Epigenetic Regulation of Presenilin 1 and 2 in the Cerebral Cortex of Mice During Development Ashish Kumar,* Mahendra Kumar Thakur* Department of Zoology, Banaras Hindu University, Varanasi 221 005, Uttar Pradesh, India
Received 13 November 2014; revised 5 January 2015; accepted 18 January 2015
ABSTRACT: Previously, we reported differential expression profile of Presenilin (PS)1 and 2 and their interacting partners in mouse cerebral cortex during development. Our findings indicated crucial involvement of these proteases in prenatal and postnatal development of mouse cerebral cortex. However, the mechanisms that precisely control their expression in stage-specific manner during brain development are still elusive. In this regard, epigenetic modifications by DNA methylation and histone acetylation during brain development deserve major attention. Therefore, we have analyzed the epigenetic regulation of PS1 and PS2 in mouse cerebral cortex during development. The data demonstrated a good correspondence of H3K9/14 Ac level in PS1 and PS2 promoter with their expression profile during cerebral cortical development. H3K9/14 Ac level was high at embryonic day (E)12.5, declined at E18.5, increased from postnatal day
INTRODUCTION Brain development is an extremely synchronized cascade of sequential and concomitant programs involving stage-specific gene expression required for Correspondence to: M.K. Thakur ([email protected]). Contract grant sponsor: Department of Science and Technology, Government of India; contract grant number: SR/SO/HS-54/2008. Contract grant sponsor: Department of Biotechnology, Government of India; contract grant number: BT/PR3996/MED/97/57/ 2011 (Government of India, to M.K.T.). *Present address: Department of Zoology, Banaras Hindu University, Varanasi, 221 005, Uttar Pradesh, India The authors do not have any actual or potential conflicts of interest including financial, personal, or academic. Ó 2015 Wiley Periodicals, Inc. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/dneu.22274
(P)0 to P45 and decreased again at 20 weeks (w) in PS1 promoter. For PS2, H3K9/14 Ac level was high at E12.5, thereafter, reduced upto P20 and increased at P45 and 20 weeks. DNA methylation sites also varied in number and position at different developmental stages, and some of them are putative sites for binding of transcription factors like HSF-1, Ets-1, and Sp1 that are crucial for brain developmental processes, as revealed by in silico analysis. Though MeCP2 level also altered during development, they did not correlate with PS1 and PS2 expression profile. Taken together, our findings provide the first evidence of epigenetic regulation of PS1 and PS2 by H3K9/ 14 histone acetylation and DNA methylation in mouse cerebral cortex during development. VC 2015 Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2015
Keywords: Presenilin; brain development; DNA methylation; histone acetylation
proliferation, migration, differentiation of neurons and glia, establishment of synapses, and myelin sheathing (Stiles and Jernigan, 2010). In our previous report, we demonstrated that the expression of Presenilin (PS)1 and PS2, polytopic transmembrane aspartyl proteases, significantly varied in mouse cerebral cortex during development (Kumar and Thakur, 2012). They also interacted with a wide range of proteins involved in cell division, glycolysis, cell adhesion, and protein trafficking in the cerebral cortex of mice during development (Kumar and Thakur, 2014). Our findings indicated that PS1 and PS2 gene expression in the cerebral cortex might be tightly regulated during different developmental stages for their proper functioning. Accumulating evidences highlight the role of epigenetic mechanisms, DNA methylation, and histone modifications in regulation of gene 1
2
Kumar and Thakur
expression during brain development (Phillips, 2008). The epigenetic regulation of PS1 and PS2 gene has not been studied in details. There are a few studies showing binding of p300 (histone acetyltransferase) and chromodomain helicase DNA binding protein 3 (histone deacetylase) to human PS1 promoter (Das, 2008), suggesting that histone acetylation might be a crucial epigenetic marker of PS gene regulation. DNA methylation analysis of PS1 promoter at nucleotide position (nt) 451, 612, 681, and 735 showed that SAM treatment dowregulates PS1 expression and modulates DNA methylation at nt 451 (Scarpa et al., 2003). Folate and vitamin B12 supplementation increased PS1 expression by reducing S-adenosyl methionine (SAM) levels (Fuso et al., 2011). However, the epigenetic regulation of PS gene expression during brain development has not been investigated. In the light of above background, we have studied the epigenetic regulation of PS1 and PS2 in the cerebral cortex of mice during development. We have analyzed histone acetylation status of PS promoter through chromatin immunoprecipitation (ChIP) using H3K9/14 acetylated (Ac) antibody. Further, we have examined another mechanism of transcriptional control, DNA methylation by sodium bisulfite sequencing of PS promoter in the mouse cerebral cortex during the critical period of brain development. As methylation suppresses gene transcription via methyl CpG binding protein MeCP2; we determined MeCP2 binding by ChIP in the cerebral cortex of mice during development.
