"Sall4 Interacts with Nanog and Co-occupies Nanog Genomic Sites in Embryonic Stem Cells*",

Qiang Wu ¹, Xi Chen ^{1, 2}, Jinqiu Zhang ³, Yuin-Han Loh ^{1, 2}, Teck-Yew Low ⁴, Weiwei Zhang ^{1, 2}, Wensheng Zhang ¹, Siu-Kwan Sze ^{4, 5}, Bing Lim ³, and Huck-Hui Ng ^{1, 2, @}

¹ Gene Regulation Laboratory, Genome Institute of Singapore, Singapore 138672,
² Department of Biological Sciences, National University of Singapore, Singapore 117543,
³ Stem Cell & Developmental Biology and ⁴ Proteomics, Genome Institute of Singapore, Singapore 138672,
⁵ School of Biological Sciences, Nanyang Technological University, Singapore 637551

^@ To whom correspondence should be addressed: Genome Institute of Singapore, 60 Biopolis St., #02-01, Genome Bldg., Singapore 138672. Tel.: 65-6478-8145; Fax: 65-6478-9004;
E-mail: nghh@gis.a-star.edu.sg

The abbreviations used are: ICM, inner cell mass; ES cell, embryonic stem cell; LC-MS/MS, liquid
chromatography-mass spectrometry/mass spectrometry; co-IP, co-immunoprecipitation; GFP, green
fluorescence protein; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation

Embryonic stem (ES) cells are pluripotent cells with self-renewing property. Nanog is a homeobox transcription factor required to maintain ES cells in a non-differentiated state. Using affinity purification coupled to liquid chromatography-tandem mass spectrometry analysis, we identified Sall4 as a Nanog co-purified protein. Co-immunoprecipitation and glutathione S-transferase pulldown experiments confirmed the interaction between Nanog and Sall4. We showed that Nanog and Sall4 co-occupied Nanog and Sall4 enhancer regions in living ES cells. Knockdown of Nanog or Sall4 by RNA interference led to a reduction in Nanog and Sall4 enhancer activities, providing evidence that these factors are positively regulating these enhancers. Importantly, co-transfection of Sall4 with these ES cell-specific enhancers led to transactivation in heterologous somatic cells. Chromatin immunoprecipitation experiments also showed that Sall4 co-occupied many Nanog binding sites in ES cells. Our data implicate Sall4 as an important component of the transcription regulatory networks in ES cells by cooperating with Nanog. We suggest that Sall4 and Nanog form a regulatory circuit similar to that of Oct4 and Sox2. This study highlights the extensive regulatory loops connecting genes, which encode for key transcription factors in ES cells.

Embryonic stem (ES) cells are isolated from the inner cell mass (ICM) of blastocysts and they can be propagated in vitro for an extended period of time. These cells are pluripotent as they exhibit the ability to differentiate into many of the specialized cell types (1, 2). The maintenance of ES cells in a pluripotent state is mediated by a group of transcription factors that are preferentially expressed in the non-differentiated state (3, 4, 5, 6, 7). Nanog, a homeodomain transcription factor, was identified as a factor that is able to sustain pluripotency in murine ES cells even in the absence of leukemia inhibitory factor (LIF) (6, 7). The expression of Nanog is controlled by at least 3 transcription factors. The proximal
promoter of Nanog contains an oct-sox motif and is bound by Oct4 and Sox2 (8, 9). Oct4 and Sox2
are also key regulators for self-renewal of pluripotent ES cells (4, 5). Furthermore, the tumor suppressor protein, p53, can also bind to the Nanog promoter after DNA damage to ES cells (10). Hence, it is proposed that upon DNA damage, p53 suppresses Nanog expression and triggers differentiation in order to maintain genomic stability. More recently, we identified a major Nanog binding locus within 5 kb upstream region of Nanog and this provides evidence for a positive feedback loop (11). It is however not clear if this Nanog-bound region plays any regulatory role.

The transcription regulatory network for Nanog has been mapped in both murine and human ES cells (11, 12). Both studies showed that Nanog binds to genes with diverse functions. While there is considerable amount of information on the downstream target genes of Nanog, little is known about the co-factor(s) that interact with Nanog to mediate transcription in ES cells.

