The myelination of axons is a crucial step during vertebrate central nervous system (CNS) development, allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly, the differentiation of oligodendrocytes, the myelinating cells of the CNS, and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor, Myelin Regulatory Factor (Myrf), as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial, however, whether Myrf directly regulates transcription, with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly, this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins, the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination.

Oligodendrocytes are a highly specialized cell type that surround axons of the vertebrate central nervous system with myelin, electrically insulating them and allowing rapid and energy-efficient propagation of nerve signals. We previously identified a protein, MYRF, that is required for the final stages of oligodendrocyte differentiation and myelination. Although we proposed that MYRF might act as a transcription factor, it remains uncertain whether this is true, given that MYRF and related proteins contain a transmembrane domain that might preclude localization to the nucleus. Here, we show that the MYRF protein undergoes an activating cleavage event to release the functional transcription factor from the transmembrane domain that otherwise anchors it to the endoplasmic reticulum. Unexpectedly, this cleavage event is mediated by a portion of MYRF that is related to a self-cleaving domain found in bacteriophage proteins. This distinguishes it from other membrane-associated transcription factors that are cleaved via regulated proteolysis within the membrane bilayer. We find that the N-terminal product of MYRF cleavage directly binds to a wide range of genes involved in myelination, stimulating their expression. Many of these MYRF binding sites identify previously uncharacterized enhancers for these myelin genes.

Funding: This work was supported by the Myelin Repair Foundation, grants from the Australian National Health and Medical Research Council (NHMRC project grant 1009095), Multiple Sclerosis Research Australia, the Trish Multiple Sclerosis Research Foundation, and NIH R01 EY10257 to BAB. BE is supported by a NHMRC CDF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we investigate the molecular mechanisms by which Myrf mediates oligodendrocyte differentiation and myelination. We find that the MYRF protein is subject to autoproteolytic cleavage within a domain related to bacteriophage tail spike proteins. This cleavage yields an N-terminal nuclear-targeted fragment containing the DBD, and is required for MYRF's promotion of myelin gene expression. Through ChIP-Seq analysis and luciferase assays we show that MYRF binds the cis-regulatory elements of multiple oligodendrocyte-specific genes involved in myelination. This binding occurs via a defined DNA consensus sequence and strongly promotes transcription from these elements. These findings establish Myrf as a membrane-associated transcription factor with a direct role in stimulating myelin gene expression.

While these results identified a vital role for Myrf in the generation and maintenance of CNS myelin, they did not address the molecular mechanisms by which it acts. Notably, the assignment of Myrf as a transcription factor was recently questioned based on a lack of nuclear localization of the C. elegans ortholog, pqn-47, with pqn-47 and Myrf instead proposed to have a role in secretion of proteins from the endoplasmic reticulum/Golgi [15] . Consistent with this, the MYRF protein contains at least one hydrophobic region that originally led to the human ortholog MYRF/C11Orf9 being classed as a probable transmembrane protein [16] . Together, these findings raise the question of whether Myrf and its orthologs promote myelination through the direct regulation of key myelin genes, or whether they may act via other mechanisms involving the membrane and myelin protein trafficking system previously implicated in myelination [17] .

We recently identified Myelin Regulatory Factor (Myrf; previously known as Gene Model 98 and MRF) as a transcript that is highly induced during oligodendrocyte differentiation and absent in other CNS cell types [10] . Based on the MYRF protein containing a putative DNA-binding domain (DBD) with homology to that of the yeast transcription factor Ndt80 [11] , [12] , we proposed that Myrf might act as a direct transcriptional regulator of CNS myelination. Consistent with this hypothesis, conditional ablation of Myrf causes severe CNS dysmyelination, with oligodendrocytes stalling at the pre-myelinating stage and showing severe deficits in myelin gene expression [13] . Inducible ablation of Myrf in mature oligodendrocytes of the adult CNS also causes a rapid down-regulation of myelin gene expression followed by a gradual degeneration of CNS myelin [14] . Unlike previously described transcription factors Olig1, Olig2, Sox10, Nkx2.2, and Ascl1, Myrf is expressed only at the postmitotic stage of the oligodendrocyte lineage, suggesting that its induction is a key step in the regulation of myelination.

