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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Stem Cells. Author manuscript; available in PMC Apr 16, 2012.
Published in final edited form as:
PMCID: PMC3327128
NIHMSID: NIHMS366822

Lsh Participates in DNA Methylation and Silencing of Stem Cell Genes

Abstract

Transcriptional control of stem cell genes is a critical step in differentiation of embryonic stem cells and in reprogramming of somatic cells into stem cells. Here we report that Lsh, a regulator of repressive chromatin at retrotransposons also plays an important role in silencing of stem cell specific genes such as Oct4. We found that CpG methylation is gained during in vitro differentiation of several stem cell specific genes (in 11 out of 12 promoter regions) and thus appears to be a common epigenetic mark. Lsh depletion prevents complete silencing of stem cell gene expression and moreover promotes the maintenance of stem cell characteristics in culture. Lsh is required for establishment of DNA methylation patterns at stem cell genes during differentiation in part by regulating access of Dnmt3b to its genomic targets. Our results indicate that Lsh is involved in the control of stem cell genes and suggests that Lsh is an important epigenetic modulator during early stem cell differentiation.

Keywords: DNA methylation, chromatin, epigenetics, differentiation, stem cell genes

Introduction

During development cells traverse from totipotent, to pluripotent to terminally differentiated cells and gain a stable gene expression profile that is characteristic of the tissue specific cell type. Considerable evidence suggests that epigenetic states can influence chromatin structure and ultimately the accessibility of transcription factors and the transcriptional machinery to their genomic targets1. Thus epigenetic changes that are dependent on histone modifications and CpG methylation reflect progression of developmental stages and are thought to be characteristic for lineage committed or undifferentiated cells.

Embryonic stem cells and embryonal carcinoma cells can be used as a model to study early events during differentiation since they are capable of differentiation into many distinct cell types in vitro 2. Several reports have identified unique epigenetic features at genes that are poised for transcription in ES cells but committed to activation or repression upon differentiation 3, 4. In contrast, another subset of genes is subject to a different scheme of regulation being uniquely expressed in embryonal stem cells and later silenced in any terminally differentiated cells. Several of these ‘stem cell specific’ genes such as Oct4 and Nanog have been shown to be critical for stem cell function5, 6. Nuclei of terminally differentiated cells can be reprogrammed to a pluripotent state by applying distinct approaches such as nuclear transfer, cell fusion, exposure to nuclear extracts or the transduction with specific factors (among them Oct4)2, 7-11. An important feature for evaluating the efficiency of reprogramming has been the re-activation of stem cell specific genes such as Oct4. Revealing the molecular mechanisms that are involved in repression of stem cell genes is important for better understanding of early events of cellular differentiation and epigenetic reprogramming.

DNA methylation is known to have profound effects on embryogenesis, influencing X-inactivation, genomic imprinting and playing a role in repression of retroviral elements12-15. The transcriptional activity of the Oct4 gene is tightly correlated with its CpG methylation levels. The Oct4 promoter is unmethylated at the blastula stage but gains CpG methylation at day 6.5 of gestation and remains methylated in somatic tissues 16. Mouse and human embryonal stem cells show DNA methylation at the Oct4 promoter region upon differentiation in culture corresponding to transcriptional repression 17-19. Methylated Oct4 transgenes are silent in vivo 16 and the cloning efficiency after nuclear transfer is enhanced when using hypomethylated donor nuclei 20, 21.Moreover, the success of epigenetic reprogramming by transcription factors has been in part monitored by induced DNA hypomethylation of the Oct4 promoter 8. Thus cytosine methylation is thought to be a critical epigenetic modification that is involved in Oct4 expression during differentiation or reprogramming.

Several major DNA methyltransferases have been identified in mammals22, 23. Dnmt1 localizes at the replication fork and shows a preference for hemimethylated substrates over that of unmethylated substrate22, 23. Together with NP95 (UHFR1), a protein that also recognizes hemimethylated CpG sites and tethers Dnmt1 to chromatin, Dnmt1 preserves methylation patterns during cell division by specific methylation of hemimethylated CpG dinucleotides24, 25. Two other DNA methyltransferases, Dnmt3a and 3b, prefer unmethylated substrates in vitro and are thought to play a role predominantly in de novo methylation during embryonic development 26. Dnmt3L, related to Dnmt3a and Dnmt3b lacks catalytic activity and functions as a co-regulator for a subset of genomic imprinted sites and repeats13, 22.

