Mammalian Gene Silencing Research Articles

Modified siRNA Structure With a Single Nucleotide Bulge Overcomes Conventional siRNA-mediated Off-target Silencing

Off-target gene silencing is a major concern when using RNA interference. Imperfect pairing of the antisense strand with unintended mRNA targets is one of the main causes of small interfering RNA (siRNA) off-target silencing. To overcome this, we have developed "bulge-siRNA," a modified siRNA backbone structure with a single nucleotide (nt) bulge placed in the antisense strand. We found that siRNAs with a bulge at position 2 of the antisense strand were able to discriminate better between perfectly matched and mismatched targets, with no loss in silencing of the intended target. Genome-wide analysis also revealed that the bulge-siRNAs significantly reduced off-target silencing of transcripts with complementarity to the seed region of the siRNA antisense strand. When compared to 2'-methoxy ribosyl (2'-OMe) modified siRNAs previously developed to alleviate antisense off-target silencing; the bulge modification could better discriminate between on- versus off-targets. Our results suggest that the bulge-siRNA structure is a simple, yet superior alternative to chemical modifications for minimizing off-target silencing triggered by conventional siRNA structures.

PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing.

Mammalian gene silencing is established through methylation of histones and DNA, although the order in which these modifications occur remains contentious. Using the human beta-globin locus as a model, we demonstrate that symmetric methylation of histone H4 arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for subsequent DNA methylation. H4R3me2s serves as a direct binding target for the DNA methyltransferase DNMT3A, which interacts through the ADD domain containing the PHD motif. Loss of the H4R3me2s mark through short hairpin RNA-mediated knockdown of PRMT5 leads to reduced DNMT3A binding, loss of DNA methylation and gene activation. In primary erythroid progenitors from adult bone marrow, H4R3me2s marks the inactive methylated globin genes coincident with localization of PRMT5. Our findings define DNMT3A as both a reader and a writer of repressive epigenetic marks, thereby directly linking histone and DNA methylation in gene silencing.

Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect

Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2–8 from the 5′end of the guide strand; its complementary sequence; the 5′end of the guide strand and the 3′overhang of the passenger strand. However, most part of the 3′ two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3′end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA–RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA–RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.

Silencing of mammalian genes by tetracycline-inducible shRNA expression

Conditional gene silencing in mammalian cells, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying gene function, particularly if the gene is essential for cell survival or development. Here we describe a simple and rapid protocol for the generation of tetracycline (Tet)-inducible vectors that express shRNAs in a time- and dosage-dependent manner. Tet-operator (TetO) sequences responsive to occupation by the Tet-repressor (TetR) were inserted at alternative positions within the wild-type H1 promoter and cloned into a eukaryotic expression vector. Additional cloning sites downstream of the promoter enable the insertion of shRNA sequences. This Tet-inducible shRNA expression system can be used for both transient and stable RNA interference (RNAi) approaches to control gene function in a spatiotemporal fashion. The entire protocol (preparation of constructs, generation of stable cell lines and functional analysis) can be completed in 3 months.

The A, B, Gs of silencing: Figure 1.

