Yota Murakami

A WW domain‐containing Yes‐associated protein (YAP) is a novel transcriptional co‐activator

A protein module called the WW domain recognizes and binds to a short oligopeptide called the PY motif, PPxY, to mediate protein–protein interactions. The PY motif is present in the transcription activation domains of a wide range of transcription factors including c‐Jun, AP‐2, NF‐E2, C/EBPα and PEBP2/CBF, suggesting that it plays an important role in transcriptional activation. We show here that mutation of the PY motif in the subregion of the activation domain of the DNA‐binding subunit of PEBP2, PEBP2α, abolishes its transactivation function. Using yeast two‐hybrid screening, we demonstrate that Yes‐associated protein (YAP) binds to the PY motif of PEBP2α through its WW domain. The C‐terminal region of YAP fused to the DNA‐binding domain of GAL4 showed transactivation as strong as that of GAL4–VP16. Exogenously expressed YAP conferred transcription‐stimulating activity on the PY motif fused to the GAL4 DNA‐binding domain as well as to native PEBP2α. The osteocalcin promoter was stimulated by exogenous PEBP2αA and a dominant negative form of YAP strongly inhibited this activity, suggesting YAP involvement in this promoter activity in vivo. These results indicate that the PY motif is a novel transcription activation domain that functions by recruiting YAP as a strong transcription activator to target genes.

Characterization of a Fission Yeast SUMO-1 Homologue, Pmt3p, Required for Multiple Nuclear Events, Including the Control of Telomere Length and Chromosome Segregation

ABSTRACT Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. Here we describe the identification of the SUMO-1 homologue of fission yeast, which we show to be required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of thepmt3 + gene, the Schizosaccharomyces pombe homologue of SMT3, was not lethal, but mutant cells carrying the disrupted gene grew more slowly. Thepmt3Δ cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, we found thatpmt3 + is required for telomere length maintenance. Loss of Pmt3p function caused a striking increase in telomere length. When Pmt3p synthesis was restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase- and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation.

DNA–RNA hybrid formation mediates RNAi-directed heterochromatin formation

Certain noncoding RNAs (ncRNAs) implicated in the regulation of chromatin structure associate with chromatin. During the formation of RNAi‐directed heterochromatin in fission yeast, ncRNAs transcribed from heterochromatin are thought to recruit the RNAi machinery to chromatin for the formation of heterochromatin; however, the molecular details of this association are not clear. Here, using RNA immunoprecipitation assay, we showed that the heterochromatic ncRNA was associated with chromatin via the formation of a DNA–RNA hybrid and bound to the RNA‐induced transcriptional silencing (RITS) complex. The presence of DNA–RNA hybrid in the cell was also confirmed by immunofluorescence analysis using anti‐DNA–RNA hybrid antibody. Over‐expression and depletion of RNase H in vivo decreased and increased the amount of DNA–RNA hybrid formed, respectively, and both disturbed heterochromatin. Moreover, DNA–RNA hybrid was formed on, and over‐expression of RNase H inhibited the formation of, artificial heterochromatin induced by tethering of RITS to mRNA. These results indicate that heterochromatic ncRNAs are retained on chromatin via the formation of DNA–RNA hybrids and provide a platform for the RNAi‐directed heterochromatin assembly and suggest that DNA–RNA hybrid formation plays a role in chromatic ncRNA function.

The Capacity of Polyomavirus Enhancer Binding Protein 2αB (AML1/Cbfa2) To Stimulate Polyomavirus DNA Replication Is Related to Its Affinity for the Nuclear Matrix

ABSTRACT The nuclear matrix is thought to play an important role in the DNA replication of eukaryotic cells, although direct evidence for such a role is still lacking. A nuclear matrix-associated transcription factor, polyomavirus (Py) enhancer binding protein 2αB1 (PEBP2αB1) (AML1/Cbfa2), was found to stimulate Py replication through its cognate binding site. The minimal replication activation domain (RAD) was identified between amino acid (aa) 302 and aa 371 by using a fusion protein containing the GAL4 DNA binding domain (GAL4-RAD). In addition, the region showed affinity for the nuclear matrix and, on the basis of competition studies, binding activity for one or more proteins involved in the initiation of Py DNA replication. A leukemogenic chimeric protein, AML1/ETO(MTG8), which does not contain this region of PEBP2αB1/AML1, was also localized in the nuclear matrix fraction and competed for nuclear matrix association with PEBP2αB1 and GAL4-RAD. Moreover, AML1/ETO inhibited Py DNA replication stimulated by PEBP2αB1 and GAL4-RAD. The inhibition was specific for replication mediated by PEBP2αB1 and GAL4-RAD, and proportional to the degree of loss of these activators from the nuclear matrix, suggesting a requirement for nuclear matrix targeting in the stimulation of Py DNA replication by RAD. These results are the first to suggest a molecular link between the initiation of DNA replication and the nuclear matrix compartment.

Phosphorylation of Swi6/HP1 regulates transcriptional gene silencing at heterochromatin

Heterochromatin protein 1 (HP1) recruits various effectors to heterochromatin for multiple functions, but its regulation is unclear. In fission yeast, a HP1 homolog Swi6 recruits SHREC, Epe1, and cohesin, which are involved in transcriptional gene silencing (TGS), transcriptional activation, and sister chromatid cohesion, respectively. We found that casein kinase II (CK2) phosphorylated Swi6. Loss of CK2-dependent Swi6 phosphorylation alleviated heterochromatic TGS without affecting heterochromatin structure. This was due to the inhibited recruitment of SHREC to heterochromatin, accompanied by an increase in Epe1. Interestingly, loss of phosphorylation did not affect cohesion. These results indicate that CK2-dependent Swi6 phosphorylation specifically controls TGS in heterochromatin.

