Molecular and Cellular Biology

Sumoylation is an essential post-translational modification that functions in multiple cellular processes, including transcriptional regulation. Indeed, transcription factors represent one of the largest groups of proteins that are modified by the SUMO peptide. Multiple roles have been identified for sumoylation of transcription factors, including regulation of their activity, interaction with chromatin, and binding site selection. Here, we examine how Cst6, a bZIP-containing sequence-specific transcription factor in Saccharomyces cerevisiae, is regulated by sumoylation. Cst6 is required for survival during ethanol stress and has roles in the utilization of carbon sources other than glucose. We find that Cst6 is sumoylated to appreciable levels in normally growing yeast at Lys residues 139, 461 and 547, and that its sumoylation level increases in ethanol and oxidative stress conditions, but decreases if ethanol is used as the sole carbon source. To understand the role of Cst6 sumoylation during ethanol stress, we generated a yeast strain that expresses a non-sumoylatable mutant form of Cst6. Cellular levels of the mutant protein are moderately reduced compared to the wild-type form, implying that sumoylation promotes Cst6 stability. Although the mutant can bind DNA, chromatin immunoprecipitation (ChIP) analysis shows that its occupancy level is significantly reduced on promoters of some ethanol stress-regulated genes, suggesting that Cst6 recruitment is attenuated or delayed if it can not be sumoylated. Furthermore, impaired Cst6 sumoylation in the mutant strain correlates with elevated expression of some target genes, either constitutively or during induction by ethanol stress. This is most striking for RPS3, which shows dramatically increased expression in the mutant strain. Together, our results suggest that sumoylation controls multiple properties of Cst6 to limit the expression of its target genes.

Previously, we showed that Papss2 expression is regulated through a Sox9-dependent pathway. Here we explore molecular mechanisms whereby Sox9 regulates mouse Papss2. A 509bp Sox9-responsive DNA element (Region C) was identified upstream of the Papss2 second start site using co-transfection and luciferase reporter assays. A Sox9 responsive element was narrowed down to 32bps within Region C (Sox9RE). Putative SoxE and C/EBP{beta} binding sites were identified within S9RE. C/EBP{beta} was identified as a repressor for Sox9-mediated activity. In cells transfected with expression vectors for C/EBP{beta} and Sox9, increasing amounts of C/EBP{beta} resulted in attenuation of Sox9-mediated activation of Region C while increasing amounts of Sox9 activated transcription in the presence of C/EBP{beta}. Using electromobility shift assays, three protein complexes were identified on S9RE after incubation with nuclear extracts from ATDC5 cells. Super shift assays indicated that under basal conditions C/EBP{beta} was present in the DNA-protein complexes observed. Unlabeled S9RE with point mutations in the predicted SoxE binding site competed with protein complex formation on the S9RE while excess oligo corresponding to the predicted SoxE binding site did not, suggesting that proteins do not bind to SoxE motiff under basal conditions. Under conditions of high Sox9 expression, the formation of protein-DNA complexes on S9RE was inhibited. We then showed by western blot that increasing Sox9 protein resulted in reduced C/EBP{beta} protein levels. Co-immunoprecipitation indicated interaction of Sox9 and C/EBP{beta} proteins. We propose that Sox9 acts to derepress C/EBP{beta}-inhibited transcription of Papss2 by first interacting with C/EBP{beta} to prevent it from binding DNA, then reducing C/EBP{beta} expression.

The centromeric protein-A (CENP-A) is an evolutionary conserved histone H3 variant that marks the identity of the centromeres. Several mechanisms regulate the centromeric deposition of CENP-A as its mislocalization causes erroneous chromosome segregation, leading to aneuploidy-based diseases, including cancers. The most crucial deposition factor is a CENP-A specific chaperone, HJURP (Scm3 in budding yeast), which specifically binds to CENP-A. However, the discovery of HJURP as a DDR (DNA damage repair) protein and evidence of its binding to Holliday junctions in vitro indicate a CENP-A-deposition-independent role of these chaperones. In this study, using budding yeast, we demonstrate that Scm3 is crucial for the DDR pathway as scm3 cells are sensitive to DNA damage. We further observe that the scm3 mutant interacts with the rad52 DDR mutant and is compromised in activating DDR-mediated arrest. We demonstrate that Scm3 associates with the DNA damage sites and undergoes posttranslational modifications upon DNA damage. Overall, from this report and earlier studies on HJURP, we conclude that DDR functions of CENP-A chaperones are conserved across eukaryotes. Thus, the revelation that these chaperones confer genome stability in more than one pathway has clinical significance.

