Global epigenomic analysis indicates that Epialleles contribute to Allele-specific expression via Allele-specific histone modifications in hybrid rice

Background

For heterozygous genes, alleles on the chromatin from two different parents exhibit histone modification variations known as allele-specific histone modifications (ASHMs). The regulation of allele-specific gene expression (ASE) by ASHMs has been reported in animals. However, to date, the regulation of ASE by ASHM genes remains poorly understood in higher plants.

Results

We used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to investigate the global ASHM profiles of trimethylation on histone H3 lysine 27 (H3K27me3) and histone H3 lysine 36 (H3K36me3) in two rice F1 hybrids. A total of 522 to 550 allele-specific H3K27me3 genes and 428 to 494 allele-specific H3K36me3 genes were detected in GL × 93-11 and GL × TQ, accounting for 11.09% and 26.13% of the total analyzed genes, respectively. The epialleles between parents were highly related to ASHMs. Further analysis indicated that 52.48% to 70.40% of the epialleles were faithfully inherited by the F1 hybrid and contributed to 33.18% to 46.55% of the ASHM genes. Importantly, 66.67% to 82.69% of monoallelic expression genes contained the H3K36me3 modification. Further studies demonstrated a significant positive correlation of ASE with allele-specific H3K36me3 but not with H3K27me3, indicating that ASHM-H3K36me3 primarily regulates ASE in this study.

Conclusions

Our results demonstrate that epialleles from parents can be inherited by the F1 to produce ASHMs in the F1 hybrid. Our findings indicate that ASHM-H3K36me3, rather than H3K27me3, mainly regulates ASE in hybrid rice.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1454-z) contains supplementary material, which is available to authorized users.     

Molecular recognition of H3/H4 histone tails by the tudor domains of JMJD2A: a comparative molecular dynamics simulations study

Background

Histone demethylase, JMJD2A, specifically recognizes and binds to methylated lysine residues at histone H3 and H4 tails (especially trimethylated H3K4 (H3K4me3), trimethylated H3K9 (H3K9me3) and di,trimethylated H4K20 (H4K20me2, H4K20me3)) via its tandem tudor domains. Crystal structures of JMJD2A-tudor binding to H3K4me3 and H4K20me3 peptides are available whereas the others are not. Complete picture of the recognition of the four histone peptides by the tandem tudor domains yet remains to be clarified.

Methodology/Principal Findings

We report a detailed molecular dynamics simulation and binding energy analysis of the recognition of JMJD2A-tudor with four different histone tails. 25 ns fully unrestrained molecular dynamics simulations are carried out for each of the bound and free structures. We investigate the important hydrogen bonds and electrostatic interactions between the tudor domains and the peptide molecules and identify the critical residues that stabilize the complexes. Our binding free energy calculations show that H4K20me2 and H3K9me3 peptides have the highest and lowest affinity to JMJD2A-tudor, respectively. We also show that H4K20me2 peptide adopts the same binding mode with H4K20me3 peptide, and H3K9me3 peptide adopts the same binding mode with H3K4me3 peptide. Decomposition of the enthalpic and the entropic contributions to the binding free energies indicate that the recognition of the histone peptides is mainly driven by favourable van der Waals interactions. Residue decomposition of the binding free energies with backbone and side chain contributions as well as their energetic constituents identify the hotspots in the binding interface of the structures.

Conclusion

Energetic investigations of the four complexes suggest that many of the residues involved in the interactions are common. However, we found two receptor residues that were related to selective binding of the H3 and H4 ligands. Modifications or mutations on one of these residues can selectively alter the recognition of the H3 tails or the H4 tails.     

KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

Background

Pluripotency of embryonic stem (ES) cells is controlled in part by chromatin-modifying factors that regulate histone H3 lysine 4 (H3K4) methylation. However, it remains unclear how H3K4 demethylation contributes to ES cell function.