MATERIALS AND METHODS Animals Swiss albino strain mice were inbred and maintained in 12 h light and dark schedule with ad libitum drinking water and standard mice feed in the animal house of Zoology Department, Banaras Hindu University, Varanasi, India. To study the epigenetic regulation of PS gene, mice of E12.5, E18.5, P0, P20, P45, and 20 weeks were used according to guidelines of the institutional animal ethical committee, Banaras Hindu University, Varanasi, India. For E12.5, telencephalon and for other developmental stages the cerebral cortex was dissected out.
A260/280 1.8 were used further. Total DNA from different groups was resolved on 0.7% agarose gel.
ChIP Analysis Chromatin was isolated from telencephalon and cerebral cortex of mice of different developmental stages. Briefly, the tissue was chopped and held in phosphate-buffered saline (PBS) with 1 mM protease inhibitors cocktail (SigmaAldrich). The tissue was incubated with 1% formaldehyde in PBS at 25 C for 15 min. It was homogenized in lysis buffer (5 mM pipes-KOH [pH 8.0], 85 mM KCl, 0.5% NP-40 with 1 mM protease inhibitors) and centrifuged at 1000g at 4 C for 5 min. The pellet was resuspended in nuclei lysis buffer (50 mM Tris-Cl [pH 8.0], 10 mM ethylenediaminetetraacetic acid (EDTA) [pH 8.0], 1% SDS with 1 mM protease inhibitors [Sigma-Aldrich]), incubated at 4 C for 20 min and sonicated for 10 s at 50% output, with 2 min refractory period at 4 C between sonications (repeated for five times). The sonicated chromatin solution was further centrifuged at 12,000g at 4 C for 15 min. The supernatant was saved and 50 mL of chromatin was taken and DNA was isolated by phenol–choloroform method. The size of sonicated DNA was checked by electrophoresis on 1% agarose gel to assure that DNA is well-sonicated (DNA length between 1500 and 2000 bp) and no DNA is present in wells of the gel. The protein concentration was measured by Bradford (1976) method. The sonicated chromatin (200 mg DNA) was diluted in a final volume of 2 mL in dilution buffer (100 mM NaCl, 20 mM Tris-Cl [pH 8.0], 2 mM EDTA [pH 8.0], 1% Triton X-100, with 1 mM protease inhibitors). The chromatin suspension was precleared with Protein A-sepharose-bead slurry at 4 C for 2 h with rotation and then centrifuged at 3500g at 4 C for 10 min. The supernatant fraction was collected and divided in two tubes labeled as input and immunoprecipitation. Histone H3K9/14 antibody (4 mg) or MeCP2 antibody was added to the imunoprecipitation tube and rotated at 4 C overnight. Next day, 50 lL of Protein Asepharose-bead slurry was added to it and incubated at 4 C for 2 h with rotation. Thereafter, it was centrifuged at 3500g at 4 C for 10 min and the supernatant was removed carefully. After washing the pellet in low salt, high salt, LiCl, and TE buffer, the immune complex was eluted by adding 250 lL of elution buffer (100 mM NaHCO3 and 1% sodium dodecyl sulfate (SDS)) to the pellet. To reverse the protein–DNA cross links, 200 mM NaCl was added to the immunoprecipitated chromatin and it was incubated at 65 C for 4 h. Thereafter, DNA was isolated by phenol–chloroform method and dissolved in 100 mL of TE buffer. The sequences of interest were amplified by semiquantitative polymerase chain reaction (PCR) method using specific primers and conditions as mentioned in Table 1.
