Gametes constitute a critical stage of the plant life cycle during which the genome undergoes reprogramming in preparation for embryogenesis. Here, we examined genome-wide distributions of small RNAs in the sperm and egg cells of rice. We found that 24-nt siRNAs, which are a hallmark of RNA-directed DNA methylation (RdDM) in plants, were depleted from heterochromatin boundaries in both gametes relative to vegetative tissues, reminiscent of siRNA patterns in DDM1type nucleosome remodeler mutants. In sperm cells, 24-nt siRNAs were spread across heterochromatic regions, while in egg cells, 24-nt siRNAs were concentrated at a smaller number of heterochromatic loci throughout the genome, especially at loci which also produced siRNAs in other tissues. In both gametes, patterns of CHH methylation, typically a strong indicator of RdDM, were similar to vegetative tissues, although lower in magnitude. These findings indicate that the small RNA transcriptome undergoes large-scale redistribution in both male and female gametes, which is not correlated with recruitment of DNA methyltransferases in gametes and suggestive of unexplored regulatory activities of gamete small RNAs.
Until recently, little knowledge existed about the molecular mechanisms regulating male gamete development. This was mainly due to the low transcriptional activity and the cellular inaccessibility of the generative and sperm cells that are enclosed by the vegetative cell in pollen. In order to study sperm cell development and possible preferential fusion during double fertilization, we have constructed a cDNA library of mRNA isolated from pure tobacco sperm cells. An initial screen of 396 clones from this library has yielded 2 cDNAs representing sperm-cell-expressed transcripts, designated NtS1 and NtS2. A preliminary characterization of these two clones showed that they accumulate in both the generative and sperm cells (i.e. the male gamete) indicating that gene expression programs between these two cell types overlap. In addition, we found that NtS1 codes for a polygalacturonase suggesting a role for this enzyme in wall degradation during differentiation of the male germ cells in tobacco. Together, these results show that with the construction of this sperm-cell cDNA library we now have a powerful tool to investigate male gamete development and function.
Transcriptionally silent chromatin in Saccharomyces cerevisiae is associated with histone hypoacetylation and is formed through the action of the Sir histone deacetylase complex. A histone acetyltransferase (HAT) targeted near silent chromatin can overcome silencing at a distance by increasing histone acetylation in a sizable region. However, how a tethered HAT acetylates distant nucleosomes has not been resolved. We demonstrate here that targeting the histone H3-specific HAT Gcn5p promotes acetylation of not only histone H3 but also histone H4 in a broad region. We also show that long range anti-silencing and histone acetylation by targeted HATs can be blocked by nucleosome-excluding sequences. These results are consistent with the contention that a tethered HAT promotes stepwise propagation of histone acetylation along the chromatin. Because histone hypoacetylation is key to the formation and maintenance of transcriptionally silent chromatin, it is believed that acetylation promoted by a targeted HAT disrupts silent chromatin thereby overcoming silencing. However, we show that the acetylated and transcriptionally active region created by a tethered HAT retains structural hallmarks of Sir-dependent silent chromatin and remains associated with Sir proteins indicating that tethered HATs overcome silencing without completely dismantling silent chromatin.Eukaryotic DNA is packed into chromatin through the formation of nucleosomes composed of a histone octamer around which 147 bp of DNA is wrapped (1, 2). Chromatin plays a pivotal role in the regulation of gene expression. It is subject to various modifications that differentially affect its compaction and accessibility to transcriptional regulators. The two major types of modifications of chromatin structure are the remodeling of nucleosome patterning along the chromatin fiber and covalent modifications of histones, including acetylation, methylation, phosphorylation, and ubiquitination (3, 4). Increasing evidence indicates that histone modifications create a histone code (surface) that is read (recognized) by other proteins to bring about various downstream events (5-7).Acetylation of the N-terminal lysine residues of histones neutralizes the positive charge they carry, which is believed to alter the property of nucleosomes resulting in a reduction in the degree of chromatin compaction (8). Moreover, as an important part of the histone code, acetyllysines on histones serve as recognition sites for regulatory factors (5, 6, 9). Promoter-specific localized hyperacetylation of histones may lead to activation of individual genes, whereas coordinated acetylation of histones across a sizable chromosomal region may help maintain a transcriptionally poised chromatin state (10, 11). Histone acetylation is carried out by histone acetyltransferases (HATs) 2 (12, 13). Known HATs fall into distinct families with specific substrate preferences (13). For example, yeast Gcn5p is a member of the GNAT (Gcn5-related N-acetyltransferase) family that prefers histones H3 and H2B as sub...
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