatal spinal cord, LRF was expressed in only about 10% of OP cells in contrast to over 70% expression of LRF in oligodendrocytes (Dobson et al., 2012) . Regardless of lineage stage, LRF was localized in nuclei. In vitro, viral transduction of LRF promoted OP differentiation, while LRF knockdown impaired OP differentiation (Dobson et al., 2012) . Furthermore, in vivo deletion of LRF inhibited OP differentiation and the generation of mature oligodendrocytes during postnatal myelination (Dobson et al., 2012) . The present study characterizes the expression and role of LRF in OP cells and mature oligodendrocytes during remyelination. Given the context-dependent activity of LRF noted above in other cell types, we characterize LRF expression using a murine hepatitis virus (MHV) model to produce focal demyelinating lesions in the spinal cord with gliosis, inflammation, and breakdown of the blood-brain barrier that reflects the complex pathology of multiple sclerosis lesions (Armstrong, Redwine, & Messersmith, 2005; Messersmith, Murtie, Le, Frost, & Armstrong, 2000; Redwine & Armstrong, 1998; Vana, Lucchinetti, Le, & Armstrong, 2007b) . We cross floxed Zbtb7a mice with Plp/CreER T and NG2CreER T lines for tamoxifen-induced conditional deletion of LRF in oligodendrocyte lineage cells following cuprizone demyelination. We have previously used this system with the cuprizone model to identify molecular interactions contributing to effective remyelination (Zhou, Pannu, Le, & Armstrong, 2012) . In vitro promoter assays demonstrate the potential for LRF to Significance Regulation of oligodendrocyte progenitor (OP) differentiation and oligodendrocyte myelination is critical for effective remyelination and recovery of function in multiple sclerosis and other demyelinating diseases. We previously showed that leukemia/lymphoma-related factor (LRF) promotes OP differentiation during postnatal myelination. We now examine LRF during CNS remyelination. We show that LRF is rarely detected in OP cells yet is present in nuclei of the majority of oligodendrocytes in healthy and remyelinating CNS. In vitro, LRF regulates transcription of myelin-related genes and notch target genes. In vivo, LRF is not required for oligodendroglial repopulation of demyelinated lesions yet modulates remyelination. regulate transcription of myelin genes and notch target genes during OP differentiation. Our studies demonstrate that LRF expression in the oligodendrocyte lineage is mainly found in oligodendrocytes during remyelination and that LRF can modulate the extent of remyelination. Animals were housed and handled according to the guidelines of the National Institutes of Health and the Institutional Animal Care and Use Committee of the Uniformed Services University of the Health Sciences. 2.1 | MHV model of spinal cord demyelination and remyelination in C57BL/6 mice C57BL/6 mice (4-week old females; Jackson Laboratories) were infected with 1000 plaque forming units (PFU) of MHV strain A59 diluted in 10 mL of sterile PBS by intracranial injection as previously described (Armstrong et al., 2005; Redwine & Armstrong 1998; Vana et al., 2007b) . Control mice were injected with 10 mL of sterile PBS. Only female mice were used because males have a high mortality rate after infection with this MHV A59 strain (Armstrong et al., 2005) . Behavioral data was previously published for this cohort of mice and demonstrated neurological impairment (hang times and paralysis/paresis) of MHV-infected mice by 2 weeks post-infection (wpi) prior to random assignment to either a 4-or 8-wpi survival time point (Redwine & Armstrong, 1998) . Spinal cord tissue analysis included a cohort of 12 mice (MHV 4 wpi, n 5 3; MHV 8 wpi, n 5 3; PBS vehicle 4 wpi, n 5 3; PBS 8 wpi, n 5 3). 