Lsh, a member of the SNF2 family of chromatin remodeling protein27, 28, has been previously demonstrated to be an important factor in setting DNA methylation patterns during mouse development at repeat sequences29-32. Lsh is crucial for de novo methylation of reporter plasmids containing retroviral sequences, and Lsh can interact directly with Dnmt3a and Dnmt3b or indirectly with Dnmt1 via Dnmt3b32, 33. In addition, Lsh plays a role in transcriptional silencing of the developmentally regulated Hox genes, a finding that is also accompanied by alterations in DNA methylation levels34. Thus Lsh plays an important role in CpG methylation and loss of Lsh perturbs heterochromatin structure leading to multiple physiologic defects29, 35-37.

In the current study, we addressed the question whether DNA methylation mediated by Lsh plays a role in stem cell gene expression. We tested the idea of whether CpG methylation is a general epigenetic mark in silencing of stem cell genes during differentiation of pluripotent cells and if Lsh is involved in this process. Furthermore, we determined whether Lsh plays a role in maintaining the stem cell phenotype and if DNA methylation is critical for silencing of stem cell specific genes in somatic tissue.

Materials and Methods

Cell culture

Mouse ES cells (V6.4) were cultured on gelatin-coated dishes in Knockout DMEM (Invitrogen/GIBCO) with 15% Knockout Serum Replacement and 1000 U/ml ESGRO (Chemicon). P19 mouse embryonal carcinoma (EC) cells were grown in alpha-MEM with 10% fetal bovine serum. For differentiation, cells were placed with to 1 μM all-trans retinoic acid (RA) (Sigma) in Petri dishes to allow for aggregation (only in supplemental Fig2a, P19 cells were kept as monolayers). Cells were transfected with siRNA-Lsh oligonucleotides using Lipofectamine 2000 (Invitrogen) before RA treatment. For the clonal growth assay limited dilution was performed. P19 cells were split into 96 well plates to achieve an approximate concentration of 5-10 cells/ml. Clonal growth was assessed after 10 days in culture. For alkaline phosphatase staining cells were fixed with 4% paraformaldehyde for 1 minute and stained using the Alkaline Phosphatase Detection kit (Chemicon). For intracellular staining (Santa Cruz Biotech) cells were incubated with anti-Oct4 antibody (Santa-Cruz) or affinity purified rabbit anti-Lsh antibody raised against recombinant Lsh34 followed by secondary goat anti-mouse IgG2B-PE and donkey anti-rabbit IgG-FITC (Santa Cruz). Cells were analyzed by FACS for separate and dual PE/FITC staining. For immunohistochemistry staining the same antibodies were used followed by secondary staining with biotinylated goat anti-mouse IgG or biotinylated goat anti-rabbit IgG (Santa Cruz). After incubation with peroxidase-conjugated streptavidin, cells were exposed to 3,3%-diaminobenzidine for signal development.

Western blot analysis

Nuclear extracts were generated as previously described 34. Samples were separated on 4-12% Tris–glycine SDS–PAGE gels and blotted onto Immobilon P membrane (Millipore) and proteins detected using ECL detection reagents (Amersham). Antibodies used for Western analysis were affinity purified rabbit anti-Lsh antibody raised against recombinant protein34, anti-Dnmt3b antibody (Alexis), and PCNA antibody (Santa Cruz).