Isolation of Genomic DNA Genomic DNA was extracted from the telencephalon and cerebral cortex of mice of different developmental stages by standard phenol–chloroform method. It was estimated by taking absorbance at 260 nm and DNA samples with Developmental Neurobiology
Sodium Bisulfite Sequencing Sodium bisulfite modification of genomic DNA was done using D-5001 kit (Zymo-research) in compliance with the
Epigenetic Regulation of PS1 and PS2 in Cerebral Cortex of Mice
3
Table 1 Gene Specific Primer Sequences And Corresponding Amplification Conditions Primer
Sequence
PS1 sense Antisense
50 -GGAGAAATGGGAAGTGTTGG-30 50 -CGGGGTTCCTCGATCTCC-30
PS1 BSP-1 sense Antisense
50 -GTTTTGAGGAATTGTTTAAA TAAATAT-30 30 -CTAAAACTAACCTAATCCAC TCRCATA-50
PS1 BSP-2 sense Antisense
50 -ATGTGAGTGGATTAGGTTAGTTT-30
PS2 sense Antisense
50 -CCTCCACCCCTTCAGTACTTC-30 50 -AGAGATGCAAGCTGCTCACC-30
PS2 BSP sense Antisense
50 -TTTTGTGTTGGTTTTTGGAAT-30
50 -CCTCGAAAACACAAACCC-30
30 -AAAAAAACACAAAAACCCCATA-50
b-Actin sense 50 -CTGAGTATGTCGTGGAGTCTACTGG-30 Antisense 50 -GTCATATTTCTCGTGGTTCACACC-30
manufacturer’s instruction. Briefly, 2 lg of genomic DNA was denatured with M-dilution buffer at 37 C for 10 min and treated with conversion reagent for 16 h at 50 C. Thereafter, DNA was purified using a column and desulfonated with buffer at 37 C for 15 min. Finally, the column was washed twice with wash buffer and eluted in elution buffer. PCR amplification was performed using modified DNA as a template. The sequence of interest in the bisulfitetreated DNA was amplified with bisulfite specific primers. PCR primer sequence and conditions are mentioned in Table 1. The PCR amplicons were run on 2% agarose gel. The bands were cut and purified by PCR clean up kit (Nucleopore, India) and sequenced using forward and reverse primers at Eurofins Scientific, India.
In Silico Analysis of DNA Methylation Data The chromatograms were converted in FASTA sequence format by Chromas (ver 2.4.3). The modified sequences were compared with unmodified normal sequence obtained from sequencing using Bio QT analyzer (ver 2.00) and
Product Size (bp)
Conditions Initial denaturation- 94 C (5 min), 25 cycles (94 C for 45 s, 55 C for 45 s, 72 C for 45 s) and final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 10 cycles (94 C for 30 s, 65 C for 30 s, 72 C for 30 s), 20 cycles (94 C for 30 s, 58 C for 30 s, 72 C for 30 s),10 cycles (94 C for 30 s, 52 C for 30 s, 72 C for 30 s) and 10 cycles (94 C for 30 s, 45 C for 30 s, 72 C for 30 s) with final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 10 cycles (94 C for 30 s, 65 C for 30 s, 72 C for 30 s), 20 cycles (94 C for 30 s, 58 C for 30 s, 72 C for 30 s),10 cycles (94 C for 30 s, 52 C for 30 s, 72 C for 30 s) and 10 cycles (94 C for 30 s, 45 C for 30 s, 72 C for 30 s) with final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 25 cycles (94 C for 45 s, 61 C for 45 s, 72 C for 1 min) and final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 10 cycles (94 C for 30 s, 65 C for 30 s, 72 C for 30 s), 20 cycles (94 C for 30 s, 58 C for 30 s, 72 C for 30 s),10 cycles (94 C for 30 s, 52 C for 30 s, 72 C for 30 s) and 10 cycles (94 C for 30 s, 45 C for 30 s, 72 C for 30 s) with final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 20 cycles (94 C for 30 s, 51.5 C for 30 s, 72 C for 30 s) and final extension at 72 C (5 min)
656
300
345
1225
357
149
methylated sites were identified. Further analysis by TFSEARCH: Searching Transcription Factor Binding Sites (ver 1.3) software revealed the cis-acting elements and corresponding putative transcription factors binding to the methylated sites.
