Epigenetics
the organism, a pattern that is not observed with protein coding DNA regions. For example, the per- centage of the genome coding for proteins is ~90% in prokaryotes, ~68% in yeast, ~25% in nema- todes, ~17% in insects, ~9% in pufferfish, ~2% in chickens and 1% in mammals15. These data strongly implicate ncRNA in the evolution of com- plex organisms. In addition, as genome-wide asso- ciation studies continue to identify regions of the genome associated with complex diseases, it is
Figure 5 Genome-wide profiling of
DNA-protein interactions by chromatin
immunoprecipitation (ChIP)
Cross-link Chromatin Sonication to Shear Chromatin
becoming increasingly apparent that many varia- tions that correlate with disease state occur in non- protein coding regions of the genome, some of which likely express regulatory RNAs16. Although the functional mechanisms remain poorly understood, there is growing evidence that many ncRNA transcripts are involved in epigenet- ic regulation, playing important roles in DNA methylation, chromatin modification, X-chromo- some inactivation, genomic imprinting and para- mutation. The fact that most ncRNAs, including small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-inter- acting RNAs (piRNAs), are expressed at signifi- cantly lower levels than mRNAs further suggests that these transcripts fulfill regulatory functions. The deregulation of several ncRNAs has been linked to many types of solid cancers, as well as lymphomas and leukaemias.
Immunoprecipitate • Anti-Histone • Anti-Transcription Factor
RNA-binding protein immunoprecipitation (RIP), an RNA analogue of the ChIP application described above, can be used to identify the associ- ation of any type of RNA molecule with specific nuclear or cytoplasmic binding proteins (Figure 6). These experiments involve immunoprecipitation of endogenously formed RNA-protein complexes, identification of mRNAs (and potentially non-cod- ing RNAs associated with them) and direct meas- urement using quantitative RT-PCR, microarray analysis (RIP-chip) and second-generation sequencing based platforms (RIP-Seq).
Stem cells and ageing
Reverse Cross-links Purify DNA
Detection • Quantitative PCR • Promoter Microarray
Epigenetic regulation is a critical mechanism for determining the fate of embryonic and adult stem cells. The pluripotent nature of embryonic stem cells is regulated by epigenetic mechanisms, relying on Polycomb group proteins to reversibly silence genes required for differentiation into specialised tissue17. Induced pluripotent stem cells (iPSCs), obtained by genetic reprogramming of somatic cells to an embryonic stem cell-like state, were gen- erally assumed to be functionally equivalent to their ESC counterparts. A number of studies have demonstrated that this is not the case, however, showing that iPSCs retain ‘epigenetic memory’ of the donor tissue from which they were derived. Adult stem cells, which reside in most mammalian tissues, maintain tissue homeostasis and help repair and regenerate tissue in response to damage. Adult stem cells have their own unique epigenetic signature, which changes upon cellular differentia- tion18. The normal process of ageing results in a decline of stem cell function.
32 Drug Discovery World Fall 2011
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