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Heterochromatin Reprogramming, Small Interfering RNA, and Germ Cell Fate

Research Summary

Rob Martienssen is investigating the role of heterochromatin reprogramming and RNA interference in plant germ cells. These mechanisms reveal and regulate transposable elements, but they also play important roles in promoting meiosis and influencing reproductive fate.

Heterochromatin and transposable elements (TEs) were both discovered in plants but make up the majority of most eukaryotic genomes. When active, TEs can disrupt genes and regulatory regions and promote chromosomal rearrangements. To suppress this mutagenic potential, surveillance systems have evolved that target active transposons; for example, small interfering RNAs (siRNAs) target transposons for post-transcriptional silencing or for the deposition of repressive chromatin modification. These modifications include DNA methylation and histone modifications, such as H3 lysine-9 methylation (H3K9me2).

In plants, transcriptional silencing via RNA-directed DNA methylation is mediated by 24-nucleotide siRNAs, resulting in cytosine methylation at asymmetric CHH sites. However, DNA methylation can be maintained independently of RNA interference (RNAi) by replication-dependent DNA methyltransferases and can lead to transgenerational inheritance.

Epigenetic changes are reversible, making them amenable to environmental as well as developmental modulation, and germline reprogramming must erase these epigenetic marks without losing control of TEs. We have previously proposed that controlled germline transposon activity in pollen can first reveal and then silence TEs via reprogramming and RNAi. A similar mechanism has since been proposed in early seed development. Reprogramming in animals may have the same function, as transposons in the germline are expressed and processed into small RNAs that can influence DNA methylation. Imprinting, hybrid lethality, and epigenetic inheritance are potential outcomes in the next generation.

Remarkably, this same pathway controls the mode of sexual reproduction in plants, by inhibiting the differentiation of asexual (aposporic) female gametophytes in the ovule. Apomixis is sometimes called the "holy grail" of plant breeding, as it allows for clonal production of hybrid seed, and this is an exciting prospect for plant breeders.

Heterochromatin Reprogramming with Small RNA in Pollen
Following meiosis, haploid microspores undergo two rounds of mitosis, first giving rise to the vegetative nucleus (VN) and then to two sperm cells. The VN supports growth of the pollen tube, while the sperm cells fuse with the egg and central cells during fertilization, initiating the development of the embryo and endosperm, respectively. We found that TEs are derepressed in the VN and generate pollen-specific, "epigenetically activated" 21-nt siRNAs (easiRNAs) that subsequently accumulate in the sperm cells. Thus active transposons are "unmasked" in the VN via the production of small RNAs, which find their way into sperm.

Derepression in the VN coincides with the loss of two key regulators of heterochromatic TE silencing, the chromatin remodeler DECREASE IN DNA METHYLATION1 (DDM1) and DNA METHYLTRANSFERASE1 (MET1), which methylates symmetric CG dinucleotides. Although lost from the VN, both DDM1 and MET1 are expressed in the uninucleate microspore, and as this cell only divides once to give rise to the VN, CG sites in TEs are fully methylated. Pollen mitosis I is a classic model for plant stem cell division, and we are sequencing the methylome of purified VN, sperm cells, and uninucleate microspores to explore the inheritance of methylation.

DDM1 is a master regulator of heterochromatin formation in Arabidopsis, via DNA methylation, histone tail modifications, and chromatin condensation, and prevents transposition of TEs. Sperm cells undergo reprogramming via histone exchange, in both plants and animals. Arabidopsis MGH3 (male germline histone H3, or HTR10) is a histone 3.3 (H3.3)-like protein that is expressed specifically in sperm Figure 1), resembling DDM1 in this respect. DDM1 belongs to the SNF2 family of chromatin-remodeling ATPases, and in ddm1 mutant leaves, H3K9me2 is replaced by H3K4me2, consistent with replacement of H3.1 by H3.3 variants. Along with sperm cell–specific expression, this makes DDM1 a prime candidate for mediating reprogramming in the germline. Consistent with this idea, ddm1 mutants transmit active TEs to heterozygous progeny (a form of transgenerational inheritance). We plan to use super-resolution OMX structured illumination microscopy to examine accumulation of histone variants in ddm1 mutants (Figure 1). We will follow up with ChIP-seq to determine the dynamics of histone exchange.

Figure 1: Heterochromatin reprogramming in pollen...

In my lab we will investigate the role of small RNA––binding Argonaute proteins in meiosis and in the differentiation of gametophytes from microspores and megaspores. We will explore the ability of small RNA to move between cells in the gametophyte and to influence transposon activity and reproductive fate following fertilization. We hope to define target genes responsible for these defects. One possibility is that reprogramming of the VN and reprogramming of the sperm cells are coupled, in that genome reprogramming in sperm cells makes TE movement more likely. TE-derived easiRNAs from the VN would then act as a surveillance mechanism, by accumulating in sperm cells and silencing TEs, during the short time window while the epigenome is reorganized.

Epigenetically Activated Small RNA and the Plant Genome
My laboratory participates in several large-scale projects supported by the Plant Genome Research Program at the National Science Foundation, including "Epigenome Dynamics During DNA replication," "Functional Genomics of Plant Polyploids," and "Genomics of Comparative Seed Evolution." We have found that easiRNA plays a key role in each area. For example, easiRNAs accumulate in dividing cells in culture, which lose methylation in some but not all transposable elements. Sperm cells are arrested in S phase and microspores can themselves be cultured, resulting in activation of TEs, indicating easiRNAs may be linked to the cell cycle, accounting for these observations.

Unexpectedly, easiRNAs are also inherited from pollen parents in allopolyploid hybrids between related species, reflecting activation of specific TEs. This likely reflects mismatches between siRNA in one parent and TE in the other, resembling hybrid dysgenesis. Finally, phylogenomic analysis indicates that genes in the easiRNA pathway are critical for the evolution of seeds in gymnosperms and angiosperms, coinciding with the maternal endosperm lineage in gymnosperms. We are planning to use what we learn about easiRNAs to test many of these ideas at the mechanistic and genetic levels.

Use of Fission Yeast as a Model System
Our work on fission yeast continues to provide inspiration as a model for RNAi and heterochromatin in plants. My laboratory was one of the first to isolate alleles of ARGONAUTE1 in Arabidopsis, and we soon realized that the fission yeast Schizosaccharomyces pombe possessed only a single Argonaute gene, in contrast with the 10 found in plants. We showed that ago1 in S. pombe, along with homologs of Dicer and RNA-dependent RNA polymerase (RdRP), are required for heterochromatic histone modification. We found that spreading of heterochromatic silencing requires the catalytic endonuclease activity of Argonaute. More recently, we found that siRNA in S. pombe accumulates in S phase and helps to release polymerase II, allowing completion of replication in a mechanism reminiscent of DNA repair. Retrotransposons also have a role in S-phase progression by controlling the direction of DNA replication via the highly conserved centromere-binding protein CENP-B.

This work, as well as our work on transposons and heterochromatin in plants, is funded by grants from the National Institutes of Health. Our work on plant genomes is supported by the National Science Foundation and the Department of Energy.

As of May 30, 2012

Scientist Profile

Cold Spring Harbor Laboratory
Genetics, Plant Biology