2.2 | Cuprizone model of demyelination and remyelination in Zbtb7a fl/fl mice Initial heterozygous breeding pairs of the floxed Zbtb7a mouse line were provided by Dr. Pier Paola Pandolfi (Beth Israel Deaconess Medical Center; Maeda et al., 2007) . Floxed Zbtb7a mice were crossed to Plp/CreER T mice, in which the proteolipid (Plp) promoter drives conditional oligodendrocyte expression of Cre recombinase fused to a mutated estrogen receptor. Breeding pairs of Plp/CreER T mice (B6.Cg- Doerflinger, Macklin, & Popko, 2003) and Zhu et al., 2011) were purchased from Jackson Laboratories. Mice were genotyped by PCR analysis of tail genomic DNA to identify wild type and floxed alleles of Zbtb7a and the presence or absence of the Cre allele (Doerflinger et al., 2003; Maeda et al., 2007) . Cuprizone, also known as bis(cyclohexanone)oxaldihydrazone, was purchased as a fine powder (Sigma-Aldrich) that was sent to Harlan Teklad to mix as 0.2% or 0.3% (w/v) to form chow pellets (diet TD.01453; Harlan Teklad). Male mice were fed cuprizone pellets for a period of 6 weeks followed by 0, 3, or 6 weeks on normal chow. Since cuprizone causes toxicity resulting in estrus cycle disruption in female C57BL/6 mice, male mice were used (Taylor, Gilmore, Ting, & Matsushima, 2010) . At the start of cuprizone feeding, mice were 8 weeks of age and weighed 22.88 6 1.14 g (s.d.). To accommodate the precuprizone wheel testing, mice in the wheel running cohorts were between 10-14 weeks of age and weighed 25.54 6 1.46 g (s.d.) at the start of cuprizone feeding. To maximize demyelination and further differentiate effects on oligodendrocyte repopulation during remyelination, mice were fed 0.2% cuprizone in the initial wheel running cohort and then 0.3% cuprizone for the subsequent wheel running cohort. To induce Cre-mediated deletion of Zbtb7a, 10mg of tamoxifen (Sigma-Aldrich) in corn oil (Sigma-Aldrich) was administered by oral gavage after 4, 5, or 6 weeks of cuprizone demyelination (as noted in the text) or in age-matched control mice. Control mice received oral gavage with the corn oil vehicle in parallel with the mice who were administered tamoxifen. The Cre deletion of Zbtb7a analysis used 65 mice, which, as noted in figure legends, included 5-7 mice per condition and time point. Sample size was based on prior data and the feasibility of breeding matched cohorts of mice to run together in a given experiment. Mice were randomly assigned to tamoxifen or corn oil conditions. Mice were perfused with 4% paraformaldehyde (Sigma-Aldrich) and post-fixed in 4% paraformaldehyde overnight at 4 8C. Segments of brain or spinal cord were cut as cryosections (15 mm thickness) and mounted onto Superfrost Plus slides (Fisher) for immunohistochemical analysis and in situ hybridization. Antibody information is provided in Table 1 . Tissue sections were immunostained for LRF using two different antibodies, which have been characterized in our previous developmental study (Dobson et al., 2012) . LRF immunolabeling was further tested to ensure specificity in lesion areas of MHV spinal cord sections. LRF immunostaining produced a similar pattern of nuclear immunofluorescence with the two different primary antibodies, and was eliminated with either omission of the primary antibody or incubation of the primary antibody with a 100 3 excess of competitive blocking peptide (Bethyl Laboratories; Supplemental Figure S1 ). Oligodendrocyte lineage cells were identified by expression of Olig2. OP cells were immunolabeled with an antibody to the external domain of NG2 (Goretzki, Burg, Grako, & Stallcup, 1999; Jones, Yamaguchi, Stallcup, & Tuszynski, 2002) . Oligodendrocytes were identified by antibodies against glutathione S-transferase pi (GSTpi) or adenomatous polyposis coli. Myelin was immunostained for 2',3'-cyclicnucleotide 3'-phosphodiesterase (CNP) or myelin oligodendrocyte glycoprotein (MOG; Breithaupt et al., 2003; Linnington, Webb, & Woodhams, 1984) . Organization of the node of Ranvier was detected by immunolabeling Nav1.6 sodium channels in the node along with contact in associated protein (Caspr) in the flanking paranodal regions. The LRF primary antibodies were detected with goat anti-hamster IgG F(ab')2 fragment conjugated with Cy3, or donkey anti-rabbit IgG F(ab')2 fragment conjugated with Cy3 (both from Jackson Immunoresearch). Donkey anti mouse IgG F(ab')2 fragment conjugated with Cy3 (Jackson Immunoresearch) was used to detect MOG. Olig2, NG2, GSTpi, CNP, and Hes5 were detected by goat anti-rabbit or donkey anti-rabbit IgG F (ab')2 fragment conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, DAVIDSON ET AL. | 2393 