Oligodendrocytes are the myelinating cells of the vertebrate CNS; their development and the ensheathment of receptive neuronal axons are vital for the rapid propagation of nerve impulses. Accordingly, the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes and their subsequent myelination of axons are highly regulated processes. At the transcriptional level, the factors involved in the development of the oligodendrocyte lineage have been relatively well characterized. The transcription factor Olig2 is required for specification of OPCs from subventricular zone precursor cells, at least within ventral regions of the CNS [1] , [2] . Olig2 is continually expressed in the lineage and has later roles in directing the chromatin-remodeling enzyme Brg1 to regulatory elements of target genes during differentiation [3] . A number of other transcription factors are subsequently required for the successful differentiation of OPCs into myelinating oligodendrocytes including Olig1 [4] , Nkx2.2 [5] , Ascl1/Mash1 [6] , Zfp191 [7] , and Sox10 [8] , [9] .

Results

In Silico Prediction of MYRF Features In spite of its clear role in regulating CNS myelination, little is known about Myrf at the protein level. To learn more about the features and likely function of the MYRF protein, we identified functional domains in its 1,139 aa sequence [13] by homology analysis (see Materials and Methods). Predicted features (illustrated in Figure 1A) include the previously described proline-rich region (residues 60–330) and putative Ndt80-like DBD (residues 393–540). In agreement with Stohr et al. [16], a transmembrane region was predicted by a number of programs at residues 767–789. In addition, several targeting motif predictors (ELM and NucPred) identified a likely nuclear localization signal (NLS) at residues 252–258. A putative coiled coil domain was identified at residues ∼685–706, though with only moderate confidence. HHpred searches as well as the NCBI conserved domain search function identified a region of similarity to the intramolecular chaperone domain (ICD) of the bacteriophage tail spike proteins at residues 587–647 (see below for further details). These predicted features and the confidence scores associated with them are listed in Table S1. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. The MYRF protein is subject to posttranslational cleavage. (A) Schematic of the MYRF protein showing positions of the predicted NLS, Ndt80-like DBD, ICD, coiled coil region, and transmembrane region. (B) Western blot analysis of a double tagged (N-terminal Myc, C-terminal FLAG tagged) or untagged MYRF expression construct in 293T cells. Probing with anti-Myc or anti-FLAG reveals the presence of a ∼140 kDa full-length fragment and truncated cleavage products. (C) Western blot of control and Myrf CKO cultured mouse oligodendrocyte lysates with anti-C-terminal MYRF mAb. (D) Cell fractionation experiment showing that the majority of Myc-tagged N-terminal cleaved MYRF product is present in the nucleus of 293T cells (Anti-Hsp60 and anti-Parp used for cytoplasmic and nuclear controls, respectively). (E) CG-4 cells transfected with the Myc-MYRF-FLAG construct were co-stained for anti-FLAG and either anti-Myc, anti-Calnexin, or anti-golgi matrix protein 130 (gm130). (F–H) Adult mouse optic nerve stained with the anti-N-terminal-MYRF and anti-C-terminal-MYRF-mab antibodies (F), anti-C-terminal-MYRF-mab and anti-Sox10 (G), or anti-N-terminal-MYRF-mab and CC1 (H). https://doi.org/10.1371/journal.pbio.1001625.g001