Here we report that Sall4, a member of spalt-like protein family, interacts with Nanog in vitro and exists as a complex with Nanog. Sall4 binds to the upstream enhancer of Nanog gene and can activate the enhancer in heterologous somatic cells. Depletion of Sall4 down-regulates this activity. Interestingly, Sall4 also binds to an intronic region of Sall4 and can activate this enhancer. Furthermore, we demonstrated that Sall4 also binds to genomic sites that are bound by Nanog. We suggest that the reciprocal regulation of Sall4 and Nanog by the Sall4 / Nanog complex is important in governing pluripotency and self-renewal of ES cells.

Materials and Methods

Co-immunoprecipitation –

Transfected cells were lysed in cell lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol with protease inhibitor cocktail) for 1 h. Whole cell extracts were collected and precleared. The beads coated with antibodies were incubated with the precleared whole cell extracts 4 ^o C for overnight. The beads were washed with cell lysis buffer 4 times. Finally, the beads were incubated
in the FLAG-peptides or the beads were boiled for 10 min. The eluents were analyzed by either protein staining or Western blot. Rabbit polyclonal antibody against Sall4 was raised against the first
200 amino-acids of Sall4.

ChIP and RNA expression analysis –

ChIP was performed as described previously (11, 22). RNA extraction, reverse transcription and quantitative realtime PCR were carried out as described previously (11, 22). The sequences of the ChIP
primers can be found in Supplemental Table 2.

siRNA mediated knockdown –

The 19 nucleotides targeted by the siRNAs are
GCAACCTGAAGGTACACTA (for Sall4-1);
GCCTTTCGTGTGTAACATA (for Sall4-2); and
GAACTATTCTTGCTTACAA (for Nanog). We obtained similar results for the 2 RNAi constructs
for Sall4 knockdown. The RNAi experiments were carried out as described in (22).

RESULTS

To further understand the mechanism of how Nanog regulates transcription, we sought to identify the proteins which interact with Nanog in ES cells. We have previously generated a stable ES cell-line that expresses FLAG-tagged Nanog (11). The tagged Nanog appears to be functional as this line is resistant to differentiation upon LIF withdrawal or RA treatment. Cell extracts were prepared from this line and subjected to affinity purification using M2 FLAG antibodies. The eluant was fractionated by SDS-PAGE. Several protein bands were excised from the gel. After in-gel digestion, the tryptic peptides extracted from each gel band were subjected to LC-MS/MS analysis. Proteins in three of the bands were successfully identified (Fig. 1A and Supplemental Table 1).

Fig. 1. Sall4 interacts with Nanog.

Fig. 1. Sall4 interacts with Nanog.

A, FLAG-tagged Nanog and associated proteins were affinity purified, separated by SDS-PAGE and detected by Coomassie Blue. Protein bands containing Sall4 and Nanog peptides are indicated by arrows. Protein marker in kilodalton is shown.

B, Co-IP using ES cell nuclear extracts using anti-Nanog antibody. Western blot was carried out with anti-Sall4 antibody. Control IP was performed using anti-GST antibody.

C, Reverse co-IP confirmed the interaction between Sall4 and Nanog. Co-IP was performed with anti-Sall4 or anti-GST antibody. Western blot was carried out with anti-Nanog antibody.

D, Schematic diagram of wild type and deletion forms of Sall4 protein. Ovals represent C2HC and C2H2 motifs. The sizes of the Sall4 proteins were shown as numbers of amino acids.

E, FLAG-tagged Nanog and V5-tagged Sall4 (full-length and deletion forms) were co-expressed in 293T cells. Co-IP was performed with M2 anti-FLAG antibody. Western blot was carried out with anti-V5
antibody. Empty vector was used as negative control.

F, Schematic diagram of wild type and deletion forms of Nanog protein. ND: N-terminal domain; HD: homeobox domain; CD1: C-terminal domain 1; WR: tryptophan repeat domain; CD2: C-terminal domain 2.