Transcriptional silencing of mammalian genes is mediated by at least two modes of methylation: (1) cytosine methylation of DNA, and (2) methylation of the histones tails—both of which play an important role in transcriptional silencing from chromosomal DNA. Although there is some evidence that these pathways are interdependent, experimentally, they have largely been treated as two distinct processes. In this issue of Genes & Development, Smallwood et al. (2007) have taken a pioneering step toward defining the molecular mechanism by which these two types of methylations can cooperate to silence a euchromatic gene, and demonstrate that the DNA methyltransferase 1 (DNMT1) cytosine methyltransferase physically and functionally interacts with three Heterochromatin Protein 1 (HP1) family members ( , , and ) to implement gene silencing. DNA methylation is required for normal development in mammals. It is brought about by the de novo methyltransferases DNMT3a and DNMT3b and is propagated by the maintenance methyltransferase DNMT1 (for review, see Goll and Bestor 2005). DNA methylation is associated with silencing repetitive elements in the genome, X-chromosome inactivation in female mammals, and silencing of individual genes during development (for review, see Li and Bird 2007). Furthermore, DNA methylation is misregulated in cancer cells such that tumor suppressor genes are silenced to favor the growth of the tumor while repetitive regions of the genome are desilenced, which can contribute to genomic instability (for review, see Baylin and Jones 2007). However, the exact molecular mechanism of DNA methylation in transcriptional regulation and during the pathogenesis of cancer remains unclear. The core histone proteins are wrapped by 147 base pairs of DNA forming the intact nucleosome. The histone N termini, or tails, extend away from the core of the nucleosome and are available for interactions with the DNA, histone-modifying enzymes, and other proteins, which alter the nucleosome structure (Shilatifard 2006). There are multiple modification sites on each histone tail, and some amino acids in the histone tail can be modified in two or more ways. Histone methylation at Lys 9 of H3 is associated with silencing at both heterochromatin and euchromatic sequences in a variety of systems (for review, see Ebert et al. 2006; Shilatifard 2006). Recent evidence demonstrates that it is also associated with transcribed regions of euchromatic genes, indicating that this modification can have distinct meanings in different contexts (Vakoc et al. 2005; Eissenberg and Shilatifard 2006). Not only can the lysine residues within histones be modified by methylation, this modification can occur in three different states. Lysine residues can accept up to three methyl groups and therefore be mono-, di-, or trimethylated. For H3K9, the pattern of mono-, di-, and trimethylation is mediated by the catalytic properties of different enzymes. For example, the G9a methyltransferase mediates dimethylation of H3K9 and is associated with silencing of euchromatic genes (Tachibana et al. 2002; Peters et al. 2003). Not only can G9a regulate the pattern of H3K9 methylation, it also functions in some way in regulating DNA methylation at multiple sites (Xin et al. 2003; Feldman et al. 2006). Furthermore, it has been demonstrated in Arabidopsis that loss of DNA methyltransferase activity can result in alteration in H3K9 methylation patterns (Soppe et al. 2002). The attachment of methyl groups to histone proteins occurs predominantly at specific lysine or arginine residues, and each site requires the enzymatic activity of a specific class of enzymes. Furthermore, methylated lysine residues within histones can serve as a specific binding site for different proteins. For example, HP1 can recognize and bind to diand trimethylated H3K9 via its chromodomain (Bannister et al. 2001; Lachner et al. 2001). HP1 was first characterized in Drosophila as a nonhistone chromosomal protein that localized to heterochromatin and was required for gene silencing (Eissenberg and Elgin 2000). Both flies and mammals have more than one HP1-like protein, with each having distinct localizations (for review, see Hediger and Gasser 2006). Mammals have three HP1 isoforms: HP1 , HP1 , and HP1 . HP1 localizes primarily to pericentric heterochromatin, HP1 localizes primarily to promoters of silent euchromatic genes, and HP1 localizes primarily to coding regions of transcribed genes (Hediger and Gasser 2006). The basis for this localization is unknown, but in Drosophila it appears to depend, in part, on the hinge region and chromo shadow domain of each protein (Smothers and Henikoff 2001). Diversification of function of different HP1s could explain the recent finding of Blobel and colleagues (Vakoc et al. 2005) that H3K9 methylation is found in the coding regions of actively Corrresponding author. E-MAIL ASH@Stowers-Institute.org; FAX (816) 926-4112. Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1559407.

Mechanisms of small RNA mediated mammalian gene silencing

Mechanisms of small RNA mediated mammalian gene silencing

Recent Assembly of an Imprinted Domain from Non-Imprinted Components

Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how—and especially why—epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105–180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B′. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing.