PCR analysis

Total RNA was prepared using Trizol reagent (Invitrogen) and genomic DNA was eliminated with TURBO DNA-free Kit (Ambion). About 1μg of total RNA was reverse transcribed using iScript reverse transcriptase (Bio-Rad). Omission of reverse transcriptase served as a negative control. cDNA was amplified using Platinum PCR SuperMix (Invitrogen). The -PCR was performed as follows: 5 min at 94°C, 30 cycles of 60 s at 94°C, 60 s at 57-60°C, and 60 s at 72°C, followed by one cycle 5 min at 72°C. For real-time PCR analysis, the MyiQTM Single-Color Real-Time PCR machine (Bio-Rad) and Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) were used: one cycle of 50°C for 2 min, one cycle of 95°C for 5 min, followed by 45 cycles of 95°C for 30 s, 57-60°C for 30 s, 72°C for 30 s, and finally followed by a melting curve analysis. A negative control without template was carried out for each PCR analysis. For quantification, standard titrations were performed for each template and primer set and linear regression equation and the calculation for DNA amounts were established using Prism 3.0 software (GraphPad Software, Inc.) and Microsoft Excel. ChIPs PCR conditions were as follows: : 94°C for 4 min; 94°C for 1 min; 55°C for 1 min; 72°C for 1 min (35 cycles) and 72°C for 7 min. All primers are listed in supplemental Table 2.

Chromatin immunoprecipitation

For chromatin immunoprecipitations (ChIPs) cells were crosslinked with 1% formaldehyde, lysed and sonicated on ice to generate DNA fragments with an average length of 200–800 bp 31. After pre-clearing, 1% of each sample was saved as input fraction. Immunoprecipitation was performed using specific antibodies against the indicated proteins or IgG as control. After reversal of crosslinking, DNA was prepared for PCR analysis.

MeDIP assay

Genomic DNA was sonicated to produce fragments ranging in size from 300 to 1,000 bp. Five μg of fragmented DNA was used for a standard MeDIP assay38 and precipitated with 10μl monoclonal antibody against 5-methylcytidine (Eurogentec, Belgium) followed by incubation with protein A agarose beads. DNA was recovered by phenol-chloroform extraction followed by ethanol precipitation. Primers (Supplemental Table 2) were chosen within −1000bp upstream of the transcriptional start sites and the amplicon sizes varied between 200 to 350 bp.

Bisulphite Sequencing

Genomic DNA was subjected to bisulphite treatment by using CpGenome DNA modification kit (Chemicon International, Temecula, CA). The PCR products were separated in agarose gels, purified,and subcloned as described34.

Southern blot analysis

Digested genomic DNA was separated by 1.5 % agarose gels. After transfer, Nytran plus membranes (Schleicher and Schuell) were hybridized overnight at 42o C in hybridization buffer (Amersham) with a 32P-labeled probe for satellite or Line1 sequences 30 or a probe covering 8.5 kb upstream of the TSS of Oct4 (kindly provided by Dr. Matsui). The hybridized membranes were washed and visualized by autoradiography.

Results

Silencing of stem cell specific genes during differentiation

To address the question whether Lsh mediated DNA methylation plays a role during silencing of ‘stem cell specific’ genes we first used P19 cells which are widely used as well established model for cellular differentiation. These embryonal carcinoma (EC) cells are pluripotent with respect to their capacity to form embryonic chimeras39. EC cells differentiate in culture into different lineages similar to ES cells, such that dimethyl sulphoxide treatment supports the differentiation into cardiac and skeletal muscle, aggregates with retinoic acid (RA) treatment lead to development of neuronal and glial tissue, and RA treatment in monolayers leads to epithelial tissues 40, 41. We used ECs as a model system for studying the molecular mechanism of transcriptional silencing during differentiation. Since Western analysis had shown Lsh expression in ECs (data not shown) we first determined Lsh expression at a single cell level using immunohistochemistry. Almost all of ECs stained positive for Lsh, similar to Oct4 staining that was used as a control (Suppl Fig1). To determine the precise frequency of Lsh and Oct4 expressing cells, we performed intracellular staining followed by FACS analysis (Fig1a). Approximately 86.3% of undifferentiated cells were double positive for Lsh (FITC) and Oct4 (PE) indicating that the majority of ECs express Lsh and the pluripotency marker Oct4. This is consistent with a recent report that performed a meta-analysis of 38 transcriptome studies and found Lsh (human Hells) to be one of a list of 40 genes that were specifically expressed in human ES cells and therefore served as signature gene for ES cells42. Next, ECs were treated with RA and then examined for expression of the pluripotent genes Oct4 and Nanog by RT-PCR analysis (Fig1b). Both genes were expressed in undifferentiated cells (day 0) and suppressed after 4 and 8 days of RA treatment, whereas Nestin and Mash1 mRNA levels increase during culture indicating a lineage commitment toward neuronal and glial cells. Likewise, RA treatment decreases transcription of Oct4 and Nanog genes in monolayer cultures (Suppl Fig2a). Using the Geo expression data base another 21 genes were selected for analysis that showed strict expression in pre-implantation embryo and silencing in somatic tissues. Those genes, termed ‘stem cell specific’ genes in this study, where also found to be reduced after RA treatment in EC cells (Fig1b and Suppl Table 1). Real-time PCR analysis confirmed that complete repression for Oct4, Nanog, Gdf3, Dppa3 and Tdgf1 was established by day 4 after RA treatment (Suppl Fig2b). Thus the ‘stem cell specific’ genes that were investigated in this study showed expression in undifferentiated EC cells and became silenced upon differentiation.

