ISSN 1990519X, Cell and Tissue Biology, 2014, Vol. 8, No. 1, pp. 27–32. © Pleiades Publishing, Ltd., 2014. Original Russian Text © I.B. Nazarov, V.A. Krasnoborova, A.G. Mitenberg, E.V. Chikhirzhina, A.P. DavidovSinitzin, M.A. Liskovykh, A.N. Tomilin , 2013, published in Tsi tologiya, 2013, Vol. 55, No. 10, pp. 697–702.

Transcription Regulation of Oct4 (Pou5F1) Gene by Its Distal Enhancer I. B. Nazarov, V. A. Krasnoborova, A. G. Mitenberg, E. V. Chikhirzhina, A. P. DavidovSinitzin, M. A. Liskovykh, and A. N. Tomilin Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia email: [email protected], [email protected] Received May 31, 2013

Abstract—The study of Oct4 gene regulation in murine embryonic stem cells revealed that distal enhancer (DE) was its key element. DE includes two functional elements, DEa and DEb. Both elements are required for Oct expression in pluripotent cells. The most feasible nominee for binding with the DEb site is Oct4 pro tein heterodimerized with Sox2. It is still unclear what transcription proteins bind to the DEa site and how cooperation between DEa and DEb sites is exerted. Using biotinylated oligonucleotides, we developed a sen sitive EMSA for DNAprotein complexes. This method, as well as protein chromatographic fractionation, was used to isolate proteins specifically bound with the DEa site of Oct4 DE in extracts of murine embryonic stem cells and tissues. Keywords: Oct4, distal enhancer, regulation of transcription DOI: 10.1134/S1990519X14010106

Oct4 is a major transcription factor required to sus tain pluripotency of embryonic stem (ES) cells. Of the transcription factors used for generation of induced pluripotent stem cells, only Oct4 is a permanent con tributor (Takahashi and Yamanaka, 2006; Maherali and Hochedlinger, 2008).

DNA methylation (Li et al., 2007) which, in turn, are under control of microRNA (miR290–miR295), as well as transcription repressor Rbl2 (Sikkonen et al., 2008). During somaticcell dedifferentiation and reaching pluripotency, Oct4 is activated concurrently with DNA demethylation (Byrne et al., 2003; Kimura et al., 2004; Maherali et al., 2007; Okita et al., 2007). Mouse ES cells differentiation is accompanied with posttranslational modifications of some histones. Active gene markers (H3K9ac, H3K14ac, H3Kme) are substituted by inactive gene markers (H3K9me and H3Kme3) (Feldman et al., 2006). Lysine methy lation recruits heterochromatin protein 1 (HP1), het erochromatin formation and complete Oct4 silencing. The most required enzyme for histone methylation is methyltransferase G9 (Tachibana et al., 2002; Feld man et al., 2006; Kellner and Kikyo, 2010). Paf1 complex binds with Oct4 gene PP site, but binding is not observed during ES cells differentiation. Knockdown of each from five complex subunits inhib its Oct4 mRNA and induces ES cells differentiation. Conversely, enhanced expression of Paf1 complex sub units supports Oct4 expression and suppresses ES cells differentiation (Ding et al., 2009). The first data on gene activation by sequences dis tant for promoters were reported about 30 years ago (Banerji et al., 1981; Moreau et al., 1981). Regardless of certain successes in understanding of diversity in functioning of distal regulatory elements, most funda mental problems are still unresolved. It has been pro posed that up to a million enhancers may be impli

Oct4 gene expression is under complex regulation. Oct4 concentration reduced to 50% triggers stemcell differentiation into trophoblasts. Its twofold increase also results in cell differentiation, but into primitive endoderm and mesoderm (Niwa et al., 2000). Oct4 gene transcription initiation site has three reg ulatory elements: DE, proximal enhancer (PE), and proximal promoter (PP) (Okazawa et al., 1991; Minucci et al., 1996; Yeom et al., 1996). Enhancer activity depends on the development stage of the mouse embryo. DE regulates Oct4 expression in inner cell mass, ES cells and in germ cell precursors; PE activates the expression in epiblast cells (Pesce and Scholer, 2001; Chambers and Smith, 2004; Boiani and Scholer, 2005). These three elements are the targets for the binding of regulatory proteins and DNA meth ylation is included in the regulation. DNA methyla tion of these elements shows the Oct4 transcriptional status. They are not methylated in ES cells, whereas they are methylated in somatic cells not expressing Oct4. During ES differentiation, de novo DNAmeth ytransferases Dnmt3a and Dnmt3b are responsible for Abbreviations: DE—distal enhancer, ES—embryonic stem, PE—proximal enhancer, PP—proximal promoter.

