Identifying direct targets of a given transcription factor is a key step in dissecting the regulatory network wherein the detailed function of the transcription factor can be understood. Early attempts included ectopic over-expression followed by expression profiling for identification of candidate genes which in turn entailed extensive sequence analyses of the promoter region [1]. Another strategy was direct cloning of DNA fragments associated with transcription factors which also entailed extensive sequence analyses for identification of nearby genes [2].

Several recently developed molecular tools and technical advances have been paving ways for achieving the same goal in a more efficient high throughput manner. For example, microarray expression profiling has dramatically improved the differential screening, and completion of genome sequencing for several species now allows a rapid identification of genes in the genomic context as well as the application of comparative genomics analysis for conserved DNA elements. Furthermore, RNA interference (RNAi) revolutionized somatic cell genetics making it possible to perform loss-of-function experiments conveniently while chromatin immunoprecipitation (ChIP) assay provides a reasonably efficient means of testing direct interaction between proteins and DNA within cellular context. It had been envisioned that these tools, in variable combinations [3, 4], can be utilized for identifying direct regulatory targets of transcription factors, and successful cases are being reported [5, 6].

Sox10 is an HMG box family transcription factor expressed in neural crest stem cells and a subset of neural crest derived lineages [7–9]. At the stem cell stage, Sox10 plays the role of a stem cell factor maintaining multipotency and inhibiting premature neurogenesis [8]. In glial cells of the peripheral nervous system (PNS) and melanocytes, Sox10 continues to be expressed after their lineage segregation and promotes differentiation by targeting cell type specific genes [10]. The function of Sox10 has been extensively studied in vivo with gene-targeted and spontaneously occurring mutant murine models [7, 11, 12]. Heterozygous and homozygous loss-of-function mutations of Sox10 result in severe compromises in the development of PNS via glial developmental failure as well as in the proliferation and differentiation of melanocytic lineages. The human patients of Waardenburg-Shah syndrome with mutations in Sox10 show strikingly similar phenotypes to heterozygous mutant murine models typified by megacolon and hypopigmentation [13].

To date, only a handful of direct regulatory targets of Sox10 are known. In the glial lineage, genes such as the P0, myelin basic protein (MBP), ciliary neurotrophic factor (CNTF) and connexin 32 (Cx32) have been shown to be direct targets of Sox10 [14–17]. In melanocytes, Sox10 has been shown to directly regulate at least two genes, microopthalmia (MITF) and dopachrome tautomerase (DCT) [18–20]. It is likely that Sox10 has additional direct target genes in both glial and melanocytic cells during differentiation and in mature stage.

Here, we describe a stream-lined procedure in identifying such direct regulatory targets. We took advantage of RNAi technique to screen for genes in a Schwannoma cell line that are down-regulated upon introducing small interfering RNA (siRNA) specific for Sox10. Genes with significant level of down-regulation were subjected to comparative genomics analysis for identification of potential Sox10 binding sites that are conserved in multiple mammalian species. Next, we confirmed the physical interaction between Sox10 and the predicted target binding sites by chromatin immunoprecipitation (ChIP) assay leading to identification of at least 4 novel direct regulatory targets, proteolipid protein (PLP), Sox10, extracellular superoxide dismutase (SOD3), and pleiotrophin (Ptn). We further confirm the direct regulation of SOD3 by Sox 10 through the identified cis-element using electrophoretic mobility shift assay (EMSA) and reporter assay.

We have previously reported a case of apparent transdifferentiation of RT4D6 Schwannoma cells into smooth muscle cells upon introduction of an siRNA specific to the transcription factor Sox10 [21]. Accompanying gene expression profiling showed apparent down-regulation of several glial specific marker genes such as GFAP and p75 and up-regulation of smooth muscle specific genes including α-smooth muscle actin confirming the phenomenon of transdifferentiation. Sox10 is known to be a direct transcriptional activator of genes specific to glial lineage including P0 and MBP [16, 17]. We reasoned that several of the genes down-regulated by Sox10 specific siRNA are likely to represent additional direct regulatory targets. We further reasoned that such direct regulatory targets have appropriate cis-elements, Sox10 protein binding sites that are conserved across mammalian if not vertebrate species.

Gene expression profiling was carried out with two independent pairs of RT4D6 cell samples each treated with wild type and mutant siRNAs, and 88 affy probes that showed at least 4-fold down-regulation upon introduction of Sox10 specific siRNA were identified (see Methods; additional data file 1). Among these, 44 genes had RefSeq Ids and were subjected to subsequent promoter and comparative genomics analyses (additional data file 1). We examined the genomic region of each gene from -2 kb upstream of the transcription start site to the end of the first intron to identify the conserved potential Sox10 binding sites. Although systematic analyses for preferred binding sites have been carried out for several Sox proteins, no such study has been reported for Sox10 [22–25]. In addition, these sites have been determined through in vitro binding assays which may not fully reflect the in vivo binding preference or potential. We therefore took as the core element the 5 nucleotide sequence, 5'ACAAT3' (or the complementary sequence 5'ATTGT3'), that is targeted by all tested Sox proteins. From the rat genomic sequences examined, profile search using the TRANSFAC database led to the identification of 1328 sites.