Alternative polyadenylation (APA) generates mRNA isoforms with different lengths of the 3 untranslated region (3 UTR). The tissue inhibitor of metalloproteinase 2 (TIMP2) plays a key role in extracellular matrix remodeling under various developmental and disease conditions. Both human and mouse genes encoding TIMP2 contain two highly conserved 3UTR APA sites, leading to mRNA isoforms that differ substantially in 3UTR size. APA of Timp2 is one of the most significantly regulated events in multiple cell differentiation lineages. Here we show that Timp2 APA is highly regulated in transformation of NIH3T3 cells by the oncogene HRASG12V. Perturbations of isoform expression with long 3UTR isoform-specific knockdown or genomic removal of the alternative UTR (aUTR) region indicate that the long 3UTR isoform contributes to the secreted Timp2 protein much more than the short 3UTR isoform. The short and long 3UTR isoforms differ in subcellular localization to endoplasmic reticulum (ER). Strikingly, Timp2 aUTR enhances secreted protein expression but no effect on intracellular proteins in reporter assays. Furthermore, downregulation of Timp2 long isoform mitigates gene expression changes elicited by HRASG12V. Together, our data indicate that regulation of Timp2 protein expression through APA isoform changes is an integral part of RAS-mediated cell transformation and 3UTR isoforms of Timp2 can have distinct impacts on expression of secreted vs. intracellular proteins.

MeCP2 is a DNA-binding transcriptional regulator that is present at near-histone levels in mammalian cortical neurons. Originally identified as a DNA methylation reader, MeCP2 has been proposed to repress transcription by recruiting corepressors to methylated DNA. While some genome-wide occupancy studies support a preference for methylated DNA, others suggest that MeCP2 binding is more influenced by DNA sequence and accessibility than methylation status. Moreover, multiple studies also suggest a role for MeCP2 in gene activation. To clarify MeCP2 function we expressed MeCP2 in Saccharomyces cerevisiae, which lacks DNA methylation and known MeCP2 corepressors. We find that MeCP2 is toxic to yeast and globally inhibits transcription, indicating that MeCP2 can have significant functional impacts without DNA methylation or mammalian corepressors. A subset of MeCP2 mutations that cause the neurodevelopmental disorder Rett syndrome, particularly those that map to the DNA binding domain, alleviate the toxicity of MeCP2 in yeast. Consistent with the importance of DNA binding for toxicity in yeast, we show that MeCP2 binds to the yeast genome, with increased occupancy at GC-rich, nucleosome-depleted sequences. These findings present yeast as a useful tool for analyzing MeCP2 and reveal MeCP2 properties that are not strictly dependent on DNA methylation or mammalian corepressors. SummaryMeCP2, a transcription regulator found in vertebrates, is proposed to repress transcription by recruiting corepressors to methylated DNA. In this work Brown et al. show that MeCP2 expressed in Saccharomyces cerevisiae, which lacks DNA methylation and mammalian co-repressors, binds promoters and inhibits transcription. A subset of MeCP2 mutations that cause the neurodevelopmental disorder, Rett syndrome, rescues MeCP2-induced phenotypes in yeast, supporting the relevance of these results to MeCP2 function in mammals.