Statistical Analysis Each experiment was repeated three times (n 5 3 3 3 mice/ group). The signals were measured in the form of integrated density value (IDV) by spot densitometry tool of AlphaEaseFC software (Alpha Innotech Corp.). The IDV for PS1 and PS2 were normalized against signal intensity of b-actin and represented as a histogram with the mean 6 standard error of mean (SEM) of three values calculated as Relative Density Value, RDV (IDV of PS1/b-actin and PS2/b-actin). Statistical analysis was performed using one-way analysis of variance, followed by post hoc test of Student–Newman–Keuls method through Jindel Scientific SigmaPlot for Windows (standard version 2.0). Values were reported as mean 6 SEM and p values
Received 13 November 2014; revised 5 January 2015; accepted 18 January 2015
ABSTRACT: Previously, we reported differential expression profile of Presenilin (PS)1 and 2 and their interacting partners in mouse cerebral cortex during development. Our findings indicated crucial involvement of these proteases in prenatal and postnatal development of mouse cerebral cortex. However, the mechanisms that precisely control their expression in stage-specific manner during brain development are still elusive. In this regard, epigenetic modifications by DNA methylation and histone acetylation during brain development deserve major attention. Therefore, we have analyzed the epigenetic regulation of PS1 and PS2 in mouse cerebral cortex during development. The data demonstrated a good correspondence of H3K9/14 Ac level in PS1 and PS2 promoter with their expression profile during cerebral cortical development. H3K9/14 Ac level was high at embryonic day (E)12.5, declined at E18.5, increased from postnatal day
INTRODUCTION Brain development is an extremely synchronized cascade of sequential and concomitant programs involving stage-specific gene expression required for Correspondence to: M.K. Thakur ([email protected]). Contract grant sponsor: Department of Science and Technology, Government of India; contract grant number: SR/SO/HS-54/2008. Contract grant sponsor: Department of Biotechnology, Government of India; contract grant number: BT/PR3996/MED/97/57/ 2011 (Government of India, to M.K.T.). *Present address: Department of Zoology, Banaras Hindu University, Varanasi, 221 005, Uttar Pradesh, India The authors do not have any actual or potential conflicts of interest including financial, personal, or academic. Ó 2015 Wiley Periodicals, Inc. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/dneu.22274
(P)0 to P45 and decreased again at 20 weeks (w) in PS1 promoter. For PS2, H3K9/14 Ac level was high at E12.5, thereafter, reduced upto P20 and increased at P45 and 20 weeks. DNA methylation sites also varied in number and position at different developmental stages, and some of them are putative sites for binding of transcription factors like HSF-1, Ets-1, and Sp1 that are crucial for brain developmental processes, as revealed by in silico analysis. Though MeCP2 level also altered during development, they did not correlate with PS1 and PS2 expression profile. Taken together, our findings provide the first evidence of epigenetic regulation of PS1 and PS2 by H3K9/ 14 histone acetylation and DNA methylation in mouse cerebral cortex during development. VC 2015 Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2015
Keywords: Presenilin; brain development; DNA methylation; histone acetylation
proliferation, migration, differentiation of neurons and glia, establishment of synapses, and myelin sheathing (Stiles and Jernigan, 2010). In our previous report, we demonstrated that the expression of Presenilin (PS)1 and PS2, polytopic transmembrane aspartyl proteases, significantly varied in mouse cerebral cortex during development (Kumar and Thakur, 2012). They also interacted with a wide range of proteins involved in cell division, glycolysis, cell adhesion, and protein trafficking in the cerebral cortex of mice during development (Kumar and Thakur, 2014). Our findings indicated that PS1 and PS2 gene expression in the cerebral cortex might be tightly regulated during different developmental stages for their proper functioning. Accumulating evidences highlight the role of epigenetic mechanisms, DNA methylation, and histone modifications in regulation of gene 1
2
Kumar and Thakur
expression during brain development (Phillips, 2008). The epigenetic regulation of PS1 and PS2 gene has not been studied in details. There are a few studies showing binding of p300 (histone acetyltransferase) and chromodomain helicase DNA binding protein 3 (histone deacetylase) to human PS1 promoter (Das, 2008), suggesting that histone acetylation might be a crucial epigenetic marker of PS gene regulation. DNA methylation analysis of PS1 promoter at nucleotide position (nt) 451, 612, 681, and 735 showed that SAM treatment dowregulates PS1 expression and modulates DNA methylation at nt 451 (Scarpa et al., 2003). Folate and vitamin B12 supplementation increased PS1 expression by reducing S-adenosyl methionine (SAM) levels (Fuso et al., 2011). However, the epigenetic regulation of PS gene expression during brain development has not been investigated. In the light of above background, we have studied the epigenetic regulation of PS1 and PS2 in the cerebral cortex of mice during development. We have analyzed histone acetylation status of PS promoter through chromatin immunoprecipitation (ChIP) using H3K9/14 acetylated (Ac) antibody. Further, we have examined another mechanism of transcriptional control, DNA methylation by sodium bisulfite sequencing of PS promoter in the mouse cerebral cortex during the critical period of brain development. As methylation suppresses gene transcription via methyl CpG binding protein MeCP2; we determined MeCP2 binding by ChIP in the cerebral cortex of mice during development.