MYRF Protein Is Cleaved to Yield a Nuclear N-Terminal Fragment The presence of both predicted transmembrane and DBDs raised the possibility that MYRF may be subject to proteolytic cleavage in a similar manner to membrane-associated transcription factors such as Notch and the SREBPs [18],[19]. We therefore performed Western blot analysis on a double-tagged MYRF expression construct (Myc-MYRF-FLAG) in 293T cells. Probing with both anti-Myc and anti-FLAG detected a faint ∼140 kda band corresponding to a full-length protein as well as more intense bands at ∼75 kda and 70 kda, respectively, corresponding to cleavage products (Figure 1B). Analysis of a number of truncated MYRF constructs indicated that this cleavage occurred several kDa past the C-terminal end of the predicted DBD (see Figure S1). To confirm that the cleavage was not an artifact of overexpression of the tagged protein or the cell line, we assessed the cleavage of the endogenous protein in primary mouse oligodendrocytes using a monoclonal antibody mapped to the C-terminal cleavage product of MYRF (anti-C-terminal-MYRF-mab). We found that the endogenous MYRF protein undergoes the same cleavage event, giving two bands of equivalent sizes to the anti-FLAG immunoblotting of the Myc-MYRF-FLAG construct. These bands were absent in lysates from MYRF conditional knockout (CKO; MyrfFL/FL; Olig2WT/Cre) oligodendrocytes (Figure 1C), confirming their identity. Subcellular fractionation experiments indicated that the Myc-tagged N-terminal cleavage product was predominantly located in the nucleus (Figure 1D). To further investigate the subcellular localization of the cleavage products we transfected the CG-4 oligodendroglial cell line [20] with the Myc-MYRF-FLAG construct. Immunostaining confirmed that the Myc-tagged N-terminal product predominantly localized to the nucleus (though at higher exposure additional extranuclear staining was also apparent). In contrast, the FLAG-tagged C-terminus was excluded from the nucleus, predominantly co-localizing with the endoplasmic reticulum marker calnexin (Figure 1E). To determine localization in vivo, mouse optic nerve sections were stained with the anti-C-terminal-MYRF-mab and a rabbit polyclonal raised against the N-terminal region of MYRF (anti-N-terminal-MYRF). Co-staining with the two antibodies resulted in labeling of the same cells, with the anti-N-terminal antibody staining the nucleus and the anti-C-terminal monoclonal resulting in extranuclear staining of the same cells and in the majority of Sox10+ cells (Figure 1F and G). Double-staining with anti-N-terminal-MYRF and the mature oligodendrocyte marker CC1 confirmed that the MYRF-expressing cells were oligodendrocytes (Figure 1H). These results show that endogenous MYRF is subject to cleavage both in vitro and in vivo, resulting in nuclear translocation of the N-terminal domain only.

