Theodore P. Rasmussen

Chromosomal silencing and localization are mediated by different domains of Xist RNA

The gene Xist initiates the chromosomal silencing process of X inactivation in mammals. Its product, a noncoding RNA, is expressed from and specifically associates with the inactive X chromosome in female cells. Here we use an inducible Xist expression system in mouse embryonic stem cells that recapitulates long-range chromosomal silencing to elucidate which Xist RNA sequences are necessary for chromosomal association and silencing. We show that chromosomal association and spreading of Xist RNA can be functionally separated from silencing by specific mutations. Silencing requires a conserved repeat sequence located at the 5′ end of Xist. Deletion of this element results in Xist RNA that still associates with chromatin and spreads over the chromosome but does not effect transcriptional repression. Association of Xist RNA with chromatin is mediated by functionally redundant sequences that act cooperatively and are dispersed throughout the remainder of Xist but show little or no homology.

BRCA1 Supports XIST RNA Concentration on the Inactive X Chromosome

BRCA1, a breast and ovarian tumor suppressor, colocalizes with markers of the inactive X chromosome (Xi) on Xi in female somatic cells and associates with XIST RNA, as detected by chromatin immunoprecipitation. Breast and ovarian carcinoma cells lacking BRCA1 show evidence of defects in Xi chromatin structure. Reconstitution of BRCA1-deficient cells with wt BRCA1 led to the appearance of focal XIST RNA staining without altering XIST abundance. Inhibiting BRCA1 synthesis in a suitable reporter line led to increased expression of an otherwise silenced Xi-located GFP transgene. These observations suggest that loss of BRCA1 in female cells may lead to Xi perturbation and destabilization of its silenced state.

The Putative Nucleic Acid Helicase Sen1p Is Required for Formation and Stability of Termini and for Maximal Rates of Synthesis and Levels of Accumulation of Small Nucleolar RNAs in Saccharomyces cerevisiae

ABSTRACT Sen1p from Saccharomyces cerevisiae is a nucleic acid helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and some small nuclear RNAs. In this report, we show that cells carryingsen1-1 exhibit altered accumulation of several small nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using Northern blotting, RNase H cleavage, primer extension, and base compositional analysis, we detected three forms of the snoRNA snR13 in wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of ∼1,400-nt 3′-extended forms (snR13R) and a 108-nt 5′-truncated form (snR13T) that is missing 16 nt at the 5′ end. A subpopulation of snR13R contains the same 5′ truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F following temperature shift of sen1-1 cells, suggesting a possible precursor-product relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that Sen1p stabilizes the 5′ end and promotes maturation of the 3′ end. snR13F contains canonical C and D boxes common to many snoRNAs. The 5′ end of snR13T and the 3′ end of snR13F reside within C2U4 sequences that immediately flank the C and D boxes. A mutation in the 5′ C2U4 repeat causes underaccumulation of snR13F, whereas mutations in the 3′ C2U4 repeat cause the accumulation of two novel RNAs that migrate in the 500-nt range. At the restrictive temperature, double mutants carrying sen1-1 and mutations in the 3′ C2U4 repeat show reduced accumulation of the novel RNAs and increased accumulation of snR13R RNA, indicating that Sen1p and the 3′ C2U4 sequence act in a common pathway to facilitate 3′ end formation. Based on these findings, we propose that Sen1p and the C2U4 repeats that flank the C and D boxes promote maturation of the 3′ terminus and stability of the 5′ terminus and are required for maximal rates of synthesis and levels of accumulation of mature snR13F.

HDAC3 overexpression and colon cancer cell proliferation and differentiation.

An immunohistochemical analysis of human colorectal adenocarcinomas showed that cancer cells express widely varying levels of HDAC3. The SW480 colon cancer cell line was found to express high levels of HDAC3 compared to other colon cancer cell lines. p21 was poorly induced in SW480 cells relative to the lower HDAC3‐expressing HT‐29 cells. RNAi‐induced reduction of HDAC3 in SW480 cells increased their constitutive, butyrate‐, TSA‐, and TNF‐α‐induced expression of p21, but did not cause all the gene expression changes induced upon general histone deacetylase (HDAC) inhibition. SW480 cells with lower HDAC3 expression appeared to be poised for gene expression responses with increased histone H4‐K12 acetylation, but not K5, K8, or K16 acetylation. Even though p21 was readily activated in HT29 cells, HDAC3 siRNA nonetheless stimulated p21 expression in these cells to a greater degree than HDAC1 and HDAC2 siRNA. SW480 cells with lower HDAC3 levels displayed an enhanced cell cycle arrest and growth inhibition by butyrate, but without changes in apoptosis or sensitivity to chemotherapeutic agents. As reported for other colon cancer cell lines, butyrate induced the rapid downregulation of the secretory cell differentiation markers mucin 2 and intestinal trefoil factor in SW480 cells. Interestingly, selective HDAC3 inhibition was sufficient to downregulate these genes. Our data support a central role for HDAC3 in regulating the cell proliferation and differentiation of colon cancer cells and suggest a potential mechanism by which colon cancers may become resistant to luminal butyrate.

