Silenced genes

Hormone response elements (for steroids, T3, retinoic acid, peptides, etc) act as—or in conjunction with— enhancers or silencers (Chapter 43). Other processes that enhance or silence gene expression—such as the response to heat shock, heavy metals (Cd and Zn +), and some toxic chemicals (eg, dioxin)—are mediated through specific regulatory elements. Tissue-specific expression of genes (eg, the albumin gene in liver, the hemoglobin gene in reticulocytes) is also mediated by specific DNA sequences. 

Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, Jenuwein T, Livingston DM, Rajewsky K (2005) Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev 19 489—501... 

Local chromatin-modifying activities Acetylation of histones increases gene expression (many genes) Methylation of DNA silences genes in genetic imprinting (Prader-Willi and Angelman syndromes)... 

The methylation of DNA at CpG islands has also turned out to be an important regulator for cell development, the differentiated proteome and the regulation of cell survival [237,238]. Indeed the implications of this chemical modification have been linked to DNA accessibility, chromatin fluidity and cell transformation [239,240]. DNA methylation is required for genomic stability and believed to act as an inert epigenetic marker in germinal cells and preimplantation embryos [238]. Presumably, DNA methylation is required for the heritable transmission of chromatin structure, which prevents the expression of terminally silenced genes in differentiated tissues, and provides a host-defense mechanism against parasitic transposable elements [241]. 

The connection between DNA methylation and gene expression was first noticed when undifferentiated cells in culture were treated with the methylation inhibitor 5-azacytidine inhibition of methylation led to the appearance of stably inherited differentiated cell types and activation of previously silenced genes [107,108]. Ectopic DNA methylation inhibits gene expression [109], whereas drug-inhibition of methylation induces the expression of certain genes [110]. Transfection experiments... 

The covalent trapping of the enzyme leads to a depletion of the cellular pool of DNMTs and subsequent DNA hypomethylation. This in turn results in activation with respect to the reactivation of silenced genes. Additionally, the covalently trapped DNMT may inhibit RNA and DNA polymerases, which leads to an inhibition of protein biosynthesis and DNA strand breaks. This may lead to apoptosis and hence cytotoxicity. Thus, it is not easy to dissect the reasons for the clinical efficacy of these inhibitors in terms of real epigenetic and plain cytotoxic effects [81]. 

These inhibitors act directly on the enzyme without the necessity for incorporation into the DNA. In theory, this should shift the dual activity of reactivation of silenced genes and cytotoxicity towards the epigenetic effects which may result in reduced side-effects. But the proof for that remains yet to be established. 

Fang, M.Z., Wang, Y, Ai, N Hou, Z Sun, Y, Lu, H., Welsh, W. and Yang, C.S. (2003) Tea polyphenol (—)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. Cancer Research, 63, 7563-7570. 

Gottlieb, S. and Esposito, R.E. (1989) A new role for a yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA. Cell, 56, 771-776. 

A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33 401-406... 

Lee Y-W, Klein CB, Kargacin B, et al. 1995. Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation A new model for epigenetic carcinogens. Molec Cell Biol 15 2547-2557. 

Loss of tumor suppressor gene function can occur through mutation but also promoter methylation, which silences genes. 

I., et al. (2000) Reactivation of heritably silenced gene expression in mice. Mamm Genome 11, 347-355. 

Cheng JC, Matsen CB, Gonzales FA, Ye W, Greer S, Marquez VE, Jones PA, Selker EU. Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst 2003 95 399-409. 

Although methods for silencing gene expression in worms and invertebrates were well established, researchers had few options for targeting specific genes within mammalian genomes. Prior to the advent of RNAi, an effective but costly method was to generate knockout mice that were used to produce knockout mouse embryonic fibroblasts (MEFs). 

A DNA modification which changes gene transcription is methylation of nucleotide bases. Heterochromatin contains heavily methylated DNA which cannot be transcribed. Genes in euchromatin are less methylated and are transcribable. Methylation at C5 of cytosine in CpG sequences by a methyltransferase, with 5-adenosyl methionine (SAM) as methyl donor, silences genes in heterochromatin. Methylation may affect the higher-order structure of DNA and may impede access of the transcriptional machinery to DNA. Members of the histone Hj family have been associated with transcriptional repression, because these histones bind preferentially to methylated CpG sequences (Fig. 9.10).4i... 

A protein s role in a signaling pathway can be assessed by introducing mutations that disrupt interactions, localization, or activity or by silencing gene expression. The dynamics of engineered proteins can be moiutored via selective placement of fluorescent tags within the protein sequence. 

Dykxhoom DM, Novina CD, Sharp PA. Killing the messenger short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 2003 4 457-467. 

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