Physical coupling of activation and derepression activities to maintain an active transcriptional state at FLC

Abstract

Establishment and maintenance of gene expression states is central to development and differentiation. Transcriptional and epigenetic mechanisms interconnect in poorly understood ways to determine these states. We explore these mechanisms through dissection of the regulation of Arabidopsis thaliana FLOWERING LOCUS C (FLC). FLC can be present in a transcriptionally active state marked by H3K36me3 or a silent state marked by H3K27me3. Here, we investigate the trans factors modifying these opposing histone states and find a physical coupling in vivo between the H3K36 methyltransferase, SDG8, and the H3K27me3 demethylase, ELF6. Previous modeling has predicted this coupling would exist as it facilitates bistability of opposing histone states. We also find association of SDG8 with the transcription machinery, namely RNA polymerase II and the PAF1 complex. Delivery of the active histone modifications is therefore likely to be through transcription at the locus. SDG8 and ELF6 were found to influence the localization of each other on FLC chromatin, showing the functional importance of the interaction. In addition, both influenced accumulation of the associated H3K27me3 and H3K36me3 histone modifications at FLC. We propose the physical coupling of activation and derepression activities coordinates transcriptional activity and prevents ectopic silencing.epigenetic regulation | histone modifications | histone methyltransferase | histone demethylase | FLOWERING LOCUS C C hromatin-based transcriptional activity is particularly important in multicellular organisms, where cells differentiate into very different developmental states. Transcriptional states are induced by internal developmental signals or triggered by environmental conditions and are then epigenetically maintained through many cell divisions. Considerable evidence suggests that opposing histone modifications mark different transcriptional states (1-6). However, there is still great debate as to how histone-based transcriptional states are initially established and maintained through development (7,8). We used Arabidopsis FLOWERING LOCUS C (FLC) as a model gene to explore histone-based transcriptional regulation and epigenetic memory. Local chromatin states are critical for quantitatively controlling FLC expression (9). Active FLC expression requires functional FRIGIDA (FRI) and a set of histone modifying Trithorax homologs, Complex Proteins Associated with Set1 (COMPASS), RNA polymerase II Associated Factor 1 complex (PAF1C), and SET DOMAIN GROUP 8 (SDG8), the major H3K36 methyltransferase (10-17). Repression of FLC requires either the autonomous pathway, which coordinately influences transcriptional initiation and elongation with associated chromatin modulation, or vernalization, the cold-induced Polycomb silencing (18-21). Both of these involve accumulation of high levels of H3K27me3 at FLC (20-22). A series of FLC antisense transcripts (COOLAIR) contribute to these repression pathways (18,(23)(24)(25)(26) Opposing H3K36me3 and H3K27me3 states are particularly ...

Arabidopsis SDG8 Potentiates the Sustainable Transcriptional Induction of the Pathogenesis-Related Genes PR1 and PR2 During Plant Defense Response

“…This together with the enrichment in H3K36me3 toward the 3end of genes spearheaded the link between H3K36 methylation and transcription elongation in animals and yeast (Wang et al, 2009). In Arabidopsis, we and others (Yang et al, 2016;Zhong et al, 2019) demonstrate that SDG8, like its orthologs, can bind RNAPII. However, while SDG8 orthologs preferentially bind the phosphorylated CTD of elongating RNAPII, we found that SDG8 can interact with both the inactive (non-phosphorylated) and active (Ser5P and/or Ser2P) forms of RNAPII (Figure 6).…”

“…DNA was purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey−Nagel, Düren, Germany) and analyzed by real-time PCR (LightCycler 480II; Roche in conjunction with the SYBR Green Master mix) using gene-specific primers listed in Supplementary Table S1. Data were analyzed as described in Zhao et al, 2019 for H3K4me3 andH3K36me3 andin Yang et al, 2016 for RNAPII. A mock control was done using uncoupled magnetic beads (Supplementary Figure S5).…”

“…Coimmunoprecipitation assays of SDG8 and the RNAPII proteins were performed using the Arabidopsis SDG8:FLAG sdg8 transgenic line (Ko et al, 2010). Total proteins were extracted as previously described (Yang et al, 2016). In brief, 10 g of 10-dayold seedlings grown on 1/2 MS-agar plate was grinded in liquid nitrogen and thawed in lysis buffer: 100 mM Tris-HCl, pH 8, 150 mM of NaCl, 2 mM EDTA pH 8, 1 mM MgCl 2 , 1% Nonidet P-40 (74385, Sigma-Aldrich) and 1 mM AEBSF (A8456, Sigma-Aldrich) supplemented with complete TM , EDTA-free Protease Inhibitor Cocktail (5056489001, Roche).…”

Interactive and noninteractive roles of histone H2B monoubiquitination and H3K36 methylation in the regulation of active gene transcription and control of plant growth and development

“…Because SDG8 does not possess the G-box, TGGGCC/T and TAATTA binding domain, it is tempting to speculate that it may act in a combination with other transcription factors that are able to bind G-box and/or FORC A motifs directly to regulate downstream genes. Finally, other processes may enable SDG8 recruitment, especially when considering its interaction with the RNA PolII, the zinc finger domain-containing H3K27 demethylase ELF6 or even H3K4me1 through its CW domain (Hoppmann et al, 2011;Yang et al, 2016;Liu & Huang, 2018).…”

State of the Art: trxG Factor Regulation of Post-embryonic Plant Development

“…SDG8 is the major H3K36me3 methyltransferase in Arabidopsis ( Yang et al, 2014 ) and is required for H3K36me3 deposition at the FLC locus ( Shafiq et al, 2014 ; Yang et al, 2014 ). SDG8 associates with components of the transcription machinery, including RNA Pol II and PAF1, as well as with the H3K27 demethylase EARLY FLOWERING 6 (ELF6) ( Yang et al, 2016 ). These physical associations ( Figure 2 ) couple removal of repressive histone marks with deposition of active marks and transcription initiation/elongation to sustain high levels of FLC expression.…”