Global Epiproteomic Signatures Distinguish Embryonic Stem Cells from Differentiated Cells

Complex organisms contain a variety of distinct cell types but only a single genome. Therefore, cellular identity must be specified by the developmentally regulated expression of a subset of genes from an otherwise static genome. In mammals, genomic DNA is modified by cytosine methylation, resulting in a pattern that is distinctive for each cell type (the epigenome). Because nucleosomal histones are subject to a wide variety of post‐translational modifications (PTMs), we reasoned that an analogous “epiproteome” might exist that could also be correlated with cellular identity. Here, we show that the quantitative evaluation of nucleosome PTMs yields epiproteomic signatures that are useful for the investigation of stem cell differentiation, chromatin function, cellular identity, and epigenetic responses to pharmacologic agents. We have developed a novel enzyme‐linked immunosorbent assay‐based method for the quantitative evaluation of the steady‐state levels of PTMs and histone variants in preparations of native intact nucleosomes. We show that epiproteomic responses to the histone deacetylase inhibitor trichostatin A trigger changes in histone methylation as well as acetylation, and that the epiproteomic responses differ between mouse embryonic stem cells and mouse embryonic fibroblasts (MEFs). ESCs subjected to retinoic acid‐induced differentiation contain reconfigured nucleosomes that include increased content of the histone variant macroH2A and other changes. Furthermore, ESCs can be distinguished from embryonal carcinoma cells and MEFs based purely on their epiproteomic signatures. These results indicate that epiproteomic nucleosomal signatures are useful for the investigation of stem cell identity and differentiation, nuclear reprogramming, epigenetic regulation, chromatin dynamics, and assays for compounds with epigenetic activities.

The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein degradation in vivo.

The yeast Sen1 protein was discovered by virtue of its role in tRNA splicing in vitro. To help determine the role of Sen1 in vivo, we attempted to overexpress the protein in yeast cells. However, cells with a high-copy SEN1-bearing plasmid, although expressing elevated amounts of SEN1 mRNA, show little increase in the level of the encoded protein, indicating that a posttranscriptional mechanism limits SEN1 expression. This control depends on an amino-terminal element of Sen1. Using a genetic selection for mutants with increased expression of Sen1-derived fusion proteins, we identified mutations in a novel gene, designated SEN3. SEN3 is essential and encodes a 945-residue protein with sequence similarity to a subunit of an activator of the 20S proteasome from bovine erythrocytes, called PA700. Earlier work indicated that the 20S proteasome associates with a multisubunit regulatory factor, resulting in a 26S proteasome complex that degrades substrates of the ubiquitin system. Mutant sen3-1 cells have severe defects in the degradation of such substrates and accumulate ubiquitin-protein conjugates. Most importantly, we show biochemically that Sen3 is a subunit of the 26S proteasome. These data provide evidence for the involvement of the 26S proteasome in the degradation of ubiquitinated proteins in vivo and for a close relationship between PA700 and the regulatory complexes within the 26S proteasome, and they directly demonstrate that Sen3 is a component of the yeast 26S proteasome.

Epigenetic regulatory mechanisms during preimplantation development

Following fertilization, the newly formed zygote faces several critical decisions regarding cell fate and lineage commitment. First, the parental genomes must be reprogrammed and reset for the zygotic genome to assume responsibility for gene expression. Second, blastomeres must be committed to form either the inner cell mass or trophectoderm before implantation. A variety of epigenetic mechanisms underlies each of these steps, allowing for proper activation of transcriptional circuits which function to specify a cells identity and maintain or adjust that state as developmental and environmental conditions dictate. These epigenetic mechanisms encompass DNA methylation, post-translational histone modification, chromatin remodeling, and alterations in nuclear architecture. In recent years, stem cells derived from the inner cell mass have been used to examine the epigenetic pathways that regulate pluripotency, differentiation, and lineage commitment. From a technical standpoint, embryonic stem cells provide an easier system to work with compared to preimplantation embryos; however, it is currently unknown how closely the epigenetic mechanisms of cultured stem cells resemble their counterparts in the intact embryo. Furthermore, it remains unclear how similar the reprogramming pathways in artificially created systems, such as nuclear transfer-derived embryos and induced pluripotent stem cells, are to those in naturally created embryos. In this review, we summarize the current knowledge of epigenetic influences during preimplantation development and shed light on the extent to which these pathways are conserved in cultured pluripotent cells in vitro. In doing so, we demonstrate the critical role that epigenetic mechanisms play in the establishment of cell fate during the earliest stages of mammalian development.