G, GST pulldown was carried out using GST-tagged Nanog proteins and cell lysates of 293 cells over-expressing V5-tagged full-length Sall4. Western blot was performed with anti-V5 antibody. GST served as negative control.

Sall4 was identified as a candidate Nanog-interacting protein. The other two bands contained peptide sequences from Nanog.

Next, we performed co-IP experiments using ES cell nuclear extracts to confirm the interaction. Sall4 was found to co-precipitate with Nanog (Fig. 1B). Reciprocal co-IP experiment also showed that Nanog can be found in the Sall4 precipitate (Fig. 1C). In addition, we generated a stable ES cell-line that expresses Sall4 tagged with 3 copies of hemagglutinin (HA) epitope (3HA-Sall4). Cell extracts from this cell-line and control ES cells without epitope-tagged Sall4 were prepared. Immunoprecipitation using an anti-HA monoclonal antibody showed that Nanog co-immunoprecipitated with Sall4 (Supplemental Fig. 1A). The reverse immunoprecipitation using anti-Nanog antibodies was also able to bring down HA-tagged Sall4 (Supplemental Fig. 1B). These results confirmed that Sall4 indeed interacts with Nanog in murine ES cells.

Sall4 contains three C2H2 zinc fingers and the N-terminus has a C2HC motif (Fig. 1D). To determine which region of Sall4 interacts with Nanog, we co-transfected FLAG-tagged Nanog with each of the four Sall4 deletion constructs containing a V5 epitope tag into 293T cells. Co-IP assay with M2 FLAG antibodies showed that Sall4-FL and Sall4-N800 could interact with Nanog, whereas Sall4-C767 and Sall4-C567 failed to interact with Nanog (Fig. 1E). This indicates that the N-terminal region of Sall4 is required for the interaction with Nanog. All four Sall4 deletion mutants were localized in the nucleus (data not shown), hence this excludes the possibility that the lack of interaction is due to mislocalization of the Sall4 mutant protein to the cytoplasm.

To determine the region that interacts with Sall4, we expressed and purified recombinant Nanog and fragments of Nanog as GST-fusion proteins (Fig. 1F) (13). These proteins were immobilized onto GSH-sepharose beads and incubated with extracts harvested from 293T cells over-expressing V5 tagged Sall4. The fragment containing only the N-terminal domain failed to pulldown Sall4, whereas recombinant protein containing homeobox domain could interact with Sall4 (Fig. 1G).

We have shown that Nanog binds to a region 5 kb upstream of the transcription start site of Nanog gene (Fig. 2A) (11). To ascertain whether this region can serve as an enhancer, we cloned a 404 bp genomic fragment and inserted it upstream or downstream of a luciferase reporter driven by an Oct4 minimal promoter. The 428 bp Oct4 minimal promoter did not show ES cell-specific expression and had only very weak activity (data not shown). The two reporters with Nanog genomic sequence exhibited robust
activities in ES cells, but showed only background activities in human embryonic kidney 293T cells (Fig. 2, A and B). Hence, this sequence could function as an enhancer that operates both upstream and downstream of a promoter. We identified a sequence within this enhancer that interacts with Nanog in vitro (Supplemental Fig. 2). Deletion of a 16 bp sequence containing this Nanog binding site abolished the enhancer activity (Fig. 2C).

Fig. 2. Nanog and Sall4 regulate Nanog enhancer.

Fig. 2. Nanog and Sall4 regulate Nanog enhancer.

A, Reporter constructs used to assay for enhancer activity are shown. A 401bp region 5kb upstream of Nanog transcription start site (in red) was inserted either upstream (Nanog Enh-Luc) or downstream (Luc-Nanog Enh) of a luciferase gene driven by Oct4 minimal promoter. Red oval signifies Nanog binding to the upstream region. The proximal promoter of Nanog is also targeted by Oct4 (blue oval) and Sox2 (green oval).

B, The reporters were transiently transfected in ES cells or 293T cells. Vector is the parental vector construct without the enhancer insert.

C, Nanog binding site was deleted from the Nanog enhancer (NanogEnh-Luc Mut) and tested for
enhancer activity in ES cells.

