Transcript profiling of the hypomethylated hog1 mutant of Arabidopsis

Abstract

Transcript profiling was used to look for genes that differ in expression between the SAH hydrolase deficient and hypomethylated hog1-1 mutant and the parental (HOG1) line. This analysis identified a subset of gene transcripts that were up-regulated in hog1-1 plants. The majority of these transcripts were from genes located in the pericentromeric heterochromatin. About a third of the genes are annotated as transposons or having transposon homology. Subsequent experiments using Northern blots, RT-PCR and real-time RT-PCR confirmed the up-regulation of 19 of the genes and identified a set of molecular probes for genes that are up-regulated in the hog1-1 background. Six (of six genes tested) of the hog1-1 up-regulated genes are also up-regulated in the hypomethylated ddm1 mutant, three in the hypomethylated met1 mutant and three in the dcl3 mutant. The results suggest that the hypomethylation in the mutant lines may have a causal role in the up-regulation of these transcripts.

Cooperation of Multiple Chromatin Modifications Can Generate Unanticipated Stability of Epigenetic States inArabidopsis

“…By contrast, hog1 mutations have so far only been analyzed for their effects on specific targets (Rocha et al, 2005;Mull et al, 2006) and general gene expression (Jordan et al, 2007). Nevertheless, the precise functional annotation of the gene product and the biochemical evidence for its role in regulating SAH levels (Rocha et al, 2005) make it easier to speculate about its mode of action.…”

“…This degradation is essential for recycling of the methyl-group donor S-adenosyl-L-methionine (SAM) and prevents inhibition of trans-methylation reactions through increased levels of SAH (Weretilnyk et al, 2001). HOG1 is involved in maintaining transcriptional gene silencing at numerous targets (Furner et al, 1998;Rocha et al, 2005;Mull et al, 2006;Jordan et al, 2007), while another SAHH-related gene (SAHH2, At3g23810) has no role in silencing or DNA methylation (Rocha et al, 2005). The DDM1 alleles were named ddm1-11 to ddm1-13, in continuation of the already available mutant alleles (Jeddeloh et al, 1999;Jordan et al, 2007): ddm1-11 has a 38-bp deletion in exon V, ddm1-12 has a 30-bp deletion in exon XIV, and ddm1-13 has a T-DNA insertion in exon VII (Figure 2A).…”

“…HOG1 is involved in maintaining transcriptional gene silencing at numerous targets (Furner et al, 1998;Rocha et al, 2005;Mull et al, 2006;Jordan et al, 2007), while another SAHH-related gene (SAHH2, At3g23810) has no role in silencing or DNA methylation (Rocha et al, 2005). The DDM1 alleles were named ddm1-11 to ddm1-13, in continuation of the already available mutant alleles (Jeddeloh et al, 1999;Jordan et al, 2007): ddm1-11 has a 38-bp deletion in exon V, ddm1-12 has a 30-bp deletion in exon XIV, and ddm1-13 has a T-DNA insertion in exon VII (Figure 2A). In contrast with the widely used alleles ddm1-2, with a point mutation generating a G-to-A transition in the splice donor site of intron XI (Jeddeloh et al, 1999), and ddm1-5, with an 82-bp insert in exon II (Mittelsten Scheid et al, 1998;Jeddeloh et al, 1999), the new mutations are all in conserved signature motifs that are characteristic of SWI2/SNF2 family proteins (Bork and Koonin, 1993) and affect the domains that are important for ATP-dependent chromatin remodeling, namely, SNF2_N and DEAD/DEAH (Figure 2A).…”

“…This raised the question of why and how the new mutations in DDM1 and HOG1 proved to be exceptions and whether this could hint at an underlying mechanism. Both mutants have been reported to interfere with transcriptional gene silencing at many other targets in the Arabidopsis genome (Lippman et al, 2004;Jordan et al, 2007), but many of these were also expressed in those other mutants that did not reactivate the HPT gene. However, mutations in DDM1 and HOG1 have in common that they reduce DNA methylation and heterochromatic histone modifications at the HPT transgene.…”

DNA Methylation and Demethylation in Arabidopsis

“…The cytosine base is flipped out of the DNA helix into the enzyme active site, where an S-adenosyl-L-methionine (SAM) cofactor donates a methyl group to the carbon 5 position. Disruptions of enzymes that affect the SAM cycle reduce DNA methylation in Arabidopsis (Tanaka et al, 1997;Rocha et al, 2005;Mull et al, 2006;Jordan et al, 2007).…”