Cell division in the shoot apical meristem is a trigger for miR156 decline and vegetative phase transition in Arabidopsis

What determines the rate at which a multicellular organism matures is a fundamental question in biology. In plants, the decline of miR156 with age serves as an intrinsic, evolutionarily conserved timer for the juvenile-to-adult phase transition. However, the way in which age regulates miR156 abundance is poorly understood. Here, we show that the rate of decline in miR156 is correlated with developmental age rather than chronological age. Mechanistically, we found that cell division in the apical meristem is a trigger for miR156 decline. The transcriptional activity of MIR156 genes is gradually attenuated by the deposition of the repressive histone mark H3K27me3 along with cell division. Our findings thus provide a plausible explanation of why the maturation program of a multicellular organism is unidirectional and irreversible under normal growth conditions and suggest that cell quiescence is the fountain of youth in plants.

AGO1 regulates major satellite transcripts and H3K9me3 distribution at pericentromeric regions in mESCs

In the past years, several studies reported nuclear roles for the Argonaute (AGO) proteins associating them with transcriptional activation or repression, alternative splicing and, chromatin organization. However, as most of these experiments have been conducted in human cancer cell lines, the nuclear functions of the AGO proteins in mouse early embryonic development still remains elusive. In this study, we investigated possible nuclear functions of the AGO proteins in mouse Embryonic Stem Cells (mESCs). By biochemical assays, we observed that AGO1 and AGO2 are present in a small fraction in the nucleus and even less on chromatin in mESCs. To profile the nuclear interactome of the AGO proteins, we performed immunoprecipitation followed by Mass Spectrometry and identified three novel nuclear interactors for AGO1, namely DNMT3a, HP1a;, and ATRX. These interactors are well-known proteins involved in the establishment and maintenance of heterochromatin at pericentromeric regions. Indeed, upon depletion of Ago1, we observed a specific redistribution of the heterochromatin protein HP1a; and the repressive histone mark H3K9me3, away from pericentromeric regions. Furthermore, these regions are characterized by AT-rich tandem repeats known as major satellite sequences. We demonstrated that major satellite transcripts are strongly upregulated in Ago1_KO mESCs. Interestingly, this phenotype was not caused by the loss of genome integrity at pericentromeres, as these could still form normally in Ago1_KO mESCs. Lastly, we showed that specific microRNAs loaded in AGO1, regulate the expression of the major satellite transcripts. Overall, our results demonstrate for the first time a novel role for AGO1 in regulating major satellite transcripts and localization of HP1a; and H3K9me3 at pericentromeres in mESCs.

Histone H3K27 methylation perturbs transcriptional robustness and underpins dispensability of highly conserved genes in fungi

Epigenetic modifications are key regulators of gene expression and underpin genome integrity. Yet, how epigenetic changes affect the evolution and transcriptional robustness of genes remains largely unknown. Here, we show how the repressive histone mark H3K27me3 influences the trajectory of highly conserved genes in fungi. We first performed transcriptomic profiling on closely related species of the plant pathogen Fusarium graminearum species complex. We determined transcriptional responsiveness of genes across environmental conditions to determine expression robustness. To infer evolutionary conservation of coding sequences, we used a comparative genomics framework of 23 species across the Fusarium genus. We integrated histone methylation data from three Fusarium species across the phylogenetic breadth of the genus. Gene expression variation is negatively correlated with gene conservation confirming that highly conserved genes show higher expression robustness. Furthermore, we show that highly conserved genes marked by H3K27me3 deviate from the typical housekeeping gene archetype. Compared to the genomic background, H3K27me3 marked genes encode smaller proteins, exhibit lower GC content, weaker codon usage bias, higher levels of hydrophobicity and are enriched for functions related to regulation and membrane transport. The evolutionary age of conserved genes with H3K27me3 histone marks falls typically within the origins of the Fusarium genus. We show that highly conserved genes marked by H3K27me3 are more likely to be dispensable for survival. Lastly, we show that conserved genes exposed to repressive H3K27me3 marks across distantly related fungi predict transcriptional perturbation at the microevolutionary scale in Fusarium fungi. In conclusion, we establish how repressive histone marks determine the evolutionary fate of highly conserved genes across evolutionary timescales.

Epigenetic effects induced by the ectopic expression of Pax7 in 3T3-L1

Although skeletal muscle cells and adipocytes are derived from the same mesoderm, they do not transdifferentiate in vivo and are strictly distinct at the level of gene expression. To elucidate some of the regulatory mechanisms underlying this strict distinction, Pax7, a myogenic factor, was ectopically expressed in 3T3-L1 adipose progenitor cells to perturb their adipocyte differentiation potential. Transcriptome analysis showed that ectopic expression of Pax7 repressed the expression of some adipocyte genes and induced expression of some skeletal muscle cell genes. We next profiled the epigenomic state altered by Pax7 expression using H3K27ac, an activating histone mark, and H3K27me3, a repressive histone mark, as indicators. Our results show that ectopic expression of Pax7 did not result in the formation of H3K27ac at loci of skeletal muscle-related genes, but instead resulted in the formation of H3K27me3 at adipocyte-related gene loci. These findings suggest that the primary function of ectopic Pax7 expression is the formation of H3K27me3, and muscle gene expression results from secondary regulation.

