Mmi1 RNA surveillance machinery directs RNAi complex RITS to specific meiotic genes in fission yeast.

RNA interference (RNAi) silences gene expression by acting both at the transcriptional and post-transcriptional levels in a broad range of eukaryotes. In the fission yeast Schizosaccharomyces pombe the RNA-Induced Transcriptional Silencing (RITS) RNAi complex mediates heterochromatin formation at non-coding and repetitive DNA. However, the targeting and role of RITS at other genomic regions, including protein-coding genes, remain unknown. Here we show that RITS localizes to specific meiotic genes and mRNAs. Remarkably, RITS is guided to these meiotic targets by the RNA-binding protein Mmi1 and its associated RNA surveillance machinery that together degrade selective meiotic mRNAs during vegetative growth. Upon sexual differentiation, RITS localization to the meiotic genes and mRNAs is lost. Large-scale identification of Mmi1 RNA targets reveals that RITS subunit Chp1 associates with the vast majority of them. In addition, loss of RNAi affects the effective repression of sexual differentiation mediated by the Mmi1 RNA surveillance machinery. These findings uncover a new mechanism for recruiting RNAi to specific meiotic genes and suggest that RNAi participates in the control of sexual differentiation in fission yeast.

GeBP/GPL transcription factors regulate a subset of CPR5-dependent processes.

The CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene of Arabidopsis (Arabidopsis thaliana) encodes a putative membrane protein of unknown biochemical function and displays highly pleiotropic functions, particularly in pathogen responses, cell proliferation, cell expansion, and cell death. Here, we demonstrate a link between CPR5 and the GLABRA1 ENHANCER BINDING PROTEIN (GeBP) family of transcription factors. We investigated the primary role of the GeBP/GeBP-like (GPL) genes using transcriptomic analysis of the quadruple gebp gpl1,2,3 mutant and one overexpressing line that displays several cpr5-like phenotypes including dwarfism, spontaneous necrotic lesions, and increased pathogen resistance. We found that GeBP/GPLs regulate a set of genes that represents a subset of the CPR5 pathway. This subset includes genes involved in response to stress as well as cell wall metabolism. Analysis of the quintuple gebp gpl1,2,3 cpr5 mutant indicates that GeBP/GPLs are involved in the control of cell expansion in a CPR5-dependent manner but not in the control of cell proliferation. In addition, to our knowledge, we provide the first evidence that the CPR5 protein is localized in the nucleus of plant cells and that a truncated version of the protein with no transmembrane domain can trigger cpr5-like processes when fused to the VP16 constitutive transcriptional activation domain. Our results provide clues on how CPR5 and GeBP/GPLs play opposite roles in the control of cell expansion and suggest that the CPR5 protein is involved in transcription.

ATAD2 controls chromatin-bound HIRA turnover.

Taking advantage of the evolutionary conserved nature of ATAD2, we report here a series of parallel functional studies in human, mouse, and Schizosaccharomyces pombe to investigate ATAD2's conserved functions. In S. pombe, the deletion of ATAD2 ortholog, abo1, leads to a dramatic decrease in cell growth, with the appearance of suppressor clones recovering normal growth. The identification of the corresponding suppressor mutations revealed a strong genetic interaction between Abo1 and the histone chaperone HIRA. In human cancer cell lines and in mouse embryonic stem cells, we observed that the KO of ATAD2 leads to an accumulation of HIRA. A ChIP-seq mapping of nucleosome-bound HIRA and FACT in Atad2 KO mouse ES cells demonstrated that both chaperones are trapped on nucleosomes at the transcription start sites of active genes, resulting in the abnormal presence of a chaperone-bound nucleosome on the TSS-associated nucleosome-free regions. Overall, these data highlight an important layer of regulation of chromatin dynamics ensuring the turnover of histone-bound chaperones.

Gene expression profiling of the different stages of Arabidopsis thaliana trichome development on the single cell level.

Leaf hairs (trichomes) of Arabidopsis thaliana are a model system for studying cell development, differentiation and cell cycle regulation. To exploit this model system with ultimate spatial resolution we applied single cell sampling, thus avoiding the averaging effect induced by complex tissue mixtures. In particular, we analysed gene expression profiles of two selected stages of the developing trichome: trichome initial cells and mature trichomes, as well as pavement cells. Ten single cells per sample were collected by glass microcapillaries and used for the generation of radioactive probes for subsequent hybridization to nylon filters representing approximately 8000 genes of A. thaliana. Functional categorization of genes transcribed in trichome initials, mature trichomes and pavement cells demonstrated involvement of these surface cells in the stress response. In silico promoter analysis of genes preferentially expressed in trichome initials revealed enrichment in MYB-binding sites and presence of elements involved in hormonal, metal, sulphur response and cell cycle regulation. Three candidate genes preferentially expressed in trichome initials were selected for further analysis: At3g16980 (putative RNA polymerase II), At5g15230 (GASA4) and At4g27260 (GH3.5, WES1). Promoter:GUS studies confirmed expression of the putative RNA polymerase II and the gibberellin responsive GASA4 in trichome initials and partially in mature trichomes. Functional implication of the three selected candidates in trichome development and hence in cell cycle regulation in A. thaliana is discussed. We suggest that these genes are involved in differentiation and initiation of endocycling during trichome development.

Lessons from yeast on emerging roles of the ATAD2 protein family in gene regulation and genome organization.

