Molecular and Cellular Biology

Andersen, Ditte Skovaa; Raja, Sunil Jayaramaiah; Colombani, Julien; Shaw, Rachael Louise; Langton, Paul Francis; Akhtar, Asifa; Tapon, Nicolas

doi: 10.1128/MCB.01586-09pmid: 20679484

Drosophila MCRS2 (dMCRS2; MCRS2/MSP58 and its splice variant MCRS1/p78 in humans) belongs to a family of forkhead-associated (FHA) domain proteins. Whereas human MCRS2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 has been largely uncharacterized. Recent data show that MCRS2 is purified as part of a complex containing the histone acetyltransferase MOF (males absent on first) in both humans and flies. MOF mediates H4K16 acetylation and regulates the expression of a large number of genes, suggesting that MCRS2 could also have a function in transcription regulation. Here, we show that dMCRS2 copurifies with RNA polymerase II (RNAP II) complexes and localizes to the 5' ends of genes. Moreover, dMCRS2 is required for optimal recruitment of RNAP II to the promoter regions of cyclin genes. In agreement with this, dMCRS2 is required for normal levels of cyclin gene expression. We propose a model whereby dMCRS2 promotes gene transcription by facilitating the recruitment of RNAP II preinitiation complexes (PICs) to the promoter regions of target genes.

Cuchalova, Lucie; Kouba, Tomas; Herrmannova, Anna; Danyi, Istvan; Chiu, Wen-ling; Valasek, Leos

doi: 10.1128/MCB.00430-10pmid: 20679478

Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro . A triple-Ala substitution of conserved residues in the RRM of g/Tif35 ( g/tif35-KLF ) or a single-point mutation in the WD40 repeat 6 of i/Tif34 ( i/tif34-Q258R ) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo . Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.

Sanders, Steven L.; Arida, Ahmad R.; Phan, Funita P.

doi: 10.1128/MCB.00404-10pmid: 20679488

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability.

Sofueva, Sevil; Du, Li-Lin; Limbo, Oliver; Williams, Jessica S.; Russell, Paul

doi: 10.1128/MCB.00413-10pmid: 20679485

Relocalization of checkpoint proteins to chromatin flanking DNA double-strand breaks (DSBs) is critical for cellular responses to DNA damage. Schizosaccharomyces pombe Crb2, which mediates Chk1 activation by Rad3 ATR , forms ionizing radiation-induced nuclear foci (IRIF). Crb2 C-terminal BRCT domains (BRCT 2 ) bind histone H2A phosphorylated at a C-terminal SQ motif by Tel1 ATM and Rad3 ATR , although the functional significance of this interaction is controversial. Here, we show that polar interactions of Crb2 serine-548 and lysine-619 with the phosphate group of phospho-H2A ( -H2A) are critical for Crb2 IRIF formation and checkpoint function. Mutations of these BRCT 2 domain residues have additive effects when combined in a single allele. Combining either mutation with an allele that eliminates the threonine-215 cyclin-dependent kinase phosphorylation site completely abrogates Crb2 IRIF and function. We propose that cooperative phosphate interactions in the BRCT 2 -H2A-binding pocket of Crb2, coupled with tudor domain interactions with lysine-20 dimethylation of histone H4, facilitate stable recruitment of Crb2 to chromatin surrounding DSBs, which in turn mediates efficient phosphorylation of Chk1 that is required for a sustained checkpoint response. This mechanism of cooperative interactions with the -H2A/X phosphate is likely conserved in S. pombe Brc1 and human Mdc1 genome maintenance proteins.

Molzan, Manuela; Schumacher, Benjamin; Ottmann, Corinna; Baljuls, Angela; Polzien, Lisa; Weyand, Michael; Thiel, Philipp; Rose, Rolf; Rose, Micheline; Kuhenne, Philipp; Kaiser, Markus; Rapp, Ulf R.; Kuhlmann, Jurgen; Ottmann, Christian

doi: 10.1128/MCB.01636-09pmid: 20679480

Du, Zhiqiang; Crow, Emily T.; Kang, Hyun Seok; Li, Liming

doi: 10.1128/MCB.00225-10pmid: 20679490

We have recently reported that the yeast chromatin-remodeling factor Swi1 can exist as a prion, SWI + , demonstrating a link between prionogenesis and global transcriptional regulation. To shed light on how the Swi1 conformational switch influences Swi1 function and to define the sequence and structural requirements for SWI + formation and propagation, we functionally dissected the Swi1 molecule. We show here that the SWI + prion features are solely attributable to the first 327 amino acid residues (N), a region that is asparagine rich. N was aggregated in SWI + cells but diffuse in swi – cells; chromosomal deletion of the N-coding region resulted in SWI + loss, and recombinant N peptide was able to form infectious amyloid fibers in vitro , enabling SWI + de novo formation through a simple transformation. Although the glutamine-rich middle region (Q) was not sufficient to aggregate in SWI + cells or essential for SWI/SNF function, it significantly modified the Swi1 aggregation pattern and Swi1 function. We also show that excessive Swi1 incurred Li + /Na + sensitivity and that the N/Q regions are important for this gain of sensitivity. Taken together, our results provide the final proof of "protein-only" transmission of SWI + and demonstrate that the widely distributed "dispensable" glutamine/asparagine-rich regions/motifs might have important and divergent biological functions.

