The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis.

Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.

ACTIN-RELATED PROTEIN6 Regulates Female Meiosis by Modulating Meiotic Gene Expression in Arabidopsis.

In flowering plants, meiocytes develop from subepidermal cells in anthers and ovules. The mechanisms that integrate gene-regulatory processes with meiotic programs during reproductive development remain poorly characterized. Here, we show that Arabidopsis thaliana plants deficient in ACTIN-RELATED PROTEIN6 (ARP6), a subunit of the SWR1 ATP-dependent chromatin-remodeling complex, exhibit defects in prophase I of female meiosis. We found that this meiotic defect is likely due to dysregulated expression of meiotic genes, particularly those involved in meiotic recombination, including DMC1 (DISRUPTED MEIOTIC cDNA1). Analysis of DMC1 expression in arp6 mutant plants indicated that ARP6 inhibits expression of DMC1 in the megasporocyte and surrounding nonsporogeneous ovule cells before meiosis. After cells enter meiosis, however, ARP6 activates DMC1 expression specifically in the megasporocyte even as it continues to inhibit DMC1 expression in the nonsporogenous ovule cells. We further show that deposition of the histone variant H2A.Z, mediated by the SWR1 chromatin-remodeling complex at the DMC1 gene body, requires ARP6. Therefore, ARP6 regulates female meiosis by determining the spatial and temporal patterns of gene expression required for proper meiosis during ovule development.

DNA LIGASE I exerts a maternal effect on seed development in Arabidopsis thaliana.

Maternal effects are defined by mutations that affect the next generation when they are maternally inherited. To date, most indepth studies of maternal effects in plants have attributed their origin to genomic imprinting that restricts expression to the maternal allele. The DNA glycosylase DEMETER (DME) removes methylated cytosine residues, causing transcriptional activation of the maternal allele of imprinted genes. In this study, we show that loss-of-function of the major DNA LIGASE I (AtLIG1) in Arabidopsis thaliana causes maternal effects in the endosperm, which is the seed tissue that nurtures embryo development. AtLIG1 expression is not imprinted and has a limited impact on imprinted gene expression. Genetic interaction analyses further indicate that AtLIG1 acts downstream of DME. The removal of methylated cytosine residues by DME involves the creation of DNA single-strand breaks and our results suggest that AtLIG1 repairs these breaks.

AtCTF7 is required for establishment of sister chromatid cohesion and association of cohesin with chromatin during meiosis in Arabidopsis.

BACKGROUND: The establishment of sister chromatid cohesion followed by its controlled release at the metaphase to anaphase transition is necessary for faithful segregation of chromosomes in mitosis and meiosis. Cohesion is established by the action of Ctf7/Eco1 on the cohesin complex during DNA replication following loading of cohesin onto chromatin by the Scc2-Scc4 complex. Ctf7 is also required for sister chromatid cohesion during repair of DNA double strand breaks. Ctf7 contains an acetyltransferase domain and a zinc finger motif and acetylates conserved lysine residues in the Smc3 subunit of cohesin. In Arabidopsis CTF7 is encoded by a single gene and mutations in AtCTF7 cause embryo lethality indicating that the gene is essential. RESULTS: To study the function of Ctf7 in plants and to determine its role in sister chromatid cohesion, we constructed a conditional allele of AtCTF7 in Arabidopsis using an inducible RNA interference (RNAi) strategy, so as to avoid the embryo lethality caused by mutations in AtCTF7. We found that induction of RNAi against AtCTF7 caused severe inhibition and defects in growth during vegetative and reproductive stages as well as sterility. AtCTF7-RNAi plants displayed chromosome fragmentation and loss of sister chromatid cohesion during meiosis. Immunostaining for the cohesion subunit AtSCC3 showed a marked reduction in association of cohesin with chromatin during meiosis in AtCTF7-RNAi plants. CONCLUSIONS: We find that AtCTF7 is essential for sister chromatid cohesion during meiosis in Arabidopsis and is required for association of cohesin with chromatin in prophase of meiosis.

