A systematic quantitative approach comprehensively defines domain-specific functional pathways linked to Schizosaccharomyces pombe heterochromatin regulation.

Heterochromatin plays a critical role in regulating gene expression and maintaining genome integrity. While structural and enzymatic components have been linked to heterochromatin establishment, a comprehensive view of the underlying pathways at diverse heterochromatin domains remains elusive. Here, we developed a systematic approach to identify factors involved in heterochromatin silencing at pericentromeres, subtelomeres, and the silent mating type locus in Schizosaccharomyces pombe. Using quantitative measures, iterative genetic screening, and domain-specific heterochromatin reporters, we identified 369 mutants with different degrees of reduced or enhanced silencing. As expected, mutations in the core heterochromatin machinery globally decreased silencing. However, most other mutants exhibited distinct qualitative and quantitative profiles that indicate domain-specific functions. For example, decreased mating type silencing was linked to mutations in heterochromatin maintenance genes, while compromised subtelomere silencing was associated with metabolic pathways. Furthermore, similar phenotypic profiles revealed shared functions for subunits within complexes. We also discovered that the uncharacterized protein Dhm2 plays a crucial role in maintaining constitutive and facultative heterochromatin, while its absence caused phenotypes akin to DNA replication-deficient mutants. Collectively, our systematic approach unveiled a landscape of domain-specific heterochromatin regulators controlling distinct states and identified Dhm2 as a previously unknown factor linked to heterochromatin inheritance and replication fidelity.

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery.

Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is not well understood. Here we demonstrate that Schizosaccharomyces pombe Lem2, an NE protein, regulates nuclear-exosome-mediated RNA degradation. Lem2 deletion causes accumulation of RNA precursors and meiotic transcripts and de-localization of an engineered exosome substrate from the nuclear periphery. Lem2 does not directly bind RNA but instead interacts with the exosome-targeting MTREC complex and its human homolog PAXT to promote RNA recruitment. This pathway acts largely independently of nuclear bodies where exosome factors assemble. Nutrient availability modulates Lem2 regulation of meiotic transcripts, implying that this pathway is environmentally responsive. Our work reveals that multiple spatially distinct degradation pathways exist. Among these, Lem2 coordinates RNA surveillance of meiotic transcripts and non-coding RNAs by recruiting exosome co-factors to the nuclear periphery.

Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase.

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.

Crosstalk between H2A variant-specific modifications impacts vital cell functions.

Selection of C-terminal motifs participated in evolution of distinct histone H2A variants. Hybrid types of variants combining motifs from distinct H2A classes are extremely rare. This suggests that the proximity between the motif cases interferes with their function. We studied this question in flowering plants that evolved sporadically a hybrid H2A variant combining the SQ motif of H2A.X that participates in the DNA damage response with the KSPK motif of H2A.W that stabilizes heterochromatin. Our inventory of PTMs of H2A.W variants showed that in vivo the cell cycle-dependent kinase CDKA phosphorylates the KSPK motif of H2A.W but only in absence of an SQ motif. Phosphomimicry of KSPK prevented DNA damage response by the SQ motif of the hybrid H2A.W/X variant. In a synthetic yeast expressing the hybrid H2A.W/X variant, phosphorylation of KSPK prevented binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and impaired response to DNA damage. Our findings illustrate that PTMs mediate interference between the function of H2A variant specific C-terminal motifs. Such interference could explain the mutual exclusion of motifs that led to evolution of H2A variants.

A Synthetic Approach to Reconstruct the Evolutionary and Functional Innovations of the Plant Histone Variant H2A.W.

Diversification of histone variants is marked by the acquisition of distinct motifs and functional properties through convergent evolution.1-4 H2A variants are distinguished by specific C-terminal motifs and tend to be segregated within defined domains of the genome.5,6 Whether evolution of these motifs pre-dated the evolution of segregation mechanisms or vice versa has remained unclear. A suitable model to address this question is the variant H2A.W, which evolved in plants through acquisition of a KSPK motif7 and is tightly associated with heterochromatin.4 We used fission yeast, where chromatin is naturally devoid of H2A.W, to study the impact of engineered chimeras combining yeast H2A with the KSPK motif. Biochemical assays showed that the KSPK motif conferred nucleosomes with specific properties. Despite uniform incorporation of the engineered H2A chimeras in the yeast genome, the KSPK motif specifically affected heterochromatin composition and function. We conclude that the KSPK motif promotes chromatin properties in yeast that are comparable to the properties and function of H2A.W in plant heterochromatin. We propose that the selection of functional motifs confer histone variants with properties that impact primarily a specific chromatin state. The association between a new histone variant and a preferred chromatin state can thus provide a setting for the evolution of mechanisms that segregate the new variant to this state, thereby enhancing the impact of the selected properties of the variant on genome activity.

