Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure.

Paramutation is a form of non-Mendelian inheritance in which the expression of a paramutable allele changes when it encounters a paramutagenic allele. This change in expression of the paramutable alleles is stably inherited even after segregation of both alleles. While the discovery of paramutation and studies of its underlying mechanism were made with alleles that change plant pigmentation, paramutation-like phenomena are known to modulate the expression of other traits and in other eukaryotes, and many cases have probably gone undetected. It is likely that epigenetic mechanisms are responsible for the phenomenon, as paramutation forms epialleles, genes with identical sequences but different expression states. This could account for the intergenerational inheritance of the paramutated allele, providing profound evidence that triggered epigenetic changes can be maintained over generations. Here, we use a case of paramutation that affects a transgenic selection reporter gene in tetraploid Arabidopsis thaliana. Our data suggest that different types of small RNA are derived from paramutable and paramutagenic epialleles. In addition, deletion of a repeat within the epiallele changes its paramutability. Further, the temperature during the growth of the epiallelic hybrids determines the degree and timing of the allelic interaction. The data further make it plausible why paramutation in this system becomes evident only in the segregating F2 population of tetraploid plants containing both epialleles. In summary, the results support a model for polyploidy-associated paramutation, with similarities as well as distinctions from other cases of paramutation.

Genetic rearrangements can modify chromatin features at epialleles.

Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3' regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles.

Analysis of DNA methylation in plants by bisulfite sequencing.

Methylation of cytosines is a very important epigenetic modification of genomic DNA in many different eukaryotes, and it is frequently involved in transcriptional regulation of genes. In plants, DNA methylation is regulated by a complex interplay between several methylating and demethylating enzymes. Analysis of the resulting cytosine methylation patterns with the highest resolution is achieved after sodium bisulfite treatment, deaminating nonmethylated cytosines to uracil. Subsequent PCR and sequence analysis of individual amplicons displays the degree, position, and sequence context of methylation of every cytosine residue in individual genomic sequences. We describe the application of bisulfite sequencing for the analysis of DNA methylation at defined individual sequences of plant genomic DNA.

Analysis of bisulfite sequencing data from plant DNA using CyMATE.

Amplifying and sequencing DNA after bisulfite treatment of genomic DNA reveals the methylation state of cytosine residues at the highest resolution possible. However, a thorough analysis is required for statistical evaluation of methylation at all sites in each genomic region. Several software tools were developed to assist in quantitative evaluation of bisulfite sequencing data from complex methylation patterns occurring in plants. This chapter describes the application of Cytosine Methylation Analysis Tool for Everyone (CyMATE). From aligned sequences, CyMATE quantifies and illustrates general and pattern-specific methylation at CG, CHG, and CHH (H = A, C, or T) sites, both per sequence and per position. CyMATE is also able to perform a quality control of sequences and to detect redundancy among individual clones. The software is able to reveal methylation patterns on complementary strands by handling data from hairpin bisulfite sequencing. The tool is freely available for non-commercial use at http://www.cymate.org .

Identification of a perinuclear positioning element in human subtelomeres that requires A-type lamins and CTCF.

The localization of genes within the nuclear space is of paramount importance for proper genome functions. However, very little is known on the cis-acting elements determining subnuclear positioning of chromosome segments. We show here that the D4Z4 human subtelomeric repeat localizes a telomere at the nuclear periphery. This perinuclear activity lies within an 80 bp sequence included within a region known to interact with CTCF and A-type Lamins. We further show that a reduced level of either CTCF or A-type Lamins suppresses the perinuclear activities of D4Z4 and that an array of multimerized D4Z4 sequence, which has lost its ability to bind CTCF and A-type Lamins, is not localized at the periphery. Overall, these findings reveal the existence of an 80 bp D4Z4 sequence that is sufficient to position an adjacent telomere to the nuclear periphery in a CTCF and A-type lamins-dependent manner. Strikingly, this sequence includes a 30 bp GA-rich motif, which binds CTCF and is present at several locations in the human genome.

The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy.

Both genetic and epigenetic alterations contribute to Facio-Scapulo-Humeral Dystrophy (FSHD), which is linked to the shortening of the array of D4Z4 repeats at the 4q35 locus. The consequence of this rearrangement remains enigmatic, but deletion of this 3.3-kb macrosatellite element might affect the expression of the FSHD-associated gene(s) through position effect mechanisms. We investigated this hypothesis by creating a large collection of constructs carrying 1 to >11 D4Z4 repeats integrated into the human genome, either at random sites or proximal to a telomere, mimicking thereby the organization of the 4q35 locus. We show that D4Z4 acts as an insulator that interferes with enhancer-promoter communication and protects transgenes from position effect. This last property depends on both CTCF and A-type Lamins. We further demonstrate that both anti-silencing activity of D4Z4 and CTCF binding are lost upon multimerization of the repeat in cells from FSHD patients compared to control myoblasts from healthy individuals, suggesting that FSHD corresponds to a gain-of-function of CTCF at the residual D4Z4 repeats. We propose that contraction of the D4Z4 array contributes to FSHD physio-pathology by acting as a CTCF-dependent insulator in patients.

CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulphite sequencing.

Cytosine methylation is a hallmark of epigenetic information in the DNA of many fungi, vertebrates and plants. The technique of bisulphite genomic sequencing reveals the methylation state of every individual cytosine in a sequence, and thereby provides high-resolution data on epigenetic diversity; however, the manual evaluation and documentation of large amounts of data is laborious and error-prone. While some software is available for facilitating the analysis of mammalian DNA methylation, which is found nearly exclusively at CG sites, there is no software optimally suited for data from DNA with significant non-CG methylation. We describe CyMATE (Cytosine Methylation Analysis Tool for Everyone) for in silico analysis of DNA sequences after bisulphite conversion of plant DNA, in which methylation is more divergent with respect to sequence context and biological relevance. From aligned sequences, CyMATE includes and distinguishes methylation at CG, CHG and CHH (where H = A, C or T), and can extract both quantitative and qualitative data regarding general and pattern-specific methylation per sequence and per position, i.e. data for individual sites in a sequence and the epigenetic divergence within a sample. In addition, it can provide graphical output from alignments in either an overview or a 'zoom-in' view as pdf files. Detailed information, including a quality control of the sequencing data, is provided in text format. We applied CyMATE to the analysis of DNA methylation at transcriptionally silenced promoters in diploid and polyploid Arabidopsis and found significant hypermethylation, high stability of the methylated state independent of chromosome number, and non-redundant patterns of mC distribution. CyMATE is freely available for non-commercial use at http://www.gmi.oeaw.ac.at/CyMATE.