D, ChIP assays were performed using anti-Sall4 antibody with extracts derived from ES cells (white bar) or ES cells transfected with Sall4 RNAi construct (grey bar). Fold enrichment represents the abundance of enriched DNA fragments over a control region. GFP ChIP served as mock ChIP (black bar).

E, Nanog enhancer luciferase reporter construct or control vector was co-transfected with Sall4 or Nanog RNAi plasmid into murine ES cells.

F, Nanog enhancer reporter or control vector was co-transfected with Sall4 expression plasmid into 293T cells and the luciferase activities were assayed. All luciferase activities were measured relative to the Renilla luciferase internal control.

As Nanog was found to associate with Sall4 in our co-immunoprecipitation experiments, we examined whether Sall4 binds to this Nanog bound enhancer region by chromatin immunoprecipitation (ChIP). Real-time PCR was performed using Nanog gene specific primers to quantify the ChIP-enriched DNA. We found that Sall4 binds to the distal enhancer of Nanog gene (Fig. 2D). Mock GFP ChIP did not show any significant enrichment along this region. Importantly, the binding of Sall4 was reduced in cells expressing shRNA targeting Sall4 (Fig. 2D). Furthermore, we also confirmed the binding of Sall4 to this region in an ES stable cell line that expressed 3HA-tagged Sall4 (Supplemental Fig. 3A, B). This result suggests that Sall4 may regulate Nanog transcription in ES cells.

Having established the interaction between Sall4 and the Nanog enhancer in vivo, we sought to understand the functional roles of Nanog and Sall4 on this enhancer. The Nanog enhancer-luciferase
construct was co-transfected with either Sall4 or Nanog RNAi construct into ES cells (11). RNAi was effective at reducing Sall4 levels in ES cells (Supplemental Fig. 4). The luciferase assays showed that Sall4 or Nanog depletion could reduce the Nanog enhancer activity by more than 75% whereas GFP and empty RNAi controls had no effect (Fig. 2E). We conclude that the Nanog enhancer activity is regulated by both Sall4 and Nanog. To address whether Nanog or Sall4 can independently activate the Nanog enhancer, we co-transfected Sall4 or Nanog expression construct along with the Nanog enhancer reporter in a heterologous cell-type that does not support ES cell-specific expression (Fig. 2B). Interestingly, Sall4 was able to transactivate this reporter in the 293T cells (Fig. 2F). However, the reporter could not be activated by Nanog (data not shown). We also did not observe synergistic activation of the reporter by Sall4 and Nanog in 293T cells (data not shown). Thus, it is likely that Sall4 and Nanog function independently to activate Nanog. Taken together, the data show that Nanog positively regulates its own enhancer and Sall4 is a novel regulator of Nanog in ES cells.

Our previous genome-wide Nanog location study also identified a Nanog binding locus within the first intron of Sall4 (Fig. 3A). This raised the possibility that Nanog may regulate the Sall4 gene through the intronic site.

Fig. 3. Nanog and Sall4 regulate Sall4 enhancer.

Fig. 3. Nanog and Sall4 regulate Sall4 enhancer.

A, A screen shot of genome browser showing Nanog Paired-End-diTag (PET) clusters at Sall4 locus. Each horizontal green line represents a DNA fragment mapped to the mouse genome. Real-time PCR primer pairs along a 2.5 kb region covering the peak of Nanog binding (highlighted in red) were used to quantify the ChIP-enriched DNA.

B, ChIP was performed using anti-Nanog, anti-Sall4 and control antibodies.

C, Transcription activity of the luciferase reporters which contains Sall4 intronic sequence upstream (Sall4 Enh-Luc) or downstream (Luc-Sall4 Enh) of luciferase gene in ES cells and 293T cells.

D, Nanog binding site was deleted from the Sall4 enhancer (Sall4 Enh-Luc Mut) and tested for enhancer activity in ES cells.

E, Sall4 enhancer luciferase reporter construct or control vector was co-transfected with Sall4 or Nanog RNAi vector.

F, Sall4 enhancer reporter or control vector was co-transfected with Sall4 into 293T cells.