Fig 1
Acquisition of DNA methylation during EC differentiation

Induction of CpG methylation at promoter regions of stem cell specific genes

To determine whether transcriptional silencing of ‘stem cell specific’ genes is associated with CpG methylation, we first performed Southern analysis of the Oct4 promoter region using the methylation sensitive restriction enzyme HpaII. The Oct4 locus showed signs of hypomethylation in undifferentiated EC cells (Fig1c) but gained DNA methylation upon differentiation consistent with previous reports16, 18, 19. Minor satellite repeats (Fig1d) and Line1 elements (Suppl Fig3) served as negative controls since these regions maintain DNA methylation after RA treatment. Next, we used MeDIP analysis38 to examine DNA methylation for a dozen selected ‘stem cell specific’ genes (among them Oct4 and Nanog) that were silenced during EC differentiation. At first, we examined several genomic sites that are expected to show a stable DNA methylation pattern during culture in order to validate the immunoprecipitated material. Repetitive elements such as minor and major satellites or IAP show signs of DNA methylation in undifferentiated and in differentiated cells (Fig1e). Genomic imprinted sites such as the differentially methylated regions of Igf2R, two regions in the imprinting center upstream of H19 and KVDMR135, are expected to maintain DNA methylation during differentiation and showed signs of DNA methylation before and after RA treatment (Fig1e).. In contrast, ‘stem cell specific’ genes (altogether 11 out of 12) were enriched after immunoprecipitation indicating an acquisition of DNA methylation in the promoter regions during the differentiation process (among them Oct4, Nanog, Dppa2, Dppa3, Dppa4, Dppa5, Fbx15, Gdf3, Ndp52, Tdgf1 and Rex1). As negative controls served the promoter regions of seven housekeeping genes and Nestin and Mash1 that remained unmethylated. Thus the ‘stem cell specific’ genes investigated in this study (with the exception of Sox2) showed a gain of DNA methylation as a stable epigenetic mark acquired upon differentiation.

Lsh is involved in transcriptional silencing of stem cell specific genes

To evaluate the role of Lsh in silencing of ‘stem cell specific’ genes, we attempted to deplete Lsh protein by RNA interference. To study the short term effects of Lsh depletion, we used a mixture containing four different siLsh oligonucleotides. Treatment with 20 nM of siRNA-Lsh oligonucleotide could effectively decrease Lsh protein levels by day 4 (Fig2a,b). The treatment reduced Lsh protein levels in the majority of cells and did not affect Oct4 gene expression (Fig2c,d). Moreover, the Lsh decrease did not affect the cell cycle compared to control siRNA treated ECs (siRNA-Ctrl). In addition, retroviral expression (such as the intracisternal A particle) was not altered after pre-treatment with siRNA-Lsh consistent with the previous observations that Lsh was not required for maintenance of DNA methylation at repeat sequences32 (Suppl Fig4). Next we examined the effect of RA treatment and Lsh depletion on the expression of ten stem cell genes that had shown methylation gains during differentiation. Whereas in control cultures stem cell genes such as Oct4, Nanog, Gdf3, Dppa3 and Tdgf1 were fully silenced, siRNA-Lsh treatment resulted in incomplete repression (Fig2e and Suppl Fig5) and mRNA expression was maintained after differentiation, albeit at reduced levels as compared to day 0 control cells. This indicated that Lsh is required for complete silencing of those examined stem cell genes. The partial repression despite Lsh depletion may be due to low Lsh levels that remained after siRNA-Lsh treatment or alternative repressive pathways (as discussed below).

