In plants, flowering time is controlled by environmental signals such as day-length and temperature, which regulate the floral pathway integrators, including FLOWERING LOCUS T (FT), by genetic and ...epigenetic mechanisms. Here, we identify an H3K27me3 demethylase, JUMONJI 13 (JMJ13), which regulates flowering time in Arabidopsis. Structural characterization of the JMJ13 catalytic domain in complex with its substrate peptide reveals that H3K27me3 is specifically recognized through hydrogen bonding and hydrophobic interactions. Under short-day conditions, the jmj13 mutant flowers early and has increased FT expression at high temperatures, but not at low temperatures. In contrast, jmj13 flowers early in long-day conditions regardless of temperature. Long-day condition and higher temperature induce the expression of JMJ13 and increase accumulation of JMJ13. Together, our data suggest that the H3K27me3 demethylase JMJ13 acts as a temperature- and photoperiod-dependent flowering repressor.
Transposable elements (TEs) are mobile genetic elements that can proliferate in their host genomes. Because of their robust amplification, TEs have long been considered 'selfish DNA', harmful ...insertions that can threaten host genome integrity. The idea of TEs as junk DNA comes from analysis of epigenetic silencing of their mobility in plants and animals. This idea contrasts with McClintock's characterization of TEs as 'controlling elements'. Emerging studies on the regulatory functions of TEs in plant genomes have updated McClintock's characterization, indicating exaptation of TEs for genetic regulation. In this review, we summarize recent progress in TE silencing, particularly in Arabidopsis and rice, and show that TEs provide an abundant, natural source of regulation for the host genome.
Despite extensive studies on mammalian neurogenesis, its post-transcriptional regulation remains under-explored. Here we report that neural-specific inactivation of two murine post-transcriptional ...regulators, Pumilio 1 (Pum1) and Pum2, severely reduced the number of neural stem cells (NSCs) in the postnatal dentate gyrus (DG), drastically increased perinatal apoptosis, altered DG cell composition, and impaired learning and memory. Consistently, the mutant DG neurospheres generated fewer NSCs with defects in proliferation, survival, and differentiation, supporting a major role of Pum1 and Pum2 in hippocampal neurogenesis and function. Cross-linking immunoprecipitation revealed that Pum1 and Pum2 bind to thousands of mRNAs, with at least 694 common targets in multiple neurogenic pathways. Depleting Pum1 and/or Pum2 did not change the abundance of most target mRNAs but up-regulated their proteins, indicating that Pum1 and Pum2 regulate the translation of their target mRNAs. Moreover, Pum1 and Pum2 display RNA-dependent interaction with fragile X mental retardation protein (FMRP) and bind to one another's mRNA. This indicates that Pum proteins might form collaborative networks with FMRP and possibly other post-transcriptional regulators to regulate neurogenesis.
Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using ...single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone
. Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.
Transposable elements (TEs) and repetitive sequences make up over 35% of the rice (Oryza sativa) genome. The host regulates the activity of different TEs by different epigenetic mechanisms, including ...DNA methylation, histone H3K9 methylation, and histone H3K4 demethylation. TEs can also affect the expression of host genes. For example, miniature inverted repeat TEs (MITEs), dispersed high copy-number DNA TEs, can influence the expression of nearby genes. In plants, 24-nt small interfering RNAs (siRNAs) are mainly derived from repeats and TEs. However, the extent to which TEs, particularly MITEs associated with 24-nt siRNAs, affect gene expression remains elusive. Here, we show that the rice Dicer-like 3 homolog OsDCL3a is primarily responsible for 24-nt siRNA processing. Impairing OsDCL3a expression by RNA interference caused phenotypes affecting important agricultural traits; these phenotypes include dwarfism, larger flag leaf angle, and fewer secondary branches. We used small RNA deep sequencing to identify 535,054 24-nt siRNA clusters. Of these clusters, ∼82% were OsDCL3a-dependent and showed significant enrichment of MITEs. Reduction of OsDCL3a function reduced the 24-nt siRNAs predominantly from MITEs and elevated expression of nearby genes. OsDCL3a directly targets genes involved in gibberellin and brassinosteroid homeostasis; OsDCL3a deficiency may affect these genes, thus causing the phenotypes of dwarfism and enlarged flag leaf angle. Our work identifies OsDCL3a-dependent 24-nt siRNAs derived from MITEs as broadly functioning regulators for fine-tuning gene expression, which may reflect a conserved epigenetic mechanism in higher plants with genomes rich in dispersed repeats or TEs.
Protein ubiquitination is involved in most cellular processes. In Arabidopsis (Arabidopsis thaliana), ubiquitin-mediated protein degradation regulates the stability of key components of the circadian ...clock feedback loops and the photoperiodic flowering pathway. Here, we identified two ubiquitin-specific proteases, UBP12 and UBP13, involved in circadian clock and photoperiodic flowering regulation. Double mutants of ubp12 and ubp13 display pleiotropic phenotypes, including early flowering and short periodicity of circadian rhythms. In ubp12 ubp13 double mutants, CONSTANS (CO) transcript rises earlier than that of wild-type plants during the day, which leads to increased expression of FLOWERING LOCUS T. This, and analysis of ubp12 co mutants, indicates that UBP12 and UBP13 regulate photoperiodic flowering through a CO-dependent pathway. In addition, UBP12 and UBP13 regulate the circadian rhythm of clock genes, including LATE ELONGATED HYPOCOTYL, CIRCADIAN CLOCK ASSOCIATED1, and TIMING OF CAB EXPRESSION1. Furthermore, UBP12 and UBP13 are circadian controlled. Therefore, our work reveals a role for two deubiquitinases, UBP12 and UBP13, in the control of the circadian clock and photoperiodic flowering, which extends our understanding of ubiquitin in daylength measurement in higher plants.