Results

Here, we show that KDM5B, which demethylates lysine 4 of histone H3, co-localizes with H3K4me3 near promoters and enhancers of active genes in ES cells; its depletion leads to spreading of H3K4 methylation into gene bodies and enhancer shores, indicating that KDM5B functions to focus H3K4 methylation at promoters and enhancers. Spreading of H3K4 methylation to gene bodies and enhancer shores is linked to defects in gene expression programs and enhancer activity, respectively, during self-renewal and differentiation of KDM5B-depleted ES cells. KDM5B critically regulates H3K4 methylation at bivalent genes during differentiation in the absence of LIF or Oct4. We also show that KDM5B and LSD1, another H3K4 demethylase, co-regulate H3K4 methylation at active promoters but they retain distinct roles in demethylating gene body regions and bivalent genes.

Conclusions

Our results provide global and functional insight into the role of KDM5B in regulating H3K4 methylation marks near promoters, gene bodies, and enhancers in ES cells and during differentiation.     

Natural variation of H3K27me3 distribution between two Arabidopsis accessions and its association with flanking transposable elements

Background

Histone H3 lysine 27 tri-methylation and lysine 9 di-methylation are independent repressive chromatin modifications in Arabidopsis thaliana. H3K27me3 is established and maintained by Polycomb repressive complexes whereas H3K9me2 is catalyzed by SUVH histone methyltransferases. Both modifications can spread to flanking regions after initialization and were shown to be mutually exclusive in Arabidopsis.

Results

We analyzed the extent of natural variation of H3K27me3 in the two accessions Landsberg erecta (Ler) and Columbia (Col) and their F1 hybrids. The majority of H3K27me3 target genes in Col were unchanged in Ler and F1 hybrids. A small number of Ler-specific targets were detected and confirmed. Consistent with a cis-regulatory mechanism for establishing H3K27me3, differential targets showed allele-specific H3K27me3 in hybrids. Five Ler-specific targets showed the active mark H3K4me3 in Col and for this group, differential H3K27me3 enrichment accorded to expression variation. On the other hand, the majority of Ler-specific targets were not expressed in Col, Ler or 17 other accessions. Instead of H3K27me3, the antagonistic mark H3K9me2 and other heterochromatic features were observed at these loci in Col. These loci were frequently flanked by transposable elements, which were often missing in the Ler genome assembly.

Conclusion

There is little variation in H3K27me3 occupancy within the species, although H3K27me3 targets were previously shown as overrepresented among differentially expressed genes. The existing variation in H3K27me3 seems mostly explained by flanking polymorphic transposable elements. These could nucleate heterochromatin, which then spreads into neighboring H3K27me3 genes, thus converting them to H3K9me2 targets.     

Ring-like distribution of constitutive heterochromatin in bovine senescent cells

Background

Cells that reach “Hayflick limit” of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining.

Methods

We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH).

Results

Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli.

Conclusions

Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.     

An High-Throughput In Vivo Screening System to Select H3K4-Specific Histone Demethylase Inhibitors

Background

Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes.

Methodology/Principal Findings

In order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which tests the effects of candidate small molecule inhibitors on a S.cerevisiae strain which requires Jhd2 demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we screened a library of 45 structurally different compounds designed as competitive inhibitors of α -ketoglutarate (α-KG) cofactor of the enzyme, and found that one of them inhibited Jhd2 activity in vitro and in vivo. The same compound effectively inhibits human Jumonji AT-Rich Interactive Domain (JARID) 1B and 1D in vitro and increases H3K4 tri-methylation in HeLa cell nuclear extracts (NEs). When added in vivo to HeLa cells, the compound leads to an increase of tri-methyl-H3K4 (H3K4me3) but does not affect H3K9 tri-methylation. We describe the cytostatic and toxic effects of the compound on HeLa cells at concentrations compatible with its inhibitory activity.

Conclusions/Significance

Our screening system is proved to be very useful in testing putative H3K4-specific HDM inhibitors for the capacity of acting in vivo without significantly altering the activity of other important 2-oxoglutarate oxygenases.     

Structural basis of the chromodomain of Cbx3 bound to methylated peptides from histone h1 and G9a

Background

HP1 proteins are highly conserved heterochromatin proteins, which have been identified to be structural adapters assembling a variety of macromolecular complexes involved in regulation of gene expression, chromatin remodeling and heterochromatin formation. Much evidence shows that HP1 proteins interact with numerous proteins including methylated histones, histone methyltransferases and so on. Cbx3 is one of the paralogues of HP1 proteins, which has been reported to specifically recognize trimethylated histone H3K9 mark, and a consensus binding motif has been defined for the Cbx3 chromodomain.