Fig 2
Lsh is involved in transcriptional silencing of stem cell specific genes

Lsh depletion maintains stemness of ECs

To investigate whether Lsh depletion inhibits the differentiation process and maintains stem cell properties we further characterized siRNA-Lsh treated EC cells. First we determined whether expression of stem cell genes in siRNA-Lsh treated EC cells was transient or continuous in culture. Extension to 20 days in culture after retinoic acid treatment did not change their ability to maintain expression of Oct4, Nanog, Dppa3, Tdgf1 and Gdf3 in Lsh depleted EC cells (Suppl Fig6).

After that we monitored soft agar growth in dependence of Lsh since loss of anchorage-independent growth is one of the hallmarks of differentiation 43. WhereassiRNA-Lsh treated cells were able to grow in soft agar after RA treatment similar to undifferentiated EC cells (Fig3a), control siRNA treated cells did not show anchorage independent growth. In addition, the capacity for clonal growth, assessed by limiting dilution analysis, was increased in siRNA-Lsh treated cells after RA treatment as compared with control siRNA treated cells (Fig3b). The Lsh depleted ECs that were selected from soft agar stained positive for alkaline phosphatase (AP), a characteristic mark of stem cells that is typically lost in differentiated cells (Fig3c). In addition, siRNA-Lsh treated cell lines derived from soft agar colonies after RA treatment continued expression of ‘stem cell specific’ genes (Fig3d).

Fig 3
Lsh depletion maintains ‘stemness’ of ECs

Taken together, Lsh depleted EC cells partially escaped the silencing program and maintained stem cell characteristics of undifferentiated EC cells pointing to an important role of Lsh during early cellular differentiation.

Reduced CpG methylation at stem cell specific genes in the absence of Lsh

Next, we sought to resolve the question of whether Lsh depletion leads to reduced de novo DNA methylation at stem cell genes which in turn may be responsible for maintenance of stem cell characteristics. Bisulphite sequencing of a region about 350 bp upstream of the transcriptional start site of Oct4 revealed an increase in DNA methylation from 6.6% to 51.6 % after RA induced differentiation (Fig4a) whereas Lsh depleted cells showed decreased CpG methylation levels (51.6% compared to 21.6%). This suggests that Lsh is required for normal DNA methylation levels at the Oct4 promoter region. Subsequently, we analyzed eleven more ‘stem cell specific’ genes for gain of CpG methylation using MeDIP (Fig4b). As controls, we used repeat sequences and imprinted sites that do not require Lsh for maintenance of DNA methylation32, 35 and that showed signs of DNA methylation in siRNA-Lsh and control siRNA treated cells. In contrast, the ‘stem cell specific’ genes showed reduced DNA methylation at their promoter regions upon Lsh depletion. Housekeeping genes, serving as negative controls, showed no signs of DNA methylation during RA treatment and Lsh depletion. Taken together, the data suggests that Lsh is required to achieve normal DNA methylation level during differentiation at the selected ‘stem cell specific’ genes.

Fig 4
Reduced CpG methylation at stem cell specific genes in the absence of Lsh

Lsh depletion interferes with stem cell gene silencing and differentiation in ES cells

To further confirm Lsh’s role in stem cell gene silencing we used genuine embryonic stem (ES) cells. Using immunohistochemistry we found Lsh and Oct4 expression in a majority of ES cells (Suppl Fig7). FACS analysis (Fig5a) exhibited about 96.7% cells that stained positive for Lsh (80.5% for Oct4). Next, ES cells were induced to differentiate after LIF removal and RA treatment. ES cells showed a substantial downregulation of Lsh and Dnmt3b protein levels upon differentiation (Fig5b) supporting the idea that both proteins have a primary function in stem cells and not in somatic cells. Depletion of Lsh by siRNA-Lsh treatment resulted in barely detectable Lsh protein levels up to day 12 of culture as assessed by Western analysis (Fig5c,d). Using Real-time PCR analysis, we examined the expression of ten ‘stem cell specific’ genes after RA treatment. Whereas control siRNA treated ES cells repressed stem cell genes within four to eight days after RA treatment, Lsh depleted ES cells only partially decreased Oct4, Nanog, Gdf3, Dppa3, Tdgf1, and other mRNAs (Fig5e). This indicated that Lsh is required for complete repression of the selected stem cell genes in ES cells similar to our observation in EC cells. Moreover, when RA treated ES cell cultures were stained for stem cell markers, siRNA-Lsh treated cells partially maintained alkaline phosphatase expression, whereas control siRNA treated ES cells failed to do so (Fig5f). Thus ES cells similar to ECs exhibited an important role for Lsh in stem cell gene silencing and maintenance of ES stem cell characteristics.