We next applied the cross-species conservation criteria (see Methods) reasoning that functional Sox10 binding sites are likely conserved across mammalian species. This led to further refinement of putative targets to 95 candidate sites belonging to 24 genes (additional data file 2). For the subsequent validation analyses using 'wet-lab' experimental procedures, further selection was carried out. Specifically, only the genes which showed 6-fold or higher levels of change by specific siRNA and whose core elements were perfectly conserved among all mammalian species with accessible genome were selected. This led to the final 10 genes with 23 putative SOX10 binding sites (table 1). The computational pipeline is summarized in figure 1, and figure 2 shows the gene structure, conserved genomic regions, and nucleotide sequence surrounding the putative binding site of one of the 10 genes, SOD3.

gene symbol	RefSeq ID	target cis element	chromosome	start	end	Location	primer pair #
Cmkor1	NM_053352	tttATTGTtctc	9	89344237	89344250	Intron	1
		agcACAATgg	9	89346538	89346547	Intron	2
Gda	NM_031776	ttgACAATtt	1	224640748	224640757	Intron	3
PLP	NM_030990	cctATTGTgttt	X	124488088	124488101	5'enhancer	4
		taATTGTttt	X	124492945	124492954	intron	5
		ctATTGTata	X	124493179	124493188	Intron	6
		gaATTGTgtg	X	124493238	124493247	Intron	6
		ggtACAATtg	X	124493249	124493258	Intron	6
		ccATTGTtta	X	124494810	124494819	Intron	7
Sox10	NM_019193	agATTGTcca	7	117150489	117150498	5'enhancer	8
		caATTGTctg	7	117150400	117150409	5'enhancer	8
SOD3	NM_012880	ccATTGTgcc	14	63387776	63387785	5'enhancer	9
Erbb3	NM_017218	tcATTGTggg	7	1874401	1874410	Intron	10
		gacACAATag	7	1874335	1874344	Intron	10
Ptn	NM_017066	caATTGTtgg	4	64147810	64147819	Intron	11
		cacACAATag	4	64147702	64147711	Intron	11
		taATTGTgag	4	64136510	64136519	Intron	12
		atATTGTaac	4	64127690	64127699	Intron	13
		attACAATaa	4	64126376	64126385	Intron	14
Ngfr	NM_012610	tggACAATgg	10	84280255	84280264	Intron	15
		cagACAATgg	10	84280237	84280246	Intron	15
Gzmb	NM_138517	cgATTGTgat	15	35199893	35199902	5'enhancer	16
Gas7	NM_053484	ccATTGTatg	10	54304002	54304011	Intron	17

The number in the last column indicates the primer pair used to test each site by ChIP assay (see additional data file 3).

We next sought to confirm the regulatory relationship between Sox10 and the proposed 10 target genes. cDNA preparations were made from RT4D6 cells treated with Sox10 specific and control siRNAs. Real time PCR analyses showed that all the proposed 10 target genes were clearly down-regulated as the result of Sox10 down-regulation confirming the microarray screen results (figure 3).

Chromatin immunoprecipitation assay was performed for the 23 putative Sox10 binding sites (table 1) with RT4D6 cells transfected with a Flag epitope tagged Sox10 construct. Ectopic expression of epitope tagged Sox10 was necessary due to the absence of an antibody that effectively immunoprecipitated the endogenous Sox10 from RT4D6 cells. As some of the potential binding sites were located close to one another, only 17 pairs of primer were used to examine the 23 sites. As a control, each of the sites was examined after precipitation with IgG antibody. We also used β-actin locus and a cis-locus located 10 kb away from the putative Sox10 binding site of SOD3 gene as additional negative controls. The result suggests that at least 4 genes, PLP, Sox10, SOD3, and Ptn contain binding sites that SOX10 interacts with clearly detectable affinity, and Ngfr and Cmkor1 may represent additional Sox10 target genes (figure 4).

We chose SOD3 for further validation as the direct regulatory target of Sox10. The unique candidate binding site, conveniently located in the 5' enhancer region, shows a perfect conservation between rat, mouse, human, dog, and opossum (figure 2).

We first confirmed that Sox10 protein can bind to the candidate site by EMSA (figure 5). The DNA binding domain of Sox10 readily formed a protein-DNA complex with a probe spanning the candidate binding element. Importantly, the binding between the protein and the labeled probe was effectively abrogated by specific unlabeled probe but not by non-specific probe which contains mutations in the core SOX binding sequence.

Next, we examined the role of the candidate binding site in a reporter assay (figure 6). Two reporter constructs were designed. The wild type SOD3 reporter, pGL3-SOD3, has luciferase gene ligated to the 5' enhancer and UTR region of SOD3 inclusive of the candidate cis-element while mutant promoter pGL3-SOD3-mut is identically designed except for mutations in the core SOX binding element. These reporter plasmids were co-transfected with various combinations of wild type and mutant siRNAs for Sox10. Importantly, at all concentrations of the wild type siRNA, the wild type reporter showed consistently higher activity than the mutant reporter demonstrating the importance of the intact core Sox binding element. The activity of the wild type reporter showed a concentration dependent down-regulation by the wild type siRNA while the mutant reporter showed little variation of the low level activity. Taken together with the results from ChIP assay and EMSA, these data demonstrate that SOD3 is a direct regulatory target of Sox10 which imparts its activity through the conserved Sox binding element in the 5' enhancer region.