ESCRT recruitment by the S. cerevisiae inner nuclear membrane protein Heh1 is regulated by Hub1-mediated alternative splicing.

Misassembled nuclear pore complexes (NPCs) are removed by sealing off the surrounding nuclear envelope (NE), which is conducted by the endosomal sorting complexes required for transport (ESCRT) machinery. Recruitment of ESCRT proteins to the NE is mediated by the interaction between the ESCRT member Chm7 and the inner nuclear membrane protein Heh1, which belongs to the conserved LEM family. Increased ESCRT recruitment results in excessive membrane scission at damage sites but its regulation remains poorly understood. Here, we show that Hub1-mediated alternative splicing of HEH1 pre-mRNA, resulting in production of its shorter form Heh1-S, is critical for the integrity of the NE in Saccharomyces cerevisiae ESCRT-III mutants lacking Hub1 or Heh1-S display severe growth defects and accumulate improperly assembled NPCs. This depends on the interaction of Chm7 with the conserved MSC domain, which is only present in the longer variant Heh1-L. Heh1 variants assemble into heterodimers, and we demonstrate that a unique splice segment in Heh1-S suppresses growth defects associated with the uncontrolled interaction between Heh1-L and Chm7. Together, our findings reveal that Hub1-mediated splicing generates Heh1-S to regulate ESCRT recruitment to the NE.This article has an associated First Person interview with the first author of the paper.

ESCRTing Heterochromatin Out of the Nuclear Periphery.

ESCRT proteins fulfill an important function in membrane remodeling and scission. In this issue of Developmental Cell, Pieper et al. reveal that the ESCRT machinery releases the inner nuclear membrane protein Lem2 from heterochromatin, thus ensuring its function in nuclear envelope resealing after mitosis.

The euchromatic histone mark H3K36me3 preserves heterochromatin through sequestration of an acetyltransferase complex in fission yeast.

Maintaining the identity of chromatin states requires mechanisms that ensure their structural integrity through the concerted actions of histone modifiers, readers, and erasers. Histone H3K9me and H3K27me are hallmarks of repressed heterochromatin, whereas H3K4me and H3K36me are associated with actively transcribed euchromatin. Paradoxically, several studies have reported that loss of Set2, the methyltransferase responsible for H3K36me, causes de-repression of heterochromatin. Here we show that unconstrained activity of the acetyltransferase complex Mst2C, which antagonizes heterochromatin, is the main cause of the silencing defects observed in Set2-deficient cells. As previously shown, Mst2C is sequestered to actively transcribed chromatin via binding to H3K36me3 that is recognized by the PWWP domain protein Pdp3. We demonstrate that combining deletions of set2 + and pdp3 + results in an epistatic silencing phenotype. In contrast, deleting mst2 + , or other members of Mst2C, fully restores silencing in Set2-deficient cells. Suppression of the silencing defect in set2Δ cells is specific for pericentromeres and subtelomeres, which are marked by H3K9me, but is not seen for loci that lack genuine heterochromatin. Mst2 is known to acetylate histone H3K14 redundantly with the HAT Gnc5. Further, it is involved in the acetylation of the non-histone substrate and E3 ubiquitin ligase Brl1, resulting in increased H2B-K119 ubiquitylation at euchromatin. However, we reveal that none of these mechanisms are responsible for the Set2-dependent silencing pathway, implying that Mst2 targets another, unknown substrate critical for heterochromatin silencing. Our findings demonstrate that maintenance of chromatin states requires spatial constraint of opposing chromatin activities.

Field-grown transgenic wheat expressing the sunflower gene HaHB4 significantly outyields the wild type.