The Plasminogen-Apple-Nematode (PAN) domain, with a core of four to six cysteine residues, is found in > 28,000 proteins across 959 genera but its role in protein function is not fully understood. The PAN domain was initially characterized to be present in numerous proteins including hepatocyte growth factor (HGF). Dysregulation of HGF-mediated signaling results in numerous deadly cancers. All biological impacts of HGF in cell proliferation are triggered by binding of HGF to its cell surface receptor, cellular mesenchymal-epidermal transition (c-MET). Here, we show that four PAN domain cysteine residues are essential for HGF/c-MET signaling. Mutating these residues resulted in retardation of perinuclear localization, cellular internalization of HGF and its receptor, c-MET, and c-MET ubiquitination. Our observations indicate that the PAN domain of HGF is required for the c-MET binding and subsequent c-MET autophosphorylation and phosphorylation of its downstream targets, protein kinase B (AKT), extracellular signal-regulated kinase (ERK), and signal transducer and activator of transcription 3 (STAT3). Furthermore, transcriptional activation of HGF/c-MET signaling-related genes including matrix metalloproteinase-9 (MMP9), ETS translocation variant 1, 4, and 5 (ETV1, ETV4, ETV5), and early growth response 1 (EGR1) was impaired and cell proliferation was attenuated. These results suggest that core cysteine residues in the PAN domain are critical for HGF/c-MET interaction, c-MET mediated signal transduction, and cell survival. Thus, targeting the PAN domain of HGF may represent a mechanism for selectively regulating the binding and activation of the c-MET pathway. SignificanceHGF/c-MET signaling induces multifunctional cellular responses. Dysregulation of HGF/c-MET signaling cascade can lead to tumorigenesis by transforming normal cells to tumor cells. This work defines the importance of core cysteine residues in the PAN domain of HGF in downstream activation of HGF/c-MET signaling. To understand the role of cysteines in the PAN domain, PAN mutants of HGF were used to stimulate c-MET signaling in cells and the impact was delineated by determining phosphorylation and transcription of downstream targets. Mutations in core cysteines in the HGF-PAN domain completely blocked downstream phosphorylation and perinuclear accumulation of c-MET. These results suggest an indispensable role for the cysteine-rich PAN domain in HGF/c-MET interaction and could set the stage for future therapies that selectively disrupt the MET signaling cascade with limited off-target effects in tumors overexpressing HGF/c-MET.

The transcription coactivators CREB binding protein (CBP) and p300 are highly homologous acetyltransferases that mediate histone 3 lysine 27 acetylation (H3K27ac) at regulatory elements such as enhancers and promoters. Although in most cases, CBP and p300 are considered to be functionally identical, both proteins are indispensable for development and there is evidence of tissue-specific nonredundancy. However, characterization of chromatin and transcription states regulated by each protein is lacking. In this study we analyze the individual contribution of p300 and CBP to the H3K27ac landscape, chromatin accessibility, and transcription in mouse embryonic stem cells (mESC). We demonstrate that p300 is responsible for the majority of H3K27ac in mESCs and that loss of acetylation in p300-depleted mESCs is more pronounced at enhancers compared to promoters. While loss of either CBP or p300 has little effect on the open state of chromatin, we observe that distinct gene sets are transcriptionally dysregulated upon depletion of p300 or CBP. Transcriptional dysregulation is generally correlated with dysregulation of promoter acetylation upon depletion of p300 (but not CBP) and appears to be relatively independent of dysregulated enhancer acetylation. Interestingly, both our transcriptional and genomic analyses demonstrate that targets of the p53 pathway are stabilized upon deletion of p300, suggesting that this pathway is highly prioritized when p300 levels are limited.

Yeast flocculation relies on cell surface flocculin proteins encoded by the FLO1 gene. The expression of FLO1 is antagonistically regulated by the Tup1-Cyc8 and the Swi-Snf complexes. The Post translational modifications of core histones regulate the transcription of Tup1-Cyc8-regulated genes. However, the mechanisms by which the physical presence of tail residues regulate FLO1 transcription process and flocculation is yet to be completely understood. Through screening we have identified a new region within the N-terminal tail of histone H3 regulating the transcription of FLO1 and FLO5. One of the histone H3 N-terminal truncation mutants H3{Delta}(17-24) showed higher FLO1 expression compared to wild-type H3. Results revealed that in absence of 17-24 stretch the occupancy of Cyc8 decreases from the upstream regions of FLO1. Additionally, analysis suggests that Hda1 is required for the Cyc8-mediated repression of FLO1. Altogether we demonstrate that 17-24 stretch is essential for the Tup1 independent binding of Cyc8 at the promoters assisted by Hda1, leading to the strong repression of FLO1 transcription. In the absence of the 17-24 stretch, Cyc8 cannot bind, resulting in uncontrolled transcription of FLO1.