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DEa:

DEa m1:

DEa m2:

DEa m3:

Fig. 1. DNA sequences used in the experiments. DEawild type sequence. Distal enhancer DEa of Oct4 gene is shown with lowercase letters. DEa m1, DEa m2, and DEa m3 are sequences with inserted mutations. Mutations are underlined.

cated in the regulation of dozens of thousands of genes (Bulger and Croudine, 2010). Enhancers are not only important for cell differentiation, but have a key value in sustaining stem cell pluripotency, the mechanisms of many diseases, and evolution. Enhancers are involved in the interrelations between transcription, structural and epigenetic chromatin changes, noncod ing RNA and nuclear organelles (Bulger and Croudine 2011; Palstra and Grosveld 2012). DE of the mouse Oct4 gene is composed of two functional elements, DEa and DEb (Site 2A and Site 2B). Both sites are necessary for valid Oct4 gene expression in pluripotent cells. The most feasible can didate for binding with the DEb site is Oct4 protein heteromerized with Sox2 (OkumuraNakanishi et al., 2005). Sequence CCCCTCCCCCC (Fig. 1) of the DEa site was first identified by specific resistance to chemical modifications in vivo in ES cells expressing Oct4 (Minucci et al., 1996). Later, it was demon strated that 50–70% deletions in DEa area inhibit enhancer activity if they are located inside, but not outside the DE area (OkumuraNakanishi et al., 2005).However, a series of mutations in DE sequence did not affect its activity. DEa significance is also fol lowed from the high conservatism of this sequence revealed in human, murine and bovine Oct4 genes (Nordoff et al., 2001). Currently, it remains unclear what transcription factors are bound with the DEa site and how coopera tion between DEa and DEb occurs. The aim of this study was to isolate and specify proteins interacting with DEa. MATERIALS AND METHOS Extract isolation. Cells and tissues were washed with ice cold PBS and homogenized with Dawn’s homogenizer in two buffer volumes (20 mM TrisHCl,

pH 8.0, 25% glycerol, 150 mM NaCl, 1 mM EDTA, 5 mM DTT, 1× protease inhibitor cocktail (Complete, Roche, Germany)). Homogenates were centrifuged at 14 000 g for 10 min. Supernatants were stored at ⎯70°C. Gel shift assay (EMSA). Ninety femtomole (2 ng) of biotinylated DNA probe was mixed with 1 to 5µL protein sample in 10–15 µL TrisHCl buffer, pH 7.6, with 10% glycerol, 0.14 M NaCl, 0.5 mM EDTA, 0.05% Triton X100, 0.1 mg/mL bovine serum albu min (BSA), 0.1 mg/mL poly(dIdC) (Amersham, Germany), 5 mM DDT, 1× protease inhibitor cocktail (Complete, Roche, Germany) and incubated for 20 min the room temperature. The samples were sep arated by electrophoresis in 6% polyacrylamide gel in 0.5× TBE buffer and transferred on the nitrocellulose membrane. The membrane was blocked in 2.5% BSA in PBS for 30 min, incubated with streptavidinHRP (Sigma, United States), dilution 1 : 20000, with block ing buffer for 1 h at room temperature. The membrane was washed with blocking buffer for 30 min, twice with PBST (PBS with 0.2% Tween20) for 30 min, and then with PBS for 10 min. Chemiluminescence was visual ized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, United States) using ChemiDoc XRC equipment (Bio Rad, United States). The signal intensity was quantified using Quantity One software. Mouse brain extract chromatography on a phenyl sepharose column. Phenyl Sepharose CL 4B 8 × 0.8cm column (Pharmacia, Sweden) was equili brated by 50 mM ThisHCl buffer, pH 8.0, with 0.5 M NaCl, 1 mM EDTA, 5 mM DDT. Elution was per formed by 70 mL buffer with linear gradient of NaCL concentrations from 0.5 up to 0.1 M and ethylene gly col from 0 to 50%). The elution rate was 3 mL/h, and the fraction volume was 2 mL. CELL AND TISSUE BIOLOGY

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+ biotinDEa + extract, µL 1

+ DEa

0.3 0.1 0.03 ×150 ×50 ×10 ×2 ×0.5

complex

biotinDEa

Fig. 2. Minimal quantity of murine ES cell extract required to visualize a complex between biotinylated DEa oligonucleotide (+ DEabiotin) and extract proteins estimated by titration (left). (Right) EMSA showing a decrease in the complex production caused by addition of unlabeled DEa (+ DEa) in a molar excess of from 0.5 to 150 times.

