Identification of the oligodendrocyte-enriched transcription factor Sip1 as a common target of Olig1 and Olig2
To identify the target genes directly regulated by Olig2, we carried out whole-genome ChIP sequencing using purified rat oligodendrocytes. Due to the lack of ChIP-grade anti-Olig1 antibody, gene-chip microarray transcriptome analysis was used, in this case, to screen for Olig1-regulated genes. ChIP-sequencing data revealed 5,439 genes carrying candidate Olig2 binding sites with four-fold enrichment over control (Figure S1A
). We compared these candidates with Olig1-regulated genes that are downregulated in the optic nerve of Olig1 null mutants (Chen et al., 2009b
) and identified 398 genes (Figure S1A
) as common candidate targets of Olig2 and Olig1 (Table S1
). The majority of them are involved in biological processes that connect to myelination (Figure S1B
). By focusing on oligodendrocyte-enriched transcriptional regulators regulated by both Olig1 and Olig2, we identified the zinc finger homeobox transcription factor Sip1/Zfhx1b. Olig2 was found to bind strongly to multiple sites around and within the Sip1
gene that are highly conserved in vertebrates (Figure S1C
). The Sip1
transcript is highly enriched in the spinal white matter, and substantially downregulated in Olig2
null mice at embryonic day (E) E18.5 and postnatal day (P) P14, respectively (). In addition, overexpression of Olig1 and Olig2, individually or in combination, was found to activate Sip1
expression in adult rat hippocampus-derived early oligodendrocyte progenitor cells () (Chen et al., 2009b
; Hsieh et al., 2004
). Collectively, these data suggest that the Sip1
gene is a common downstream target regulated by both Olig1 and Olig2.
Identification of Olig1/2-regulated Oligodendrocyte-enriched Transcription Factor Sip1
To identify Sip1-expressing cell types, we performed immunohistochemistry analysis of Sip1 and co-stained for the oligodendrocyte lineage marker Olig2. Sip1 was detected in the majority, if not all, of Olig2-positive (+) cells in the white matter of the spinal cord at P14 (). We determined the developmental state of Sip1+ cells in the oligodendrocyte lineage, by co-labeling Sip1 with the stage-specific markers for differentiated oligodendrocytes (CC1+ or MBP+) or their precursors (PDGFRα+) in the spinal cord and in cultured oligodendrocytes. High Sip1 protein levels were detected in mature oligodendrocytes, in contrast to low levels in OPCs (). In addition, the majority of Sip1+ cells in the oligodendrocyte lineage were differentiated oligodendrocytes in the corpus callosum, cortex and spinal cord (). The proportions of CC1+ and Olig2+ cells among the Sip1+ cells in the spinal white matter at P14 are 82.5±5.8% and 96.0±4.0%, respectively (> 500 cell count; n=3). We did not observe Sip1 expression in GFAP+ astrocytes in white matter tracts of the CNS (data not shown). These observations suggest that Sip1 is largely confined to oligodendrocytes in the developing white matter.
Sip1 is required for oligodendrocyte maturation and myelination
To assess the functional role of Sip1 in oligodendrocyte development in vivo
, we generated oligodendrocyte-lineage specific Sip1
knockout mice. Conditional Sip1flox/flox
mice (Higashi et al., 2002
) were bred with an Olig1-Cre
line, in which Cre recombinase is produced in the oligodendrocyte lineage (Xin et al., 2005
; Ye et al., 2009
) (). We observed that all resulting mutant Sip1flox/flox;Olig1Cre+/−
mice (referred to as Sip1cKO), but not their control littermates, developed generalized tremors, hindlimb paralysis and seizures from postnatal week 2 (, upper panel), although they were born at a normal Mendelian ratio. Sip1cKO mice exhibited the phenotypes reminiscent of myelin-deficient mice (Nave, 1994
), and died around postnatal week 3, in contrast to the normal lifespan of wild-type and Sip1 conditional heterozygous control (Sip1flox/+;Olig1Cre+/−
) mice (). The optic nerve, a well-characterized CNS white matter tract, from Sip1cKO mice was translucent compared to the control (, lower panels), which is a sign of severe deficiency in myelin formation.
