Physical coupling of activation and derepression activities to maintain an active transcriptional state at FLC
Wednesday, 2016/08/17 | 08:00:24
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Hongchun Yang, Martin Howard, and Caroline Dean SignificanceHow chromatin influences transcription is a major question in biology. Chromatin states and transcriptional activity are correlated, but the interconnections and feedbacks in chromatin regulation that lead to quantitative transcriptional regulation are poorly understood. Previous mathematical modeling has shown the importance of dynamic, opposing histone modifications to enable maintenance of one chromatin state through many cell divisions, yet retain the ability to switch from one state to the other. Here, we validate a prediction of that modeling—physical association between the histone modifiers: the methyltransferase specific for the active state with the demethylase specific for the silent state. Such physical coupling of activities regulating opposing chromatin states is likely to be a general feature in chromatin-based transcriptional regulation. AbstractEstablishment 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.
See: http://www.pnas.org/content/113/33/9369.full PNAS August 16 2016; vol.113; no.33: 9369–9374
Fig. 1. SDG8 interacts with PAF1 complex and ELF6. (A) SDG8 was enriched after GFP pull-down. SDG8 was not detected in the input sample. FRI seedlings without any transgene were used for the negative control. Empty beads were used in the CTL IP. (B) SDG8-associated proteins. The protein material eluted from A was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total number of protein peptides is listed. (C) SDG8 interacts with ELF6 in a N. benthamiana transient assay. SDG8:Flag and ELF6:GFP were coinfiltrated into N. benthamiana leaves. Protein was extracted and GFP pull-down performed. Infiltrated SDG8:Flag was used as a negative control. The arrow indicates the SDG8:Flag. (D) ELF6 and Pol II were copurified with SDG8 in vivo. Pol II was detected using the 8WG16 anti-Pol II antibody. ELF6:GFP/elf6 and SDG8:Flag/sdg8 transgenic lines were generated and crossed together, and the experiment was performed on F2 seedlings. |
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