CTC1-STN1-TEN1 (CST) is a heterotrimeric, RPA-like complex that binds single-stranded DNA, stimulates DNA polymerase -primase, and functions in several genome maintenance pathways, including telomere maintenance and DNA replication/repair. During telomere replication, CST prevents telomerase from overextending the G-rich single-stranded overhang (G-OH) and promotes fill-in of the C-rich strand by stimulating DNA polymerase -primase. Previous work characterized the effects of CST loss by deleting CTC1 or TEN1. Interestingly, CTC1 knockout (KO) caused severe proliferation defects and telomeric damage signaling, whereas these phenotypes were absent following TEN1 KO. Molecular analysis revealed that, while loss of CTC1 or TEN1 leads to defective C-strand fill-in, only CTC1 KO exhibited excessive G-OH lengthening. Here, we characterized conditional STN1 KO cells and determined that STN1 KO leads to proliferation defects and telomeric damage signaling. Moreover, STN1 KO caused genome instability in the form of anaphase bridges and micronuclei. Interestingly, these phenotypes and growth inhibition were largely dependent on telomerase activity. Our findings indicate that STN1 KO closely resembles CTC1 versus TEN1 KO and that excessive G-OH extension underlies the genome instability caused by STN1 deletion. SUMMARY STATEMENTThe STN1 subunit of the single-stranded DNA binding protein CST prevents telomeric damage signaling, genome instability, and proliferation defects by limiting telomerase activity.

The positive coactivator 4 or PC4 is a chromatin-associated protein whose role in gene regulation by wild-type p53 is now well-known. During tumorigenesis, p53 is often mutated resulting in its loss of function. A sub-class of these mutants gain new pro-proliferation properties which occur largely due to the upregulation of many pro-proliferation genes. Little is known about the roles of PC4 in tumor cells bearing mutant p53 genes. In this article, we show that PC4 associates with one of the tumor-associated gain-of-function p53 mutants, R273H. This association drives its recruitment to two promoters, UBE2C, and MDR1, known to be responsible for imparting aggressive growth and resistance to many drugs. A previously reported peptide that disrupts PC4-wild-type p53 interaction also disrupts the PC4-R273Hp53 protein-protein interaction. The introduction of this peptide to tumor cells bearing the R273HTP53 gene resulted in a lowering of MDR1 expression and abrogation of drug resistance. Interestingly, cells bearing another gain-of-function mutant R248W do not show the same type of response, suggesting that the action of PC4 on mutant p53s may differ for different GOF mutants. The results presented here suggest that PC4-R273H interaction may be a promising target for reducing proliferation and tumor drug resistance.

Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging DNA strands, respectively. Single Dpb3 deletion (dpb3{Delta}) or Mcm2 mutation (mcm2-3A), which each disrupt one parental histone transfer pathway, leads to the others predominance. However, the impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3{Delta}/mcm2-3A double mutant did not exhibit the single dpb3{Delta} and mcm2-3A mutants asymmetric parental histone patterns, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3{Delta}, mcm2-3A, and dpb3{Delta}/mcm2-3A mutants relative to the wild-type strain, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones to the leading and lagging strands during DNA replication is essential for maintaining chromatin structure and that high levels of free histones due to parental histone transfer defects are detrimental to cells.

The p53 family of transcription factors regulate numerous organismal processes including the development of skin and limbs, ciliogenesis, and preservation of genetic integrity and tumor suppression. p53 family members control these processes and gene expression networks through engagement with DNA sequences within gene regulatory elements. Whereas p53 binding to its cognate recognition sequence is strongly associated with transcriptional activation, p63 can mediate both activation and repression. How the DNA sequence of p63-bound gene regulatory elements is linked to these varied activities is not yet understood. Here, we use massively parallel reporter assays (MPRA) in a range of cellular and genetic contexts to investigate the influence of DNA sequence on p63-mediated transcription. Most regulatory elements with a p63 response element motif (p63RE) activate transcription, with those sites bound by p63 more frequently or adhering closer to canonical p53 family response element sequences driving higher transcriptional output. The most active regulatory elements are those also capable of binding p53. Elements uniquely bound by p63 have varied activity, with p63RE-mediated repression associated with lower overall GC content in flanking sequences. Comparison of activity across cell lines suggests differential activity of elements may be regulated by a combination of p63 abundance or context-specific cofactors. Finally, changes in p63 isoform expression dramatically alters regulatory element activity, primarily shifting inactive elements towards a strong p63-dependent activity. Our analysis of p63-bound gene regulatory elements provides new insight into how sequence, cellular context, and other transcription factors influence p63-dependent transcription. These studies provide a framework for understanding how p63 genomic binding locally regulates transcription. Additionally, these results can be extended to investigate the influence of sequence content, genomic context, chromatin structure on the interplay between p63 isoforms and p53 family paralogs.