Next, we investigated whether Nanog and Sall4 bind to this region by ChIP. Real-time PCR using Sall4 gene-specific primers showed that both Nanog and Sall4 have an enrichment peak at the same region while a control ChIP showed no enrichment (Fig. 3B and Supplemental Fig. 3C). We conclude that Nanog and Sall4 bind within the first intron of Sall4 gene in living ES cells. To further characterize this
Nanog and Sall4 bound region, we cloned a 441 bp Sall4 intronic region that showed the highest level of Nanog and Sall4 occupancies and fused it upstream or downstream of a luciferase gene driven by the Oct4 minimal promoter. We transfected these constructs into ES cells and 293T cells. Interestingly, this fragment showed robust activity in ES cells but only background activity in 293T cells, indicating that it can function as an enhancer in an ES cell-specific manner (Fig. 3C). A sequence within the Sall4 enhancer interacted with Nanog as revealed by EMSA (Supplemental Fig. 2). Deletion of a 20 bp sequence containing this Nanog binding site abolished the enhancer activity (Fig. 3D).

To confirm the regulatory role of Nanog and Sall4 on this Sall4 enhancer, RNAi experiment was performed. The Sall4 enhancer reporter construct was co-transfected with Nanog or Sall4 RNAi plasmid into ES cells. The depletion of both Nanog and Sall4 reduced the Sall4 enhancer activity dramatically whereas empty vector and GFP RNAi showed no effect (Fig. 3E). Finally, we co-expressed Sall4 or Nanog with the Sall4 enhancer construct in 293T cells (Fig. 3F). The luciferase reporter was activated by Sall4, while Nanog had no effect (data not shown). Hence, Sall4 alone is sufficient to activate this enhancer in a heterologous system. In summary, we showed the binding of Nanog and Sall4 to a Sall4 intronic region. This region is important for the activation of transcription and its enhancer activity is regulated by Nanog and Sall4 in ES cells.

The co-binding of Nanog and Sall4 to both Nanog and Sall4 enhancer regions prompted us to examine more genomic sites for co-occupancies (Fig. 4A).

Fig. 4. Transcription circuits for Nanog and Sall4.

Fig. 4. Transcription circuits for Nanog and Sall4.

A, ChIP was performed using anti-Sall4 antibodies to detect Sall4 binding at Nanog bound regions (11).

B, Model for auto-regulation and reciprocal regulation of Nanog and Sall4 by Nanog and Sall4. Ovals denote the transcription factors. Rectangles denote the genes. Solid arrows indicate the binding of transcription factor to regulatory sites. Dotted arrows signify the synthesis of transcription factor by the respective gene.

C, Model for transcription circuits involving Oct4, Sox2, Nanog and Sall4.

We tested 22 known Nanog binding loci (11) for the presence of Sall4 by ChIP. Real-time PCR results showed that all these loci were bound by Sall4 as well while control ChIP using anti-GFP antibody gave only background enrichment. The binding of Sall4 to these genomic loci was also confirmed by anti-HA ChIP using extracts derived from a stable ES cell-line that expresses HA-tagged Sall4 (Supplemental Fig. 3D).

We next compared the effects of RNAi-mediated Sall4 knockdown with knockdown of Oct4, Sox2 or Nanog in ES cells (Supplemental Fig. 5A, B). Depletion of Oct4 or Sox2 by siRNA induced morphological changes in ES cells. The resultant cells were fibroblastic with extensive cytoplasmic projections and some cells had enlarged nuclei typically of trophoblast giant-like cells (Supplemental Fig. 5B). In contrast,
knockdown of Sall4 and Nanog produced stellate shaped cells with different morphologies compared to those Oct4 or Sox2 depleted cells. This observation suggests that Nanog and Sall4 are more functionally related to each other than Oct4 or Sox2. Analysis of expression of marker genes also revealed that Sall4 knockdown cells showed reduced levels of ES cell-specific gene expression and increased expression of
differentiation-associated marker genes (Supplemental Fig. 5C). The results indicate that Sall4 is important for maintaining the non-differentiated state of murine ES cells.