Morel, Caroline; Standen, Claire L.; Jung, Dae Young; Gray, Susan; Ong, Helena; Flavell, Richard A.; Kim, Jason K.; Davis, Roger J.

doi: 10.1128/MCB.00585-10pmid: 20679483

The c-Jun NH 2 -terminal kinase (JNK) interacting protein 1 (JIP1) has been proposed to act as a scaffold protein that mediates JNK activation. However, recent studies have implicated JIP1 in multiple biochemical processes. Physiological roles of JIP1 that are related to the JNK scaffold function of JIP1 are therefore unclear. To test the role of JIP1 in JNK activation, we created mice with a germ line point mutation in the Jip1 gene (Thr 103 replaced with Ala) that selectively blocks JIP1-mediated JNK activation. These mutant mice exhibit a severe defect in JNK activation caused by feeding of a high-fat diet. The loss of JIP1-mediated JNK activation protected the mutant mice against obesity-induced insulin resistance. We conclude that JIP1-mediated JNK activation plays a critical role in metabolic stress regulation of the JNK signaling pathway.

Tanji, Masahiro; Ishizaki, Toshimasa; Ebrahimi, Saman; Tsuboguchi, Yuko; Sukezane, Taiko; Akagi, Tsuyoshi; Frame, Margaret C.; Hashimoto, Nobuo; Miyamoto, Susumu; Narumiya, Shuh

doi: 10.1128/MCB.00197-10pmid: 20679479

The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. mDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from mDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-mDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced mDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in mDia1-deficient cells with impaired tyrosine phosphorylation. mDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo . Given overexpression of c-Src in various cancers, these findings suggest that Rho-mDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.

Welford, Scott M.; Dorie, Mary Jo; Li, Xiaofeng; Haase, Volker H.; Giaccia, Amato J.

doi: 10.1128/MCB.01618-09pmid: 20679489

Loss of the VHL tumor suppressor is regarded as an initiating event in the development of clear-cell renal carcinoma. Surprisingly, loss of VHL induces senescence in mouse fibroblasts in vitro , a response that would restrict development of renal carcinoma in vivo . Typical in vitro cell culture levels of oxygen, however, are significantly higher than physiological levels of oxygen, which have been shown to abrogate senescence induced by many stimuli. Therefore, we investigated the oxygen dependence of VHL loss-induced senescence. Using mouse fibroblasts and primary renal epithelial cells in vitro , we found that VHL loss leads to senescence under atmospheric conditions (21% O 2 ), partly through increasing p27 levels, but not under physiological oxygenation (2% to 5% O 2 ), despite maintaining increased p27 expression. This suggests that VHL inactivation sensitizes cells to oxidative stress. In support of this concept, senescence following VHL loss depends on p53 activity, which decreases under the less stressful conditions of mild hypoxia. We confirmed these observations in vivo by treating kidney-specific VHL knockout animals with the potent oxidizer paraquat and observed a robust induction of cellular senescence. Together, these data demonstrate that in vivo oxygenation promotes tolerance of VHL loss in renal epithelia, which may promote the development of renal carcinoma.

Eroglu, Binnur; Moskophidis, Demetrius; Mivechi, Nahid F.

doi: 10.1128/MCB.01493-09pmid: 20679486

Accumulation of tau into neurofibrillary tangles is a pathological consequence of Alzheimer's disease and other tauopathies. Failures of the quality control mechanisms by the heat shock proteins (Hsps) positively correlate with the appearance of such neurodegenerative diseases. However, in vivo genetic evidence for the roles of Hsps in neurodegeneration remains elusive. Hsp110 is a nucleotide exchange factor for Hsp70, and direct substrate binding to Hsp110 may facilitate substrate folding. Hsp70 complexes have been implicated in tau phosphorylation state and amyloid precursor protein (APP) processing. To provide evidence for a role for Hsp110 in central nervous system homeostasis, we have generated hsp110 – / – mice. Our results show that hsp110 – / – mice exhibit accumulation of hyperphosphorylated-tau (p-tau) and neurodegeneration. We also demonstrate that Hsp110 is in complexes with tau, other molecular chaperones, and protein phosphatase 2A (PP2A). Surprisingly, high levels of PP2A remain bound to tau but with significantly reduced activity in brain extracts from aged hsp110 – / – mice compared to brain extracts from wild-type mice. Mice deficient in the Hsp110 partner (Hsp70) also exhibit a phenotype comparable to that of hsp110 – / – mice, confirming a critical role for Hsp110-Hsp70 in maintaining tau in its unphosphorylated form during aging. In addition, crossing hsp110 – / – mice with mice overexpressing mutant APP (APPβsw) leads to selective appearance of insoluble amyloid β42 (Aβ42), suggesting an essential role for Hsp110 in APP processing and Aβ generation. Thus, our findings provide in vivo evidence that Hsp110 plays a critical function in tau phosphorylation state through maintenance of efficient PP2A activity, confirming its role in pathogenesis of Alzheimer's disease and other tauopathies.