The 19S proteasome regulates subtelomere silencing and facultative heterochromatin formation in fission yeast.

Accumulating evidence shows that non-proteolytic functions of the proteasome are as crucial as its well-known proteolytic function in regulating cellular activities. In our recent work, we showed that the 19S proteasome mediates the heterochromatin spreading of centromeric heterochromatin in non-proteolytic manner. However, the involvement of the proteasome in other heterochromatin regions remained largely unknown. In the present study, we investigated the non-proteolytic role of the 19S proteasome in subtelomere and facultative heterochromatin regions. Using the non-proteolytic mutant, rpt4-1, we show that the 19S proteasome is involved in regulating subtelomere silencing and facultative heterochromatin formation in fission yeast. In addition to this proteasome-related regulation, we also observed a distinct pathway that regulates subtelomere silencing and facultative heterochromatin formation through the Paf1 complex subunit, Leo1. Our comparison of the two pathways revealed a new group of heterochromatin domains that are regulated exclusively by the proteasome pathway. Taken together, our findings reveal that the proteasome is involved in the global regulation of facultative and constitutive heterochromatin.

Both H4K20 mono-methylation and H3K56 acetylation mark transcription-dependent histone turnover in fission yeast.

Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5' promoter and 3' termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNA production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast.

Human histone H3K79 methyltransferase DOT1L protein [corrected] binds actively transcribing RNA polymerase II to regulate gene expression.

Histone-modifying enzymes play a pivotal role in gene expression and repression. In human, DOT1L (Dot1-like) is the only known histone H3 lysine 79 methyltransferase. hDOT1L is associated with transcriptional activation, but the general mechanism connecting hDOT1L to active transcription remains largely unknown. Here, we report that hDOT1L interacts with the phosphorylated C-terminal domain of actively transcribing RNA polymerase II (RNAPII) through a region conserved uniquely in multicellular DOT1 proteins. Genome-wide profiling analyses indicate that the occupancy of hDOT1L largely overlaps with that of RNAPII at actively transcribed genes, especially surrounding transcriptional start sites, in embryonic carcinoma NCCIT cells. We also find that C-terminal domain binding or H3K79 methylations by hDOT1L is important for the expression of target genes such as NANOG and OCT4 and a marker for pluripotency in NCCIT cells. Our results indicate that a functional interaction between hDOT1L and RNAPII targets hDOT1L and subsequent H3K79 methylations to actively transcribed genes.

Vagococcus acidifermentans sp. nov., isolated from an acidogenic fermentation bioreactor.

A Gram-staining-positive, coccus-shaped, non-spore-forming, facultatively anaerobic bacterium, designated AC-1(T), was isolated from an acidogenic fermentation bioreactor treating food wastewater. On the basis of 16S rRNA gene sequence analysis, strain AC-1(T) was shown to belong to the genus Vagococcus. The closest phylogenetic relatives were Vagococcus elongatus PPC9(T) (97.4 % 16S rRNA gene sequence similarity), Vagococcus penaei CD276(T) (96.7 %) and Vagococcus carniphilus ATCC BAA-640(T) (96.6 %). The major fatty acids were C(18 : 1)ω9c (24.8 %) and C(16 : 0) (19.5 %) and the G+C content of genomic DNA was 44.2 mol%, which supported the affiliation of strain AC-1(T) to the genus Vagococcus. Strain AC-1(T) and V. elongatus DSM 21480(T) exhibited 11 % DNA-DNA relatedness. Physiological and biochemical tests differentiated strain AC-1(T) from the type strains of recognized species of the genus Vagococcus. Therefore, strain AC-1(T) is considered to represent a novel species, for which the name Vagococcus acidifermentans sp. nov. is proposed. The type strain is AC-1(T) ( = KCTC 13418(T)  = LMG 24798(T)).

Arabidopsis ING and Alfin1-like protein families localize to the nucleus and bind to H3K4me3/2 via plant homeodomain fingers.

