Emily Bernstein

Role for a bidentate ribonuclease in the initiation step of RNA interference

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells

In a diverse group of organisms that includes Caenorhabditis elegans , Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that ‘loss-of-function’ phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, ∼25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.

Dicer is essential for mouse development

To address the biological function of RNA interference (RNAi)-related pathways in mammals, we disrupted the gene Dicer1 in mice. Loss of Dicer1 lead to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with our inability to generate viable Dicer1-null embryonic stem (ES) cells, this suggests a role for Dicer, and, by implication, the RNAi machinery, in maintaining the stem cell population during early mouse development.

Epigenetics : A landscape takes shape

Epigenetics has recently evolved from a collection of diverse phenomena to a defined and far-reaching field of study. In this Essay, we examine the epistemology of epigenetics, provide a brief overview of underlying molecular mechanisms, and suggest future challenges for the field.

DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA.

Mammals use DNA methylation for the heritable silencing of retrotransposons and imprinted genes and for the inactivation of the X chromosome in females. The establishment of patterns of DNA methylation during gametogenesis depends in part on DNMT3L, an enzymatically inactive regulatory factor that is related in sequence to the DNA methyltransferases DNMT3A and DNMT3B. The main proteins that interact in vivo with the product of an epitope-tagged allele of the endogenous Dnmt3L gene were identified by mass spectrometry as DNMT3A2, DNMT3B and the four core histones. Peptide interaction assays showed that DNMT3L specifically interacts with the extreme amino terminus of histone H3; this interaction was strongly inhibited by methylation at lysine 4 of histone H3 but was insensitive to modifications at other positions. Crystallographic studies of human DNMT3L showed that the protein has a carboxy-terminal methyltransferase-like domain and an N-terminal cysteine-rich domain. Cocrystallization of DNMT3L with the tail of histone H3 revealed that the tail bound to the cysteine-rich domain of DNMT3L, and substitution of key residues in the binding site eliminated the H3 tail–DNMT3L interaction. These data indicate that DNMT3L recognizes histone H3 tails that are unmethylated at lysine 4 and induces de novo DNA methylation by recruitment or activation of DNMT3A2.

Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin.

ABSTRACT The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. Here we have investigated the five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, we find that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. We propose that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites.

The histone variant macroH2A suppresses melanoma progression through regulation of CDK8

Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm.

MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation

Summary The Polycomb Group (PcG) of chromatin modifiers regulates pluripotency and differentiation. Mammalian genomes encode multiple homologs of the Polycomb repressive complex 1 (PRC1) components, including five orthologs of the Drosophila Polycomb protein (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8). We have identified Cbx7 as the primary Polycomb ortholog of PRC1 complexes in embryonic stem cells (ESCs). The expression of Cbx7 is downregulated during ESC differentiation, preceding the upregulation of Cbx2, Cbx4, and Cbx8, which are directly repressed by Cbx7. Ectopic expression of Cbx7 inhibits differentiation and X chromosome inactivation and enhances ESC self-renewal. Conversely, Cbx7 knockdown induces differentiation and derepresses lineage-specific markers. In a functional screen, we identified the miR-125 and miR-181 families as regulators of Cbx7 that are induced during ESC differentiation. Ectopic expression of these miRNAs accelerates ESC differentiation via regulation of Cbx7. These observations establish a critical role for Cbx7 and its regulatory miRNAs in determining pluripotency.

MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency

The chromatin template imposes an epigenetic barrier during the process of somatic cell reprogramming. Here, using fibroblasts derived from macroH2A double knockout mice we show that these histone variants act cooperatively as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, we further demonstrate that macroH2A2 is the predominant barrier to reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together with H3K27me3, co-occupy pluripotency genes in wild type fibroblasts. In particular, we find macroH2A isoforms to be highly enriched at target genes of the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming. Finally, while macroH2A double knockout induced pluripotent cells are able to differentiate properly in vitro and in vivo, such differentiated cells retain the ability to return to a stem-like state. Therefore, we propose that macroH2A isoforms provide a redundant silencing layer or terminal differentiation ‘lock’ at critical pluripotency genes that presents as an epigenetic barrier when differentiated cells are challenged to reprogram.

Smarcc1/Baf155 Couples Self‐Renewal Gene Repression with Changes in Chromatin Structure in Mouse Embryonic Stem Cells

Little is known about the molecular mechanism(s) governing differentiation decisions in embryonic stem cells (ESCs). To identify factors critical for ESC lineage formation, we carried out a functional genetic screen for factors affecting Nanog promoter activity during mESC differentiation. We report that members of the PBAF chromatin remodeling complex, including Smarca4/Brg1, Smarcb1/Baf47, Smarcc1/Baf155, and Smarce1/Baf57, are required for the repression of Nanog and other self‐renewal gene expression upon mouse ESC (mESC) differentiation. Knockdown of Smarcc1 or Smarce1 suppressed loss of Nanog expression in multiple forms of differentiation. This effect occurred in the absence of self‐renewal factors normally required for Nanog expression (e.g., Oct4), possibly indicating that changes in chromatin structure, rather than loss of self‐renewal gene transcription per se, trigger differentiation. Consistent with this notion, mechanistic studies demonstrated that expression of Smarcc1 is necessary for heterochromatin formation and chromatin compaction during differentiation. Collectively, our data reveal that Smarcc1 plays important roles in facilitating mESCs differentiation by coupling gene repression with global and local changes in chromatin structure. STEM CELLS 2009;27:2979–2991