Methodology/Principal Findings

Here, we found that the Cbx3 chromodomain can bind to H1K26me2 and G9aK185me3 with comparable binding affinities compared to H3K9me3. We also determined the crystal structures of the human Cbx3 chromodomain in complex with dimethylated histone H1K26 and trimethylated G9aK185 peptides, respectively. The complex structures unveil that the Cbx3 chromodomain specifically bind methylated histone H1K26 and G9aK185 through a conserved mechanism.

Conclusions/Significance

The Cbx3 chromodomain binds with comparable affinities to all of the methylated H3K9, H1K26 and G9aK185 peptides. It is suggested that Cbx3 may regulate gene expression via recognizing both histones and non-histone proteins.     

Analysis of Jmjd6 cellular localization and testing for its involvement in histone demethylation

Background

Methylation of residues in histone tails is part of a network that regulates gene expression. JmjC domain containing proteins catalyze the oxidative removal of methyl groups on histone lysine residues. Here, we report studies to test the involvement of Jumonji domain-containing protein 6 (Jmjd6) in histone lysine demethylation. Jmjd6 has recently been shown to hydroxylate RNA splicing factors and is known to be essential for the differentiation of multiple tissues and cells during embryogenesis. However, there have been conflicting reports as to whether Jmjd6 is a histone-modifying enzyme.

Methodology/Principal Findings

Immunolocalization studies reveal that Jmjd6 is distributed throughout the nucleoplasm outside of regions containing heterochromatic DNA, with occasional localization in nucleoli. During mitosis, Jmjd6 is excluded from the nucleus and reappears in the telophase of the cell cycle. Western blot analyses confirmed that Jmjd6 forms homo-multimers of different molecular weights in the nucleus and cytoplasm. A comparison of mono-, di-, and tri-methylation states of H3K4, H3K9, H3K27, H3K36, and H4K20 histone residues in wildtype and Jmjd6-knockout cells indicate that Jmjd6 is not involved in the demethylation of these histone lysine residues. This is further supported by overexpression of enzymatically active and inactive forms of Jmjd6 and subsequent analysis of histone methylation patterns by immunocytochemistry and western blot analysis. Finally, treatment of cells with RNase A and DNase I indicate that Jmjd6 may preferentially associate with RNA/RNA complexes and less likely with chromatin.

Conclusions/Significance

Taken together, our results provide further evidence that Jmjd6 is unlikely to be involved in histone lysine demethylation. We confirmed that Jmjd6 forms multimers and showed that nuclear localization of the protein involves association with a nucleic acid matrix.     

Structural cooperativity in histone H3 tail modifications

Post-translational modifications of histone H3 tails have crucial roles in regulation of cellular processes. There is cross-regulation between the modifications of K4, K9, and K14 residues. The modifications on these residues drastically promote or inhibit each other. In this work, we studied the structural changes of the histone H3 tail originating from the three most important modifications; tri-methylation of K4 and K9, and acetylation of K14. We performed extensive molecular dynamics simulations of four types of H3 tails: (i) the unmodified H3 tail having no chemical modification on the residues, (ii) the tri-methylated lysine 4 and lysine 9 H3 tail (K4me3K9me3), (iii) the tri-methylated lysine 4 and acetylated lysine 14 H3 tail (K4me3K14ace), and (iv) tri-methylated lysine 9 and acetylated lysine 14 H3 tail (K9me3K14ace). Here, we report the effects of K4, K9, and K14 modifications on the backbone torsion angles and relate these changes to the recognition and binding of histone modifying enzymes. According to the Ramachandran plot analysis; (i) the dihedral angles of K4 residue are significantly affected by the addition of three methyl groups on this residue regardless of the second modification, (ii) the dihedral angle values of K9 residue are similarly altered majorly by the tri-methylation of K4 residue, (iii) different combinations of modifications (tri-methylation of K4 and K9, and acetylation of K14) have different influences on phi and psi values of K14 residue. Finally, we discuss the consequences of these results on the binding modes and specificity of the histone modifying enzymes such as DIM-5, GCN5, and JMJD2A.