Fig 5
Lsh depletion interferes with stem cell silencing in embryonal stem cells

Effect of Lsh depletion in Lsh−/− embryonic tissue

Subsequently, we tested whether Lsh depletion can influence Oct4 silencing during embryonic development in vivo. Lsh−/− depletion in mice leads to perinatal death, reduced birth weight, renal abnormalities, hematopoietic deficiencies and germ cell defects as well as mitotic problems27, 29, 35-37, 44. We examined Oct4 expression around day 8.5 of gestation by comparing Lsh−/− embryos with Lsh+/+ littermates controls. Enhanced Oct4 and Nanog expression was detected in five different Lsh−/− embryos in comparison to litter mate wild type controls at day 8.5 of gestation (Fig6a) implying an in vivo role for Lsh in silencing of Oct4. However, Lsh−/− somatic tissue at day 18.5 such as brain, whole embryo or MEFs do not show sustained Oct4 expression (data not shown). These results leave the possibilities that either Lsh is dispensable for DNA methylation at the Oct4 gene during embryonic development or that alternative silencing mechanisms exist to compensate for loss of DNA methylation. To discriminate between the two options we performed bisulphite sequencing analysis of the Oct4 promoter region spanning about 1700 bp using genomic DNA derived from Lsh−/− embryonic tissue compared to Lsh+/+ embryonic tissue (Fig6b). The methylation state of 25 CpG sites was greatly reduced in the absence of Lsh (82.8 % compared to 10.4 %). This confirms that Lsh regulates in vivo DNA methylation at the Oct4 promoter. Furthermore, it suggests that additional silencing mechanisms independent of DNA methylation participate in the transcriptional control of Oct4 gene expression.

Fig 6
Effect of Lsh depletion in vivo in somatic tissue

Lsh controls association of Dnmt3b with stem cell genes in ES cells

In order to reveal more about the molecular mechanisms of how Lsh controls DNA methylation we performed chromatin immunoprecipitations (ChIPs) in ES cells. Lsh is specifically bound to the Oct4 and Nanog promoter regions, suggesting that Lsh is directly involved in the control of Oct4 and Nanog expression (Fig7a). Lsh as well as Dnmt3b were both already found associated with the stem cell genes in undifferentiated cells (Fig7a,b). Therefore RA treatment does not induce recruitment of Lsh/Dnmt3b to the Oct4 or Nanog promoter regions but must activate another mechanism that controls DNA methylation activity. ChIPs analysis showed a gradual decline of promoter bound Lsh and Dnmt3b over time during differentiation (Fig7c,d) which may be in part due to downregulation of both proteins after RA treatment (Fig5b). To address the question of whether Lsh is required for Dnmt3b recruitment, we performed Lsh depletion in ES cells by treatment with siRNA-Lsh (Fig7c,d). Lsh binding to Oct4 and Nanog was undetectable in si-Lsh treated cells in contrast to control siRNA treated cells, confirming efficient reduction of Lsh protein levels after siRNA-Lsh treatment. Moreover, Dnmt3b association with either stem cell gene was undetectable after Lsh depletion (Fig7d). Similar results could be demonstrated in EC (P19) cells (Suppl Fig8). Taken together, the data suggests that Lsh is necessary but not sufficient to control Dnmt3b activity at a specific genomic site.

Fig 7
Lsh controls association of Dnmt3b with stem cell specific genes in ES cells

Discussion

We report here that Lsh plays a role in silencing of stem cell genes and this is accompanied by acquisition of DNA methylation. We furthermore give evidence that DNA hypomethylation, though preceding reprogramming in somatic cells is not sufficient for reactivation of Oct4 gene transcription. Finally, we demonstrate that Lsh depletion supports maintenance of the stem cell phenotype, suggesting that Lsh is involved as co-factor in the control of cellular differentiation.