In yeast and animals, tri- and dimethylation of histone H3 at lysine 4 (H3K4me3/2) are markers of transcriptionally active genes that have recently been shown to be primary ligands for the plant homeodomain (PHD) finger. However, PHD fingers able to bind to H3K4me3/2 have not been identified in plants. Here, we identify 83 canonical PHD fingers in the Arabidopsis proteome database that are supported by both SMART and Pfam prediction. Among these, we focus on PHD fingers in ING (inhibitor of growth) homologues (AtING) and Alfin1-like (AL) proteins, which are highly similar to those in human ING2 and bromodomain PHD finger transcription factor (BPTF), based on predicted tertiary structures. ING proteins are found in yeast, animals and plants, whereas AL proteins exist only in plants. In vitro binding experiments indicated that PHD fingers in AtING and AL proteins in Arabidopsis can bind to H3K4me3, and, to a lesser extent, to H3K4me2. In addition, mutational analysis confirmed that a predicted aromatic cage and a specific conserved acidic residue are both crucial for binding to H3K4me3/2. Finally, we demonstrate that AtING and AL proteins are nuclear proteins that are expressed in various tissues of the Arabidopsis plant. Thus, we propose that ING and AL proteins are nuclear proteins that are involved in chromatin regulation by binding to H3K4me3/2, the active histone markers, in plants.

Histone occupancy-dependent and -independent removal of H3K27 trimethylation at cold-responsive genes in Arabidopsis.

Trimethylation of histone H3 at lysine 27 (H3K27me3) is a histone marker that is present in inactive gene loci in both plants and animals. Transcription of some of the genes with H3K27me3 should be induced by internal or external cues, yet the dynamic fate of H3K27me3 in these genes during transcriptional regulation is poorly understood in plants. Here we show that H3K27me3 in two cold-responsive genes, COR15A and ATGOLS3, decreases gradually in Arabidopsis during exposure to cold temperatures. We found that removal of H3K27me3 can occur by both histone occupancy-dependent and -independent mechanisms. Upon cold exposure, histone H3 levels decreased in the promoter regions of COR15A and ATGOLS3 but not in their transcribed regions. When we returned cold-exposed plants to normal growth conditions, transcription of COR15A and ATGOLS3 was completely repressed to the initial level before cold exposure in 1 day. In contrast, plants still maintained the cold-triggered decrease in H3K27me3 at COR15A and ATGOLS3, but this decrease did not enhance transcriptional induction of the two genes upon re-exposure to cold. Taken together, these results indicate that gene activation is not inhibited by H3K27me3 itself but rather leads to removal of H3K27me3, and that H3K27me3 can be inherited at a quantitative level, thereby serving as a memory marker for recent transcriptional activity in Arabidopsis.

Unwinding chromatin for development and growth: a few genes at a time.

SWI/SNF chromatin remodeling ATPases control accessibility of the information stored in the genome. However, the in vivo role of these remodelers has remained poorly understood because null mutations in these result in embryonic lethality in most organisms. Recently, the study of conditional mutants in mammals and viable null mutants in plants, combined with genome wide expression studies in mammals, flies and plants, have implicated chromatin remodeling ATPases in the regulation of many developmental pathways in multicellular eukaryotes. In addition, these studies reveal striking functional specificity for chromatin remodeling in individual developmental processes.

A lysine-rich region in Dot1p is crucial for direct interaction with H2B ubiquitylation and high level methylation of H3K79.

Dot1p is involved in maintenance of the heterochromatin boundary, the DNA damage response, and transcriptional regulation in yeast and animals. Dot1p is a histone H3 lysine 79 (H3K79) methyltransferase, but H3K79 trimethylation (H3K79me3) by Dot1p requires histone H2B monoubiquitylation (H2Bub) as a pre-requisite. The underlying mechanism for H2Bub requirement has not been well elucidated. In this work, we found that nucleosomes containing H2Bub stimulate the yeast Dot1p to produce H3K79me3. A pulldown assay showed that the yeast Dot1p directly binds to ubiquitin. In addition, we demonstrate that a lysine-rich region (aa 101-140) in the first half of DNA binding domain of the Dot1p is critical in interaction with ubiquitin as well as binding to nucleosome core. Consistent with this, either deletion or point mutation of the lysine-rich region resulted in defect in global H3K79me3 accumulation and subtelomeric gene silencing in vivo. Taken together, our results indicate that a direct interaction between the lysine-rich region of Dot1p and the ubiquitin of H2Bub is required for H2Bub-mediated trans-tail regulation.

A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis.

