Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis

TL;DR: Short genomic fragments, known as Polycomb response elements (PREs), that direct Polycomb repressive complex 2 (PRC2) placement at developmental genes regulated by silencing in Arabidopsis thaliana are defined.

Abstract: Disruption of gene silencing by Polycomb protein complexes leads to homeotic transformations and altered developmental-phase identity in plants. Here we define short genomic fragments, known as Polycomb response elements (PREs), that direct Polycomb repressive complex 2 (PRC2) placement at developmental genes regulated by silencing in Arabidopsis thaliana. We identify transcription factor families that bind to these PREs, colocalize with PRC2 on chromatin, physically interact with and recruit PRC2, and are required for PRC2-mediated gene silencing in vivo. Two of the cis sequence motifs enriched in the PREs are cognate binding sites for the identified transcription factors and are necessary and sufficient for PRE activity. Thus PRC2 recruitment in Arabidopsis relies in large part on binding of trans-acting factors to cis-localized DNA sequence motifs.

Summary

Introduction

• Here the authors define short genomic 29 fragments, Polycomb Response Elements (PREs), that direct Polycomb Repressive 30 Complex 2 (PRC2) placement at developmental genes regulated by silencing in 31 Arabidopsis.
• To determine which sequence-specific binding proteins associate with the five functional PREs, the authors 72 performed high-throughput DNA binding assays using a library of 1956 Arabidopsis TFs19.
• R.A. performed the motif analysis, C.H. contributed to functional PRE analysis and R.J. and M.S. 185 to transcription factor/PRC2 interaction tests.
• Shown are mean ± SEM of three ChIP 367 experiments (red dots).

Test of PRE activity.

• (a) Construct to test ability of candidate PREs or control DNA fragments (NC) to recruit PRC2 and H3K27me3.
• Below: regions tested: PRE or a distal site (Dist).
• (b) H3K27me3/H3 abundance (top) and occupancy of PRC2 components MSI1 and EMF2 assessed by ChIP-qPCR.
• (d) Fluorometric assay of beta-glucuronidase (GUS) activity of 15 independent transformants in the wild type (WT) (top) or a PRC2 mutant (clf-28) .
• Violin plot of GUS activity in PREs (left) or negative controls : range, median= white circle, mean= white line, lower to higher quartile= vertical black line.

GA repeat and telobox motifs are bound by class I BPC and C1-2iD Zn Finger transcription factors.

• (a) Electrophoretic mobility shift assay to test association of BPC1 with the GA repeats or with mutated versions thereof.
• WT, M1 and M2 were also used as cold competitors.
• This, combined with the presence of two GA repeats in the tested DNA fragment, may explain why multiple shifted bands are observed when the protein is complexed with the DNA.
• (b) Motif-based yeast one hybrid screen identified members of the C1-2iD family of Zn-finger TFs as telobox binding transcription factors.
• The thin white line indicates where the plate image was cut.

FIE, AZF1, BPC1 and H3K27me3 ChIPseq analysis.

• (a, b) Principal component analysis (PCA) of RPM-normalized ChIP and input DNA reads for narrow (a) and broad (b) peak calling.
• (d) Enrichment (p-values, converted from the Z-scores of the motif enrichment calculations using a normal distribution) and frequency (%) of PRE cis motifs under FIE-bound peaks.
• (e) Functional classification of PRC2 (FIE), C1-2iD ZnF (AZF1) and class I BPC (BPC1) peak associated genes.
• Enrichment of Gene Ontology terms (FDR<10-5 in at least one of the datasets) for the genes associated with FIE, BPC1 and AZF1 peaks (Q<10-10).
• Enriched GO terms include regulation of transcription, postembryonic development (reproductive development, shoot development, gynoecium development) and hormone response.

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Cis and trans determinants of epigenetic silencing by polycomb repressive complex 2 in arabidopsis" ?

Xiao et al. this paper investigated the determinants of epigenetic silencing by polycomb repressive complex 2 in Arabidopsis. 

Q2. What is the GO term for clfR?

Enriched GO terms include regulation of transcription, postembryonic development (reproductive development, shoot development, gynoecium development) and hormone response. 

Q3. What is the role of alanine zippers in the telobox?

Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus. 

Q4. What are the components of the PRC2 complex?

Complex components: one of two SET domain methyltransferases (CURLY LEAF (CLF) or SWINGER (SWN)), one of two VEFS domain proteins (EMBRYONIC FLOWER2 (EMF2) or VERNALIZATION2 (VRN2)), a WD40 domain protein that can recognize H3K27me3 (FERTILIZATION INPENDPENT ENDOSPERM (FIE)) and a histone binding protein (MSI1)3.2(a) 

Q5. How many genes were considered high confidence?

132 of the 851 genes were significantly upregulated in prc2 mutants15,33 or strongly developmentally regulated48 and thus considered high confidence PRC2 regulated genes. 

Q6. What is the phenotype of the gus activity in the wild type?

(e) Herbicide resistance (survival rate) conferred by the BAR gene product in n=60 independent T1 plants in the wild-type (top) or in the prc2 (clf-28) mutant (bottom) background. 

Q7. What is the effect of knockdown on the class The authorBPC TFs?

Light green shading highlights the class The authorBPC TFs and light purple shading the C1-2iD Zn-finger proteins targeted for knockdown. 

Q8. What is the phenotype of the clfR mutant?

(a) Leaf curling (inset) and flowering time in wild type (WT), the weak clfR mutant, class The authorBPC knockdown in clfR, C1-2iD ZnF knock down in clfR and in clf-50 RNA null mutant, bar = 1cm. 

Q9. What is the qRT-PCR effect of the TFs?

(c) Simultaneous knockdown of Class The authorand C1-2iD C2H2 Zn TFs by RNAi (BPC + ZnFKD) in the wild type tested as described in (a, b) in 2 independent lines. 

Q10. What is the significance of the test?

P -value (one-tailed Mann– Whitney U test): ns, not significant; P> 0.07; * P<0.05; ** P<0.01; *** P<0.001 relative to NC_1.3Physical interaction of PRE-binding transcription factors with PRC2. 

Q11. What is the significant term of the GO term of the clfR?

The majority of the significant BPC1 and AZF1 target gene Gene Ontology terms are also significant Gene Ontology terms of FIE targets. 

Q12. What is the mean SEM of two independent TF lines?

d) Leaf curling in wild type (WT), two independent lines (L1 and L2) of 35S:BPC1 clfR and of 35S:AZF1 clfR and the hypomorph clfR mutant. 

Q13. What is the phenotype of the clfR TFs?

(b) Leaf curling phenotype of independent BPCKD clfR (top) and ZnFKD clfR (bottom) transgenic lines, bar = 1cm. (c) Phenotype quantification of genotypes in (b). 

Q14. What is the p-value of the cis motifs?

(d) Enrichment (p-values, converted from the Z-scores of the motif enrichment calculations using a normal distribution) and frequency (%) of PRE cis motifs under FIE-bound peaks. 

Q15. How many genes were identified in the Arabidopsis sporophyte?

The authors identified 1504 genomic regions marked by at least 3 of the following: H3K27me3, FIE, CLF or EMF115-17 (CLF ChIP-chip data: GSE7065) and linked these to 851 genes as previously described23.