To date, epithelial-to-mesenchymal transition (EMT) has been observed in cultured hepatocytes, but not in vivo. TGF-{beta} is supposed to initiate EMT in hepatocytes by inhibiting HNF4 through the SMAD2/3 complex. We report that TGF-{beta} does not directly inhibit HNF4, but contributes to its transcriptional regulation by SMAD2/3 recruiting acetyltransferase CBP/p300 to the HNF4 promoter. The recruitment of CBP/p300 is indispensable for C/EBPa binding, another essential requirement for constitutive HNF4 expression in hepatocytes. In contrast to the observed induction of HNF4, SMAD2/3 inhibits C/EBP transcription. Therefore, long-term TGF-{beta} incubation results in C/EBP depletion, which abrogates HNF4 expression. Intriguingly, SMAD2/3 inhibitory binding to the C/EBP promoter is abolished by insulin. Thus, maintaining a high insulin concentration in culture medium ensures constitutive HNF4 and thereby prevents TGF-{beta}-induced hepatocyte EMT. Furthermore, insulin inhibits TGF-{beta}-induced SMAD2/3 binding to the promoters of core EMT transcription factors e.g., SNAI1. SNAI1 transcription requires both SMAD2/3 and FOXO1 in nuclei. Insulin inhibits SNAI1 transcription through impeding SMAD2/3 binding to its promoter and inducing FOXO1 phosphorylation. Hence, insulin is the key factor that prevents TGF-{beta}-induced EMT in hepatocytes.

Histone post-translational modifications (PTMs) promote genome segregation into heterochromatin and euchromatin by recruiting reader proteins, including HP1 proteins that bind repressive H3 lysine 9 methylation (H3K9me). However, recent studies suggest that H3K9me readers and writers also have PTM-independent activities. Here we examine the PTM-independent roles of the key H3K9 methyltransferase, MET-2/SETDB1, and the C. elegans HP1 orthologue, HPL-2, in organogenesis. The two factors co-localize in sub-nuclear foci independently of each other, and their combined loss additively disrupts gene repression and vulval organogenesis. HPL-2 remains functional in the absence of H3K9me, and HPL-2 lacking its H3K9me-binding chromodomain still localizes to heterochromatin foci, restricting vulva formation and repressing developmental genes. While MET-2 requires the disordered protein LIN-65 to form foci and silence genes, HPL-2 functions together with LIN-13, which is bound through HPL-2s chromoshadow domain. In conclusion, HPL-2 and MET-2 repress in parallel on largely H3K9me-independent pathways, requiring distinct cofactors; while H3K9me reinforces both.

Chromodomain helicase DNA binding protein 8 (CHD8) is a chromatin remodeler of the SNF2 family involved in gene transcription regulation. It has been shown that CHD8 is required for cell proliferation, cell differentiation and central nervous system development. In fact, CHD8 haploinsufficiency causes a human syndrome characterized by autism, macrocephaly, gastrointestinal complaints and some other clinical characteristics. However, the mechanism by which CHD8 controls transcription and how it is recruited to its targets in the chromatin is still unclear. We have previously shown that serum depletion causes that CHD8 detaches from chromatin. Here we demonstrate that serum-dependent recruitment of CHD8 to promoters requires the extracellular signal-regulated kinase (ERK)/ ETS-like (ELK) branch of the mitogen-activated protein kinase (MAPK) pathway. Our analysis of genomic occupancy data shows that CHD8 binding sites were strongly enriched in ELK1 and ELK4 DNA binding motifs and that CHD8 and ELK1 co-occupy multiple transcription start sites. We show that ELK1 and ELK4 are required for normal recruitment of CHD8 to the promoters of CCNA2, CDC6, CCNE2, BRCA2 and MYC genes. However, CHD8 is dispensable for ELK1 and ELK4 binding. Genome wide transcriptomic analysis evidenced that serum-dependent activation of a subset of immediate early genes, including the well-known ELK1 target gene FOS, was impaired upon depletion of CHD8. In summary, our results uncover the role of the ERK/ELK pathway in CHD8 recruitment to chromatin and provide evidences indicating a role of CHD8 in regulating serum-dependent transcription.

