We recently reported that transforming growth factor (TGF)-␤ induced the neural crest stem cell line Monc-1 to differentiate into a spindle-like contractile smooth muscle cell (SMC) phenotype and that Smad signaling played an important role in this phenomenon. In addition to Smad signaling, other pathways such as mitogenactivated protein kinase (MAPK), phosphoinositol-3 kinase, and RhoA have also been shown to mediate TGF-␤ actions. The objectives of this study were to examine whether these signaling pathways contribute to TGF-␤-induced SMC development and to test whether Smad signaling cross-talks with other pathway(s) during SMC differentiation induced by TGF-␤. We demonstrate here that RhoA signaling is critical to TGF-␤-induced SMC differentiation. RhoA kinase (ROCK) inhibitor Y27632 significantly blocks the expression of multiple SMC markers such as smooth muscle ␣-actin, SM22␣, and calponin in TGF-␤-treated Monc-1 cells. In addition, Y27632 reversed the cell morphology and abolished the contractility of TGF-␤-treated cells. RhoA signaling was activated as early as 5 min following TGF-␤ addition. Dominant negative RhoA blocked nuclear translocation of Smad2 and Smad3 because of the inhibition of phosphorylation of both Smads and inhibited Smaddependent SBE promoter activity, whereas constitutively active RhoA significantly enhanced SBE promoter activity. Consistent with these results, C3 exotoxin, an inhibitor of RhoA activation, significantly attenuated SBE promoter activity and inhibited Smad nuclear translocation. Taken together, these data point to a new role for RhoA as a modulator of Smad activation while regulating TGF-␤-induced SMC differentiation.

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells -/-(helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation.

Lsh, a chromatin remodeling protein of the SNF2 family, is critical for normal heterochromatin structure. In particular, DNA methylation at repeat elements, a hallmark of heterochromatin, is greatly reduced in Lsh−/− (KO) cells. Here, we examined the presumed nucleosome remodeling activity of Lsh on chromatin in the context of DNA methylation. We found that dynamic CG methylation was dependent on Lsh in embryonic stem cells. Moreover, we demonstrate that ATP function is critical for de novo methylation at repeat sequences. The ATP binding site of Lsh is in part required to promote stable association of the DNA methyltransferase 3b with the repeat locus. By performing nucleosome occupancy assays, we found distinct nucleosome occupancy in KO ES cells compared to WT ES cells after differentiation. Nucleosome density was restored to wild-type level by re-expressing wild-type Lsh but not the ATP mutant in KO ES cells. Our results suggest that ATP-dependent nucleosome remodeling is the primary molecular function of Lsh, which may promote de novo methylation in differentiating ES cells.

DNA methylation is critical for normal development and plays important roles in genome organization and transcriptional regulation. Although DNA methyltransferases have been identified, the factors that establish and contribute to genome-wide methylation patterns remain elusive. Here, we report a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences. We provide evidence that Lsh also controls genome-wide cytosine methylation at nonrepeat sequences and relate those changes to alterations in H4K4me3 modification and gene expression. Deletion of Lsh alters the allocation of cytosine methylation in chromosomal regions of 50 kb to 2 Mb and, in addition, leads to changes in the methylation profile at the 5′ end of genes. Furthermore, we demonstrate that loss of Lsh promotes—as well as prevents—cytosine methylation. Our data indicate that Lsh is an epigenetic modulator that is critical for normal distribution of cytosine methylation throughout the murine genome.

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.

DNA cytosine methylation is an important epigenetic mechanism that is involved in transcriptional silencing of developmental genes. Several molecular pathways have been described that interfere with Pol II initiation, but at individual genes the molecular mechanism of repression remains uncertain. Here, we study the molecular mechanism of transcriptional regulation at Hox genes in dependence of the epigenetic regulator Lsh that controls CpG methylation at selected Hox genes. Wild type cells show a nucleosomal deprived region around the transcriptional start site at methylated Hox genes and mediate gene silencing via Pol II stalling. Hypomethylation in Lsh−/− cells is associated with efficient transcriptional elongation and splicing, in part mediated by the chromodomain protein Chd1. Dynamic modulation of DNA methylation in Lsh−/− and wild type cells demonstrates that catalytically active DNA methyltransferase activity is required for Pol II stalling. Taken together, the data suggests that DNA methylation can be compatible with Pol II binding at selected genes and Pol II stalling can act as alternate mechanism to explain transcriptional silencing associated with DNA methylation.