CA). Donkey anti-rabbit IgG F(ab)2 fragment conjugated to Alexa Fluor 594 was used to detect Nav1.6 while Caspr was detected by donkey anti-mouse IgG F(ab')2 fragment conjugated to Alexa Fluor 488 (both from Jackson Immunoresearch). Protocols were optimized to ensure that no signal was observed when primary antibody was not applied. All sections were incubated with DAPI (Sigma-Aldrich) to stain nuclei prior to mounting with Vectashield (Vector Labs). In situ hybridization was performed using a previously described riboprobe to hybridize to PLP mRNA transcripts as a marker of oligodendrocytes (Armstrong, Le, Frost, Borke, & Vana, 2002) . After hybridization, labeling was detected with alkaline phosphatase-conjugated sheep anti-digoxigenin, followed by reaction with substrate solution (nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate [NBT/BCIP]; DAKO). As detailed previously, OP cultures were prepared and grown in defined medium to induce differentiation or with mitogens added to inhibit differentiation (Armstrong, 1998; Zhou & Armstrong, 2007) . Briefly, brains from P2 rats were dissociated and plated in tissue culture flasks to produce stratified "primary" cultures. After shaking to dislodge microglia, flasks were shaken overnight to yield OP cells. Preliminary studies were performed using the previously characterized CNP promoter construct (Nielsen et al., 2004) to optimize the number of cells to plate per well, the ratio of transfection reagents, and the time point for luciferase analysis (data not shown). The same protocol was then followed for all subsequent experiments. Cells were plated at a concentration of 120,000 cells per well in 24-well plates in defined medium, supplemented with platelet-derived growth factor-AA (PDGF) and fibroblast growth factor 2 (FGF2; both 10 ng/ml; R&D Systems) to induce proliferation and prevent differentiation. One day after plating, the cells were transiently transfected by incubation for 5 hours with plasmid constructs and a 9:1 ratio of FuGENE®6 (Promega). The cultures were maintained for 1 day in defined medium (with PDGF and FGF2), and then transferred to defined medium (either with or without PDGF and FGF2) for one more day. Cells were then lysed and harvested for luciferase analysis at a total of 72 hours, post-plating, to examine promoter activity relative to continued OP proliferation or during OP differentiation. The LRF-expressing plasmid (Lenti-TomLRF) contains full length murine Zbtb7a, followed by an IRES sequence, and a tdTomato fluorescent reporter (pLVX-IRES-tdTomato; Clontech); the pLVX-IRES-tdTomato backbone without the LRF insert was used as a reference control (Dobson et al., 2012) . The OP cultures were additionally transfected with one of several firefly-luciferase reporter plasmids, each driven by a unique promoter sequence. Plasmids with firefly-luciferase expression, controlled by the promoters for Hes1 (pHes1; 2467 to 1 46) and Hes5 (pHes5; 2800 to 1 73) were used to study the response of these Notch targets (kind gifts from Dr. Ryoichiro Kageyama, Kyoto University; Ohtsuka et al., 1999) . Transfection with the Notch1 intracellular domain (Notch1IC; kind gift from Dr. Nye, Pharmacia) inserted into the PMX vector was used to increase the level of expression of both Hes1 and Hes5 (Nye, Kopan, & Axel, 1994; Zhou & Armstrong, 2007) . The All images were acquired with a Spot 2 digital camera mounted to an IX-70 microscope (Olympus), with the exception of the node of Ranvier, which was imaged as fluorescent image stacks acquired using a We first asked whether LRF was differentially expressed in the oligodendrocyte lineage during the progression of demyelinating disease with spontaneous remyelination. MHV infection results in oligodendrocyte loss over 2-3 wpi that causes focal demyelination throughout the spinal cord followed by OP amplification by 4 wpi, which then advances to oligodendrogenesis and remyelination by 8 wpi (Redwine & Armstrong, 1998; Vana et al., 2007b) . Immunohistochemistry for LRF, along with CNP to detect myelin, demonstrates the pattern of LRF Many cells immunolabeled for CNP (arrows in C') or Olig2 (arrows in F') exhibit nuclear LRF at 8 wpi. Olig2 cell