Endoribonuclease E (RNase E) is a regulator of global gene expression in Escherichia coli and is the best studied member of the RNase E/G ribonuclease family. Homologues are present in other bacteria but the roles of plant RNase E/G-like proteins are not known. Arabidopsis thaliana contains a single nuclear gene (At2g04270) encoding a product with the conserved catalytic domain of RNase E/G-like proteins. At2g04270 and the adjacent At2g04280 gene form converging transcription units with a approximately 40 base overlap at their 3' ends. Several translation products were predicted from the analyses of At2g04270 cDNAs. An antibody raised against a recombinant A. thaliana RNase E/G-like protein recognized a 125 kDa protein band in purified chloroplast preparations fractionated by SDS-PAGE. The 125 kDa RNase E/G-like protein was detected in cotyledons, rosette and cauline leaves. T-DNA insertions in exon 6 or intron 11 of At2g04270 result in loss of the 125 kDa band or truncation to a 110 kDa band. Loss of At2g04270 function resulted in the arrest of chloroplast development, loss of autotrophic growth, and reduced plastid ribosomal, psbA and rbcL RNA levels. Homozygous mutant plants were pale-green, contained smaller plastids with fewer thylakoids and shorter granal stacks than wild-type chloroplasts, and required sucrose at all growth stages following germination right up to flowering and setting seeds. Recombinant A. thaliana RNase E/G-like proteins rescued an E. coli RNase E mutant and cleaved an rbcL RNA substrate. Expression of At2g04270 was highly correlated with genes encoding plastid polyribonucleotide phosphorylase, S1 RNA-binding, and CRS1/YhbY domain proteins.

Dot1 regulates nucleosome dynamics by its inherent histone chaperone activity in yeast.

Dot1 (disruptor of telomeric silencing-1, DOT1L in humans) is the only known enzyme responsible for histone H3 lysine 79 methylation (H3K79me) and is evolutionarily conserved in most eukaryotes. Yeast Dot1p lacks a SET domain and does not methylate free histones and thus may have different actions with respect to other histone methyltransferases. Here we show that Dot1p displays histone chaperone activity and regulates nucleosome dynamics via histone exchange in yeast. We show that a methylation-independent function of Dot1p is required for the cryptic transcription within transcribed regions seen following disruption of the Set2-Rpd3S pathway. Dot1p can assemble core histones to nucleosomes and facilitate ATP-dependent chromatin-remodeling activity through its nucleosome-binding domain, in vitro. Global analysis indicates that Dot1p appears to be particularly important for histone exchange and chromatin accessibility on the transcribed regions of long-length genes. Our findings collectively suggest that Dot1p-mediated histone chaperone activity controls nucleosome dynamics in transcribed regions.

Biogenesis and regulation of the let-7 miRNAs and their functional implications.

The let-7 miRNA was one of the first miRNAs discovered in the nematode, Caenorhabditis elegans, and its biological functions show a high level of evolutionary conservation from the nematode to the human. Unlike in C. elegans, higher animals have multiple isoforms of let-7 miRNAs; these isoforms share a consensus sequence called the 'seed sequence' and these isoforms are categorized into let-7 miRNA family. The expression of let-7 family is required for developmental timing and tumor suppressor function, but must be suppressed for the self-renewal of stem cells. Therefore, let-7 miRNA biogenesis must be carefully controlled. To generate a let-7 miRNA, a primary transcript is produced by RNA polymerase II and then subsequently processed by Drosha/DGCR8, TUTase, and Dicer. Because dysregulation of let-7 processing is deleterious, biogenesis of let-7 is tightly regulated by cellular factors, such as the RNA binding proteins, LIN28A/B and DIS3L2. In this review, we discuss the biological functions and biogenesis of let-7 miRNAs, focusing on the molecular mechanisms of regulation of let-7 biogenesis in vertebrates, such as the mouse and the human.

Characterization of two homologs of Ire1p, a kinase/endoribonuclease in yeast, in Arabidopsis thaliana.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits an ER-to-nucleus signaling pathway known as the unfolded protein response (UPR) in eukaryotes. In yeast, Ire1p, a kinase/endoribonuclease in the ER membrane, plays a key role in the UPR signaling. We isolated two cDNA homologs of IRE1 gene from Arabidopsis (AtIre1a, AtIre1b). The two IRE1 homologs were predicted to form a type I transmembrane protein structure and contain kinase/endoribonuclease domains at their C-terminal halves. The expressions of the two genes were detected in various organ tissues of the Arabidopsis plant. The C-terminal half of the AtIre1a protein showed in vitro autophosphorylation activity. However, we could not detect endoribonuclease activity of the AtIre1a protein when we used yeast HAC1 RNA as the substrate in vivo.

Expression of an evolutionarily distinct novel BiP gene during the unfolded protein response in Arabidopsis thaliana.