DNA methylation patterns are established in early embryogenesis and are critical for cellular differentiation. To investigate the role of CG methylation in potential enhancer formation, we assessed H3K4me1 modification in murine embryonic fibroblasts (MEFs) derived from the DNA methylation mutant Lsh −/− mice. We report here de novo formation of putative enhancer elements at CG hypomethylated sites that can be dynamically altered. We found a subset of differentially enriched H3K4me1 regions clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. C ytosine methylation is an epigenetic mechanism conserved among diverse eukaryotes whereby a family of conserved DNA methyltransferases (DNMTs) is responsible for cytosine methylation at cytosine-phosphate-guanosine (CpG) sites. Deletion of DNMTs in mammals alters DNA methylation patterns and results in early embryonic lethality, indicating a critical role of DNMTs in development (1). Genome-wide methylation changes have been reported after deletion of Uhfr1, a hemimethylation binding protein supporting maintenance of DNA methylation, and after deletion of Lsh (2-4). Lsh and the Arabidopsis thaliana homolog DDM1 belong to the SNF2 family of chromatin remodeling proteins, and DDM1 can cause nucleosomal sliding in vitro (5).Nucleosome templates provide limited access to DNMTs, and the addition of SNF2 proteins can improve de novo methylation in vitro (6). DDM1 mutants show reduced cytosine methylation at genomic regions rich in the linker histone H1, suggesting that the chromatin remodeling activity of DDM1 promotes access to densely packaged chromatin (7).Deletion of Lsh in mice is perinatally lethal, indicating that Lsh is not a redundant SNF2 family member and plays a critical role in murine development (8, 9). Lsh promotes de novo methylation of exogenous retroviral sequences and endogenous genomic loci, including stem cell-associated genes (10, 11). Lsh can interact with DNMT3b in vitro and supports the association of DNMT3b to some genomic targets. In addition, deletion of Lsh alters genomewide cytosine methylation pattern, suggesting a regulatory role for Lsh in the establishment of DNA methylation (3,4,12,13).DNA methylation patterns are highly dynamic and undergo large transitions during germ cell development, early embryogenesis, and reprogramming into induced pluripotent stem (iPS) cells. Dynamic alterations in DNA methylation and H3K4me1 modifications occur at putative enhancers upon cellular differentiation (14, 15). Despite their close association, the causal relationship of CG methylation changes and the establishment of cis-regulatory regions are only partially understood. Engagement of CCCTCbinding factor or repressor element-1 silencing transcription factor to specific loci causes local CG hypomethylation, indicating that DNA-binding factors, such as transcription factors, can shape the methylome (16). Whether CG hypomethylation in turn can influence the formation of enhancer sites and affect cellul...

Inactivation of tumor suppressor genes plays an important role in tumorigenesis, and epigenetic modifications such as DNA methylation are frequently associated with transcriptional repression. Here, we show that gene silencing at selected genes with signs of DNA hypermethylation in breast cancer cells involves Pol II stalling. We studied several repressed genes with DNA hypermethylation within a region 1-kb upstream of the transcriptional start site that were upregulated after treatment with DNA demethylating agents, such as Azacytidine and several natural products. All those selected genes had stalled Pol II at their transcriptional start site and showed enhanced ser2 phosphorylated Pol II and elevated transcripts after drug treatment indicating successful elongation. In addition, a decrease of the epigenetic regulator LSH in a breast cancer cell line by siRNA treatment reduced DNA methylation and overcame Pol II stalling, whereas overexpression of LSH in a normal breast epithelial cell line increased DNA methylation and resulted in repression. Decrease of LSH was associated with reduced DNMT3b binding to promoter sequences, and depletion of DNMT3b by siRNA could release Pol II suggesting that DNMT3b is functionally involved. The release of paused Pol II was accompanied by a dynamic switch from repressive to active chromatin marks. Thus release of Pol II stalling can act as a mechanism for gene reactivation at specific target genes after DNA demethylating treatment in cancer cells.