DISCUSSION

The mammalian Sall4 gene encodes a spalt-like zinc finger transcription factor. Mutations of the human SALL4 gene are the cause of the human disease Okihiro syndrome (14, 15, 16). A recent study showed that Tbx5 regulates Sall4 gene and interacts with Sall4 protein to regulate patterning and morphogenesis of forelimb and heart (17). The heterozygous Sall4 knockout mice exhibit limb and heart defects that resemble the human disease. Sall4 is also known to be expressed predominantly in the ICM of early
mouse embryos (18) as well as in embryonic carcinoma cells (14). Immuno-staining also revealed the presence of Sall4 in the trophectoderm (19). In adult tissues, Sall4 is predominantly expressed in testis and ovary (20). Hence, Sall4 is likely to play different roles during development and in adult organism. Our data on the link between Sall4 and Nanog, a known regulator of ES cell pluripotency, suggests that
Sall4 plays an important role in early development of mouse embryo and ES cells. Indeed, siRNA-mediated depletion of Sall4 in oocytes and ES cells led to developmental defects of early embryos (21). This is in part due to the dysregulation of Oct4, which is another bona fide target of Sall4 (21). Disruption of both alleles of Sall4 leads to embryonic lethality during peri-implantation (19). The Sall4 null-ES cells also showed defect in proliferation in vitro, further highlighting the importance of Sall4 in
maintenance of ES cells (19).

In this study, we showed that Sall4 associates with Nanog. This observation is intriguing as the Sall4 / Nanog complex resembles the network configuration for the Oct4 / Sox2 complex in several aspects. Oct4 and Sox2 are known to interact physically and co-occupy many sites in ES cells (11, 12). Similarly, we found co-targeting of Nanog and Sall4 to many genomic sites. In addition, Sall4 and Nanog also bind to and regulate the respective regulatory regions of their own genes. The data suggests that Sall4 and Nanog employ autoregulatory feedforward loops to maintain their expression (Fig. 4B). This circuitry is very similar to the Oct4 / Sox2 circuit that we proposed previously for murine ES cells (22). Furthermore, we also detected the binding of Sall4 to Oct4 and Sox2 upstream regulatory sequences, which are bound by Nanog as well. Hence, our data uncovered an extensive usage of autoregulatory, feedforward connections in ES cells (Fig. 4C). It is conceivable that such circuitry confers stability in the expression of genes encoding for key transcription factors crucial for the maintenance of ES cells in the non-differentiated state. While we observed substantial levels of co-occupancy for Sall4 and Nanog, it
should also be noted that the extent of co-occupancy at the whole genome level remains to be examined. As the sequence specific DNA binding property of Sall4 has not been demonstrated in our study and is poorly documented in the literature, it is also possible that Sall4 serves a co-activator function in mediating transcription.

Oct4, Sox2, Nanog and Sall4 are important in controlling the pluripotency of ES cells and their regulatory connections with each other are extensive. It should be emphasized that the role of each factor may differ with regards to the genes that each factor regulates and the lineage controlled by each factor. For example, the morphologies of cells after Nanog or Sall4 depletion appeared similar and are distinct from
Oct4 or Sox2 depletion cells. This result suggests a parallel role of Nanog and Sall4 in maintaining murine ES cells. However, this does not exclude the possibility that Nanog and Sall4 may have distinct functions. It is likely that genome wide location profiling and microarray analysis will further dissect out the regulatory networks governed by Sall4. It is also of interest to understand the biological differences among these key transcription factors that control ES cell pluripotency.

REFERENCES

1. Smith, A. G. (2001) Ann. Rev. Cell Dev. Biol. 17, 435-462

2. Loebel, D. A., Watson, C. M., De Young, R. A., and Tam, P. P. (2003) Dev. Biol. 264, 1-14

3. Scholer, H. R., Ruppert, S., Suzuki, N., Chowdhury, K., and Gruss, P. (1990) Nature. 344, 435-439

4. Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Scholer, H.,
and Smith, A. (1998) Cell 95, 379-391

5. Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian, N., and Lovell-Badge, R. (2003) Genes
Dev. 17, 126-140

6. Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., and Smith, A. (2003) Cell
113, 643-655

7. Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M.,
Maeda, M., and Yamanaka, S. (2003) Cell 113, 631-642

8. Rodda, D. J., Chew, J. L., Lim, L. H., Loh, Y. H., Wang, B., Ng, H. H., and Robson, P. (2005) J.
Biol. Chem. 280, 24731-24737

9. Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S. Y., Suemori, H., Nakatsuji, N., and Tada,
T. (2005) Mol. Cell. Biol. 25, 2475-2485

10. Lin, T., Chao, C., Saito, S., Mazur, S. J., Murphy, M. E., Appella, E., and Xu, Y. (2005) Nat. Cell
Biol. 7, 165-171

11. Loh, Y. H., Wu, Q., Chew, J. L., Vega, V. B., Zhang, W., Chen, X., Bourque, G., George, J., Leong,
B., Liu, J., Wong, K. Y., Sung, K. W., Lee, C. W., Zhao, X. D., Chiu, K. P., Lipovich, L., Kuznetsov,
V. A., Robson, P., Stanton, L. W., Wei, C. L., Ruan, Y., Lim, B., and Ng, H. H. , "The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells", (2006) Nat. Genet.
38, 431-440.  http://www.nature.com/ng/journal/v38/n4/abs/ng1760.html

12. Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P., Guenther, M. G.,
Kumar, R. M., Murray, H. L., Jenner, R. G., Gifford, D. K., Melton, D. A., Jaenisch, R., and Young,
R. A.(2005) Cell 122, 947-956

13. Pan, G., and Pei, D. (2005) J. Biol. Chem. 280, 1401-1407

14. Kohlhase, J., Heinrich, M., Schubert, L., Liebers, M., Kispert, A., Laccone, F., Turnpenny, P.,
Winter, R. M., and Reardon, W. (2002) Hum. Mol. Genet. 11, 2979-2987

15. Kohlhase, J., Schubert, L., Liebers, M., Rauch, A., Becker, K., Mohammed, S. N., Newbury-Ecob,
R., and Reardon, W. (2003) J. Med. Genet. 40, 473-478

16. Kohlhase, J., Chitayat, D., Kotzot, D., Ceylaner, S., Froster, U. G., Fuchs, S., and Montgomery, T.,
and Rosler, B. (2005) Hum. Mutat. 26, 176-183

17. Koshiba-Takeuchi, K., Takeuchi, J. K., Arruda, E. P., Kathiriya, I. S., Mo, R., Hui, C. C., Srivastava,
D., and Bruneau, B. G. (2006) Nat. Genet. 38, 175-183

18. Yoshikawa, T., Piao, Y., Zhong, J., Matoba, R., Carter, M. G., Wang, Y., Goldberg, I., and Ko, M. S.
(2006) Gene. Expr. Patterns 6, 213-224

19. Sakaki-Yumoto, M., Kobayashi, C., Sato, A., Fujimura, S., Matsumoto, Y., Takasato, M., Kodama,
T., Aburatani, H., Asashima, M., Yoshida, N., and Nishinakamura, R. (2006) Development. Jun 21;
[Epub ahead of print]

20. Kohlhase, J., Heinrich, M., Liebers, M., Frohlich, Archangelo. L., Reardon, W., and Kispert, A.
(2002) Cytogenet. Genome Res. 98, 274-277

21. Zhang, J., Tam, W. L., Tong, G., Wu, Q., Chan, H. Y., Soh, B. S., Lou, Y., Yang, J., Ma, Y., Chai, L.,
Ng, H. H., Lufkin, T., Robson, P., and Lim, B. (2006) submitted

22. Chew, J. L., Loh, Y. H., Zhang, W., Chen, X., Tam, W. L., Yeap, L. S., Li, P., Ang, Y. S., Lim, B.,
Robson, P., and Ng, H. H. (2005) Mol. Cell. Biol. 25, 6031-6046

FOOTNOTES

* We are grateful to Katty Kuay for technical assistance. We thank Agency for Science, Technology and
Research (A*STAR) for funding. X.C. and W.Z. are supported by the National University of Singapore
research scholarships. Y-H. L is supported by the A*STAR graduate scholarship. X.C is supported by the
Singapore Millennium Foundation scholarship.

