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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 meiosis and 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, paramutation 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 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 (Figure 1). 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.

We have found that most, if not all easiRNA are triggered by initial cleavage of TE transcripts by microRNA. Cleavage is thought to promote the activity of RNA dependent RNA polymerase, and hence easiRNA. More than 50 miRNA target transposons when they are activated in the absence of the chromatin remodeler DECREASE IN DNA METHYLATION1 (DDM1) and DNA METHYLTRANSFERASE1 (MET1), which methylates symmetric CG dinucleotides. Several of these miRNA are expressed in pollen, including the pollen specific miRNA miR845, which targets retrotransposons in the highly conserved tRNA primer binding site (PBS). We are investigating the mechanism by which this might lead to easiRNA accumulation in pollen.

Figure: Pollen development in Arabidopsis...

Transposon derepression in the VN coincides with the loss of DDM1 and MET1, suggesting a potential mechanism for TE activation. However, both genes are expressed in the uninucleate microspore, and as this cell only divides once to give rise to the VN, CG sites in TEs would be expected to retain methylation. We have sequenced the methylome of purified VN, sperm cells, and uninucleate microspores to explore the inheritance of methylation. We found that CG methylation is largely maintained, except for targets of the DNA demethylases DEMETER and ROS1, which are demethylated in the VN. These targets include imprinted genes, and small RNA from nearby transposons accumulate in the sperm cells. This may help to protect sperm cells from TE activation, and loss of imprinting, as asymmetric CHH methylation is specifically lost from sperm cells, only to be regained after fertilization in the embryo.

Sperm cells also undergo reprogramming via histone exchange. Arabidopsis MGH3 (male germline histone H3, or HTR10) is a histone 3.3 (H3.3)-like protein that is expressed specifically in sperm, resembling DDM1 in this respect. DDM1 belongs to the SNF2 family of chromatin-remodeling ATPases, and in ddm1 mutant sperm MGH3 appears to invade heterochromatin. This makes DDM1 a prime candidate for mediating reprogramming in the germline.

The VN also undergoes extensive histone exchange, resulting in little or no H3.1 in the mature pollen grain. We have found that H3K27 monomethylation, which is controlled by the methyltranferases ATXR5 and ATXR6, is specific to the variant H3.1. Using a combination of structural biology and biochemistry we have shown that specificity is conferred by recognition of Alanine31, the only residue uniquely found in the N terminal tail of H3.1. Enzymatic specificity for histone variants in the sperm cells and VN likely account for dramatic changes in histone modification that occur during reprogramming, which contribute to TE activation.

We are investigating the role of reprogramming, and of small RNA, in chromosome behavior during meiosis and in the differentiation of gametophytes from microspores and megaspores. 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.

Epigenetics 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", “INvestigating TRiticeae EPIgenomes for Domestication (INTREPID)” and “MaizeCODE - An Initial Analysis of Functional Elements in the Maize Genome”. We have found that 24nt siRNA from TEs are replaced by easiRNAs in dividing cells in culture, which lose asymmetric CHH methylation as a result. We have evidence from other plants that this mechanism contributes to somaclonal variation, an important epigenetic consequence of clonal propagation in crop plants. Similar mechanisms appear to be responsible for widespread paramutation-like phenomena in the maize methylome.

RNAi guides heterochromatic silencing in Fission Yeast

Our work on fission yeast continues to provide inspiration as a model for RNAi and heterochromatin in plants. My laboratory was the first to show that RNAi guides heterochromatin modification and silencing in fission yeast. We showed that siRNA in S. pombe accumulates in S phase where it helps to release RNA polymerase II, allowing completion of replication-coupled histone modification by the leading strand DNA polymerase. In this way, RNAi helps to resolve collisions between DNA and RNA polymerases during replication. In the absence of RNAi, DNA repair by homologous recombination becomes essential, and DNA damage accumulates. 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. Recently, we have found an essential role for RNAi in quiescent cells, which lack homologous recombination repair. Suppressor screens have been used to identify mutants in RNA polymerase II and in heterochromatin modification, providing strong genetic evidence for our model.

Our work on heterochromatin in fission yeast and transposon silencing in plants is funded by the National Institutes of Health. Our work on plant genomes is supported by the National Science Foundation.

As of March 24, 2016

Scientist Profile

Cold Spring Harbor Laboratory
Genetics, Plant Biology