Myeloid Ezh2 Deficiency Limits Atherosclerosis Development

Macrophages define a key component of immune cells present in atherosclerotic lesions and are central regulators of the disease. Since epigenetic processes are important in controlling macrophage function, interfering with epigenetic pathways in macrophages might be a novel approach to combat atherosclerosis. Histone H3K27 trimethylation is a repressive histone mark catalyzed by polycomb repressive complex with EZH2 as the catalytic subunit. EZH2 is described to increase macrophage inflammatory responses by supressing the suppressor of cytokine signaling, Socs3. We previously showed that myeloid deletion of Kdm6b, an enzymes that in contrast to EZH2 removes repressive histone H3K27me3 marks, results in advanced atherosclerosis. Because of its opposing function and importance of EZH2 in macrophage inflammatory responses, we here studied the role of myeloid EZH2 in atherosclerosis. A myeloid-specific Ezh2 deficient mouse strain (Ezh2del) was generated (LysM-cre+ x Ezh2fl/fl) and bone marrow from Ezh2del or Ezh2wt mice was transplanted to Ldlr-/- mice which were fed a high fat diet for 9 weeks to study atherosclerosis. Atherosclerotic lesion size was significantly decreased in Ezh2del transplanted mice compared to control. The percentage of macrophages in the atherosclerotic lesion was similar, however neutrophil numbers were lower in Ezh2del transplanted mice. Correspondingly, the migratory capacity of neutrophils was decreased in Ezh2del mice. Moreover, peritoneal Ezh2del foam cells showed a reduction in the inflammatory response with reduced production of nitric oxide, IL-6 and IL-12. In Conclusion, myeloid Ezh2 deficiency impairs neutrophil migration and reduces macrophage foam cell inflammatory responses, both contributing to reduced atherosclerosis.

Allele-specific gene regulation by KDM6A

SUMMARYKDM6A demethylates the repressive histone mark H3K27me3 and thus plays an important role in developmental gene regulation. KDM6A expression is female-biased due to escape from X inactivation, suggesting that this protein may play a role in sex differences. Here, we report that maternal and paternal alleles of a subset of mouse genes are differentially regulated by KDM6A. Knockouts of Kdm6a in male and female embryonic stem cells derived from F1 hybrid mice from reciprocal interspecific crosses resulted in preferential downregulation of maternal alleles of a number of genes implicated in development. Moreover, the majority of these genes exhibited a maternal allele expression bias, which was observed in both reciprocal crosses. Promoters of genes downregulated on maternal but not paternal alleles demonstrated a loss of chromatin accessibility, while the expected increase in H3K27me3 levels occurred only at promoters of genes downregulated on paternal but not maternal alleles. These results illustrate parent-of-origin mechanisms of gene regulation by KDM6A, consistent with histone demethylation-dependent and -independent activities.

Premature termination codons in theDMDgene cause reduced local mRNA synthesis

Duchenne muscular dystrophy (DMD) is caused by mutations in theDMDgene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD transcript levels in DMD patient and animal model muscles when PTC are present. Nonsense-mediated decay (NMD) has been suggested to be responsible for the observed reduction, but there is no experimental evidence supporting this claim. In this study, we aimed to investigate the mechanism responsible for the drop inDMDexpression levels in the presence of PTC. We observed that the inhibition of NMD does not normalizeDMDgene expression in DMD. Additionally, in situ hybridization showed that DMD messenger RNA primarily localizes in the nuclear compartment, confirming that a cytoplasmic mechanism like NMD indeed cannot be responsible for the observed reduction. Sequencing of nascent RNA to exploreDMDtranscription dynamics revealed a lower rate ofDMDtranscription in patient-derived myotubes compared to healthy controls, suggesting a transcriptional mechanism involved in reduced DMD transcript levels. Chromatin immunoprecipitation in muscle showed increased levels of the repressive histone mark H3K9me3 inmdxmice compared to wild-type mice, indicating a chromatin conformation less prone to transcription inmdxmice. In line with this finding, treatment with the histone deacetylase inhibitor givinostat caused a significant increase in DMD transcript expression inmdxmice. Overall, our findings show that transcription dynamics across theDMDlocus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process.

Exogenously overexpressed intronic long noncoding RNAs activate host gene expression by affecting histone modification in Arabidopsis

Involvement of long non-coding RNAs (lncRNAs) in the regulation of gene expression in cis has been well studied in eukaryotes but relatively little is known whether and how lncRNAs affect gene expression in tans. In Arabidopsis thaliana, COLDAIR, a previously reported lncRNA, is produced from the first intron of FLOWERING LOCUS C (FLC), which encodes a repressor of flowering time. Our results indicated that the exogenously overexpressed COLDAIR enhances the expression of FLC in trans, resulting in a late-flowering phenotype. In 35S-COLDAIR lines, the enhanced expression of FLC is correlated with the down-regulation of the repressive histone mark H3K27me3 and with the up-regulation of the active histone mark H3K4me3 at the FLC chromatin. Furthermore, we demonstrated that overexpression of intronic lncRNAs from several other H3K27me3-enriched MADS-box genes also activates the expression of their host genes. This study suggests that the involvement of overexpressed intronic lncRNAs in gene activation may be conserved in H3K27me3-enriched genes in eukaryotes.

Functional loss of a non-canonical BCOR-PRC1.1 complex accelerates SHH-driven medulloblastoma formation

Medulloblastoma is a childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH)-subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs) and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional co-repressor BCOR are recurrent and highly enriched in SHH-medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a germline genetically engineered mouse model to delete exons 9/10 of Bcor (BcorΔE9-10) in GNPs during development. This leads to reduced expression of C-terminally truncated BCOR (BCORΔE9-10). While BcorΔE9-10 alone did not promote tumorigenesis or affect GNP differentiation, BcorΔE9-10 combined with loss of the SHH-receptor gene Ptch1 resulted in highly penetrant medulloblastomas. In Ptch1+/-;BcorΔE9-10 tumors, the growth factor gene Igf2 was aberrantly upregulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/-;BcorΔE9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.