Sip1 is Required for Oligodendrocyte Myelination
To confirm the myelin-deficient phenotypes, we examined myelin gene expression in Sip1cKO mice. In contrast to robust expression in control mice, expression of myelin genes such as Mbp (myelin basic protein) and Plp1 (proteolipid protein) is essentially undetectable in the forebrain, spinal cord and cerebellum of mutant mice at P14 (). In light of our data demonstrating that expression of mature oligodendrocyte markers was absent in Sip1cKO mice, we further examined myelin sheath assembly in the CNS of these mutants by electron microscopy. In contrast to a large number of myelinated axons that are observed in control mice at P14 (upper panels in ), they were completely absent in the optic nerve and spinal cord of Sip1cKO mutants (lower panels in ), indicating that myelin ensheathment has not begun in these animals. These results suggest that Sip1 is required for myelinogenesis in the CNS.
Normal oligodendrocyte precursor development in Sip1 mutant mice
Despite the deficiency in myelin gene expression, the OPC marker PDGFRα was detected in the brain and the spinal cord in the mutant mice (). The number of OPCs and their proliferation rate (percentage of Ki67+ proliferating OPCs) in Sip1
mutants were comparable to control mice (). We did not detect any significant cell death in the brain and spinal cord of Sip1cKO mice at P7 and P14 based on TUNEL assay and staining for the active form of Caspase-3 (n=3; data not shown). In addition, oligodendrocyte lineage specific Sip1
inactivation did not lead to obvious alterations of astrocytes and neurons marked by GFAP and NeuN, respectively, in the brain of Sip1cKO mice (Figure S2
). Our data indicate that OPCs are able to form in the CNS of Sip1cKO mice.
Normal Oligodendrocyte Precursor Development in Sip1cKO Mice
To investigate whether the differentiation capacity of OPCs in the absence of Sip1 in vitro is blocked, we carried out Cre-mediated Sip1 excision in cultures of purified OPCs. OPCs from the neonatal cortex of Sip1 flox/flox mice were transduced with lentivirus expressing GFP (lenti-GFP) and lenti-CreGFP. Two days post transduction, OPC cultures were switched to oligodendrocyte differentiation medium to promote oligodendrocyte maturation. In lenti-GFP transduced Sip1flox/flox cells, we observed an increase of mature MBP+ oligodendrocytes typically bearing a complex morphology during differentiation (). In contrast, under such differentiation conditions, no MBP+ oligodendrocytes were detected in lenti-CreGFP infected Sip1flox/flox cells (). All Sip1flox/flox cells transduced with lenti-CreGFP remained as PDGFRα+ OPCs (). As a control, infection of wildtype OPCs with lenti-CreGFP did not affect OPC differentiation (data not shown). These observations indicate that the ablation of Sip1 in the oligodendrocyte lineage in vivo and in vitro even under the differentiation-promoting condition prevents OPCs from further differentiation, suggesting that Sip1 is a key component of the intracellular machinery that is essential for OPC maturation.
Sip1 promotes OPC differentiation by modulating critical differentiation regulators
Given the essential role of Sip1 in oligodendrocyte maturation in vivo
, we then asked whether Sip1 is sufficient to promote OPC differentiation. For this, we isolated OPCs from the neonatal rat brain and cultured these cells in oligodendrocyte growth medium containing the mitogen PDGF-AA, and then transfected these cells with expression vectors carrying a GFP-control and/or Sip1 cDNA, and immunostained for the differentiated oligodendrocyte marker RIP (Friedman et al., 1989
) four days after transfection. In the control group, spontaneous OPC differentiation detected as RIP+ cells was less than 3% ( left panel, and ) in the presence of PDGF-AA mitogen. In contrast, Sip1 overexpression led to a drastic increase of RIP+ mature oligodendrocytes that harbored complex processes ( right panel, and ), while displaying a concomitant reduction of PDGFRα+ OPCs (). Similarly, there was a significant increase of galactocerebroside O1+ differentiated oligodendrocytes with Sip1 transfection (). The extent of process outgrowth measured by average circumference of O1+ oligodendrocytes with transfected Sip1 vector is significantly greater than that of spontaneous differentiated cells with control vector (179 μm ± 42 versus 119 μm ± 18, p < 0.01). These results indicate that high levels of Sip1 promote OPC maturation.