Using nonradioactive labeled oligonucleotides, we developed the sensitive gelretardation method of DNAprotein complexes. We obtained 5'biotinylated oligonucleotides. After the annealing, elongation, and purification of twostrand fragments, we estimated the conditions for their binding, transfer, and signal visu alization. DNA sequences applied in this work are pre sented in Fig. 1. Figure 1 shows unlabeled DNA frag ments of wild type and three mutant DNA fragments: DEa m1, DEa m2, and DEa m3. Figure 2 shows the production of a resistant com plex of mouse ES cell extract proteins with biotin DEa. The figure’s left part shows the estimate of the minimal amount of the protein extract required for generation of welldefined complex. It is seen that about 0.3 µL (or 1 µg of total protein) of ES cell extract is sufficient to bind the total DNA (biotinDEa) added to the reaction mixture (2 ng). In addition, no less two complexes that are distinct in electrophoretic mobility are produced. The figure’s right part illustrates com petitive exclusion of a labeled DNAprobe from the complex. The study of proteins implicated in DE function of the Oct4 gene using EMSA showed that the DEa site interacted with a proteins found in both ES D3 and NIH3T3 cells (OkumuraNakanishi et al., 2005). Thus, the interaction with proteins from only ES extract specific for the DEb site, was not reported for the DEa site. Based on these data, we assumed that the proteins interacting with DEa were expressed both in ES and differentiated cells in adult tissues. To verify CELL AND TISSUE BIOLOGY

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the idea extracts from adult mouse tissues were tested upon their interaction with DEa in EMSA. Figure 3 presents the results of these experiments. It is seen that

bi ot in D Ea 3T 3 N IH Th ym u Th s ym us Br ai n Br ai n Li ve r Li ve r

RESULTS AND DISCUSSION

Fig. 3. The presence of proteins interacting with DEa in extracts of mouse differentiated cells (3T3NIH) and tis sues. Two tissue samples were taken from different animals.

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(a) A280, AU

0.8 0.6 0.4 f27

f33

0.2

1

5

10

15 20 25 Fraction number

30

35

(b) 23 24 25 26 27 28 29 32 33 34

Fig. 4. Hydrophobic interaction chromatography of murine brain extract on phenylsepharose CL4B. (a) Chromatogram. Elution was performed by 70 mL 50 mM TrisHCl, pH8.0 with 1 mM EDTA, mM DTT and linear gradient of NaCl (0.5–0.1 M) and ethylene glycol concentrations (0–50%). f27 and f33 are peaks containing proteins specifically binding with DEa; (b) EMSA of the corresponding fractions.

complexes binding DEa common for ES cells are pro duced in all cases. Thus, it is plausible that the DEa site interacts with an similar protein (or proteins) expressed in both ES and differentiated cells. Presum ably, this is necessary for functions common to all cell types. The function may be, for example, the interac tion with constitutive chromatin structures required for the regulation of the particular gene transcription. For the following study of properties and identifi cation of the complex proteins, we purified DEa inter acting proteins by chromatography. The method of hydrophobic interaction chromatography allowed a higher purification efficiency and gave the highest yield of the proteins compared to other used methods. Two protein fractions were obtained by hydrophobic interaction chromatography of mouse brain extract able to produce complexes with DEa (f27 and f33, Fig. 4). f27 was eluted with 0.22 M NaCl and 35% eth ylene glycol; f33 was eluted with 0.13 M NaCl and 43% ethylene glycol. In search of the most adequate methods for protein purification we probed two other methods of column chromatography: gelfiltration on column biogel P200 (BioRad, United States) and ionexchange chromatography on DEAESepharose Fast Flow (Pharmacia, Sweden). Gel filtration is not considered sufficiently effective for purification of these proteins; however, it allowed determining the sizes of proteins (or complexes thereof). The protein molecular weight varied from 40 to 85 kDa. Ion exchange chromatography on DEAEsepharose coceded to the hydrophobic interaction chromatogra