ATAD2, a remarkably conserved, yet poorly characterized factor is found upregulated and associated with poor prognosis in a variety of independent cancers in human. Studies conducted on the yeast Saccharomyces cerevisiae ATAD2 homologue, Yta7, are now indicating that the members of this family may primarily be regulators of chromatin dynamics and that their action on gene expression could only be one facet of their general activity. In this review, we present an overview of the literature on Yta7 and discuss the possibility of translating these findings into other organisms to further define the involvement of ATAD2 and other members of its family in regulating chromatin structure and function both in normal and pathological situations.

GeBP and GeBP-like proteins are noncanonical leucine-zipper transcription factors that regulate cytokinin response in Arabidopsis.

Understanding the role of transcription factors (TFs) is essential in reconstructing developmental regulatory networks. The plant-specific GeBP TF family of Arabidopsis thaliana (Arabidopsis) comprises 21 members, all of unknown function. A subset of four members, the founding member GeBP and GeBP-like proteins (GPL) 1, 2, and 3, shares a conserved C-terminal domain. Here we report that GeBP/GPL genes represent a newly defined class of leucine-zipper (Leu-zipper) TFs and that they play a redundant role in cytokinin hormone pathway regulation. Specifically, we demonstrate using yeast, in vitro, and split-yellow fluorescent protein in planta assays that GeBP/GPL proteins form homo- and heterodimers through a noncanonical Leu-zipper motif located in the C-terminal domain. A triple loss-of-function mutant of the three most closely related genes gebp gpl1 gpl2 shows a reduced sensitivity to exogenous cytokinins in a subset of cytokinin responses such as senescence and growth, whereas root inhibition is not affected. We find that transcript levels of type-A cytokinin response genes, which are involved in the negative feedback regulation of cytokinin signaling, are higher in the triple mutant. Using a GPL version that acts as a constitutive transcriptional activator, we show that the regulation of Arabidopsis response regulators (ARRs) is mediated by at least one additional, as yet unknown, repressor acting genetically downstream in the GeBP/GPL pathway. Our results indicate that GeBP/GPL genes encode a new class of unconventional Leu-zipper TF proteins and suggest that their role in the cytokinin pathway is to antagonize the negative feedback regulation on ARR genes to trigger the cytokinin response.

The immunophilin-interacting protein AtFIP37 from Arabidopsis is essential for plant development and is involved in trichome endoreduplication.

The FKBP12 (FK506-binding protein 12 kD) immunophilin interacts with several protein partners in mammals and is a physiological regulator of the cell cycle. In Arabidopsis, only one specific partner of AtFKBP12, namely AtFIP37 (FKBP12 interacting protein 37 kD), has been identified but its function in plant development is not known. We present here the functional analysis of AtFIP37 in Arabidopsis. Knockout mutants of AtFIP37 show an embryo-lethal phenotype that is caused by a strong delay in endosperm development and embryo arrest. AtFIP37 promoter::beta-glucuronidase reporter gene constructs show that the gene is expressed during embryogenesis and throughout plant development, in undifferentiating cells such as meristem or embryonic cells as well as highly differentiating cells such as trichomes. A translational fusion with the enhanced yellow fluorescent protein indicates that AtFIP37 is a nuclear protein localized in multiple subnuclear foci that show a speckled distribution pattern. Overexpression of AtFIP37 in transgenic lines induces the formation of large trichome cells with up to six branches. These large trichomes have a DNA content up to 256C, implying that these cells have undergone extra rounds of endoreduplication. Altogether, these data show that AtFIP37 is critical for life in Arabidopsis and implies a role for AtFIP37 in the regulation of the cell cycle as shown for FKBP12 and TOR (target of rapamycin) in mammals.

Involvement of the R2R3-MYB, AtMYB61, in the ectopic lignification and dark-photomorphogenic components of the det3 mutant phenotype.

Overexpression of a pine MYB, PtMYB4, in Arabidopsis caused ectopic lignin deposition and allowed the plants to undergo photomorphogenesis even when they were grown in the dark. The phenotype caused by PtMYB4 overexpression was reminiscent of the previously characterised dark-photomorphogenic mutant, de-etiolated 3 (det3); consequently, we tested the hypothesis that MYB misexpression may explain aspects of the det3 phenotype. We show here that AtMYB61, a member of the Arabidopsis R2R3-MYB family, is misexpressed in the det3 mutant. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) experiments suggested that AtMYB61 was misexpressed in a det3 background relative to wild-type plants. Examination of AtMYB61 promoter activity in a det3 background showed that the spatial control of AtMYB61 expression was lost. In order to determine if such misexpression could explain the mutant phenotype, AtMYB61 was overexpressed in wild-type Arabidopsis plants. Transgenic plants that overexpressed AtMYB61 had the same ectopic lignification and dark-photomorphogenic phenotype as that of the det3 mutant. In order to test if AtMYB61 was necessary for these aspects of the det3 phenotype, AtMYB61 expression was downregulated in det3 plants in both antisense and sense suppression experiments. Suppression of AtMYB61 in a det3 mutant background restored all mutant phenotypes of the det3 mutant associated with development in the dark. Taken together, these results suggest that AtMYB61 misexpression was both sufficient and necessary to explain the ectopic lignification and dark-photomorphogenic phenotypes of the det3 mutant.