populations recover by 8 wpi and the distribution of Olig2 cells is almost evenly split among those with or without LRF (G). Olig2 nuclear immunoreactivity was used to identify oligodendrocytes, as was clearly visible even at lower magnifications (D-F). A low intensity of Olig2 immunoreactivity in processes was detectable in lesion areas at higher magnifications (E', F'), which was considered non-specific signal due to changes in the lesion tissue. One way ANOVA using n 5 3 mice for each condition; Longitudinal analysis of behavior was included in the study design to assess the behavioral effect of LRF knockdown during the subsequent remyelination period ( Figure S4 ). Running activity on wheels with a complex pattern of rungs was used to detect impaired maximal running velocity, which is associated with myelination status of the corpus callosum in longitudinal studies of mice fed cuprizone (Hibbits, Pannu, Wu, & Armstrong, 2009; Mierzwa, Zhou, Hibbits, Vana, & Armstrong, 2013 ). In the current study, mice had wheels continuously available for running and every third week the wheel was changed from a training wheel (all rungs in place) to a complex wheel (non-uniform pattern of missing rungs). The same Plp/CreER T :Zbtb7a fl/fl mice that are shown for tissue analysis in Figures 3, 4 had running wheel data collected. These mice exhibited impaired maximal running velocity during cuprizone feeding, consistent with demyelination ( Figure S4a ). However, during the recovery period, there was no functional effect of LRF knockdown, based on comparison of mice administered tamoxifen versus oil ( Figure S4a ). The effect of the 6-week 0.2% cuprizone treatment on maximal running velocity was significant, but not as marked as in our prior studies (Hibbits et al., 2009; Mierzwa et al., 2013) , which may be due to the Coronal sections from Plp/CreERT:Zbtb7a fl/fl mice were analyzed to determine the effect of LRF knockdown on the oligodendrocyte lineage stage and the extent of remyelination in the corpus callosum. Tamoxifen, or oil vehicle, was administered at the end of 6 weeks of 0.2% cuprizone and then mice were fed normal chow for 6 weeks to allow for spontaneous remyelination. A-C: Immunoreactivity for MOG shows that remyelination has progressed after 6 weeks on normal chow in mice administered oil (A) or tamoxifen (B), as compared to the demyelination observed throughout the corpus callosum at 6 weeks of 0.2% cuprizone feeding ( Figure S3 ). In mice administered oil, the remyelination is equivalent to myelination in age-matched Plp/CreERT:Zbtb7a fl/fl mice that were not fed cuprizone (no cup; C). In mice administered tamoxifen, the remyelinated area of the corpus callosum is significantly reduced compared to the oil control mice (Cohen's d effect size 5 2.08; C). D-F: PLP in situ hybridization in mice administered oil (D) or tamoxifen (E) shows similar oligodendrocyte repopulation of the corpus callosum (F), which is also similar to mice that were not fed cuprizone (no cup; F). G-I: Oligodendrocyte progenitors, identified by NG2 (green; G-H), in mice administered oil (G) or tamoxifen (H) were found in a similar density (I). J-L: Mature oligodendrocytes, identified by CC1 (red; J-K) in mice administered oil (J) or tamoxifen (K) were also present at similar levels (L). cc 5 corpus callosum, cg 5 cingulum. Scale bars 5 250 um. Analysis of n 5 5 mice per condition. ANOVA with Tukey multiple comparisons used in C, F. Student t-test used in I, L. oligodendrocytes are generated while mice are still on cuprizone at 6 weeks. LRF knockdown appeared to also reduce the density of oligodendrocytes, identified by PLP expression, but this effect was not statistically significant (Figure 6d-f ). The density of OP cells, detected with in situ hybridization for PDGF alpha-receptor (PDGFaR), was not different in tamoxifen versus vehicle treated mice (Figure 6g-i) . This result is consistent with LRF expression in only a minority of OP cells so that LRF deletion should have little effect at the OP stage. FIG URE 5 Tamoxifen administration in Plp/CreERT:Zbtb7a fl/fl mice impairs remyelination, but not oligodendrocyte repopulation, after demyelination from 0.3% cuprizone