sample-preparation-for-mass-spectrometry-based-identification-rna

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

University of Pennsylvania Perelman School of Medicine

In plants, multiple detached tissues are capable of forming a pluripotent cell mass, termed callus, when cultured on media containing appropriate plant hormones. Recent studies demonstrated that callus resembles the root-tip meristem, even if it is derived from aerial organs. This finding improves our understanding of the regeneration process of plant cells; however, the molecular mechanism that guides cells of different tissue types to form a callus still remains elusive. Here, we show that genome-wide reprogramming of histone H3 lysine 27 trimethylation (H3K27me3) is a critical step in the leaf-to-callus transition. The Polycomb Repressive Complex 2 (PRC2) is known to function in establishing H3K27me3. By analyzing callus formation of mutants corresponding to different histone modification pathways, we found that leaf blades and/or cotyledons of the PRC2 mutants curly leaf swinger (clf swn) and embryonic flower2 (emf2) were defective in callus formation. We identified the H3K27me3-covered loci in leaves and calli by a ChIP-chip assay, and we found that in the callus H3K27me3 levels decreased first at certain auxin-pathway genes. The levels were then increased at specific leaf genes but decreased at a number of root-regulatory genes. Changes in H3K27me3 levels were negatively correlated with expression levels of the corresponding genes. One possible role of PRC2-mediated H3K27me3 in the leaf-to-callus transition might relate to elimination of leaf features by silencing leaf-regulatory genes, as most leaf-preferentially expressed regulatory genes could not be silenced in the leaf explants of clf swn. In contrast to the leaf explants, the root explants of both clf swn and emf2 formed calli normally, possibly because the root-to-callus transition bypasses the leaf gene silencing process. Furthermore, our data show that PRC2-mediated H3K27me3 and H3K27 demethylation act in parallel in the reprogramming of H3K27me3 during the leaf-to-callus transition, suggesting a general mechanism for cell fate transition in plants.

Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues.

Polycomb group (PcG) proteins act in an evolutionarily conserved epigenetic pathway that regulates chromatin structures in plants and animals, repressing many developmentally important genes by modifying histones. PcG proteins can form at least two multiprotein complexes: Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2, respectively). The functions of Arabidopsis thaliana PRCs have been characterized in multiple stages of development and have diverse roles in response to environmental stimuli. Recently, the mechanism that precisely regulates Arabidopsis PcG activity was extensively studied. In this review, we summarize recent discoveries in the regulations of PcG at the three different layers: the recruitment of PRCs to specific target loci, the polyubiquitination and degradation of PRC2, and the antagonism of PRC2 activity by the Trithorax group proteins. Current knowledge indicates that the powerful activity of the PcG pathway is strictly controlled for specific silencing of target genes during plant development and in response to environmental stimuli.

Mechanisms guiding Polycomb activities during gene silencing in Arabidopsis thaliana.

Polycomb repressive complexes (PRCs) play crucial roles in transcriptional repression and developmental regulation in both plants and animals. In plants, depletion of different members of PRCs causes both overlapping and unique phenotypic defects. However, the underlying molecular mechanism determining the target specificity and functional diversity is not sufficiently characterized. Here, we quantitatively compared changes of tri-methylation at H3K27 in Arabidopsis mutants deprived of various key PRC components. We show that CURLY LEAF (CLF), a major catalytic subunit of PRC2, coordinates with different members of PRC1 in suppression of distinct plant developmental programs. We found that expression of flower development genes is repressed in seedlings preferentially via non-redundant role of CLF, which specifically associated with LIKE HETEROCHROMATIN PROTEIN1 (LHP1). In contrast, expression of embryo development genes is repressed by PRC1-catalytic core subunits AtBMI1 and AtRING1 in common with PRC2-catalytic enzymes CLF or SWINGER (SWN). This context-dependent role of CLF corresponds well with the change in H3K27me3 profiles, and is remarkably associated with differential co-occupancy of binding motifs of transcription factors (TFs), including MADS box and ABA-related factors. We propose that different combinations of PRC members distinctively regulate different developmental programs, and their target specificity is modulated by specific TFs.

Arabidopsis Flower and Embryo Developmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with Distinct Sets of Cis-regulatory Elements.

Interactions between noncoding RNAs and chromatin proteins play important roles in gene regulation, but the molecular details of most of these interactions are unknown. Using protein-RNA photocrosslinking and mass spectrometry on embryonic stem cell nuclei, we identified and mapped, at peptide resolution, the RNA-binding regions in ∼800 known and previously unknown RNA-binding proteins, many of which are transcriptional regulators and chromatin modifiers. In addition to known RNA-binding motifs, we detected several protein domains previously unknown to function in RNA recognition, as well as non-annotated and/or disordered regions, suggesting that many functional protein-RNA contacts remain unexplored. We identified RNA-binding regions in several chromatin regulators, including TET2, and validated their ability to bind RNA. Thus, proteomic identification of RNA-binding regions (RBR-ID) is a powerful tool to map protein-RNA interactions and will allow rational design of mutants to dissect their function at a mechanistic level.

High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells.

[This corrects the article DOI: 10.1371/journal.pgen.1005771.].

Correction: Arabidopsis Flower and Embryo Developmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with Distinct Sets of Cis-regulatory Elements.

A new technique called CUT&RUN can map the distribution of proteins on the genome with higher resolution and accuracy than existing approaches.

A cut above.

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.

Article	Year
Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues. PLoS genetics\| PubMed ID: 22927830	2012
Mechanisms guiding Polycomb activities during gene silencing in Arabidopsis thaliana. Frontiers in plant science\| PubMed ID: 24312106	2013
Arabidopsis Flower and Embryo Developmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with Distinct Sets of Cis-regulatory Elements... PLoS genetics\| PubMed ID: 26760036	2016
High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells. Molecular cell\| PubMed ID: 27768875	2016
Correction: Arabidopsis Flower and Embryo Developmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with Distinct Sets of Cis-regulat... PLoS genetics\| PubMed ID: 28122004	2017
A cut above. eLife\| PubMed ID: 28199181	2017
Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis. Nature genetics\| PubMed ID: 28825728	2017

Chongsheng He

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