Lies on the strong attachment of the DNA to the histones in the nucleosome and isolation of nucleosomal fragments by micrococcal nuclease digestion. nChIP has been recently adopted for Arabidopsis vegetative tissues, and reveals a highly similar pattern compared to formaldehyde-fixed material in H3K9me2 profiling [22]. For our analyses we focused on two key histone marks: H3K4me3, which is associated with transcriptionally active genes, and H3K27me3, an inhibitory mark. The deposition of both marks is mediated by trxG- and PcG SET domain proteins respectively [23], and the corresponding HMTases showed differential expression patterns during the dormancy-to-germination transition, suggesting that changes in the chromatin landscape take place (Fig. 2). We investigated a range of genes that encode proteins with a demonstrated or intimated positive regulatory role in seed maturation and/or dormancy, as well as genes encoding proteins that can be classified as 1418741-86-2 site markers of seed dormancy (Table 1). For markers and regulators of germination, we selected six genes that exhibit the strongest up-regulation during dormancy termination of Cvi seeds based on published transcriptomes (Table 1) [5,6]. These germination-associated genes bear the H3K4me3 in seedlings, and we asked whether this histone methylation isdeposited during the actual dormancy-to-germination transition (Figs. 3, S1). Indeed, all six genes showed increasing amounts of H3K4me3 from the dormant seed to the seedling stage. In contrast, these genes were generally not decorated with H3K27me3. One exception was the dehydrin COR47, which carried H3K27me3 in the dormant state. This repressive mark on COR47 was gradually lost and exchanged for increasing amounts of H3K4me3 following transfer of seeds to germination conditions (Fig. S1) Therefore the transcriptional activation during germination is consistent with an extensive reprogramming at the chromatin level.Histone Methylation Dynamics of Major Seed Maturationand Dormancy-regulators during the Environmentally Cued Transition from Dormancy to GerminationOur new nChIP protocol allowed us to focus on the dynamics of major dormancy regulators in seeds at key physiological stages (Fig. 1). We decided to follow seven central regulators and markers (Table 1): The gene products of ABI3 (24) and LEC2 [25,26] control seed maturation and dormancy [4,27]. Delay of Germination1 (DOG1) is a major dormancy QTL in Arabidopsis [27,28]. SOMNUS (SOM) positively influences signaling of the dormancy-inducing and germination-inhibiting plant hormone abscisic acid (ABA) and negatively influences signaling of its antagonist gibberellin (GA) [29]. FLC has been implicated in the regulation of temperature dependent seed germination [30], but is best known for its role as a repressor of flowering, in which context it is subject to epigenetic regulation when plants are exposed to cold temperatures during vernalization [1,2]. Our analyses also included the gene for the 34540-22-2 biological activity storage protein 1379592 2S1 (a maturation marker) as well as RAB18. RAB18 likely plays an indirect role in dormancy, as this protein is connected more to the survival of the seed in the dispersed, dormant state (e.g. tolerance of the dispersed seed to environmental stresses such as water/desiccation stress) (Table 1). The RAB18 gene is expressed during late seed maturation, and exhibits reduced expression during germination. Therefore it is most accurately considered a “dormancy marker”, and not a dormancy.Lies on the strong attachment of the DNA to the histones in the nucleosome and isolation of nucleosomal fragments by micrococcal nuclease digestion. nChIP has been recently adopted for Arabidopsis vegetative tissues, and reveals a highly similar pattern compared to formaldehyde-fixed material in H3K9me2 profiling [22]. For our analyses we focused on two key histone marks: H3K4me3, which is associated with transcriptionally active genes, and H3K27me3, an inhibitory mark. The deposition of both marks is mediated by trxG- and PcG SET domain proteins respectively [23], and the corresponding HMTases showed differential expression patterns during the dormancy-to-germination transition, suggesting that changes in the chromatin landscape take place (Fig. 2). We investigated a range of genes that encode proteins with a demonstrated or intimated positive regulatory role in seed maturation and/or dormancy, as well as genes encoding proteins that can be classified as markers of seed dormancy (Table 1). For markers and regulators of germination, we selected six genes that exhibit the strongest up-regulation during dormancy termination of Cvi seeds based on published transcriptomes (Table 1) [5,6]. These germination-associated genes bear the H3K4me3 in seedlings, and we asked whether this histone methylation isdeposited during the actual dormancy-to-germination transition (Figs. 3, S1). Indeed, all six genes showed increasing amounts of H3K4me3 from the dormant seed to the seedling stage. In contrast, these genes were generally not decorated with H3K27me3. One exception was the dehydrin COR47, which carried H3K27me3 in the dormant state. This repressive mark on COR47 was gradually lost and exchanged for increasing amounts of H3K4me3 following transfer of seeds to germination conditions (Fig. S1) Therefore the transcriptional activation during germination is consistent with an extensive reprogramming at the chromatin level.Histone Methylation Dynamics of Major Seed Maturationand Dormancy-regulators during the Environmentally Cued Transition from Dormancy to GerminationOur new nChIP protocol allowed us to focus on the dynamics of major dormancy regulators in seeds at key physiological stages (Fig. 1). We decided to follow seven central regulators and markers (Table 1): The gene products of ABI3 (24) and LEC2 [25,26] control seed maturation and dormancy [4,27]. Delay of Germination1 (DOG1) is a major dormancy QTL in Arabidopsis [27,28]. SOMNUS (SOM) positively influences signaling of the dormancy-inducing and germination-inhibiting plant hormone abscisic acid (ABA) and negatively influences signaling of its antagonist gibberellin (GA) [29]. FLC has been implicated in the regulation of temperature dependent seed germination [30], but is best known for its role as a repressor of flowering, in which context it is subject to epigenetic regulation when plants are exposed to cold temperatures during vernalization [1,2]. Our analyses also included the gene for the storage protein 1379592 2S1 (a maturation marker) as well as RAB18. RAB18 likely plays an indirect role in dormancy, as this protein is connected more to the survival of the seed in the dispersed, dormant state (e.g. tolerance of the dispersed seed to environmental stresses such as water/desiccation stress) (Table 1). The RAB18 gene is expressed during late seed maturation, and exhibits reduced expression during germination. Therefore it is most accurately considered a “dormancy marker”, and not a dormancy.