Role of DNA methylation in stem cell gene regulation

De novo methylation at the Oct4 gene and other stem cell genes is gained within 96 hours after RA treatment (Fig1c,e). In the same time span Lsh and Dnmt3b are dramatically down regulated (Fig5b) suggesting a critical role of these proteins before lineage commitment. The gain of CpG methylation is found at 11 out of 12 examined ‘stem cell specific’ genes. These findings are consistent with a recent report showing DNA methylation differences at 7 pluripotency associated genes when comparing ES cells with neuronal progenitor cells 45. Our findings support the notion that DNA methylation gain is not unique for Nanog or Oct4 gene regulation but is a frequent epigenetic mark associated with the investigated ‘stem cell specific’ genes. Since CpG methylation can be faithfully propagated over 50 cell generations it is considered a very stable epigenetic mark thus contributing to stable repression of stem cell genes in somatic cells. Our data are consistent with the model that the first step in embryonic differentiation comprises silencing of stem cell genes before subsequent steps allow for tissue specific gene expression.

Role of Lsh in transcriptional repression

Lsh is highly expressed in pluripotent cells, downregulated during embryogenesis and shows reduced expression in adult tissues. The effect of Lsh on stem cell gene expression is tightly correlated with DNA methylation. Methylated promoters are usually inactive, and associated with hypoacetylated chromatin12, 46. Proteins that selectively bind to methylated CpG sites (such as MecP2) or Dnmts can recruit histone deacetylases12 and thus may contribute to repression since histone acetylation plays an important role in the formation of a Pol II initiation complex 1. DNA methylation can also interact with other silencing pathways such as polycomb proteins 34, 45, 47 In addition, Lsh has been reported to show repressive function on reporter gene expression separate from CpG methylation33. This repressive activity was dependent on interaction of Lsh with Dnmts, but not on the catalytic activity of the methyltransferase. Though our results demonstrate an effect of Lsh on DNA methylation they do not exclude alternate possibilities of Lsh effects.

How does Lsh affect DNA methylation at stem cell genes?

Part of the Lsh effect maybe due to stabilization of Dnmt3b association with genomic loci since Lsh depletion reduces Dnmt3b binding (Fig7d). Likewise, we have found Dnmt3b association at HoxA genes and at the PU.1 locus is dependent on Lsh34, 44. This may in part be by direct interaction between Lsh and Dnmt3b or alternatively the presumed chromatin remodeling activity of Lsh might support Dnmt3b binding to DNA 32, 33. Since Lsh and Dnmt3b binding at the Oct4 gene was observed before differentiation, Lsh is necessary but not sufficient to control DNA methylation. Furthermore, neither Lsh nor Dnmt3b are rate limiting steps in DNA methylation. Dnmt3b activity might be regulated by post-translational modifications 48, 49 or an as yet unidentified co-factor for Dnmt3 mediated DNA methylation could be involved, similar to the recent discovery of NP95 supporting Dnmt1 activity24, 25. Another possibility is that simultaneous down regulation of the H3K4me3 mark at ‘stem cell specific’ genes may facilitate the intimate interaction of Dnmt3b with the nucleosome. Co-crystallization has recently shown that Dnmt3L, required for CpG methylation of retrotransposons and imprinted sites in germ cells, promotes interaction of Dnmt3a with its genomic target by binding to histone 3 tails that are unmethylated at the H3K4 residues 50, 51. Though a role for Dnmt3L has not been addressed in ES cells a similar mechanism in regulating Dnmt3b activity may play a role for stem cell gene silencing. Finally, a new study has reported a high turnover of CpG methylation marks at an estrogen responsive promoter region 52. Since our study addressed only the equilibrium of DNA methylation pattern we cannot exclude the possibility that Lsh additionally controls CpG methylation turnover rates.