Two fission yeast mitotic activators, Cdc13 and Cdc25, have been shown to increase in concentration in correlation with cell size, and have been proposed to thereby regulate cell size at division. Here, we show that the expression of both Cdc13 and Cdc25 are, in fact, size dependent, as apposed to simply sizecorrelated due to time-dependent expression. However, we also find that their size dependence is regulated by different mechanisms. Cdc25 was known to be regulated transcriptionally. Here, we show that Cdc13 is regulated translationally. Its transcript is not expression is a size-dependent manner, rather a size-dependent concentration of protein is expressed from a size-independent concentration of mRNA. Moreover, the degradation rate of Cdc13 is not size dependent, implicating size-dependent translation in its regulation. We identify a 20-amino-acid motif, which includes the APC D-box degron, as necessary and sufficient for sizedependent expression, which allowed us to construct a size-independent allele of cdc13. Using this allele, in combination with a size-independent allele of cdc25, expressed from a size-independent promoter, we show that size-dependent expression of neither Cdc13 nor Cdc25 is required for size control, nor are the redundantly required for size control.

Cyclin C is a highly conserved component of the Mediator kinase module that regulates transcription through CDK8 stimulation. In addition, the yeast (Cnc1) and human (CCNC) cyclin C exhibit stress-induced mitochondrial translocation to stimulate fission through direct interaction with the dynamin-like GTPase DRP1 (mammals) or Dnm1 (yeast). Gene ablation studies revealed that both Cnc1 and CCNC are required for cell damage-induced regulated cell death (RCD). To determine the relative contributions of cyclin Cs transcriptional and mitochondrial roles in promoting RCD, this study utilized docking simulation algorithms to predict interaction interfaces for CCNC-CDK8 and CCNC-DRP1 heterodimers. As expected, CCNC bound CDK8 through its amino terminal cyclin box domain while DRP1 associated with the second carboxyl cyclin box. Using these predictions, we used site directed mutagenesis on Cnc1 to separate these functions. Importantly, only the DRP1/Dnm1-interaction residues were important for RCD in yeast. Interestingly, although Dnm1 is required for RCD, its fission activity was not. Moreover, Dnm1 is still required for RCD even when Cnc1 is targeted to the mitochondria indicating it is not simply functioning as a tether. Finally, when expressed in yeast, the human CCNC efficiently induced fission and stimulated RCD. Moreover, these functions required predicted DRP1 interaction sites as well. In conclusion, these studies revealed that cyclin C separates its nuclear and mitochondrial activities by utilizing different cyclin box domains. Second, Dnm1-cyclin C interaction, and not transcriptional control, is critical for cyclin C-dependent RCD and this role is conserved from yeast to humans. Author SummaryThe cyclin C protein is found in all eucaryotes and exhibits two conserved functions. First cyclin C activates the CDK8 protein kinase to control transcription of genes involved in the stress response. Second, stress induces cyclin C translocation to the mitochondria where it induces fragmentation by stimulating the fission GTPase DRP1. Mitochondrial fission is an early step in the regulated cell death pathway. Importantly, cyclin C is required for stress-induced cell death raising the question of what the relative contribution of its transcription and mitochondrial roles is. To address this question, we generated mutations in the yeast cyclin C that interfered with its association with CDK8 or DRP1 without affecting the other. We found that the mitochondrial role, but not transcription, was required for cell death in yeast. In addition, when expressed in yeast, the human cyclin C also induced mitochondrial fission and cell death. Finally, although DRP1 is important for regulated cell death, its fission function is not. These results point to a highly conserved role for cyclin C in mediating regulated cell death at the mitochondria. In addition, these results point to a non-enzymatic role for DRP1 in executing the cell death pathway.