HaHB4 is a sunflower transcription factor belonging to the homeodomain-leucine zipper I family whose ectopic expression in Arabidopsis triggers drought tolerance. The use of PCR to clone the HaHB4 coding sequence for wheat transformation caused unprogrammed mutations producing subtle differences in its activation ability in yeast. Transgenic wheat plants carrying a mutated version of HaHB4 were tested in 37 field experiments. A selected transgenic line yielded 6% more (P<0.001) and had 9.4% larger water use efficiency (P<0.02) than its control across the evaluated environments. Differences in grain yield between cultivars were explained by the 8% improvement in grain number per square meter (P<0.0001), and were more pronounced in stress (16% benefit) than in non-stress conditions (3% benefit), reaching a maximum of 97% in one of the driest environments. Increased grain number per square meter of transgenic plants was accompanied by positive trends in spikelet numbers per spike, tillers per plant, and fertile florets per plant. The gene transcripts associated with abiotic stress showed that HaHB4's action was not dependent on the response triggered either by RD19 or by DREB1a, traditional candidates related to water deficit responses. HaHB4 enabled wheat to show some of the benefits of a species highly adapted to water scarcity, especially in marginal regions characterized by frequent droughts.

Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability.

Telomeres and the shelterin complex cap and protect the ends of chromosomes. Telomeres are flanked by the subtelomeric sequences that have also been implicated in telomere regulation, although their role is not well defined. Here, we show that, in Schizosaccharomyces pombe, the telomere-associated sequences (TAS) present on most subtelomeres are hyper-recombinogenic, have metastable nucleosomes, and unusual low levels of H3K9 methylation. Ccq1, a subunit of shelterin, protects TAS from nucleosome loss by recruiting the heterochromatic repressor complexes CLRC and SHREC, thereby linking nucleosome stability to gene silencing. Nucleosome instability at TAS is independent of telomeric repeats and can be transmitted to an intrachromosomal locus containing an ectopic TAS fragment, indicating that this is an intrinsic property of the underlying DNA sequence. When telomerase recruitment is compromised in cells lacking Ccq1, DNA sequences present in the TAS promote recombination between chromosomal ends, independent of nucleosome abundance, implying an active function of these sequences in telomere maintenance. We propose that Ccq1 and fragile subtelomeres co-evolved to regulate telomere plasticity by controlling nucleosome occupancy and genome stability.

A uORF Represses the Transcription Factor AtHB1 in Aerial Tissues to Avoid a Deleterious Phenotype.

AtHB1 is an Arabidopsis (Arabidopsis thaliana) homeodomain-leucine zipper transcription factor that participates in hypocotyl elongation under short-day conditions. Here, we show that its expression is posttranscriptionally regulated by an upstream open reading frame (uORF) located in its 5' untranslated region. This uORF encodes a highly conserved peptide (CPuORF) that is present in varied monocot and dicot species. The Arabidopsis uORF and its maize (Zea mays) homolog repressed the translation of the main open reading frame in cis, independent of the sequence of the latter. Published ribosome footprinting results and the analysis of a frame-shifted uORF, in which the repression capability was lost, indicated that the uORF causes ribosome stalling. The regulation exerted by the CPuORF was tissue specific and did not act in the absence of light. Moreover, a photosynthetic signal is needed for the CPuORF action, since plants with uncoupled chloroplasts did not show uORF-dependent repression. Plants transformed with the native AtHB1 promoter driving AtHB1 expression did not show differential phenotypes, whereas those transformed with a construct in which the uORF was mutated exhibited serrated leaves, compact rosettes, and, most significantly, short nondehiscent anthers and siliques containing fewer or no seeds. Thus, we propose that the uncontrolled expression of AtHB1 is deleterious for the plant and, hence, finely repressed by a translational mechanism.

A matter of quantity: Common features in the drought response of transgenic plants overexpressing HD-Zip I transcription factors.

Plant responses to water deficit involve complex molecular mechanisms in which transcription factors have key roles. Previous reports ectopically overexpressed a few members of the homeodomain-leucine zipper I (HD-Zip I) family of transcription factors from different species, and the obtained transgenic plants exhibited drought tolerance which extent depended on the level of overexpression, triggering diverse molecular and physiological pathways. Here we show that most HD-Zip I genes are regulated by drought in the vegetative and/or reproductive stages. Moreover, uncharacterized members of this family were expressed as transgenes both in Col-0 and rdr6-12 backgrounds and were able to enhance drought tolerance in host plants. The extent of such tolerance depended on the expression level of the transgene and was significantly higher in transgenic rdr6-12 than in Col-0. Comparative transcriptome analyses of Arabidopsis thaliana plants overexpressing HD-Zip I proteins indicated that many members have common targets. Moreover, the water deficit tolerance exhibited by these plants is likely due to the induction and repression of certain of these common HD-Zip I-regulated genes. However, each HD-Zip I member regulates other pathways, which, in some cases, generate differential and potentially undesirable traits in addition to drought tolerance. In conclusion, only a few members of this family could become valuable tools to improve drought-tolerance.