Sip1 Promotes OPC Differentiation by Modulating Oligodendrocyte Differentiation Regulators
To further examine Sip1 as a key regulator for oligodendrocyte differentiation, we performed qRT-PCR analysis of oligodendroglial gene alteration after Sip1 vector transfection. Our data revealed a significant upregulation of myelin genes such as Cnp, Cgt and Mbp, and of the genes encoding crucial differentiation activators such as Sox10, MRF and Olig2 in Sip1-transfected cells compared to the control (). Conversely, we observed significant downregulation of steady-sate levels of transcripts for negative regulators of differentiation, including Id2, Id4, Hes1, Hes5 and BMPR1a (). These results suggest that Sip1 promotes oligodendrocyte differentiation by activating positive regulators while repressing negative regulators of oligodendrocyte differentiation.
Sip1 antagonizes the inhibitory effect of receptor-activated BMP-Smad signaling on the oligodendrocyte differentiation program
In the presence of BMP4, expression of myelin genes Mbp
in differentiating oligodendrocyte precursors was inhibited (). However, overexpression of Sip1 was able to reverse BMP4-induced suppression of these myelin genes (). To investigate a possible link between the function of Sip1, identified as a Smad-interacting transcriptional repressor (Remacle et al., 1999
; Verschueren et al., 1999
), and BMP-Smad transcriptional activity in regulating oligodendrocyte differentiation, we examined the promoter activity of myelination-associated genes in the presence of Sip1 and activated BMP receptor signaling, which was shown to inhibit oligodendrocyte differentiation (Cheng et al., 2007
; Hall and Miller, 2004
). Expression of Smad1, and its subsequent activation by phosphorylation (p-Smad1) was achieved by co-transfection of expression vectors carrying Smad1 and constitutively activated BMP receptor 1b (mutant Q203D) (BMPRCA
), the latter obviating the need to stimulate the cells with ligand but recapitulating faithfully receptor-mediated Smad activation (Skillington et al., 2002
). This combination was found to significantly repress Mbp
reporter activity (Ye et al., 2009
), in a BMPRCA
-dependent fashion, in the oligodendrocyte cell line Oli-Neu (Kadi et al., 2006
). On the other hand, BMPRCA
-activated Smad1 significantly enhanced reporter activities directed by the promoter of differentiation inhibitory genes Id2
, acknowledged downstream target genes of BMPR-Smad signaling (Samanta and Kessler, 2004
), as well as of Hes1
, an effector of activated Notch signaling (Ogata et al., 2010
; Wu et al., 2003
). Addition of p300/CBP, a co-activator of p-Smad1 (Nakashima et al., 1999
; Pearson et al., 1999
), further reduced the Mbp
promoter activity and enhanced Hes1, Id2
reporter activities (). In contrast, overexpression of Sip1 antagonized the inhibitory effects mediated by BMPRCA
/Smad1/p300 expression on the Mbp
promoter activity while repressing the promoter activity of Id2, Id4
activated by BMP-Smad signaling (). These results suggest that Sip1 blocks p-Smad1/p300 complex mediated transcriptional activation of oligodendrocyte differentiation inhibitors.
Sip1 Antagonizes the Inhibition of BMPR-Smad Signaling in Oligodendrocyte Maturation
To determine whether Sip1 would interfere with p-Smad/p300 complexes and physically interact with p-Smad1, we introduced Smad4, the co-Smad of Smad1, and BMPRCA
individually or in combination with Sip1, and performed co-immunoprecipitation assays. In the absence of BMPRCA
, Sip1 interacted weakly with p-Smad1 as long as Smad4 was present (). This interaction increased dramatically when BMPRCA
was introduced (), suggesting that Sip1 is associated with receptor-activated Smad1 likely in a complex with Smad4. To verify the Sip1-pSmad interaction at the endogenous protein level, we carried out co-immunoprecipitation assays using mouse brain tissues at different stages. Sip1 was found to interact with p-Smad in cortical tissues at P0, P7, P14 and P60 (). The decrease of p-Smad pulled-down by Sip1 with ages might reflect a reduction of activated BMPR-Smads when OPCs differentiate into mature oligodendrocytes (Cheng et al., 2007
). To further demonstrate this interaction during oligodendrocyte differentiation, we performed a co-immunoprecipitation assay in differentiating oligodendrocytes using an antibody against p300, which was previously shown to interact with p-Smad and bridge the p-Smad transcriptional activity (Nakashima et al., 1999
). Sip1 was detected in the complex of p-Smad together with p300 (). Given that p-Smad is observed in CC1+ differentiating oligodendrocytes in the developing spinal cord at P7 (), the physical interaction Sip1 with p-Smad suggests that Sip1 inhibits the p-Smad/p300-mediated negative regulatory activity during oligodendrocyte maturation.