MATERIALS AND METHODS Animals Swiss albino strain mice were inbred and maintained in 12 h light and dark schedule with ad libitum drinking water and standard mice feed in the animal house of Zoology Department, Banaras Hindu University, Varanasi, India. To study the epigenetic regulation of PS gene, mice of E12.5, E18.5, P0, P20, P45, and 20 weeks were used according to guidelines of the institutional animal ethical committee, Banaras Hindu University, Varanasi, India. For E12.5, telencephalon and for other developmental stages the cerebral cortex was dissected out.
A260/280 1.8 were used further. Total DNA from different groups was resolved on 0.7% agarose gel.
ChIP Analysis Chromatin was isolated from telencephalon and cerebral cortex of mice of different developmental stages. Briefly, the tissue was chopped and held in phosphate-buffered saline (PBS) with 1 mM protease inhibitors cocktail (SigmaAldrich). The tissue was incubated with 1% formaldehyde in PBS at 25 C for 15 min. It was homogenized in lysis buffer (5 mM pipes-KOH [pH 8.0], 85 mM KCl, 0.5% NP-40 with 1 mM protease inhibitors) and centrifuged at 1000g at 4 C for 5 min. The pellet was resuspended in nuclei lysis buffer (50 mM Tris-Cl [pH 8.0], 10 mM ethylenediaminetetraacetic acid (EDTA) [pH 8.0], 1% SDS with 1 mM protease inhibitors [Sigma-Aldrich]), incubated at 4 C for 20 min and sonicated for 10 s at 50% output, with 2 min refractory period at 4 C between sonications (repeated for five times). The sonicated chromatin solution was further centrifuged at 12,000g at 4 C for 15 min. The supernatant was saved and 50 mL of chromatin was taken and DNA was isolated by phenol–choloroform method. The size of sonicated DNA was checked by electrophoresis on 1% agarose gel to assure that DNA is well-sonicated (DNA length between 1500 and 2000 bp) and no DNA is present in wells of the gel. The protein concentration was measured by Bradford (1976) method. The sonicated chromatin (200 mg DNA) was diluted in a final volume of 2 mL in dilution buffer (100 mM NaCl, 20 mM Tris-Cl [pH 8.0], 2 mM EDTA [pH 8.0], 1% Triton X-100, with 1 mM protease inhibitors). The chromatin suspension was precleared with Protein A-sepharose-bead slurry at 4 C for 2 h with rotation and then centrifuged at 3500g at 4 C for 10 min. The supernatant fraction was collected and divided in two tubes labeled as input and immunoprecipitation. Histone H3K9/14 antibody (4 mg) or MeCP2 antibody was added to the imunoprecipitation tube and rotated at 4 C overnight. Next day, 50 lL of Protein Asepharose-bead slurry was added to it and incubated at 4 C for 2 h with rotation. Thereafter, it was centrifuged at 3500g at 4 C for 10 min and the supernatant was removed carefully. After washing the pellet in low salt, high salt, LiCl, and TE buffer, the immune complex was eluted by adding 250 lL of elution buffer (100 mM NaHCO3 and 1% sodium dodecyl sulfate (SDS)) to the pellet. To reverse the protein–DNA cross links, 200 mM NaCl was added to the immunoprecipitated chromatin and it was incubated at 65 C for 4 h. Thereafter, DNA was isolated by phenol–chloroform method and dissolved in 100 mL of TE buffer. The sequences of interest were amplified by semiquantitative polymerase chain reaction (PCR) method using specific primers and conditions as mentioned in Table 1.