The plant adherin AtSCC2 is required for embryogenesis and sister-chromatid cohesion during meiosis in Arabidopsis.

Adherin plays an important role in loading the cohesin complex onto chromosomes, and is essential for the establishment of sister-chromatid cohesion. We have identified and analyzed the Arabidopsis adherin homolog AtSCC2. Interestingly, the sequence analysis of AtSCC2 and of other putative plant adherin homologs revealed the presence of a PHD finger, which is not found in their fungal and animal counterparts. AtSCC2 is identical to EMB2773, and mutants show early embryo lethality and formation of giant endosperm nuclei. A role for AtSCC2 in sister-chromatid cohesion was established by using conditional RNAi and examining meiotic chromosome organization. AtSCC2-RNAi lines showed sterility, arising from the following defects in meiotic chromosome organization: failure of homologous pairing, loss of sister-chromatid cohesion, mixed segregation of chromosomes and chromosome fragmentation. The mutant phenotype, which included defects in chromosome organization and cohesion in prophase I, is distinct from that of the Arabidopsis cohesin mutant Atrec8, which retains centromere cohesion up to anaphase I. Immunostaining experiments revealed the aberrant distribution of the cohesin subunit AtSCC3 on chromosomes, and defects in chromosomal axis formation, in the meiocytes of AtSCC2-RNAi lines. These results demonstrate a role for AtSCC2 in sister-chromatid cohesion and centromere organization, and show that the machinery responsible for the establishment of cohesion is conserved in plants.

p18INK4c and p27KIP1 are required for cell cycle arrest of differentiated myotubes.

Myogenic differentiation is characterized by permanent and irreversible cell cycle withdrawal and increased resistance to apoptosis. These functions correlate with changes in expression and activity of several cyclin-dependent kinase inhibitors, including p18, p21, and p27. In this study, we examined the requirements for p18, p21, and p27 in initiating growth arrest in multinucleated myotubes under differentiation conditions and in maintaining terminal arrest upon restimulation of differentiated myotubes with mitogenic signals. Under differentiation conditions, only p27(-/-) or p18(-/-)p27(-/-) myotubes are capable of reentering the cell cycle and synthesizing DNA at a very low frequency. Escape from cell cycle arrest was significantly greater in p18(-/-)p27(-/-) myotubes than in p27(-/-) myotubes. Stimulation of differentiated cultures with a mitogen-rich growth medium enhances p18(-/-)p27(-/-) myotube proliferation to encompass approximately half of the nuclei. p18(-/-)p21(-/-) and p21(-/-)p27(-/-) myotubes remain terminally arrested. Nuclei within individual restimulated p18(-/-)p27(-/-) myotubes can be found in all phases of the cell cycle, and a myotube can be multiphasic without any obvious deleterious effects. Increasing the time of differentiation or serum stimulation of p18(-/-)p27(-/-) myotubes neither increases the proliferation index of the myotube nuclei, nor does it alter the percentage of nuclei in each of the cell cycle phases. During the first 24 h of serum stimulation, the p18(-/-)p27(-/-) myotube nuclei that escape G0 arrest will rearrest in either S or G2 phase, without either mitosis or endoreplication. Apoptosis is increased in restimulated p18(-/-)p27(-/-) myotube nuclei, but is not specific for any cell cycle phase. These results suggest a collaborative role for p18 and p27 in initiating and maintaining G0 arrest during myogenic differentiation. While p18 and p27 appear to be essential in initiating G0 arrest in a proportion of postmitotic myotube nuclei, there must be another cell cycle inhibitor protein that functions with p18 and p27 in maintaining terminal arrest. We propose that the combined rate-limiting expressions of p18, p27, and this other inhibitor determine whether the myotube nuclei will remain postmitotic, or reenter the cell cycle, and if the nuclei escape G0 arrest, in which phase of the cell cycle the nuclei will ultimately rearrest.