Functional characterization of the homeodomain leucine zipper I transcription factor AtHB13 reveals a crucial role in Arabidopsis development.

AtHB13 is a homeodomain leucine zipper I transcription factor whose function in development is largely unknown. AtHB13 and AtHB23 mutant and silenced lines were characterized by expression studies, reciprocal crosses, complementation, molecular analyses, and developmental phenotypes. The athb13-1 and athb13-2 mutants, athb23 silenced, and athb13/athb23 double-silenced plants exhibited faster elongation rates of their inflorescence stems, whereas only athb13-1 and the double-knockdown athb13/athb23 exhibited shorter siliques, fewer seeds, and unfertilized ovules compared with the wild type (WT). The cell sizes of mutant and WT plants were similar, indicating that these transcription factors probably affect cell division. Reciprocal crosses between athb13-1 and the WT genotype indicated that the silique defect was male specific. Pollen hydration assays indicated that the pollen grains of the athb13-1 mutant were unable to germinate on stigmas. AtHB23-silenced plants exhibited normal siliques, whereas double-knockdown athb13/athb23 plants were similar to athb13-1 plants. Both AtHB13 and AtHB23 were able to rescue the abnormal silique phenotype. AtHB23 was upregulated in athb13-2 plants, whereas its transcript levels in athb13-1 mutants were not significantly increased. Transcriptome analysis comparing athb13-1 and WT inflorescences revealed that a large number of genes, including several involved in pollen coat formation, are regulated by AtHB13. Finally, athb13-1 complementation with mutated versions of AtHB13 confirmed that two different tryptophans in its C terminus are essential. We conclude that AtHB13 and AtHB23 play independent, negative developmental roles in stem elongation, whereas only AtHB13 is crucial for pollen germination. Furthermore, AtHB23, which does not normally exert a functional role in pollen, can act as a substitute for AtHB13.

Arabidopsis thaliana HomeoBox 1 (AtHB1), a Homedomain-Leucine Zipper I (HD-Zip I) transcription factor, is regulated by PHYTOCHROME-INTERACTING FACTOR 1 to promote hypocotyl elongation.

Arabidopsis thaliana HomeoBox 1 (AtHB1) is a homeodomain-leucine zipper transcription factor described as a transcriptional activator with unknown function. Its role in A. thaliana development was investigated. AtHB1 expression was analyzed in transgenic plants bearing its promoter region fused to reporter genes. Knock-down mutant and overexpressor plant phenotypes were analyzed in different photoperiod regimes. AtHB1 was mainly expressed in hypocotyls and roots and up-regulated in seedlings grown under a short-day photoperiod. AtHB1 knock-down mutants and overexpressors showed shorter and longer hypocotyls, respectively, than wild type (WT). AtHB1 transcript levels were lower in PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) mutants than in controls, suggesting that AtHB1 is regulated by PIF1 in hypocotyls. ß-glucuronidase (GUS) activity in Nicotiana benthamiana leaves cotransformed with PromAtHB1::GUS and 35S::PIF1 indicated that PIF1 induces AtHB1 expression. Hypocotyl lenght was measured in seedlings of athb1, pif1, or double athb1/pif1 mutants and PIF1 or AtHB1 overexpressors in WT, athb1 or pif1 backgrounds, both in short- or long-day. These analyses allowed us to determine that AtHB1 is a factor acting downstream of PIF1. Finally, a transcriptome analysis of athb1 mutant hypocotyls revealed that AtHB1 regulates genes involved in cell wall composition and elongation. The results suggest that AtHB1 acts downstream of PIF1 to promote hypocotyl elongation, especially in response to short-day photoperiods.

Arabidopsis AtHB7 and AtHB12 evolved divergently to fine tune processes associated with growth and responses to water stress.