Activating Cleavage of MYRF Occurs Via an Autoproteolytic ICD Related to Bacteriophage Tail Spike Proteins We next sought to identify the mechanism of the cleavage event. HHpred and NCBI conserved domain searches using the region of MYRF that we predicted to contain the cleavage site as input (residues 546–763) revealed a region of significant homology to RCBS structural entries 3GUD and 3GW6 (with E-values of 2.8E-17 and 4.9E-12 for 3GUD3, respectively; see Table S1). These hits represent the ICD of the bacteriophage tail spike proteins, including the GP12 neck appendage and Endo-N-acetylneuraminidase (endosialidase) proteins. Alignment of MYRF and the bacteriophage neck appendage protein (Uniprot Q9FZW3) within this region of 61 amino acids revealed an amino acid identity of 21.3%, with 49.1% similarity (Figure 2A). The ICD mediates folding and subsequent autoproteolytic cleavage of these proteins to release a functional trimeric N-terminal fragment [21]–[23]. Although this protein domain has not to our knowledge been reported to mediate proteolytic cleavage of proteins in eukaryotes, the high degree of similarity between MYRF and the ICD of the bacteriophage tail spike proteins at a site closely matching the predicted cleavage site of MYRF was striking. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Autoproteolytic processing via the ICD and a NLS are required for nuclear localization of MYRF. (A) ClustalW2 alignment of the peptide sequence of MYRF and the ICD domain of the bacteriophage GA-1 Neck appendage protein. The serine/lysine dyad residues subjected to mutagenesis are highlighted. (B) Western blot analysis of un-mutated or ICD mutant (K592R, K592H, K592M) Myc-tagged MYRF constructs in 293T cells. (C) Immunofluorescence for the N-terminal Myc tagged MYRF and the K587H mutant in CG-4 cells. Prevention of the cleavage is associated with a loss of nuclear localization of the N-terminus. (D) Primary rat OPC cultures co-transfected with GFP and either empty vector (pcDNA3) or pcDNA3 containing MYRF or the S587A and K592H mutant constructs, stained for MOG. (E) Quantification of the percentage of transfected (GFP+) cells expressing MOG 48 h posttransfection in each condition. **p<0.01 by t test. (F) Predicted NLS within the proline-rich region of MYRF showing the KKR to AAA mutation in the Myc-MYRFΔNLS construct. (G) Western analysis of the Myc-MYRF and Myc-MYRFΔNLS construct; mutation of the NLS has no effects on the cleavage of MYRF, though it routinely led to an increase in protein levels. (H) Representative images of immunostaining for the N-terminal Myc tag showing shift from nuclear to extranuclear staining in the Myc-MYRFΔNLS construct. (I) Quantification of the proportion of predominantly nuclear, mixed, or predominantly extranuclear staining seen with each construct (100 cells assessed/condition). Scale bars, (C and H) 10 µm and (D) 50 µm. https://doi.org/10.1371/journal.pbio.1001625.g002 Autoproteolytic processing within the endosialidase ICD is dependent on a serine-lysine dyad that mediates cleavage at the serine residue [21],[22]. To assess whether this domain may also mediate cleavage of MYRF, we performed site-directed mutagenesis of the equivalent amino acids in MYRF (Figure 2A). Nonconservative mutation of S587 (S587A) or mutation of K592 (K592H, K592R, and K592M) in the Myc-MYRF-FLAG construct was sufficient to block the cleavage of MYRF as assessed by Western blot analysis; in contrast, the cleavage was preserved with a conservative mutation of the S587 residue (S587C) (Figure 2B). In addition to the absence of cleavage, the S587A, K592H, or K592R mutants were blocked from the nucleus (shown for the K592H mutation in Figure 2C), demonstrating that the cleavage is a prerequisite for nuclear localization of the protein. To determine the functional consequences of blocking cleavage we co-transfected primary rat OPCs in proliferative conditions with GFP and either empty vector (pcDNA3), or pcDNA containing Myc-MYRF-FLAG or the corresponding S587A and K592H mutant constructs. As previously described [13], forced expression of MYRF results in precocious expression of the mature marker Myelin Oligodendrocyte Glycoprotein (MOG) in a subset of cells within 48 h of transfection. In contrast, the S587A and K592H mutants did not increase MOG expression relative to the pcDNA3 control transfected cells (Figure 2D–E), confirming that the uncleavable mutants were unable to promote myelin gene expression. The predicted NLS within the proline-rich region of MYRF (Figure 1A) was consistent with the observed nuclear localization of the N-terminal cleavage product. To assess whether the predicted NLS has a role in nuclear targeting of the N-terminal cleavage product, we mutated the putative NLS sequence in the Myc-MYRF construct (254KRR256 to 254AAA256; Myc-MYRFΔNLS) (Figure 2F). Unlike mutation of the ICD, mutation of this putative NLS did not inhibit the cleavage of MYRF, however total levels of the Myc-MYRFΔNLS protein invariably appeared to be higher than the unmutated protein (Figure 2G). Immunostaining for the Myc-MYRF and Myc-MYRFΔNLS proteins showed that mutation of the NLS shifted the predominant localization of the N-terminal region from nuclear to extranuclear (Figure 2H and I), indicating that this NLS largely mediates the nuclear localization of the N-terminal portion of the protein. These results demonstrate that the MYRF protein is cleaved via a domain related to the ICD chaperone domain of bacteriophage tail spike proteins to yield a nuclear-targeted N-terminal fragment consisting of the proline-rich region and DBD. The C-terminal cleavage product containing the transmembrane region is excluded from the nucleus.