ingestion. Demyelination of the corpus callosum was induced in Plp/CreERT:Zbtb7a fl/fl mice by feeding 0.3% cuprizone for 6 weeks. A higher dose of cuprizone was used to maximize demyelination and oligodendrocyte loss while allowing continuous access to either the training or complex wheel configuration. Mice were administered tamoxifen (Tam), or vehicle (Oil) after week 5 of cuprizone feeding and then perfused at the end of week 6 on cuprizone (no recovery; A, D) or after a 3 week period on a normal chow diet to allow for spontaneous remyelination (3 wks recovery; B, E). The mice analyzed for the 6 week recovery time point (6 wks recovery; C, F) are from the wheel assessments shown in Figure S3 and so were given tamoxifen at the end of the 6 week period of cuprizone demyelination. Quantification of MOG immunoreactivity was used to estimate the myelinated area of the corpus callosum (A-C). In mice administered tamoxifen to knockdown LRF, the extent of remyelination is significantly reduced at both the 3 and 6 week time points of the recovery period (3 weeks, p < 0.0364, Cohen's d effect size 5 1.35, n 5 6 oil, n 5 7 tamoxifen; 6 weeks, p < 0.0296, Cohen's d effect size 5 1.67, n 5 5 oil, n 5 5 tamoxifen). Immunolabeling for Caspr (green) and Nav1.6 (red) indicates relatively normal organization of the node of Ranvier with the progression of remyelination in mice administered oil while examples of disrupted broader nodes continued to be found in mice administered tamoxifen (C). PLP in situ hybridization shows similar oligodendrocyte repopulation of the corpus callosum in mice administered tamoxifen or oil (D-F). To gain insight as to the potential role of LRF in acting as a transcription factor relevant to remyelination, we co-transfected OP cells with an LRF expression plasmid and myelin gene promoter element plasmids driving luciferase expression (Figure 7 ). This analysis included the promoters for MBP and CNP, which are repressed by Hes5 (Liu et al., 2006) , since LRF has been associated with the notch-signaling pathway (Lee et al., 2013; Maeda et al., 2007) . Transcriptional activity from the full length MBP promoter was highest in OP cells grown in defined medium (i.e., without mitogens), which indicates appropriate transcriptional regulation during OP differentiation ( Figure 7a ). LRF significantly repressed transcription from the full length MBP promoter (Figure 7a) . LRF regulation of a truncated MBP promoter was then used to examine dependence on Sp1 transcription factor binding sites. LRF can interact with Sp1 promoter elements and Sp1 phosphorylation initiates upregulation of MBP Guo, Eviatar-Ribak, & Miskimins, 2010) . In conditions that promoted differentiation, LRF repression of the truncated MBP required an intact Sp1 site (Figure 7b,c) . The second myelin-specific gene, CNP, is expressed from an early stage in the oligodendrocyte lineage (Zhang et al., 2009) ; accordingly, the CNP promoter response was expressed similarly with and without mitogens present. CNP transcription was strongly repressed by LRF in both growth conditions (Figure 7d ). Finally, analysis was extended to the promoter for FASN, which encodes fatty acid synthase, a key enzyme in the synthesis of the fatty acid palmitate that is essential for lipids in cell membranes and myelin (Smith, Witkowski, & Joshi 2003) . In contrast to MBP and CNP, FASN promoter activity was not reduced by the presence of LRF in defined medium, which promoted differentiation (Figure 7e ). However, LRF significantly increased FASN promoter activity in mitogenic conditions that maintained OP cells in an immature, proliferative stage ( Figure 7e ). These myelin gene promoter assays indicate that LRF has the potential to regulate myelin gene transcription in a context dependent manner. FIG URE 6 Tamoxifen administration in NG2CreERT:Zbtb7a fl/fl mice impairs remyelination after demyelination from 0.3% cuprizone ingestion. Demyelination of the corpus callosum was induced in NG2CreERT:Zbtb7a fl/fl mice by feeding 0.3% cuprizone for 6 weeks. Mice were administered tamoxifen (Tam), or vehicle (Oil) after week 4 of cuprizone feeding and then perfused at the end of week 6 on cuprizone. Tamoxifen (Tam) administration significantly reduced the extent of remyelination determined by MOG immunoreactivity (A-C; p 5 0.0053, Cohen's d effect size 5 1.98, n 5 6 oil, n 5 7 tamoxifen). Tamoxifen appeared to reduce the generation of oligodendrocytes (D-F) but this difference was not statistically significant (p 5 0.1726). Tamoxifen did not change the OP cell density (G-I). cc 5 corpus callosum, cg 5 cingulum. Scale bar in H 5 100 um. The role of LRF in transcriptional regulation of the notch pathway was tested using promoter constructs of notch target genes, Hes1 and Hes5 (Figure 8 ). In cultures of OP cells in the presence of mitogens, LRF enhanced Hes1 transcription (Figure 8a ). Co-transfection with Notch1IC increases notch tone and inhibits OP differentiation (Zhou et al., 2007) . In the presence of Notch1IC, LRF enhanced transcription from the Hes1 promoter, but only in the presence of mitogens ( Figure 8b ). LRF repressed Hes5 transcription in OP cells cultured with mitogens ( Figure 8c ). In the presence of Notch1IC, LRF significantly repressed Hes5 transcriptional activity both in defined medium and in the presence of mitogens (Figure 8d) . Therefore, LRF specifically represses Hes5 transcription in OP cells across growth conditions (Figure 8c,d) . The potential for LRF and notch pathway interactions to play a role during remyelination is indicated by Notch1 and Jagged1 upregu-lation in cuprizone lesions along with LRF co-localization in cells expressing Hes5 (Supplemental Figure S6) . This time point of 5 weeks of cuprizone ingestion corresponds well with the 4-6 week cuprizone period of LRF knockdown that reduced MOG in NG2CreER T :Zbtb7a fl/fl mice (Figure 6a ). We present the first study of LRF expression and function in CNS pathology. LRF was examined in the context of demyelination, followed by oligodendrocyte regeneration and remyelination. This work shows nuclear LRF immunoreactivity in the majority of oligodendrocytes, but only rarely in OP stage cells. Therefore, LRF function was further examined in vivo with conditionally induced knockdown of LRF in FIG URE 7 LRF transcriptional regulation of myelin genes. Transcriptional activity was measured from transient transfections of OP cultures with expression plasmids either with (Tom-LRF) or without (Tom) the Zbtb7a insert encoding LRF, along with firefly luciferase plasmids containing myelin promoter elements, and Renilla luciferase as an internal control. Transcription from the full-length promoter for myelin basic protein (1323MBP) is significantly higher in defined medium (**p 5 0.0017), indicating appropriate upregulation as expected during OP differentiation in vitro (A). Co-transfection with the Tom-LRF plasmid significantly represses transcription from 1323MBP in defined medium (****p < 0.0001; A). Further, in defined medium, LRF repression of a truncated 105 bp MBP promoter requires the intact wild type Sp1 site (*p 5 0.0326; B) and is not observed with the mutation to prevent Sp1 binding (C). In the presence of mitogens, LRF does not alter transcription from the full-length 1323MBP promoter (A) yet gains repressive activity using the truncated construct with the wild type sequence (**p 5 0.0032; B) and with the Sp1 site mutated (****p < 0.0001; C). LRF expression significantly represses transcription from the 2',3'-cyclic-nucleotide 3'-phoshodiesterase (CNP) promoter in both growth conditions (****p < 0.0001; D). In the presence of LRF, transcription of fatty acid synthase from FASN is significantly increased by the addition of mitogens (**p 5 0.0037; E). Two-way ANOVA using values combined from four independent experiments. (Lunardi et al., 2013; Maeda et al., 2005; Wang et al., 2012) . Within the promoter elements tested, those that showed repression contain one or more potential LRF consensus sites in the proximal promoter region. However, LRF binding of DNA appears flexible and may involve a single site or two half sites with variable orientation and separation (Lee et al., 2013; Pessler & Hernandez, 2003) . Therefore, direct DNA binding of LRF cannot be predicted solely from a given promoter sequence. Myelin-specific genes are regulated by epigenetic marks and