Compared to mammals, little is known about the unfolded protein response (UPR) in plants. Using an oligonucleotide array comprising approximately 8200 Arabidopsis genes we investigated the effect of endoplasmic reticulum (ER) stress on gene expression. Expression of 26 genes increased, including at least nine whose products act in the ER, while their transcriptional activations were confirmed by promoter analyses. Among them, BiP-L, a novel BiP, whose expression appeared to be regulated by two promoter sequences perfectly matching mammalian ERSE. Cloning and sequencing of full-length BiP-L cDNA showed it contained a signal peptide sequence and the ER retention signal (HDEL). Interestingly, BiP-L was substantially different from the other two Arabidopsis BiP genes in genomic organization and sequence homology. Furthermore, phylogenetic analysis showed that the BiP-L protein is the most distal form among the reported plant BiP proteins. RNA levels of BiP-L were very low in various mature Arabidopsis plant organs, while significant levels of BiP-L only observed in stressed seedlings. Transcription of BiP-L during ER stress was shown to be regulated by a feedback loop.

PROTOCOLS: Chromatin Immunoprecipitation from Arabidopsis Tissues.

The ability of proteins to associate with genomic DNA in the context of chromatin is critical for many nuclear processes including transcription, replication, recombination, and DNA repair. Chromatin immunoprecipication (ChIP) is a practical and useful technique for characterizing protein / DNA association in vivo. The procedure generally includes six steps: (1) crosslinking the protein to the DNA; (2) isolating the chromatin; (3) chromatin fragmentation; (4) imunoprecipitation with antibodies against the protein of interest; (5) DNA recovery; and (6) PCR identification of factor associated DNA sequences. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP in intact Arabidopsis tissues. This protocol has been used to study association of histone modifications, of chromatin remodeling ATPases, as well as of sequence-specific transcription factors with the genomic DNA in various Arabidopsis thaliana tissues. The protocol described focuses on ChIP-qPCR, but can readily be adapted for use in ChIP-chip or ChIP-seq experiments. The entire procedure can be completed within 3 days.

Unique, shared, and redundant roles for the Arabidopsis SWI/SNF chromatin remodeling ATPases BRAHMA and SPLAYED.

Chromatin remodeling is emerging as a central mechanism for patterning and differentiation in multicellular eukaryotes. SWI/SNF chromatin remodeling ATPases are conserved in the animal and plant kingdom and regulate transcriptional programs in response to endogenous and exogenous cues. In contrast with their metazoan orthologs, null mutants in two Arabidopsis thaliana SWI/SNF ATPases, BRAHMA (BRM) and SPLAYED (SYD), are viable, facilitating investigation of their role in the organism. Previous analyses revealed that syd and brm null mutants exhibit both similar and distinct developmental defects, yet the functional relationship between the two closely related ATPases is not understood. Another central question is whether these proteins act as general or specific transcriptional regulators. Using global expression studies, double mutant analysis, and protein interaction assays, we find overlapping functions for the two SWI/SNF ATPases. This partial diversification may have allowed expansion of the SWI/SNF ATPase regulatory repertoire, while preserving essential ancestral functions. Moreover, only a small fraction of all genes depends on SYD or BRM for expression, indicating that these SWI/SNF ATPases exhibit remarkable regulatory specificity. Our studies provide a conceptual framework for understanding the role of SWI/SNF chromatin remodeling in regulation of Arabidopsis development.

Transcriptional target prediction using qualitative reasoning.

Transcription target prediction from functional genomics data often involves incorporating a conjunction of complex prior biological knowledge to the analysis. Unfortunately, typical prior hypotheses are qualitative rather than quantitative in nature. But, many qualitative biological hypotheses can be decomposed into a set of logic statements on binary outcomes. Here, we present a new method to convert qualitative statements into a collection of binary statements that in turn generates a partial ordering of outcomes, which can be tested using a semi-parametric isotonic regression. This semi-parametric approach yields a flexible but principled way of testing biological hypotheses. We applied this method to a published Arabidopsis microarray dataset to identify organ specific transcriptional target genes, and tested predictions independently using the AtGenExpress dataset. Our new algorithm performed comparably to published approaches and allowed rapid analysis of complex, multiple gene selection criteria.

A role for chromatin remodeling in regulation of CUC gene expression in the Arabidopsis cotyledon boundary.