BACKGROUND: Arabidopsis AtHB7 and AtHB12 transcription factors (TFs) belong to the homeodomain-leucine zipper subfamily I (HD-Zip I) and present 62% amino acid identity. These TFs have been associated with the control of plant development and abiotic stress responses; however, at present it is not completely understood how AtHB7 and AtHB12 regulate these processes. RESULTS: By using different expression analysis approaches, we found that AtHB12 is expressed at higher levels during early Arabidopsis thaliana development whereas AtHB7 during later developmental stages. Moreover, by analysing gene expression in single and double Arabidopsis mutants and in transgenic plants ectopically expressing these TFs, we discovered a complex mechanism dependent on the plant developmental stage and in which AtHB7 and AtHB12 affect the expression of each other. Phenotypic analysis of transgenic plants revealed that AtHB12 induces root elongation and leaf development in young plants under standard growth conditions, and seed production in water-stressed plants. In contrast, AtHB7 promotes leaf development, chlorophyll levels and photosynthesis and reduces stomatal conductance in mature plants. Moreover AtHB7 delays senescence processes in standard growth conditions. CONCLUSIONS: We demonstrate that AtHB7 and AtHB12 have overlapping yet specific roles in several processes related to development and water stress responses. The analysis of mutant and transgenic plants indicated that the expression of AtHB7 and AtHB12 is regulated in a coordinated manner, depending on the plant developmental stage and the environmental conditions. The results suggested that AtHB7 and AtHB12 evolved divergently to fine tune processes associated with development and responses to mild water stress.

Plant homeodomain-leucine zipper I transcription factors exhibit different functional AHA motifs that selectively interact with TBP or/and TFIIB.

Different members of the HD-Zip I family of transcription factors exhibit differential AHA-like activation motifs, able to interact with proteins of the basal transcriptional machinery. Homeodomain-leucine zipper proteins are transcription factors unique to plants, classified in four subfamilies. Subfamily I members have been mainly associated to abiotic stress responses. Several ones have been characterized using knockout or overexpressors plants, indicating that they take part in different signal transduction pathways even when their expression patterns are similar and they bind the same DNA sequence. A bioinformatic analysis has revealed the existence of conserved motifs outside the HD-Zip domain, including transactivation AHA motifs. Here, we demonstrate that these putative activation motifs are functional. Four members of the Arabidopsis family were chosen: AtHB1, AtHB7, AtHB12 and AtHB13. All of them exhibited activation activity in yeast and in plants but with different degrees. The protein segment necessary for such activation was different for these four transcription factors as well as the role of the tryptophans they present. When interaction with components of the basal transcription machinery was tested, AtHB1 was able to interact with TBP, AtHB12 interacted with TFIIB, AtHB7 interacted with both, TBP and TFIIB while AtHB13 showed weak interactions with any of them, in yeast two-hybrid as well as in pull-down assays. Transient transformation of Arabidopsis seedlings confirmed the activation capacity and specificity of these transcription factors and showed some differences with the results obtained in yeast. In conclusion, the differential activation functionality of these transcription factors adds an important level of functional divergence of these proteins, and together with their expression patterns, these differences could explain, at least in part, their functional divergence.

Uncharacterized conserved motifs outside the HD-Zip domain in HD-Zip subfamily I transcription factors; a potential source of functional diversity.

BACKGROUND: Plant HD-Zip transcription factors are modular proteins in which a homeodomain is associated to a leucine zipper. Of the four subfamilies in which they are divided, the tested members from subfamily I bind in vitro the same pseudopalindromic sequence CAAT(A/T)ATTG and among them, several exhibit similar expression patterns. However, most experiments in which HD-Zip I proteins were over or ectopically expressed under the control of the constitutive promoter 35S CaMV resulted in transgenic plants with clearly different phenotypes. Aiming to elucidate the structural mechanisms underlying such observation and taking advantage of the increasing information in databases of sequences from diverse plant species, an in silico analysis was performed. In addition, some of the results were also experimentally supported. RESULTS: A phylogenetic tree of 178 HD-Zip I proteins together with the sequence conservation presented outside the HD-Zip domains allowed the distinction of six groups of proteins. A motif-discovery approach enabled the recognition of an activation domain in the carboxy-terminal regions (CTRs) and some putative regulatory mechanisms acting in the amino-terminal regions (NTRs) and CTRs involving sumoylation and phosphorylation. A yeast one-hybrid experiment demonstrated that the activation activity of ATHB1, a member of one of the groups, is located in its CTR. Chimerical constructs were performed combining the HD-Zip domain of one member with the CTR of another and transgenic plants were obtained with these constructs. The phenotype of the chimerical transgenic plants was similar to the observed in transgenic plants bearing the CTR of the donor protein, revealing the importance of this module inside the whole protein. CONCLUSIONS: The bioinformatical results and the experiments conducted in yeast and transgenic plants strongly suggest that the previously poorly analyzed NTRs and CTRs of HD-Zip I proteins play an important role in their function, hence potentially constituting a major source of functional diversity among members of this subfamily.