Publications by authors named "Chi-Fu Chen"

11 Publications

  • Page 1 of 1

Centromere RNA Is Negatively Regulated by Cbf1 and Its Unscheduled Synthesis Impacts CenH3 Binding.

Genetics 2019 10 7;213(2):465-479. Epub 2019 Aug 7.

Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, New Jersey 08544

Two common features of centromeres are their transcription into noncoding centromere RNAs (cen-RNAs) and their assembly into nucleosomes that contain a centromere-specific histone H3 (cenH3). Here, we show that cen-RNA was present in low amounts in wild-type (WT) cells, and that its appearance was tightly cell cycle-regulated, appearing and disappearing in a narrow window in S phase after centromere replication. In cells lacking Cbf1, a centromere-binding protein, cen-RNA was 5-12 times more abundant throughout the cell cycle. In WT cells, cen-RNA appearance occurred at the same time as loss of Cbf1's centromere binding, arguing that the physical presence of Cbf1 inhibits cen-RNA production. Binding of the Pif1 DNA helicase, which happens in mid-late S phase, occurred at about the same time as Cbf1 loss from the centromere, suggesting that Pif1 may facilitate this loss by its known ability to displace proteins from DNA. Cen-RNAs were more abundant in Δ cells but only in mid-late S phase. However, fork pausing at centromeres was not elevated in Δ cells but rather was due to centromere-binding proteins, including Cbf1 Strains with increased cen-RNA lost centromere plasmids at elevated rates. In Δ cells, where both the levels and the cell cycle-regulated appearance of cen-RNA were disrupted, the timing and levels of cenH3 centromere binding were perturbed. Thus, cen-RNAs are highly regulated, and disruption of this regulation correlates with changes in centromere structure and function.
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http://dx.doi.org/10.1534/genetics.119.302528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781895PMC
October 2019

Two Pif1 Family DNA Helicases Cooperate in Centromere Replication and Segregation in .

Genetics 2019 01 15;211(1):105-119. Epub 2018 Nov 15.

Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, New Jersey 08544

Pif1 family helicases are found in virtually all eukaryotes. (Sc) encodes two Pif1 family helicases, ScPif1 and Rrm3 ScPif1 is multifunctional, required not only for maintenance of mitochondrial DNA but also for multiple distinct nuclear functions. Rrm3 moves with the replication fork and promotes movement of the fork through ∼1400 hard-to-replicate sites, including centromeres. Here we show that ScPif1, like Rrm3, bound robustly to yeast centromeres but only if the centromere was active. While Rrm3 binding to centromeres occurred in early to mid S phase, about the same time as centromere replication, ScPif1 binding occurred later in the cell cycle when replication of most centromeres is complete. However, the timing of Rrm3 and ScPif1 centromere binding was altered by the absence of the other helicase, such that Rrm3 centromere binding occurred later in cells and ScPif1 centromere binding occurred earlier in Δ cells. As shown previously, the modest pausing of replication forks at centromeres seen in wild-type cells was increased in the absence of Rrm3 While a lack of ScPif1 did not result in increased fork pausing at centromeres, pausing was even higher in Δ Δ cells than in Δ cells. Likewise, centromere function as monitored by the loss rate of a centromere plasmid was increased in Δ but not Δ cells, and was even higher in Δ Δ cells than in Δ cells. Thus, ScPif1 promotes centromere replication and segregation, but only in the absence of Rrm3 These data also hint at a potential post-S phase function for ScPif1 at centromeres. These studies add to the growing list of ScPif1 functions that promote chromosome stability.
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http://dx.doi.org/10.1534/genetics.118.301710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325707PMC
January 2019

PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes.

Nat Commun 2017 04 21;8:15025. Epub 2017 Apr 21.

Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544, USA.

Saccharomyces cerevisiae encodes two Pif1 family DNA helicases, Pif1 and Rrm3. Rrm3 promotes DNA replication past stable protein complexes at tRNA genes (tDNAs). We identify a new role for the Pif1 helicase: promotion of replication and suppression of DNA damage at tDNAs. Pif1 binds multiple tDNAs, and this binding is higher in rrm3Δ cells. Accumulation of replication intermediates and DNA damage at tDNAs is higher in pif1Δ rrm3Δ than in rrm3Δ cells. DNA damage at tDNAs in the absence of these helicases is suppressed by destabilizing R-loops while Pif1 and Rrm3 binding to tDNAs is increased upon R-loop stabilization. We propose that Rrm3 and Pif1 promote genome stability at tDNAs by displacing the stable multi-protein transcription complex and by removing R-loops. Thus, we identify tDNAs as a new source of R-loop-mediated DNA damage. Given their large number and high transcription rate, tDNAs may be a potent source of genome instability.
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http://dx.doi.org/10.1038/ncomms15025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413955PMC
April 2017

Multimerization domains are associated with apparent strand exchange activity in BLM and WRN DNA helicases.