Furthermore, endogenous Sip1 was found to bind to the Sip1-consensus binding sites of promoter regions of Id2 and Hes1 in OPCs and Id4 in differentiating oligodendrocytes () by ChIP assays, suggesting that Sip1 targets directly the promoter of the genes for these differentiation inhibitors. Together, these observations suggest that Sip1 interacts with activated p-Smad and directly regulates the expression of a set of genes encoding differentiation inhibitors, thereby blocking the inhibitory effects of BMPR-Smad-p300 signaling on oligodendrocyte differentiation ().
Smad7 is a downstream target gene induced by Sip1 in oligodendrocyte lineage cells
As an unbiased approach to determine the downstream genes of Sip1 that regulate oligodendrocyte differentiation, we also carried out mRNA microarray profiling analysis in the spinal cord of control and Sip1cKO mice at P14. Consistent with our in situ
hybridization analysis (), myelination-associated genes including myelin genes for mature oligodendrocytes and critical differentiation regulatory genes (such as MRF
) were found remarkably downregulated in the spinal cord of Sip1
mutants (Table S2
) (Figure S3
Besides previously known transcriptional regulators for myelination, the clustering analysis of the transcriptome for myelin genes revealed that Smad7
was drastically downregulated in Sip1
mutants (; Table S2
). Smad7, a member of I-Smads, is a negative feedback regulator of signaling by liganded TGFβ and BMP receptor complexes (Massague et al., 2005
expression appeared in the ventral spinal cord at P0, increased strongly in the spinal white matter at perinatal stages, and persisted into adulthood (). Consistent with the Sip1
expression pattern, intense Smad7 protein staining () and RNA levels () were predominantly detected in differentiated oligodendrocytes but not in OPCs in developing spinal cord and primary oligodendrocyte culture ().
Smad7 is an Oligodendrocyte-specific Downstream Target of Sip1
Analysis of the expression pattern of Sip1
in the spinal cord at early developmental stages indicates that Sip1 mRNA was detected as early as E16.5 while Smad7 was initially detected at P0 in the developing white matter (Figure S4
), suggesting that expression of Sip1
precedes that of Smad7
in the oligodendrocyte lineage. In addition, we identified Sip1 consensus binding sites (Remacle et al., 1999
) in the highly conserved Smad7
promoter (). To determine whether Smad7
is a direct target gene of Sip1, we performed ChIP on the chromatin isolated from OPCs and differentiated oligodendrocytes. Sip1 was recruited to the Smad7
promoter region that carries Sip1 consensus binding sites in differentiating oligodendrocytes but this enrichment was barely detectable in proliferating OPCs (). In addition, overexpression of Sip1 in OPCs significantly promoted Smad7 mRNA expression assayed by qRT-PCR (). Collectively, these data suggest that Smad7
is a direct Sip1-induced target gene in the oligodendrocyte lineage.