1 The abbreviations used are: ICM, inner cell mass; ES cell, embryonic stem cell; LC-MS/MS, liquid
chromatography-mass spectrometry/mass spectrometry; co-IP, co-immunoprecipitation; GFP, green
fluorescence protein; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation

Supplementary Data:

The on-line version of this article (available at:  http://www.jbc.org/cgi/content/full/C600122200/DC1 ) contains supplemental Figs. 1–5, supplemental Tables 1 and 2, and supplemental Refs. 1–4.

NetworkEditor's Perspective: "Activation of enhancers by the corresponding gene products".

In this detailed paper by Qiang Wu, Xi Chen, Jinqiu Zhang, Yuin-Han Loh, Teck-Yew Low, Weiwei Zhang, Wensheng Zhang, Siu-Kwan Sze, Bing Lim, and Huck-Hui Ng, the mechanisms of enhancer activation of gene transcription have been closely defined in murine embryonic stem cells, revealing the surprising occurence of activation of enhancers by the corresponding gene products.

1. Mollica LR, Crawley JTB, Liu K, Rance JB, Cockerill PN, Follows GA, Landry J-R, Wells DJ, and Lane DA, "Role of a 5'-enhancer in the transcriptional regulation of the human endothelial cell protein C receptor gene".

2. Richards M, Tan S-P, Chan W-K, and Bongso A,
"Reverse Serial Analysis of Gene Expression (SAGE) Characterization of Orphan SAGE Tags from Human Embryonic Stem Cells Identifies the Presence of Novel Transcripts and Antisense Transcription of Key Pluripotency Genes".

3. Jones EA, and Flavell RA.
 "Distal enhancer elements transcribe intergenic RNA in the IL-10 family gene cluster",  J Immunol. 2005 Dec 1;175(11):7437-46.

4. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

5. Ling J, Baibakov B, Pi W, Emerson BM, and Tuan D, "The HS2 Enhancer of the b-globin Locus Control Region Initiates Synthesis of Non-coding, Polyadenylated RNAs Independent of a cis-linked Globin Promoter".

6. Cho DH, Thienes CP, Mahoney SE, Analau E, Filippova GN, and Tapscott SJ,
"Antisense Transcription and Heterochromatin at the DM1 CTG Repeats Are Constrained by CTCF".

7. Navarro P, Pichard S, Ciaudo C, Avner P, and Rougeulle C, "Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation".

8. Chen J, Sun M, Rowley JD, and Hurst LD,
"The small introns of antisense genes are better explained by selection for rapid transcription than by ‘genomic design’".

9. Sun M, Hurst LD, Carmichael GG, and Chen J, "Evidence for variation in abundance of antisense transcripts between multicellular animals, but no relationship between antisense transcriptionand organismic complexity", Genome Research vol. 16: no. 7, pp. 922-933 (July, 2006).

10. Frenster JH, and Hovsepian JA, "Kissing Chromosomes and Paired Sense-Antisense RNA Synthesis". 71st Cold Spring Harbor Symposium on Quantitative Biology: Regulatory RNAs", Program page 62, May 31-June 5, 2006.

11. Xu N, Tsai C-L, and Lee JT, "Transient Homologous Pairing Marks the Onset of X Inactivation", Science vol. 311, no. 5764, pp. 1149-1152 (February 24, 2006).

12. Coudert AE, Pibouin L, Vi-Fane B, Thomas BL, Macdougall M, Choudhury A, Robert B, Sharpe PT, Berda A, and Lezot F, "Expression and regulation of the Msx1 natural antisense transcript during development", Nucleic Acid Research, vol. 33, no. 16, pp. 5208-5216 (2005).

13. Shin JT, Priest JR, Ovcharenko I, Ronco A, Moore RK, C. Burns CG, and MacRae CA,
"Human-zebrafish non-coding conserved elements act in vivo to regulate transcription".

14. Iwamoto M, and Higob K, "Accumulation of sense–antisense transcripts of the rice catalase gene CatB under dark conditions requires signals from shoots".

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