The CUP-SHAPED COTYLEDON (CUC) genes CUC1, CUC2 and CUC3 act redundantly to control cotyledon separation in Arabidopsis. In order to identify novel regulators of this process, we have performed a phenotypical enhancer screen using a null allele of cuc2, cuc2-1. We identified three nonsense alleles of AtBRM, an Arabidopsis SWI/SNF chromatin remodeling ATPase, that result in strong cotyledon fusion in cuc2-1. atbrm also enhances cotyledon fusion in loss-of-function cuc1 and cuc3 mutants, suggesting a general requirement for this ATPase in cotyledon separation. By contrast, a null allele of SPLAYED (SYD), the closest homolog of AtBRM in Arabidopsis, enhances only the loss-of-function cuc1 mutant. By investigating the activities of the CUC promoters in the cotyledon boundary during embryogenesis in sensitized backgrounds, we demonstrate that AtBRM upregulates the transcription of all three CUC genes, whereas SYD upregulates the expression of CUC2. Our results uncover a specific role for both chromatin remodeling ATPases in the formation and/or maintenance of boundary cells during embryogenesis.

The LEAFY target LMI1 is a meristem identity regulator and acts together with LEAFY to regulate expression of CAULIFLOWER.

The timing of the switch from vegetative to reproductive development is crucial for species survival. The plant-specific transcription factor and meristem identity regulator LEAFY (LFY) controls this switch in Arabidopsis, in part via the direct activation of two other meristem identity genes, APETALA1 (AP1) and CAULIFLOWER (CAL). We recently identified five new direct LFY targets as candidates for the missing meristem identity regulators that act downstream of LFY. Here, we demonstrate that one of these, the class I homeodomain leucine-zipper transcription factor LMI1, is a meristem identity regulator. LMI1 acts together with LFY to activate CAL expression. The interaction between LFY, LMI1 and CAL resembles a feed-forward loop transcriptional network motif. LMI1 has additional LFY-independent roles in the formation of simple serrated leaves and in the suppression of bract formation. The temporal and spatial expression of LMI1 supports a role in meristem identity and leaf/bract morphogenesis.

The N-terminal ATPase AT-hook-containing region of the Arabidopsis chromatin-remodeling protein SPLAYED is sufficient for biological activity.

The SNF2-like chromatin-remodeling ATPase SPLAYED (SYD) was identified as a co-activator of floral homeotic gene expression in Arabidopsis. SYD is also required for meristem maintenance and regulates flowering under a non-inductive photoperiod. SNF2 ATPases are structurally and functionally conserved from yeast to humans. In addition to the conserved protein features, SYD has a large unique C-terminal domain. We show here that SYD is present as two forms in the nucleus, full-length and truncated, with the latter apparently lacking the C-terminal domain. The ratio of the two forms of endogenous SYD differs in juvenile and in adult tissues. Furthermore, an SYD variant lacking the C-terminal domain (SYDDeltaC) rescues the syd null mutant, indicating that the N-terminal ATPase AT-hook-containing region of SYD is sufficient for biological activity. Plants expressing SYDDeltaC show molecular and morphological phenotypes opposite to those of the null mutant, suggesting that the construct results in increased activity. This increased activity is at least in part due to elevated SYD protein levels in these lines. We propose that the C-terminal domain may control SYD accumulation and/or specific activity in the context of the full-length protein. The presence of the C-terminal domain in rice SYD suggests that its role is probably conserved in the two classes of flowering plants.

Tobacco Tsip1, a DnaJ-type Zn finger protein, is recruited to and potentiates Tsi1-mediated transcriptional activation.

Tobacco stress-induced1 (Tsi1) is an ethylene-responsive-element binding protein/APETALA2-type transcription factor that plays an important role in both biotic and abiotic stress signaling pathways. We show that Tsi1-interacting protein1 (Tsip1), a DnaJ-type Zn finger protein, interacts with Tsi1 in vitro and in yeast (Saccharomyces cerevisiae). The transcript level of Tsip1 in tobacco (Nicotiana tabacum) increased upon treatment with salicylic acid (SA), ethylene, gibberellic acid, NaCl, and virus challenge. Tsip1 appeared to be physically associated with the chloroplast surface but dissociated from it after SA treatment. Tsip1 colocalized and coimmunoprecipitated with Tsi1 in plant cells following SA treatment. Tsip1 expression increased Tsi1-mediated transcription and was able to functionally compensate for loss of the Tsi1 transcriptional activation domain through a direct interaction with Tsi1. Transgenic plants simultaneously coexpressing Tsi1 and Tsip1 displayed stronger pathogen resistance and salt tolerance than did transgenic plants expressing either Tsi1 or Tsip1 alone. Concurrent with this, the expression of a subset of stress-related genes was induced in a cooperative manner in Tsi1/Tsip1 transgenic plants. These results together implied that Tsi1 recruits Tsip1 to the promoters of stress-related genes to potentiate Tsi1-mediated transcriptional activation.