phy efficiency and separation of fractions with differ ent electrophoretic mobility. The interaction specificity was verified by biotin DEa interaction with isolated fractions in the presence of a 200fold excess of unlabeled DE fragments with inserted mutations (Figs. 1, 5). The interaction of the f33 fraction with biotin DEa in the presence of DEam1 excess resulted in total inhibition of competi tion, whereas other mutations in competitive DNA suppressed the interaction. Inhibition of competitive ability of the interaction of the f27 fraction with biotin DEa was observed with the DEam1 and DEam2. These results show that DEa left and central parts and about five base pairs to the left of DEa are required for the f27 fraction protein, whereas the f33 fraction requires left and central DEa sites. Using twodimensional electrophoresis with native conditions in the first direction and denaturing condi tion in the second direction, we isolated the protein from the f33 fraction. The homogeneity and amount of this protein rendered possible its identification by mass spectrometry (Fig. 6). The protein candidate interacting with DEa is indicated with an arrow (Fig. 6). The protein meets all the needed require ments. First, its electrophoretic mobility corresponds to the protein with molecular weight 40–70 kDa that matches the molecular weight range 40–85 kDa according to gel filtration. Second, the complex and protein migration in the first direction practically coincided. Third, the protein content calculated by the amount of DNA migrating in the complex was no less than 200 ng. Since no other proteins in this quan CELL AND TISSUE BIOLOGY

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tity have been observed in the complex migration zone, the protein is the only one satisfying the require ment.

+ biotinDEa + Protein fraction + ×200 DEa wt

m1

m2

REFERENCES m3

f33

f27

Fig. 5. EMSA of biotinylated DEa complex in the presence of a 200fold excess of unlabeled oligonucleotides with wild type (wt) and modified (m1, m2, m3) DEa sequences. It is seen that mutation m1 results in elimination of inhibi tion of the complex production with biotinylated DEa in the presence of f33 and f27; mutation m2 produces this effect only in the presence of f27.

(a)

Banerji, J., Rusconi, S., and Schaffner, W., Expression of a betaglobin gene is enhanced by remote SV40 DNA sequences, Cell, 1981, vol. 27, pp. 299–308. Boiani, M., and Scholer, H.R., Regulatory networks in embryoderived pluripotent stem cells, Nat. Rev. Mol. Cell. Biol., 2005, vol. 6, pp. 872–884. Bulger, M. and Groudine, M., Enhancers: the abundance and function of regulatory sequences beyond promoters, Dev. Biol., 2010, vol. 339, pp. 250–257. Bulger, M. and Groudine, M., Functional and mechanistic diversity of distal transcription enhancers, Cell, 2011, vol. 144, pp. 327–339. Byrne, J.A., Simonsson, S., Western, P.S., and Gurdon, J.B., Nuclei of adult mammalian somatic cells are directly reprogrammed to Oct4 stem cell gene expression by amphibian oocytes, Curr. Biol., 2003, vol. 13, pp. 1206– 1213. Chambers, I., and Smith, A., Selfrenewal of teratocarci noma and embryonic stem cells, Oncogene, 2004, vol. 23, pp. 7150–7160. Ding, L., PaszkowskiRogacz, M., Nitzsche, A., Slabicki, M.M., Heninger, A.K., de, Vries, I., Kittler, R., Junqueira, M., Shevchenko, A., Schulz, H., Hubner, N, Doss, M.X., Sachinidis, A., Hescheler, J., Iacone, R., Anastassiadis, K., Stewart, A.F., Pisabarro, M.T., Cal

(b)

(c) 6% PAAG

ng 2 10 50

12% PAAG with SDS

kD 170 130 100 70 55 40 35 25

Fig. 6. Twodimensional electrophoresis of f33 fraction proteins. (a) Vertical gel fragment after electrophoresis in 6% polyacrylamide gel in 0.5× TBE buffer (first direction, native conditions), (left) the fragment is stained with Sypro Ruby Protein Stain, (right) the membrane is stained for biotin. The upper sign on the membrane is the complex migration zone; the lower is free DEa. Dotted lines show the cut in the gel containing complex. An excised gel fragment was used for electrophoresis in the second direction. (b) Membrane with biotin signal showing the complex position (upper part). Below—the gel after electrophoresis in the second direction in 12% PAAG with SDS; the gel was stained with Sypro Ruby Protein Stain. The arrow indicates the proteincandidateproducing complex with DEa. (c) Quantitation assay, the gel fragment from the second direction with 2, 10, and 50 ng BSA. CELL AND TISSUE BIOLOGY

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Translated by I. Fridlyanskaya

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