RNA-directed DNA methylation (RdDM) is required for transcriptional silencing of transposons and other DNA repeats in Arabidopsis thaliana. Although previous research has demonstrated that the SET domain-containing SU(VAR)3–9 homologs SUVH2 and SUVH9 are involved in the RdDM pathway, the underlying mechanism remains unknown. Our results indicated that SUVH2 and/or SUVH9 not only interact with the chromatin-remodeling complex termed DDR (DMS3, DRD1, and RDM1) but also with the newly characterized complex composed of two conserved Microrchidia (MORC) family proteins, MORC1 and MORC6. The effect of suvh2suvh9 on Pol IV-dependent siRNA accumulation and DNA methylation is comparable to that of the Pol V mutant nrpe1 and the DDR complex mutant dms3, suggesting that SUVH2 and SUVH9 are functionally associated with RdDM. Our CHIP assay demonstrated that SUVH2 and SUVH9 are required for the occupancy of Pol V at RdDM loci and facilitate the production of Pol V-dependent noncoding RNAs. Moreover, SUVH2 and SUVH9 are also involved in the occupancy of DMS3 at RdDM loci. The putative catalytic active site in the SET domain of SUVH2 is dispensable for the function of SUVH2 in RdDM and H3K9 dimethylation. We propose that SUVH2 and SUVH9 bind to methylated DNA and facilitate the recruitment of Pol V to RdDM loci by associating with the DDR complex and the MORC complex.

Substantial evidence shows that long non-coding RNAs (lncRNAs) participate in many biological mechanisms, and their dysregulation are also involved in the development and progression of cancers, including gastric cancer (GC). Long intergenic non-coding RNA 00324 (LINC00324), a 2115 bp ncRNA, is located on chromosome 17p13.1. The biological function and molecular mechanisms of LINC00324 in GC remains undiscovered. In this paper, we found that the expression level of LINC00324 was significantly upregulated in GC tissues compared with the corresponding normal tissues. The overexpression of LINC00324 was correlated with advanced TNM stage, larger tumor size, and lymph node metastasis as well as poor prognosis. Further experiments revealed that knockdown of LINC00324 could suppress the proliferation of GC cells. RNA transcriptome sequencing technology revealed that FAM83B may be a significant downstream target gene of LINC00324. LINC00324 could combine with the RNA-binding protein (RBP) human antigen R (HuR) and thus stabilize the expression of FAM83B. Moreover, rescue assays showed that the reduced FAM83B expression partially reversed the promotion of cell growth in GC induced by the overexpression of LINC00324. In conclusion, our study revealed that LINC00324 acted as an oncogene in tumorigenesis and progression, suggesting that it could be a new biomarker in diagnosis and prognosis of GC.

In eukaryotes, heterochromatin regions are typically subjected to transcriptional silencing. DNA methylation has an important role in such silencing and has been studied extensively. However, little is known about how methylated heterochromatin regions are subjected to silencing. We conducted a genetic screen and identified an () mutant that releases heterochromatin silencing in We demonstrated that EPCR1 functions redundantly with its paralog EPCR2 and interacts with PWWP domain-containing proteins (PWWPs), AT-rich interaction domain-containing proteins (ARIDs), and telomere repeat binding proteins (TRBs), thus forming multiple functionally redundant protein complexes named PEAT (WWPs-PCRs-RIDs-RBs). The PEAT complexes mediate histone deacetylation and heterochromatin condensation and thereby facilitate heterochromatin silencing. In heterochromatin regions, the production of small interfering RNAs (siRNAs) and DNA methylation is repressed by the PEAT complexes. The study reveals how histone deacetylation, heterochromatin condensation, siRNA production, and DNA methylation interplay with each other and thereby maintain heterochromatin silencing.