Role for DNA hypomethylation in re-activation of Oct4

Oct4 promoter analysis has revealed proximal as well as distal enhancer elements 19, 53. Multiple sites close to the transcriptional start site have been shown to undergo complete or partial CpG methylation (the HpaII sites around −1000, −700,−260 bp and the HhaI site at −130) during ES cell differentiation 16,18. Using Xenopus oocytes it was demonstrated that DNA demethylation at proximal sites (−24,−166,−289) are critical for Oct4 promoter regulation, but not at distal sites (at −754 or −1148bp) 21. Reprogramming of somatic cells is correlated to hypomethylation at proximal promoter sites. Though Lsh is involved in DNA methylation and silencing of Oct4 in vitro and in vivo, other pathways can ultimately maintain Oct4 silencing. Lsh deficiency in embryos delays Oct4 mRNA repression during development (Fig6), but nevertheless in Lsh−/− newborn tissues Oct4 mRNA is undetectable. The presence of repressors such as GCNF could in part be accountable for silencing. GCNF binds directly to the Oct4 promoter region and recruits co-repressor complexes such as SMRT and N-CoR54. On the other hand a lack of activating factors may play a role, too. Oct4 has been shown to be regulated by a self-reinforcing loop, since Oct4 can bind to its own promoter region and enhance its own expression55. Moreover, Oct4 repression effects expression of other ‘stem cell specific’ genes such as Nanog, Sox2, Rex1, Tdgf1 or Dppa4, and Nanog and Sox2 in turn have been shown to associate to the Oct4 promoter and to regulate its expression as well56, 57. Thus a lack of Oct4 and other ‘stem cell specific’ genes may contribute to a failure to re-activate Oct4 in Lsh−/− tissues. In addition, compacted, inaccessible chromatin, the presence of polycomb proteins or Pol II stalling could possibly be responsible. Thus DNA methylation may act as an additional safeguard to prevent aberrant Oct4 expression. Since hypomethylated genomes show improved cloning efficiency after somatic cell nuclear transfer20, it is yet to be examined whether Lsh depleted cells can improve nuclear reprogramming efficiency. To further unravel these molecular mechanisms will be challenging in the future and beneficial to advance our understanding of cellular differentiation as well as to improve techniques for cellular reprogramming and their applications to regenerative medicine.

Summary

We show here that Lsh is involved in DNA methylation and silencing of stem cell genes. This suggests that Lsh plays an important role in setting gene expression pattern during cellular differentiation and has the potential to serve as molecular target for epigenetic programming and reprogramming.

Supplementary Material

Supp Table S1-S2 & Figure S1-S8

Acknowledgements

We thank Drs. Jonathan Keller, Steven Hou and Nancy Colburn for critical reviewing of the manuscript. We thank Dr Takaaki Matsui for the generous gift of the mouse Oct4 promoter region. We are grateful to Jenny Mercardo and Jennifer Waters for excellent animal technical assistance.

This project has been funded in whole or part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-C0-12400. NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals in accordance with the “Guide for Care and Use of Laboratory Animals” (National Research Council; 1996; National Academic Press; Washington, D.C.).

Contributor Information

Sichuan Xi, Conception and design, collection of data, data analysis and interpretation, manuscript writing, final approval of manuscript; Laboratory of Cancer Prevention, SAIC-FCRDC, National Cancer Institute, Frederick, MD 21701.

Theresa M. Geiman, Conception and design, collection of data, data analysis and interpretation, manuscript writing, final approval of manuscript; Laboratory of Cancer Prevention, SAIC-FCRDC, National Cancer Institute, Frederick, MD 21701.

Victorino Briones, Conception and design, collection of data, data analysis and interpretation, manuscript writing, final approval of manuscript; Laboratory of Cancer Prevention, SAIC-FCRDC, National Cancer Institute, Frederick, MD 21701.

Yong Guang Tao, Conception and design, collection of data, data analysis and interpretation, manuscript writing, final approval of manuscript; Laboratory of Cancer Prevention, SAIC-FCRDC, National Cancer Institute, Frederick, MD 21701.

Hong Xu, collection of data, final approval of manuscript; Laboratory of Cancer Prevention, SAIC-FCRDC, National Cancer Institute, Frederick, MD 21701.

Kathrin Muegge, Conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript; Laboratory of Cancer Prevention, SAIC-FCRDC, National Cancer Institute, Frederick, MD 21701.

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