Isolation of Genomic DNA Genomic DNA was extracted from the telencephalon and cerebral cortex of mice of different developmental stages by standard phenol–chloroform method. It was estimated by taking absorbance at 260 nm and DNA samples with Developmental Neurobiology
Sodium Bisulfite Sequencing Sodium bisulfite modification of genomic DNA was done using D-5001 kit (Zymo-research) in compliance with the
Epigenetic Regulation of PS1 and PS2 in Cerebral Cortex of Mice
3
Table 1 Gene Specific Primer Sequences And Corresponding Amplification Conditions Primer
Sequence
PS1 sense Antisense
50 -GGAGAAATGGGAAGTGTTGG-30 50 -CGGGGTTCCTCGATCTCC-30
PS1 BSP-1 sense Antisense
50 -GTTTTGAGGAATTGTTTAAA TAAATAT-30 30 -CTAAAACTAACCTAATCCAC TCRCATA-50
PS1 BSP-2 sense Antisense
50 -ATGTGAGTGGATTAGGTTAGTTT-30
PS2 sense Antisense
50 -CCTCCACCCCTTCAGTACTTC-30 50 -AGAGATGCAAGCTGCTCACC-30
PS2 BSP sense Antisense
50 -TTTTGTGTTGGTTTTTGGAAT-30
50 -CCTCGAAAACACAAACCC-30
30 -AAAAAAACACAAAAACCCCATA-50
b-Actin sense 50 -CTGAGTATGTCGTGGAGTCTACTGG-30 Antisense 50 -GTCATATTTCTCGTGGTTCACACC-30
manufacturer’s instruction. Briefly, 2 lg of genomic DNA was denatured with M-dilution buffer at 37 C for 10 min and treated with conversion reagent for 16 h at 50 C. Thereafter, DNA was purified using a column and desulfonated with buffer at 37 C for 15 min. Finally, the column was washed twice with wash buffer and eluted in elution buffer. PCR amplification was performed using modified DNA as a template. The sequence of interest in the bisulfitetreated DNA was amplified with bisulfite specific primers. PCR primer sequence and conditions are mentioned in Table 1. The PCR amplicons were run on 2% agarose gel. The bands were cut and purified by PCR clean up kit (Nucleopore, India) and sequenced using forward and reverse primers at Eurofins Scientific, India.
In Silico Analysis of DNA Methylation Data The chromatograms were converted in FASTA sequence format by Chromas (ver 2.4.3). The modified sequences were compared with unmodified normal sequence obtained from sequencing using Bio QT analyzer (ver 2.00) and
Product Size (bp)
Conditions Initial denaturation- 94 C (5 min), 25 cycles (94 C for 45 s, 55 C for 45 s, 72 C for 45 s) and final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 10 cycles (94 C for 30 s, 65 C for 30 s, 72 C for 30 s), 20 cycles (94 C for 30 s, 58 C for 30 s, 72 C for 30 s),10 cycles (94 C for 30 s, 52 C for 30 s, 72 C for 30 s) and 10 cycles (94 C for 30 s, 45 C for 30 s, 72 C for 30 s) with final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 10 cycles (94 C for 30 s, 65 C for 30 s, 72 C for 30 s), 20 cycles (94 C for 30 s, 58 C for 30 s, 72 C for 30 s),10 cycles (94 C for 30 s, 52 C for 30 s, 72 C for 30 s) and 10 cycles (94 C for 30 s, 45 C for 30 s, 72 C for 30 s) with final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 25 cycles (94 C for 45 s, 61 C for 45 s, 72 C for 1 min) and final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 10 cycles (94 C for 30 s, 65 C for 30 s, 72 C for 30 s), 20 cycles (94 C for 30 s, 58 C for 30 s, 72 C for 30 s),10 cycles (94 C for 30 s, 52 C for 30 s, 72 C for 30 s) and 10 cycles (94 C for 30 s, 45 C for 30 s, 72 C for 30 s) with final extension at 72 C (5 min) Initial denaturation- 94 C (5 min), 20 cycles (94 C for 30 s, 51.5 C for 30 s, 72 C for 30 s) and final extension at 72 C (5 min)
656
300
345
1225
357
149
methylated sites were identified. Further analysis by TFSEARCH: Searching Transcription Factor Binding Sites (ver 1.3) software revealed the cis-acting elements and corresponding putative transcription factors binding to the methylated sites.
Statistical Analysis Each experiment was repeated three times (n 5 3 3 3 mice/ group). The signals were measured in the form of integrated density value (IDV) by spot densitometry tool of AlphaEaseFC software (Alpha Innotech Corp.). The IDV for PS1 and PS2 were normalized against signal intensity of b-actin and represented as a histogram with the mean 6 standard error of mean (SEM) of three values calculated as Relative Density Value, RDV (IDV of PS1/b-actin and PS2/b-actin). Statistical analysis was performed using one-way analysis of variance, followed by post hoc test of Student–Newman–Keuls method through Jindel Scientific SigmaPlot for Windows (standard version 2.0). Values were reported as mean 6 SEM and p values