Stress granules are RNA-protein condensates that form in response to an increase in untranslating mRNPs. Stress granules form by the condensation of mRNPs through a combination of protein-protein, protein-RNA, and RNA-RNA interactions. Several reports have suggested that G-rich RNA sequences capable of forming G-quadruplexes promote stress granule formation. Here, we provide three observations arguing that G-tracts capable of forming rG4s do not promote mRNAs partitioning into stress granules in human osteosarcoma cells. First, we observed no difference in the accumulation in stress granules of reporter mRNAs with and without G-tracts in their 3 UTRs. Second, in U-2 OS cell lines with reduced DHX36 expression, which is thought to unwind G-quadruplexes, the partitioning of endogenous mRNAs was independent of their predicted rG4-forming potential. Third, while mRNAs in stress granules initially appeared to have a higher probability of forming rG4s than bulk mRNAs, this effect disappeared when rG4 motif abundance was standardized by mRNA length. However, we observe that in a G3BP1/2 double knockout cell line, reducing DHX36 expression rescued stress granule-like foci formation. This indicates that DHX36 can limit stress granule formation, potentially by unwinding trans rG4s, or limiting other intermolecular RNA-RNA interactions that promote stress granule formation. Key Points- G-tract RNAs with quadruplex forming potential in an mRNA do not affect its partitioning into stress granules - mRNA partitioning to stress granules is dependent on mRNA length rather than rG4-forming potential - DHX36, a DEAH-box helicase that unwinds RNA G-quadruplexes, limits stress granule formation O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/659950v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@a24c58org.highwire.dtl.DTLVardef@1422ce9org.highwire.dtl.DTLVardef@1929677org.highwire.dtl.DTLVardef@d48fa6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tribbles related homolog 1 (TRIB1) contributes to lipid and glucose homeostasis by facilitating the degradation of cognate cargos by the proteasome. We previously reported that TRIB1 was unstable in non-hepatic cellular models. Moreover, inclusion of proteasome inhibitors failed to prevent TRIB1 loss, consistent with the involvement of proteasome independent degradative processes. In view of the key role of TRIB1 in liver function, we continue our exploration of TRIB1 regulation pathways in two commonly used human hepatocyte models, HuH-7 and HepG2 cells. Proteasome inhibitors potently upregulated both endogenous and recombinant TRIB1 mRNA and protein levels. Increased transcript abundance was independent of MAPK activation while ER stress was a relatively mild inducer. Despite increasing TRIB1 protein abundance and stabilizing bulk ubiquitination, proteasome inhibition failed to stabilize TRIB1, pointing to the predominance of proteasome independent protein degradation processes controlling TRIB1 protein abundance in hepatomas. Proteasome inhibition via downregulation of its PSMB3 regulatory subunit, in contrast to its chemical inhibition, had minimal impact on TRIB1 levels. Moreover, immunoprecipitation experiments showed no evidence of TRIB1 ubiquitination. Cytoplasmic retained TRIB1 was unstable, indicating that TRIB1 lability is regulated prior to its nuclear import. Substitution of the TRIB1 PEST-like region with a GST helical region or N-terminal deletions failed to fully stabilize TRIB1. Finally, inclusion of protease or autophagy inhibitors in vivo did not rescue TRIB1 stability. This work excludes proteasome-mediated degradation as a significant contributor to TRIB1 instability and identifies transcriptional regulation as a prominent mechanism regulating TRIB1 abundance in liver models in response to proteasome inhibition.

The Fanconi anemia (FA) protein FANCD2, and MCM8/9 heterohexameric helicase complex are critical for maintaining genomic integrity in response to replication stress. However, the nature of their relationship remains unclear. Here, we show that MCM8/9 physically interacts and functionally cooperates with FANCD2 during the repair of DNA interstrand crosslinks (ICLs). Using immunofluorescence and co-immunoprecipitation studies, we show that MCM8/9 interacts with FANCD2 through its core domain, independently of DNA. FANCD2 is essential for the recruitment of MCM9 to ICL damage induced nuclear foci and acts downstream the FANCD2I monoubiquitination. Although MCM8/9 foci formation requires its intact ATPase activity, BRCv motif and HROB, these are not required for FANCD2 binding, highlighting a distinction between physical interaction and functional activation. Interestingly, FANCD2 foci formation increase in MCM8 or MCM9 knockout cells, suggesting that MCM8/9 functions to mitigate replication associate stress. {gamma}H2AX DNA damage assays and cell survival assays show that combined loss of MCM9 and FANCD2 do not cause any additive DNA damage beyond individual knockouts, indicating an epistatic relationship and suggests they function in the same DNA repair pathway. Together, our findings identify MCM8/9 as a downstream effector of the FA pathway critical for resolving ICL induced DNA damage. HighlightsO_LIMCM8/9 interacts and colocalize with FANCD2 upon ICL DNA damage. C_LIO_LIFANCD2 is essential for recruitment of MCM9 to DNA damage site. C_LIO_LIMCM8/9 is epistatic to FANCD2 and within the same DNA damage response pathway. C_LI