Transposable elements (TEs) are ubiquitously present in plant genomes and often account for significant fractions of the nuclear DNA. For example, roughly 40% of the rice genome consists of TEs, many ...of which are retrotransposons, including 14% LTR- and ∼1% non-LTR retrotransposons. Despite their wide distribution and abundance, very few TEs have been found to be transpositional, indicating that TE activities may be tightly controlled by the host genome to minimize the potentially mutagenic effects associated with active transposition. Consistent with this notion, a growing body of evidence suggests that epigenetic silencing pathways such as DNA methylation, RNA interference, and H3K9me2 function collectively to repress TE activity at the transcriptional and posttranscriptional levels. It is not yet clear, however, whether the removal of histone modifications associated with active transcription is also involved in TE silencing. Here, we show that the rice protein JMJ703 is an active H3K4-specific demethylase required for TEs silencing. Impaired JMJ703 activity led to elevated levels of H3K4me3, the misregulation of numerous endogenous genes, and the transpositional reactivation of two families of non-LTR retrotransposons. Interestingly, loss of JMJ703 did not affect TEs (such as Tos17) previously found to be silenced by other epigenetic pathways. These results indicate that the removal of active histone modifications is involved in TE silencing and that different subsets of TEs may be regulated by distinct epigenetic pathways.
DNA methylation and histone H3 Lys 9 dimethylation (H3K9me2) are important epigenetic repression marks for silencing transposons in heterochromatin and for regulating gene expression. However, the ...mechanistic relationship to other repressive marks, such as histone H3 Lys 27 trimethylation (H3K27me3) is unclear. FERTILIZATION-INDEPENDENT ENDOSPERM1 (FIE1) encodes an Esc-like core component of the Polycomb repressive complex 2, which is involved in H3K27me3-mediated gene repression. Here, we identify a gain-of-function epi-allele (Epi-df) of rice (Oryza sativa) FIE1; this allele causes a dwarf stature and various floral defects that are inherited in a dominant fashion. We found that Epi-df has no changes in nucleotide sequence but is hypomethylated in the 5′ region of FIE1 and has reduced H3K9me2 and increased H3K4me3. In Epi-df, FIE1 was ectopically expressed and its imprinting was disrupted. FIE1 interacted with rice Enhancer of Zeste homologs, consistent with its role in H3K27me3 repression. Ectopic expression of FIE1 in Epi-df resulted in alteration of H3K27me3 levels in hundreds of genes. In summary, this work identifies an epi-allele involved in H3K27me3-mediated gene repression that itself is highly regulated by DNA methylation and histone H3K9me2, thereby shedding light on the link between DNA methylation and histone methylation, the two important epigenetic marks regulating rice development.
Argonaute/Piwi proteins can regulate gene expression via RNA degradation and translational regulation using small RNAs as guides. They also promote the establishment of suppressive epigenetic marks ...on repeat sequences in diverse organisms. In mice, the nuclear Piwi protein MIWI2 and Piwi‐interacting RNAs (piRNAs) are required for DNA methylation of retrotransposon sequences and some other sequences. However, its underlying molecular mechanisms remain unclear. Here, we show that piRNA‐dependent regions are transcribed at the stage when piRNA‐mediated DNA methylation takes place. MIWI2 specifically interacts with RNAs from these regions. In addition, we generated mice with deletion of a retrotransposon sequence either in a representative piRNA‐dependent region or in a piRNA cluster. Both deleted regions were required for the establishment of DNA methylation of the piRNA‐dependent region, indicating that piRNAs determine the target specificity of MIWI2‐mediated DNA methylation. Our results indicate that MIWI2 affects the chromatin state through base‐pairing between piRNAs and nascent RNAs, as observed in other organisms possessing small RNA‐mediated epigenetic regulation.
Synopsis
The nuclear Piwi protein MIWI2 regulates DNA methylation of specific genomic regions in murine spermatogenic cells. Base‐pairing between MIWI2‐bound piRNAs and nascent RNA determines the target specificity of piRNA‐dependent DNA methylation.
piRNA‐dependent regions are transcribed at the developmental stage when piRNA‐mediated DNA methylation occurs.
The MIWI2‐piRNA complex specifically interacts with RNAs transcribed from piRNA‐dependent regions.
Removing a retrotransposon sequence either in a piRNA‐dependent region or a piRNA cluster abolishes DNA methylation of the piRNA‐dependent region.
In Mili−/− prospermatogonia deficient in producing MIWI2‐bound piRNAs, de novoDNA methylation is compromised in many MIWI2‐dependent regions.
The piRNA pathway represses retrotransposons during early development. Base‐pairing between nascent RNA and piRNAs bound by mouse Piwi protein MIWI2 determines the specificity of suppressive DNA methylation.
RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) counteracts Polycomb-mediated gene silencing by removing methyl groups from trimethylated histone H3 lysine 27 (H3K27me3) in hundreds of ...genes in Arabidopsis thaliana. Here we show that REF6 function and genome-wide targeting require its four Cys2His2 zinc fingers, which directly recognize a CTCTGYTY motif. Motifs bound by REF6 tend to cluster and reside in loci with active chromatin states. Furthermore, REF6 targets CUP-SHAPED COTYLEDON 1 (CUC1), which harbors CTCTGYTY motifs, to modulate H3K27me3 levels and activate CUC1 expression. Loss of REF6 causes CUC1 repression and defects in cotyledon separation. In contrast, REF6 does not bind CUC2, encoding a close homolog of CUC1, which lacks the CTCTGYTY motif. Collectively, these results identify a new targeting mechanism of an H3K27 demethylase to counteract Polycomb-mediated gene silencing that regulates plant development, including organ boundary formation.
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