Smad7 overexpression rescues the differentiation defect of Sip1-deficient OPCs and targets inhibitory signaling pathways
is a critical target gene of Sip1 in myelination, introducing and overexpressing Smad7 should rescue the defect caused by Sip1
deletion. OPCs were isolated from cortices of control and Sip1cKO pups at P1 and transduced with GFP control or HA-tagged Smad7 encoding lentivirus. Under differentiation condition, robust MBP expression was detected in the culture derived from control OPCs; in contrast, no MBP+ oligodendrocytes were observed in Sip1
mutant OPCs (). When Sip1
mutant OPCs were transduced with Smad7
expressing lentivirus, a significant increase in MBP+ oligodendrocyte formation was detected (). Mature oligodendrocytes formed after Smad7 transduction of Sip1cKO cells were confirmed by the detection of the HA-epitope tag on Smad7 (). These observations suggest that Smad7 rescues, at least partially, the differentiation defect of OPCs in the absence of Sip1. In addition, Smad7
transduction in developing chick neural tube was able to promote ectopic expression of the OPC marker PDGFRα
and a differentiated oligodendrocyte marker Sox10
), indicating that Smad7 is capable of inducing oligodendrocyte differentiation in vivo.
Smad7 Overexpression Rescues OPC Differentiation Defects Caused by Sip1 loss
Smad7 can negatively regulate TGFβ/BMP signaling in various ways, including via forming a complex with Smurf proteins or other E3 ubiquitin ligases. The Smad7-Smurf complex was shown to target and degrade TGFβ/BMP receptors by ubiquitination, thereby attenuating TGFβ/BMP signaling at the receptor level (Kavsak et al., 2000
; Suzuki et al., 2002
). Smad7 was also reported to negatively regulate Wnt/β-catenin signaling (Han et al., 2006
; Millar, 2006
), while β-catenin stabilization inhibits oligodendrocyte myelination (Fancy et al., 2009
; Ye et al., 2009
). To investigate the effects of Smad7 and its cofactor E3 ubiquitin ligase Smurf1 on BMPR/Smad and Wnt/β-catenin signaling, we expressed Smad7 and Smurf1 individually or in combination in rat OPCs. Smad7 alone could slightly decrease BMPR1a and β-catenin protein levels. When cotransfected with Smurf1, Smad7 substantially downregulated BMPR1a and β-catenin steady-state protein levels (). Similarly, the level of p-Smad is also reduced (), indicating that a decrease of BMP-Smad signaling parallels with downregulation of the BMPR1a level, possibly underlying a reduced sensitivity to BMPs ().
Consistently, expression of Smad7 together with Smurf1 was found to reverse the inhibition of expression of myelin genes Mbp, Mag
in rat OPC culture exposed to BMP4 (). In addition, Smad7/Smurf1 expression antagonized the inhibitory effects mediated by BMPRCA
-Smad1/p300 expression on the Mbp
promoter activity while repressing the Hes1
promoter activity (). These data agree fully with other biochemical studies in the TGFβ field that inhibitory Smads negatively regulate receptor-activated Smad signaling in BMP-stimulated cells (Massague et al., 2005
). Collectively, our observations suggest that Smad7
is a critical downstream target of Sip1 and promotes oligodendrocyte differentiation indirectly by inhibiting BMP-Smad signaling and perhaps β-catenin-mediated negative regulatory pathways.
Smad7 is required for oligodendrocyte differentiation
To further determine whether Smad7 is required for oligodendrocyte development, we generated and analyzed conditional Smad7
knockout mice, with the Smad7
allele deleted in the oligodendrocyte lineage by Olig1-Cre (Chen et al., 2009a
) (). Conventional Smad7 null embryos die in utero
due to multiple defects in cardiovascular development (Chen et al., 2009a
). Although Smad7cKO (Smad7flox/flox;Olig1Cre+/−
) mice are viable, they developed tremors at postnatal week two. To determine the role of Smad7 in oligodendrocyte development, we examined expression of the markers for mature oligodendrocytes and their precursors in the CNS of Smad7cKO animals at P7. In the brain and spinal cord of Smad7cKO mice, the expression of the myelin genes Mbp
was diminished in the white matter in contrast to robust expression in control mice (). In contrast, the OPC marker PDGFR
α was detected throughout the spinal cord and the number of positive cells was comparable to that of control littermates (). We did not detect any significant alteration of astrocytic GFAP expression in the spinal cord of Smad7
mutant mice (data not shown). The severe downregulation of myelin gene expression in Smad7cKO mice suggests that Smad7 is critically required for oligodendrocyte differentiation.
Smad7 is Required for Oligodendrocyte Differentiation