IDN2/RDM12 has been previously identified as a component of the RNA–directed DNA methylation (RdDM) machinery in Arabidopsis thaliana, but how it functions in RdDM remains unknown. By affinity purification of IDN2, we co-purified two IDN2 paralogs IDP1 and IDP2 (IDN2 PARALOG 1 and 2). The coiled-coil domain between the XS and XH domains of IDN2 is essential for IDN2 homodimerization, whereas the IDN2 C-terminal XH domain but not the coiled-coil domain is required for IDN2 interaction with IDP1 and IDP2. By introducing the wild-type IDN2 sequence and its mutated derivatives into the idn2 mutant for complementation testing, we demonstrated that the previously uncharacterized IDN2 XH domain is required for the IDN2-IDP1/IDP2 complex formation as well as for IDN2 function. IDP1 is required for de novo DNA methylation, siRNA accumulation, and transcriptional gene silencing, whereas IDP2 has partially overlapping roles with IDP1. Unlike IDN2, IDP1 and IDP2 are incapable of binding double-stranded RNA, suggesting that the roles of IDP1 and IDP2 are different from those of IDN2 in the IDN2-IDP1/IDP2 complex and that IDP1 and IDP2 are essential for the functioning of the complex in RdDM.

DNA methylation in transposons and other DNA repeats is conserved in plants as well as in animals. In Arabidopsis thaliana, an RNA-directed DNA methylation (RdDM) pathway directs de novo DNA methylation. We performed a forward genetic screen for suppressors of the DNA demethylase mutant ros1 and identified a novel Zincfinger and OCRE domain-containing Protein 1 (ZOP1) that promotes Pol IV-dependent siRNA accumulation, DNA methylation, and transcriptional silencing. Wholegenome methods disclosed the genome-wide effects of zop1 on Pol IV-dependent siRNA accumulation and DNA methylation, suggesting that ZOP1 has both RdDM-dependent and -independent roles in transcriptional silencing. We demonstrated that ZOP1 is a pre-mRNA splicing factor that associates with several typical components of the splicing machinery as well as with Pol II. Immunofluorescence assay revealed that ZOP1 overlaps with Cajal body and is partially colocalized with NRPE1 and DRM2. Moreover, we found that the other development-defective splicing mutants tested including mac3a3b, mos4, mos12 and mos14 show defects in RdDM and transcriptional silencing. We propose that the splicing machinery rather than specific splicing factors is involved in promoting RdDM and transcriptional silencing.

The SU(VAR)3-9 homolog SUVH9 and the double-stranded RNA-binding protein IDN2 were thought to be components of an RNA-directed DNA methylation (RdDM) pathway in Arabidopsis. We previously found that SUVH9 interacts with MORC6 but how the interaction contributes to transcriptional silencing remains elusive. Here, our genetic analysis indicates that SUVH2 and SUVH9 can either act in the same pathway as MORC6 or act synergistically with MORC6 to mediate transcriptional silencing. Moreover, we demonstrate that IDN2 interacts with MORC6 and mediates the silencing of a subset of MORC6 target loci. Like SUVH2, SUVH9, and IDN2, other RdDM components including Pol IV, Pol V, RDR2, and DRM2 are also required for transcriptional silencing at a subset of MORC6 target loci. MORC6 was previously shown to mediate transcriptional silencing through heterochromatin condensation. We demonstrate that the SWI/SNF chromatin-remodeling complex components SWI3B, SWI3C, and SWI3D interact with MORC6 as well as with SUVH9 and then mediate transcriptional silencing. These results suggest that the RdDM components are involved not only in DNA methylation but also in MORC6-mediated heterochromatin condensation. This study illustrates how DNA methylation is linked to heterochromatin condensation and thereby enhances transcriptional silencing at methylated genomic regions.

RNA-directed DNA methylation (RdDM) is an important de novo

DNA methylation is a conserved epigenetic marker in plants and animals. In Arabidopsis, DNA methylation can be established through an RNA-directed DNA methylation (RdDM) pathway. By screening for suppressors of ros1, we identified STA1, a PRP6-like splicing factor, as a new RdDM regulator. Whole-genome bisulfite sequencing suggested that STA1 and the RdDM pathway share a large number of common targets in the Arabidopsis genome. Small RNA deep sequencing demonstrated that STA1 is predominantly involved in the accumulation of the siRNAs that depend on both Pol IV and Pol V. Moreover, the sta1 mutation partially reduces the levels of Pol V-dependent RNA transcripts. Immunolocalization assay indicated that STA1 signals are exclusively present in the Cajal body and overlap with AGO4 in most nuclei. STA1 signals are also partially overlap with NRPE1. Localization of STA1 to AGO4 and NRPE1 signals is probably related to the function of STA1 in the RdDM pathway. Based on these results, we propose that STA1 acts downstream of siRNA biogenesis and facilitates the production of Pol V-dependent RNA transcripts in the RdDM pathway.