a hierarchy of transcription factors that vary with lineage stage (Fulton, Denarier, Friedman, Wasserman, & Peterson, 2011; Hernandez & Casaccia, 2015) . The epigenetic landscape of myelin-specific promoters may be particularly important for LRF binding or the effect of LRF molecular interactions, such as recruitment of HDACs (Liu et al., 2004) , . Sp1 may also be involved in LRF regulation of FASN in oligodendrocyte lineage cells. LRF interacts with SREBP-1 at Sp1 sites to synergistically activate FASN transcription in multiple cell lines . FASN is a key enzyme in fatty acid synthesis that is essential for phospholipids in myelin and cell membranes (Smith et al., 2003) . In OP cells, Figure 2 ). This role for LRF would also be consistent with the reduced remyelination after LRF knockdown (Figures 4-6) . CNP, another myelin-specific gene, was strongly repressed by LRF, regardless of growth condition ( Figure 7d ) and is not known to be regulated by Sp1 phosphorylation (Fulton et al., 2011) . MBP and CNP have been shown to be repressed by Hes5 (Liu et al., 2006) . Therefore, LRF overexpression could be expected to release or de-repress MBP and CNP from Hes5 repression. Importantly, in vitro LRF co-transfection reduced MBP and CNP promoter activity (Figure 7) , which is not consistent with our in vivo results of reduced myelin levels with LRF knockdown during remyelination (Figures 4-6) . A potential explanation for this in vitro result is that LRF is overexpressed after transfection of the LRF plasmid. A further explanation may be that transfection at the OP cell stage is ectopic since LRF is not expressed in the majority OP cells in vivo. Furthermore, the binding sites of transfected MBP and CNP promoter constructs may not undergo the epigenetic modifications in myelin genes that occur with lineage progression. This artificial approach thus reveals a potent LRF repression of these myelin gene promoters, especially CNP, early in the lineage that must undergo as yet unknown modifications to prevent this potent repression of myelin genes in mature oligodendrocytes. Since high levels of membrane biogenesis are required for both cell proliferation and myelin formation, FASN may not be as restricted to such cell stage specific promoter modifications. Along these lines, we note that the adult brain and spinal cord exhibits strong LRF expression in neurons, which also have a high level of membrane biogenesis to maintain extensive processes and continuously synthesize vesicles for axonal transport. Two distinct models of experimental demyelination were used to exploit the advantages of each in our studies of LRF. The MHV model produces multiple sclerosis-like lesions with environmental signals associated with lytic infection of oligodendrocytes, microglial activation, astrogliosis, and lymphocytic infiltration (Armstrong et al., 2005) . After viral clearance mediated through a Th1 immune response, demyelinated areas undergo effective spontaneous remyelination (Jordan et al., 1989; Parra et al., 1999) . MHV lesions have increased expression of diverse cytokines and mitogens, including PDGF and FGF2 mitogens (Messersmith et al., 2000; Redwine & Armstrong, 1998 (Armstrong et al., 2005) . In addition, tamoxifen may alter disease progression in female mice through binding of endogenous estrogen receptors, which raises concern for using female mice in studies utilizing tamoxifen-induced recombination (Klinge, Studinski-Jones, Kulakosky, Bambara, & Hilf, 1998) . Indeed, the severity of demyelination varies with estrus stage in female mice while male mice undergo progressive disease with high mortality (Armstrong et al., 2005) . We have used the cuprizone model effectively with tamoxifen administration to reveal effects of gene deletion during remyelination (Mierzwa et al., 2013; Zhou et al., 2012) . The cuprizone model produces extensive demyelination of the corpus callosum that involves a well-documented progression of OP proliferation and differentiation leading to efficient spontaneous remyelination (Hibbits, Yoshino, Le, & Armstrong, 2012; Matsushima & Morell, 2001) . Cuprizone ingestion results in high OP proliferation after 4-5 weeks and is followed by