MYRF ChIP-Seq Peaks Identify Novel Enhancers of Myelin Gene Transcription The positioning of the MYRF binding sites relative to oligodendrocyte-specific genes indicated that MYRF binding may identify cis-regulatory elements/enhancers for these genes. We therefore cloned a number of 400–700 bp DNA sequences encompassing the MYRF peaks shown in Figure 4 into the pGL3-promoter construct upstream of the SV40 promoter and the luciferase gene (see Table S4 for genomic coordinates of regions used and expression profiles of the associated genes). When transfected into CG-4 cells, these DNA regions modestly increased luciferase activity by several-fold relative to the control vector. Co-transfection of a MYRF expression vector with these putative enhancers induced luciferase expression by a further 4–12-fold. In contrast, MYRF co-expression had no effect on luciferase expression from the pGL3 vectors lacking enhancers (pGL3-Promoter), with an irrelevant SV40 enhancer (pGL3-Control) or with a control DNA region 1 kb upstream of the MYRF binding site identified within intron 1 of the Cntn2 gene (Figure 5A). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. MYRF binding identifies enhancers of myelin gene expression. (A) DNA sequences (from 400–700 bp) encompassing the MYRF peaks proximal to the Cntn2, Trf, Mag, Mbp, and Plp1 genes (Figure 4) were cloned into pGL3 upstream of the SV40 promoter and luciferase gene and co-transfected into the CG-4 cell line with either empty (pCS6) or MYRF overexpression (pCS6-MYRF) vectors. Co-expression of MYRF strongly induces luciferase expression in the presence of these enhancers, but has no effect on luciferase activity in their absence. (B) The MYRF-bound regions identified upstream of the Mbp and Plp1 genes strongly promote luciferase expression in control oligodendrocytes relative to Myrf CKO oligodendrocytes, mirroring the loss of expression of MBP protein in the Myrf CKO cells (C). Fold inductions for all conditions are expressed relative to the pGL3-Promoter condition in control cells (pCS6 transfected in A, MyrfWt/Fl in B). Data are shown as means and SEMs from 4–5 independent experiments. *p<0.05, **p = 0.01, **p<0.001 based on two-way ANOVA with Bonferroni posttest. https://doi.org/10.1371/journal.pbio.1001625.g005 To confirm that endogenous levels of MYRF can also regulate transcriptional activity from these elements, we selected two of these constructs, one from 19.1 kb upstream from the main Mbp TSS and one 80.7 kb upstream of the Plp1 TSS, and transfected them into primary OPCs derived from either control (MyrfWT/FL; Olig2WT/Cre) or Myrf conditional knockout (MyrfFl/FL; Olig2WT/Cre) mice. These OPCs were placed in differentiating conditions for 48 h before being assayed for luciferase activity to induce the expression of endogenous MYRF. The enhancers increased luciferase expression ∼60-fold in control oligodendrocytes relative to the promoter-only constructs, indicating that these ChIP-Seq-identified regions represent powerful oligodendrocyte enhancers. In contrast, only a modest increase in luciferase activity was seen in the MYRF conditional knockout cells (Figure 5B, p<0.001 between genotypes for each construct), mimicking the loss of endogenous MBP expression in these cells (Figure 5C).