HDAC3 impacts multiple oncogenic pathways in colon cancer cells with effects on Wnt and vitamin D signaling

Histone deacetylase 3 (HDAC3) is over-expressed in approximately half of all colon adenocarcinomas. We took an RNAi approach to determine how HDAC3 influenced chromatin modifications and the expression of growth regulatory genes in colon cancer cells. A survey of histone modifications revealed that HDAC3 knockdown in SW480 cells significantly increased histone H4-K12 acetylation, a modification present during chromatin assembly that has been implicated in imprinting. This modification was found to be most prominent in proliferating cells in the intestinal crypt and in APCMin tumors, but was less pronounced in the tumors that over-express HDAC3. Gene expression profiling of SW480 revealed that HDAC3 shRNA impacted the expression of genes in the Wnt and vitamin D signaling pathways. The impact of HDAC3 on Wnt signaling was complex, with both positive and negative effects observed. However, long-term knockdown of HDAC3 suppressed β-catenin translocation from the plasma membrane to the nucleus, and increased expression of Wnt inhibitors TLE1, TLE4 and SMO. HDAC3 knockdown also enhanced expression of the TLE1 and TLE4 repressors in HT-29 and HCT116 cells. HDAC3 shRNA enhanced expression of the vitamin D receptor in SW480 and HCT116 cells, and rendered SW480 cells sensitive to 1,25-dihydroxyvitamin D3. We propose that HDAC3 over-expression alters the epigenetic programming of colon cancer cells to impact intracellular Wnt signaling and their sensitivity to external growth regulation by vitamin D.

Genome-Wide Reprogramming in Hybrids of Somatic Cells and Embryonic Stem Cells

Recent experiments demonstrate that somatic nuclei can be reprogrammed to a pluripotent state when fused to ESCs. The resulting hybrids are pluripotent as judged by developmental assays, but detailed analyses of the underlying molecular‐genetic control of reprogrammed transcription in such hybrids are required to better understand fusion‐mediated reprogramming. We produced hybrids of mouse ESCs and fibroblasts that, although nearly tetraploid, exhibit characteristics of normal ESCs, including apparent immortality in culture, ESC‐like colony morphology, and pluripotency. Comprehensive analysis of the mouse embryonic fibroblast/ESC hybrid transcriptome revealed global patterns of gene expression reminiscent of ESCs. However, combined analysis of variance and hierarchical clustering analyses revealed at least seven distinct classes of differentially regulated genes in comparisons of hybrids, ESCs, and somatic cells. The largest class includes somatic genes that are silenced in hybrids and ESCs, but a smaller class includes genes that are expressed at nearly equivalent levels in hybrids and ESCs that contain many genes implicated in pluripotency and chromatin function. Reprogrammed genes are distributed throughout the genome. Reprogramming events include both transcriptional silencing and activation of genes residing on chromosomes of somatic origin. Somatic/ESC hybrid cell lines resemble their pre‐fusion ESC partners in terms of behavior in culture and pluripotency. However, they contain unique expression profiles that are similar but not identical to normal ESCs. ESC fusion‐mediated reprogramming provides a tractable system for the investigation of mechanisms of reprogramming.

ES Cell Cycle Progression and Differentiation Require the Action of the Histone Methyltransferase Dot1L

Mouse embryonic stem cells (ESCs) proliferate with rapid cell cycle kinetics but without loss of pluripotency. The histone methyltransferase Dot1L is responsible for methylation of histone H3 at lysine 79 (H3K79me). We investigated whether ESCs require Dot1L for proper stem cell behavior. ESCs deficient in Dot1L tolerate a nearly complete loss of H3K79 methylation without a substantial impact on proliferation or morphology. However, shortly after differentiation is induced, Dot1L‐deficient cells cease proliferating and arrest in G2/M‐phase of the cell cycle, with increased levels of aneuploidy. In addition, many aberrant mitotic spindles occur in Dot1L‐deficient cells. Surprisingly, these mitotic and cell cycle defects fail to trigger apoptosis, indicating that mouse ESCs lack stringent cell cycle checkpoint control during initial stages of differentiation. Transcriptome analysis indicates that Dot1L deficiency causes the misregulation of a select set of genes, including many with known roles in cell cycle control and cellular proliferation as well as markers of endoderm differentiation. The data indicate a requirement for Dot1L function for early stages of ESC differentiation where Dot1L is necessary for faithful execution of mitosis and proper transcription of many genes throughout the genome. STEM CELLS 2009;27:1538–1547