Localized methylation in the key regulator gene endothelin- 1 is associated with cell type-specific transcriptional silencing

DNA methylation can contribute to the stable transcriptional silencing of mammalian genes. Often times, these genes are important developmental regulators, and their silencing in cell types where they are not supposed to be active is important for the phenotypic stability of the cells. To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2′-deoxycytidine, and changes in gene expression monitored by microarray analysis. Endothelin- 1 ( Et1 or Edn1), which encodes a cytokine with diverse regulatory functions, was among the genes upregulated following demethylation. We found that CpG dinucleotides within a short region in intron 1 of the gene have dramatically higher levels of methylation in Et1-non-expressing fibroblasts and chondrocytes as compared to the Et1-expressing mouse cell line, mIMCD-3. Strong evolutionary conservation of this region implies its role in the cis-regulation of Et1 transcription. To confirm that should Et1 in dermal fibroblasts become aberrantly activated, it could indeed lead to the dysregulation of many downstream genes, we exposed fibroblasts to exogenous ET1 peptide and assayed for transcriptional changes by microarray. ET1 treatment resulted in significant expression changes – primarily downregulation – of a significant number of genes. In particular, Tgfβ2 and Tgfβ3 were among the downregulated genes, which in turn alter the expression status of their many target genes. These data suggest that the stable silencing of Et1 through its associated DNA methylation in intron 1 is critical for the developmental stability of dermal fibroblasts, and perhaps other cell types as well.

Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation

Double-stranded RNA molecules targeted to gene promoter regions can induce transcriptional gene silencing in a DNA cytosine methylation-dependent manner in plants (RNA-dependent DNA methylation). Whether a similar mechanism exists in mammalian systems is a vital and controversial issue. DNA methylation is an important component in mammalian gene silencing for normal processes such as gene imprinting and X-chromosome inactivation, and aberrant CpG island hypermethylation at tumor-suppressor promoters is associated with transcriptional silencing and loss of gene function in cancer. Hence, we investigated whether RNA-dependent DNA methylation might operate in human cancers to mediate epigenetic silencing using the endogenous gene CDH1 as a potential target. The loss of this cell-cell adhesion factor facilitates the metastatic process, and its promoter is frequently hypermethylated in breast and other cancers. We found that, although small double-stranded RNAs targeted exclusively to the CDH1 promoter could effectively induce transcriptional repression with chromatin changes characteristic of inactive promoters, this was entirely independent of DNA methylation. Moreover, we could accomplish such silencing in a cancer cell line genetically modified to lack virtually any capacity to methylate DNA.

Inhibition of Xenogeneic Response in Porcine Endothelium Using RNA Interference

Rejection mediated by antibody recognition of the alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) is a major barrier in porcine-to-human xenotransplantation. Because the synthesis of alpha-Gal is dependent on alpha1,3 galactosyltransferase (alpha1,3GT), methods of blocking this enzyme are needed. RNA interference induced by small interfering RNA (siRNA) is a powerful technique for allowing the silencing of mammalian genes with great specificity and potency. In this study, we use siRNA for silencing of alpha1,3GT with the purpose of reducing expression of the alpha-Gal epitope and subsequently decreasing immunogenicity of porcine endothelial cells. alpha1,3GT-specific and control siRNAs were transfected into the porcine aortic endothelial cell line, PED. alpha-Gal expression was assessed by Western blotting, flow cytometry, and immunofluorescence. Protection from human-complement and natural killer (NK)-cell-mediated cytotoxicity was evaluated by Cr-release assays after incubation of PED with normal human serum (NHS) and NK92 cell, respectively. RNA interference was successfully achieved in PED as witnessed by the specific knock-down of alpha1,3GT mRNA levels. Flow cytometric analysis using the Griffonia simplicifolia isolectin B4 lectin confirmed the suppression of alpha1,3GT activity as evidenced by decreased alpha-Gal. Functional relevance of the knock-down phenotype was illustrated by the finding that silenced PED were protected from cytotoxicity of NHS. Protection from NK-mediated cytotoxicity was not observed. Our data are the first to demonstrate that RNA interference is a potent tool to down modulate alpha-Gal expression and to protect endothelial cells from complement-mediated cytotoxicity. Gene silencing by siRNA may represent a new approach for overcoming hyperacute and acute vascular rejection.

Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing.

Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.

The mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis.

The yeast Sir2p protein has an essential role in maintaining telomeric and mating type genes in their transcriptionally inactive state. Mammalian cells have a very large proportion of their genome inactive and also contain seven genes that have regions of homology with the yeast sir2 gene. One of these mammalian genes, sir2alpha, is the presumptive mammalian homologue of the yeast sir2 gene. We set out to determine if sir2alpha plays a role in mammalian gene silencing by creating a strain of mice carrying a null allele of sir2alpha. Animals carrying two null alleles of sir2alpha were smaller than normal at birth, and most died during the early postnatal period. In an outbred background, the sir2alpha null animals often survived to adulthood, but both sexes were sterile. We found no evidence for failure of gene silencing in sir2alpha null animals, suggesting that either SIR2alpha has a different role in mammals than it does in Saccharomyces cerevisiae or that its role in gene silencing in confined to a small subset of mammalian genes. The phenotype of the sir2alpha null animals suggests that the SIR2alpha protein is essential for normal embryogenesis and for normal reproduction in both sexes.

Cancer epigenetics takes center stage

Epigenetics is defined as modifications of the genome, heritable during cell division, that do not involve a change in the DNA sequence. Examples include methylation induced premeiotically in Ascobolus, repeat-induced gene silencing and paramutation in plants, mating type silencing and telomere silencing in yeast, position effect variegation in Drosophila, and genomic imprinting in mammals and flowering plants. There are several features that distinguish epigenetics from conventional genetic mechanisms: reversibility; position effects, i.e., the ability to act over unexpected distances larger than a single gene; apparent mutations at unexpectedly high frequency; and the involvement of gene domains. One common thread to most epigenetic phenomena is DNA methylation, a covalent modification of the C5 position of cytosine. This methylation pattern is stably maintained at CpG dinucleotides by a family of DNA methyltransferases that recognize hemimethylated CpG dinucleotides after DNA replication. DNA methyltransferases belong to multiprotein complexes, and they contain sequence motifs for multiple such interactions, including interactions with chromatin components, some of which have been directly identified (3). Another common thread to epigenetics is a link to transcriptional regulation, generally involving gene silencing. DNA methylation in particular is generally but not exclusively linked to transcriptional silencing, including methylation induced premeiotically, paramutation, and mammalian gene silencing.

Enzymic removal of 5-methylcytosine from DNA by a human DNA-glycosylase.

DNA 5-methylcytosine is a major factor in the silencing of mammalian genes; it is involved in gene expression, differentiation, embryogenesis and neoplastic transformation. A decrease in DNA 5-methylcytosine content is associated with activation of specific genes. There is much evidence indicating this to be an enzymic process, with replacement of 5-methylcytosine by cytosine. We demonstrate here enzymic release of 5-methylcytosines from DNA by a human 5-methylcytosine-DNA glycosylase activity, which affords a possible mechanism for such replacement. This activity generates promutagenic apyrimidinic sites, which can be related to the high frequency of mutations found at DNA 5-methylcytosine loci. The recovery of most released pyrimidines as thymines indicates subsequent deamination of free 5-methylcytosines by a 5-methylcytosine deaminase activity. This prevents possible recycling of 5-methylcytosine into replicative DNA synthesis via a possible 5-methyl-dCTP intermediate synthesized through the pyrimidine salvage pathway. Taken together, these findings indicate mechanisms for removal of 5-methylcytosines from DNA, hypermutability of DNA 5-methylcytosine sites, and exclusion of 5-methylcytosines from DNA during replication.