MYRF Binds DNA Via a Defined Consensus Sequence Previous work has shown that the putative DBD from the human ortholog MYRF/C11Orf9 is not functionally interchangeable with the DBD of Ndt80, suggesting the MSE DNA consensus sequence recognized by the yeast members of the family may not be conserved throughout evolution [11]. To identify a DNA consensus motif for MYRF, we first submitted the central 100 bp sequences of 80 MYRF peaks proximal to oligodendrocyte-specific genes to MEME-ChIP [29] for de novo motif analysis. This analysis revealed a consensus sequence [G/C]CTGGYAC (where Y = C or T) as the strongest candidate (Figure 6A), which did not match any known consensus sequences for other transcription factors based on analysis with Tomtom [30]. This motif was confirmed in a broader de novo motif analysis using the central 100 bp from the 500 strongest peaks as input (Figure 6B), which also identified the 7 bp core CTGGYAC in 395 of the 500 sequences (E-value 2.7e-264). Parallel analysis submitting all 2,085 Myc-MYRF peak sequences to DREME [31] also yielded the same seven base pair motif with an E-value of 7.6e-061 (Figure 6C). Suggestively, the second most enriched motif in the DREME analysis was ACAA[A/T]G (E-value 1.1e-028), a close match to the consensus sequence for the oligodendrocyte transcription factor Sox10 (Figure 6D). Central enrichment analysis of the two motifs indicated that while the Sox10 motif showed little central enrichment within the input sequences, the CTGGYAC motif showed a strong central tendency (p = 1.2e-84), consistent with it being the primary binding motif for MYRF (Figure 6E). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. De novo identification of consensus sequences from MYRF peaks. (A) De novo sequence analysis using MEME from 80 peaks identified within 100 kb of oligodendrocyte-enriched genes identifies the sequence CTGGYAC, where Y = C or T. In separate analyses, essentially the same motif was identified using the 100 bp sequences surrounding the 500 strongest peaks using MEME (B) or 500 bp sequences of all 2,085 peaks using DREME (C). The second strongest motif identified in the DREME analysis from (C) was ACAA(A/T)G (D), a strong match for the known consensus sequence for Sox10. (E) Central enrichment analysis of the CTGGYAC and ANAA(A/T)G (Sox10) motifs in the 2,085 500 bp input sequences. https://doi.org/10.1371/journal.pbio.1001625.g006 To assess the functional significance of the CTGGYAC motif, we selected six MYRF peaks with clear examples of the motif (the previously analyzed peaks from proximal to the Trf, Mag, and Cntn2 genes, an intronic peak from the Rffl gene and two peaks from the first intron of the Nfasc gene, see Table S4) and assessed them in luciferase assays. In all six cases the wild-type sequences promoted luciferase expression when co-expressed with MYRF. In four cases (Mag, Rffl, and the two Nfasc peaks) PCR mutagenesis of the CTGGYAC motif completely abolished the effect of MYRF (Figure 7A). In the other two cases (Cntn2 and Trf), mutation of the motif had no effect on the ability of MYRF to enhance transcription from these DNA regions, however other close matches for the CTGGYAC motif present in these enhancers may suggest redundancy in binding and explain the retained function. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 7. MYRF binds DNA via the CTGGYAC motif. (A) Mutational analysis of the CTGGYAC motif in six luciferase reporters containing MYRF-bound DNA regions from near the Trf, Mag, Cntn2, Rffl, and Nfasc genes in CG-4 cells. In four out of the six sequences, mutation of a single CTGGYAC sequence (Δ) was sufficient to abolish the effect of MYRF. Fold inductions for all conditions are expressed relative to the pGL3-Promoter and pCS6 co-transfected control cells. Data are shown as means and SEMs from three independent experiments. ***p<0.001. (B) DNA pulldowns using double-stranded oligonucleotides corresponding to the predicted MYRF binding site in the Rffl intronic enhancer, or equivalent oligonucleotides with the seven base pair motif mutated, conjugated to magnetic beads. The wild-type Rffl sequence efficiently captured Myc-MYRF330–1139 from cell lysates, whereas no interaction was detected for the mutated sequence or beads without DNA (C). (D) Sequence alignment between MYRF, Dictyostelium MrfA (Uniprot Q54PT9), and S. cerevisiae Ndt80 (Uniprot P38830) showing conservation of basic amino acids required for DNA binding by Ndt80 (highlighted). (E) DNA pulldown assay measuring interaction between the DNA sequence from the Rffl enhancer and the DNA binding domain of wild-type or mutant MYRFs. All detections performed with anti-Myc. https://doi.org/10.1371/journal.pbio.1001625.g007 To further confirm direct binding of MYRF to this motif, we performed a DNA pull-down assay, conjugating double-stranded 37 bp oligonucleotides, corresponding to the predicted binding site in the Rffl intronic enhancer, to magnetic beads (Figure 7B). These oligonucleotides could capture the DBD of MYRF from cell lysates. In contrast, corresponding oligonucleotides with the CTGGYAC motif mutated or beads alone showed no interaction with the DBD of MYRF (Figure 7C). MYRF and its human ortholog (MYRF/C11Orf9) were initially identified as putative transcription factors due to apparent conservation of several basic amino acids required for the DNA binding activity of yeast Ndt80 [11],[12]. As Russel et al. [15] note, the overall degree of sequence homology between Ndt80 and MYRF is quite low, however a recent report found that the key residues required for DNA binding by Ndt80 are also required for DNA binding by dictostelium MrfA [32]. To assess whether this requirement is shared by the vertebrate orthologs, we made individual point mutants of the equivalent basic residues in MYRF (K339, R454, and R478; Figure 7D) and assessed their ability to interact with the Rffl intronic enhancer sequence (Figure 7B) in DNA pull-down assays. In agreement with their vital role for DNA binding in both yeast and dictostelium, mutation of each of the residues in MYRF led to a dramatic decrease in DNA binding. These DNA pull-down experiments demonstrate a bidirectional specificity of MYRF binding to DNA, requiring both conserved residues within the DNA binding domain of MYRF as well as a specific target DNA sequence.