We have applied in combination several recently developed techniques and tools of molecular biology to identify multiple novel targets of Sox10. Rather than relying on overexpression of Sox10, we used RNAi to screen for affected genes. Overexpression of transcription factors can potentially activate non-physiological targets or even repress true targets by 'squelching'. Overexpression of transcription factors in cells already expressing the factor may also have minimal or no effects on the level of target gene expression. The loss-of-function approach followed by microarray screen should produce less non-specific side effects and thus lead to more precise profiling of direct and indirect targets. The procedure combining differential expression profiling and comparative genomics analyses represents a reasonably streamlined method accessible for most molecular biology laboratories.

There are clearly more direct target genes for Sox10 than those we have identified here. The number is limited due to several practical reasons. Most importantly, in the end we have examined by 'wet-lab' experiments only the genes that showed at least 6-fold decrease upon down-regulation of Sox10. This excludes the far greater number of genes that are less but meaningfully affected by the change in the level of Sox10. Furthermore, we have restricted our search within the span of -2 kb from the transcription initiation site to the end of the 1^st intron. It is well established that regulatory cis-elements often reside outside such span. Lastly, applying less stringent requirements for the core element sequence and the degree of conservation should allow identification of additional candidate targets. It should also be noted that Sox10 plays important roles in other cell types than Schwann cells. These include neural crest stem cells, melanocytes, and oligodendrocytes. Given that Sox10 likely regulates different genes in different cellular contexts, we should expect distinct additional direct targets of Sox10 from these cells. An important merit to our method is that all these limiting parameters can be easily adjusted for expanded search. Obviously, more comprehensive procedures such as ChIP-on-chip or ChIP-Seq screening would produce more direct targets. However, these methods also have inherent limitations and shortcomings and cannot be readily applied to all cases, and developing diverse strategies to identifying targets of transcription factors will continue to be important. An inherent requirement for identifying direct targets of transcription factors is proving through biological functional assays. It should be noted that it is thus difficult to assess the error rates during each of the filtering steps. If and when extensive biological data are collected, a retrospective assessment of the accuracy of prediction from each criterion may be possible. In a sense, our goal in the current study can be described as an application of data exploration techniques to objectively and subjectively identify the genes most likely to yield positive results in our biological assays of binding and transcriptional activation.

We propose four novel direct targets of Sox10. PLP expression has been shown to be rapidly and robustly induced by Sox10 in Tet-inducible Neuro2A cells [26], and PLP is also known to be expressed in Schwann cells [27, 28]. Sox10 has been shown to directly regulate other myelin genes including MBP and Cx32 [14, 17], and our identification of PLP as another target is consistent with the role that Sox10 plays in peripheral glial cells. Pleiotrophin is a heparin binding secreted soluble factor with multiple functions in various systems [29, 30]. Of note it is expressed in glial cells of CNS and PNS during development as well as in adult stages [29, 30]. Its up-regulation in Schwann cells after nerve resection also suggest a role in axon regeneration, a process requiring active participation of Schwann cells [31]. SOD3 is the extracellular form of superoxide dismutase that functions as the major extracellular scavenger of superoxide [32]. SOD3 gene-targeted mouse does not show clear phenotypes except for the reduced viability under hyperoxic condition [32]. Nevertheless, under various experimental conditions, the protective role of SOD3 on neural tissues has been shown suggesting that secreted SOD3 may play a role in reducing oxidative stress to neurons and Schwann cells [33, 34]. That Sox10 was among the down-regulated genes was not necessarily meaningful given that Sox10 specific siRNA had been applied to the cell. Still, results from the ChIP assay indicate that Sox10 may regulate its own expression. Whether an active positive feedback loop contributes to known functions of Sox10 such as promoting and maintaining glial phenotype should be further explored. While Ngfr (p75) is a well established marker of Schwann cell [8], the expression and function of Cmkor1, chemokine orphan receptor 1 (also known as CXCR7), in Schwann cell lineage have not been investigated thus far. Binding of Sox10 to the putative Sox binding elements belonging to these genes is repeatable, and given the change in the expression level upon down-regulation of Sox10, we would tentatively classify these genes as direct regulatory targets of Sox10. Additional characterization of their binding elements through mutation analyses and reporter assays should further validate the assignment.

We have developed and successfully applied a streamlined procedure for identification of direct targets of the transcription factor Sox10. The key elements are RNAi based expression profiling and comparative genomics analysis which led to prediction of candidate cis-elements belonging to multiple candidate genes. We subsequently confirmed direct interaction between several of these candidate cis-elements and Sox10 and identified at least 4 different genes as direct regulatory targets of Sox10. Our methodology should be applicable to other transcription factors, and genes thus discovered should be useful in deciphering physiology and function of peripheral glial cells which are critically dependent on Sox10.