Production of reactive oxygen species in response to replication stress and inappropriate mitosis in fission yeast

Previous studies have indicated that replication stress can trigger apoptosis-like cell death, accompanied (where tested) by production of reactive oxygen species (ROS), in mammalian cells and budding yeast (Saccharomyces cerevisiae). In mammalian cells, inappropriate entry into mitosis also leads to cell death. Here, we report similar responses in fission yeast (Schizosaccharomyces pombe). We used ROS- and death-specific fluorescent stains to measure the effects of mutations in replication initiation and checkpoint genes in fission yeast on the frequencies of ROS production and cell death. We found that certain mutant alleles of each of the four tested replication initiation genes caused elevated ROS and cell death. Where tested, these effects were not enhanced by checkpoint-gene mutations. Instead, when cells competent for replication but defective in both the replication and damage checkpoints were treated with hydroxyurea, which slows replication fork movement, the frequencies of ROS production and cell death were greatly increased. This was a consequence of elevated CDK activity, which permitted inappropriate entry into mitosis. Thus, studies in fission yeast are likely to prove helpful in understanding the pathways that lead from replication stress and inappropriate mitosis to cell death in mammalian cells.

Block of granulocytic differentiation of 32Dcl3 cells by AML1/ETO(MTG8) but not by highly expressed Bcl-2

The chimeric gene, AML1/ETO (MTG8), generated in t(8;21) acute myeloid leukemia enhances the expression of Bcl-2. To evaluate whether this enhancement is the primary role of AML1/ETO in leukemogenesis, effects of over-expression of Bcl-2 in the murine myeloid precursor cell line, 32Dcl3, were examined. When 32Dcl3 cells expressing exogenous Bcl-2 were induced to differentiate, the onset of morphological differentiation was delayed. However, even the cells expressing very high levels of exogenous Bcl-2 eventually underwent differentiation without a significant decrease in the synthesis of Bcl-2. On the contrary, 32Dcl3 cells stably expressing AML1/ETO were completely resistant to differentiation and continued to grow in the presence of G-CSF. These results are consistent with the interpretation that stimulation of Bcl-2 expression is not the primary target of AML1/ETO.

Fission yeast chromatin assembly factor 1 assists in the replication-coupled maintenance of heterochromatin

Chromatin assembly factor‐1 (CAF1) is a well‐conserved histone chaperone that loads the histone H3‐H4 complex onto newly synthesized DNA in vitro through interaction with the replication factor PCNA. CAF1 is considered to be involved in heterochromatin maintenance in several organisms, but the evidence is circumstantial and functional details have not been established. We identified fission yeast CAF‐1 (spCAF1), which interacts with PCNA in S phase. Depletion of spCAF1 caused defects in silencing at centromeric and mating locus heterochromatin, accompanied with a decrease in Swi6, the fission yeast HP1 homologue. Loss of spCAF1 destabilized both the silent and active states of chromatin at the meta‐stable heterochromatic region, with a more pronounced effect on the silent state, indicating that spCAF1 is involved in the maintenance of heterochromatin. Swi6 dissociated from heterochromatin during G1/S phase appears to associate with spCAF1. In early S phase, spCAF1 localized to replicating heterochromatin as well as euchromatin and remained associated with Swi6, and Swi6 then bound to heterochromatin. Taken together, we propose that spCAF1 functions in heterochromatin maintenance by recruiting dislocated Swi6 during replication to replicated heterochromatin at the replication fork.

c-Jun stimulates origin-dependent DNA unwinding by polyomavirus large Tantigen.

The AP1 protein c‐Jun has previously been shown to stimulate polyomavirus (Py) DNA replication in vivo. In order to define the mechanism, we added purified c‐Jun protein to the origin‐dependent and large T antigen (LT)‐dependent in vitro DNA unwinding assay. c‐Jun protein was found to stimulate by approximately 5‐fold the unwinding of a 290 bp linear DNA fragment containing both the Py origin and the AP1 recognition sequence to which c‐Jun binds. Efficient levels of stimulation were specifically observed at limiting concentrations of LT for unwinding. Under similar conditions, Py DNA replication was stimulated to a comparable extent by AP1 in a purified in vitro replication assay. Mobility shift and DNase I footprinting assays showed that c‐Jun stimulates the ATP‐dependent binding of LT to the origin core by approximately 7‐fold. Furthermore, c‐Jun was found to interact directly with LT, but not with replication protein A. The activities of c‐Jun to stimulate unwinding and origin binding of LT were found to be harbored within the N‐terminal region of c‐Jun, which is distinct from the DNA binding domain. We speculate that certain transcription factors may possess specific DNA replication domains that function to stimulate the loading of replication factors at the origin during the initiation of DNA synthesis.

Spt6 prevents transcription-coupled loss of posttranslationally modified histone H3

The tail of histone H3 is an ideal medium for storing epigenetic information because displacement of histone H3 is heavily restricted during transcription. To maintain the locus-specific modifications of histone H3, histone molecules should be retained locally at the original position through multiple rounds of transcription. Here, we found that fission yeast Spt6, a highly conserved RNA polymerase II-interacting histone H3–H4 chaperone, is essential for the maintenance of Lys-4 and Lys-9 methylation of histone H3 in euchromatin and heterochromatin, respectively. In euchromatin, loss of Lys-4 methylated histone H3 and deposition of newly synthesized Lys-56 acetylated histone H3 induced by Spt6 inactivation were coupled with transcription. While in heterochromatin, Spt6 prevents histone turnover and cryptic transcription in parallel with Clr3 histone deacetylase. We propose that Spt6 retains posttranslationally modified histone H3 during transcription to maintain epigenome integrity.