Towards understanding the epigenetics of transcription by chromatin structure and the nuclear matrix

Abstract

The eukaryotic nucleus houses a significant amount of information that is carefully ordered to ensure that genes can be transcribed as needed throughout development and differentiation. The genome is partitioned into regions containing functional transcription units, providing the means for the cell to selectively activate some, while keeping other regions of the genome silent. Over the last quarter of a century the structure of chromatin and how it is influenced by epigenetics has come into the forefront of modern biology. However, it has thus far failed to identify the mechanism by which individual genes or domains are selected for expression. Through covalent and structural modification of the DNA and chromatin proteins, epigenetics maintains both active and silent chromatin states. This is the "other" genetic code, often superseding that dictated by the nucleotide sequence. The nuclear matrix is rich in many of the factors that govern nuclear processes. It includes a host of unknown factors that may provide our first insight into the structural mechanism responsible for the genetic selectivity of a differentiating cell. This review will consider the nuclear matrix as an integral component of the epigenetic mechanism.

Figure 1. The nucleosome is the fundamental unit of DNA organization in the eukaryotic nucleus

Approximately 146 base pairs (bp) of DNA wraps 1.75 times around a histone octamer core composed of two of each of the H2A and H2B homodimers together with two H3–H4 dimers. Successive nucleosomes are linked by approximately 15 bp of DNA and the entire structure is stabilized associating with histone H1. In solution this arrangement forms the 10 nm “beads on a string” that is readily transcribed in the presence of all of the requisite trans-acting factors and transcription “machinery.” This form can be compacted by coiling into a solenoid form (~30 nm in diameter, in solution) that can be transcribed at basal levels. However, potentiation is a necessary transition from the compacted to the “beads on the string” conformation in order to allow higher degrees of transcription.

Figure 2. Genomic Methylation and silencing

a) Cytosine can be methylated at the carbon-5 position by any number of DNA Methyltransferases (DMNTs) with S-adenosyl-L-methionine (AdoMet) co-factor. Once modified, 5-methylcytosine is highly labile and can be readily deaminated to thymine. This modification is mutagenic yielding C≡G →5mC≡G →T=G→T=A transition essentially ablating trans-factor recognition, silencing Alu elements and other invading DNA elements. b) The primary mechanism of genomic methylation-based silencing requires the recognition of symmetrically methylated CpG dinucleotides (di-mCpG). For example, MeCP2, recognizes and binds to methyl-CpG Binding Domains (MBD) excluding transcription promoting interactions. The MBD interacts with the modified bases in the major groove and several residues in the loop domain between the second and third β-sheets have been shown to be critical to methyl-CpG recognition. Methyl-residues are shown in red, DNA phosphodiester backbone in blue. The ribbon model was adapted (Wakefield et al, 1999; Khorasanizadeh, 2004).

Figure 3. Genomic imprinting is governed by DNA methylation

Genomic methylation plays an essential role in genomic imprinting, the mono-allelic expression of specific loci. In mice, the Igf2-H19 locus provides a well-characterized model where differential methylation between alleles at various DMRs (differentially methylated regions) mediates the imprint. The unmethylated maternal allele, enables the CTCF boundary element to bind, restricting the activity of the upstream enhancers (EEE) on H19. In contrast, the paternal allele is heavily methylated at all three DMRs (red m’s). When unmethylated, DMRi, acts as an activator and could loop to physically interact with DMRiii to drive maternal H19 expression. This interaction may be mediated by CTCF, binding the unmethylated downstream DMR. DMRii acts as a methylation-sensitive repressor within intron 6 of Igf2. Upon interacting with the downstream enhancers unmethylated DMRii promotes paternal Igf2 expression.

Figure 4. RNA-dependent DNA methylation RdDM

A ~21–26 nt, double stranded RNA targets specific regions, e.g., promoters, for localized methylation of all cytosines. Only those cytosines within the short RNA are methylated to block the molecular recognition of cis-regulatory promoter elements silencing transcription. Unlike X-ist RNA targeting in mammalian X-inactivation, this process is specific and does not spread along a larger domain.

Figure 5. Post-translational histone modifications

The covalent modification of histones can serve to signal any number of cellular and genetic processes. This has been reviewed by (Fischle et al, 2003a; Lachner et al, 2003; Gill, 2004; Khorasanizadeh, 2004; de la Cruz et al, 2005; Lowndes and Toh, 2005), and (Upstate, Waltham, MA, USA). The effect is varied, depending on the residue and how it is modified. Lysines are subject to acetylation, methylation, ubiquitination and sumoylation, arginine to methylation while serine and tyrosine can be phosphorylated. Acetylation of H3 and H4 lysines are generally associated with transcriptionally active DNA, e.g. H3:K4, K9, K27. In contast, sumoylation of H4 lysine is associated with transcriptional repression, but the specific residues have not yet been identified. Arginine and lysine can be mono-, di- and tri-methylated associated with both active and silent chromatin. Specific modifications seldom exist to the exclusion of the other. In concert they form a code imparting silencing or activation.

Figure 6. RNA-mediated silencing

Post-transcriptional RNA-mediated silencing termed RNAi, RNA interference is a multi-step mechanism. It begins in the nucleus, with expression of 5′ capped, polyadenylated transcripts that are ubiquitous throughout the genome (1). These are processed in the nucleus by an RNaseIII-like complex including Drosha (2) that cleaves the primary transcripts into ~70 nucleotide hairpin pre-micro RNAs (pre-miRNAs; 3) that are exported to the cytoplasm by Exportin 5, a nuclear pore protein (4). Upon reaching the cytoplasm, these pre-miRNAs are further processed by Dicer (5), another RNaseIII family member, to produce 21–25nt miRNAs (6). Silencing is achieved upon association of these miRNAs with the RNA-Induced Silencing Complex, RISC (7). RISC is a heterogeneous complex of additional RNaseIII-like factors as well as members of the Argonaut family. This association empowers RISC to down regulate a specific message by either targeted mRNA cleavage or targeted mRNA translational inhibition (8). Silencing efficiency is dependent upon the level of miRNA and target identity as well as the type of Argonaut protein that is associated with the RISC. The greater the level of identity drives the mechanism towards cleavage and degradation as opposed to translational repression of the target.

Figure 7. Nuclear matrix association mediates molecular interactions that regulate transcription

The human β-globin cluster is composed of five genes that are temporally regulated from embryo to adult. The cluster is regulated through LCR mediated interactions localized by five DNase I hypersensitive sites labeled 1–5. Nuclear matrix association varies directly with the temporal expression of the members of the locus. a) In non-expressing cells, the pattern of binding tethers the locus to the matrix, preventing long range chromatin interactions between the locus control region and the various gene promoters. b) The cis sequence binding pattern and tethering to the nuclear matrix changes in cells that express ε, ^Gγ and ^Aγ-globins coincident with long range looping-based interactions that constitute an “active chromatin hub”.

From left to right: Yi Lu, Adrian E. Platts, Stephen A. Krawetz, Amelia Quayla, Rui Pires Martins, Jodi Gardner and Robert Goodrich