initial oligodendrocyte repopulation of lesions within 6 weeks (Armstrong et al., 2006; Armstrong et al., 2002) . Cuprizone demyelinated lesions include several factors examined in our in vitro promoter assays. Namely, cuprizone lesions exhibit upregulation of the mitogens PDGF and FGF2 (Armstrong et al., 2006; Armstrong et al., 2002; Vana, Lucchinetti, Le, & Armstrong, 2007a) . Notch1 is also upregulated in proliferating OP cells and Jagged1 is present in lesion areas ( Figure S6 ). Furthermore, Hes5 is present in OP cells and HDAC1 binding to the Hes5 promoter is increased during cuprizone demyelination, followed by normalization early in remyelination (Shen et al., 2008) . Hes5 inhibits OP differentiation into mature oligodendrocytes in the context of both developmental myelination and remyelination (Kondo & Raff, 2000; Liu et al., 2006; Shen et al., 2008; Wang et al., 1998) . Hes5 may also be a downstream partner that intersects with fibroblast growth factor signaling, which inhibits OP differentiation in demyelinated lesions (Zhou & Armstrong, 2007) . LRF function was tested in the corpus callosum using conditional deletion induced in oligodendrocyte lineage cells prior to remyelinaton in Plp/CreER T :Zbtb7a fl/fl mice. During remyelination, LRF was expressed in 94% of Olig2 cells; the LRF negative subset presumably overlapped with the NG2 1 OP population that comprised 5% of Olig2 cells (Figures 3, 4) . Tamoxifen administration in Plp/CreER T :Zbtb7a fl/fl mice knocked down LRF expression to 41% among Olig2 cells. LRF deletion in Olig2 cells did not reduce the total oligodendrocyte lineage population ( Figure 3 ). Somewhat surprisingly, LRF knockdown also did not alter the proportion of mature oligodendrocytes versus OP cells, with NG2 1 OP cells comprising 3% of the Olig2 population in mice administered tamoxifen (Figure 4 ). This finding is in contrast to our study of postnatal myelination during spinal cord development in which LRF knockdown inhibited differentiation to increase OP cells at the expense of oligodendrocytes (Dobson et al., 2012) . LRF knockdown significantly reduced the extent of remyelination in three independent cohorts of Plp/CreER T :Zbtb7a fl/fl mice, with no significant change in oligodendrocyte repopulation (Figures 4, 5) . The deficit of remyelination was not sufficient to impair running on complex wheels ( Figure S4 ). Impairment on the complex wheel assessment has corresponded well with MOG detection of myelination status in the corpus callosum in our prior cuprizone studies (Hibbits et al., 2009; Mierzwa et al., 2013) . Additional assessments may be needed to detect more subtle effects from reduced remyelination, including changing the conditions of the complex wheel assessment to test motor skill learning (McKenzie et al., 2014) . LRF knockdown in NG2CrER T :Zbtb7a fl/fl mice earlier in the oligodendrocyte lineage resulted in more dramatic reduction in myelination after 6 weeks of cuprizone ( Figure 6 ). However, during the recovery period after demyelination, the extent of remyelination remained reduced with tamoxifen administration in Plp/CreER T :Zbtb7a fl/fl mice (Figures 4-5) but recovered to vehicle levels in NG2CrER T :Zbtb7a fl/fl mice ( Figure S5 ). Together, these results indicate that LRF acts at a late OP and/or mature oligodendrocyte stage to modulate the extent of remyelination. lineage (Vacchio et al., 2014) . This work was supported by the National Multiple Sclerosis Society, grant numbers: RG 4224; RG 4675. NK performed the work as a Prince Mahidol Scholar with support from the government of Thailand. We thank those who provided plasmids and mouse lines, as noted in the text. We also thank Xiaomei Zi for technical assistance. Associate Editor: Ernesto Bongarzone No conflicting interests exist. All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the The authors, reviewers and editors affirm that in accordance to the policies set by the Journal of Neuroscience Research, this manuscript presents an accurate and transparent account of the study being reported and that all critical details describing the methods and results are present. 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