DNA Repair (Amst) 2014 Oct 3;22:137-46. Epub 2014 Sep 3.

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, United States. Electronic address:

BLM and WRN are members of the RecQ family of DNA helicases that act to suppress genome instability and cancer predisposition. In addition to a RecQ helicase domain, each of these proteins contains an N-terminal domain of approximately 500 amino acids (aa) that is incompletely characterized. Previously, we showed that the N-terminus of Sgs1, the yeast ortholog of BLM, contains a physiologically important 200 aa domain (Sgs1103-322) that displays single-stranded DNA (ssDNA) binding, strand annealing (SA), and apparent strand-exchange (SE) activities in vitro. Here we used a genetic assay to search for heterologous proteins that could functionally replace this domain of Sgs1 in vivo. In contrast to Rad59, the oligomeric Rad52 protein provided in vivo complementation, suggesting that multimerization is functionally important. An N-terminal domain of WRN was also identified that could replace Sgs1103-322 in yeast. This domain, WRN235-526, contains a known coiled coil and displays the same SA and SE activities as Sgs1103-322. The coiled coil domain of WRN235-526 is required for both its in vivo activity and its in vitro SE activity. Based on this result, a potential coiled coil was identified within Sgs1103-322. This 25 amino acid region was similarly essential for wt Sgs1 activity in vivo and was replaceable by a heterologous coiled coil. Taken together, the results indicate that a coiled coil and a closely linked apparent SE activity are conserved features of the BLM and WRN DNA helicases.
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http://dx.doi.org/10.1016/j.dnarep.2014.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174979PMC
October 2014

Wss1 is a SUMO-dependent isopeptidase that interacts genetically with the Slx5-Slx8 SUMO-targeted ubiquitin ligase.

Mol Cell Biol 2010 Aug 1;30(15):3737-48. Epub 2010 Jun 1.

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA.

Protein sumoylation plays an important but poorly understood role in controlling genome integrity. In Saccharomyces cerevisiae, the Slx5-Slx8 SUMO-targeted Ub ligase appears to be needed to ubiquitinate sumoylated proteins that arise in the absence of the Sgs1 DNA helicase. WSS1, a high-copy-number suppressor of a mutant SUMO, was implicated in this pathway because it shares phenotypes with SLX5-SLX8 mutants, including a wss1Delta sgs1Delta synthetic-fitness defect. Here we show that Wss1, a putative metalloprotease, physically binds SUMO and displays in vitro isopeptidase activity on poly-SUMO chains. Like that of SLX5, overexpression of WSS1 suppresses sgs1Delta slx5Delta lethality and the ulp1ts growth defect. Interestingly, although Wss1 is relatively inactive on ubiquitinated substrates and poly-Ub chains, it efficiently deubiquitinates a Ub-SUMO isopeptide conjugate and a Ub-SUMO fusion protein. Wss1 was further implicated in Ub metabolism on the basis of its physical association with proteasomal subunits. The results suggest that Wss1 is a SUMO-dependent isopeptidase that acts on sumoylated substrates as they undergo proteasomal degradation.
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http://dx.doi.org/10.1128/MCB.01649-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916399PMC
August 2010

An essential DNA strand-exchange activity is conserved in the divergent N-termini of BLM orthologs.

EMBO J 2010 May 13;29(10):1713-25. Epub 2010 Apr 13.

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA.

The gene mutated in Bloom's syndrome, BLM, encodes a member of the RecQ family of DNA helicases that is needed to suppress genome instability and cancer predisposition. BLM is highly conserved and all BLM orthologs, including budding yeast Sgs1, have a large N-terminus that binds Top3-Rmi1 but has no known catalytic activity. In this study, we describe a sub-domain of the Sgs1 N-terminus that shows in vitro single-strand DNA (ssDNA) binding, ssDNA annealing and strand-exchange (SE) activities. These activities are conserved in the human and Drosophila orthologs. SE between duplex DNA and homologous ssDNA requires no cofactors and is inhibited by a single mismatched base pair. The SE domain of Sgs1 is required in vivo for the suppression of hyper-recombination, suppression of synthetic lethality and heteroduplex rejection. The top3Delta slow-growth phenotype is also SE dependent. Surprisingly, the highly divergent human SE domain functions in yeast. This work identifies SE as a new molecular function of BLM/Sgs1, and we propose that at least one role of SE is to mediate the strand-passage events catalysed by Top3-Rmi1.
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http://dx.doi.org/10.1038/emboj.2010.61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2876966PMC
May 2010

Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre-mRNA splicing.

Mol Cell 2007 Nov;28(4):665-76

Department of Biochemistry, New York University School of Medicine, New York, NY 10016, USA.

Trimethylation of histone H3 on lysine 4 (H3K4me3) localizes near the 5' region of genes and is tightly associated with active loci. Several proteins, such as CHD1, BPTF, JMJD2A, and the ING tumor suppressor family, directly recognize this lysine methyl mark. However, how H3K4me3 recognition participates in active transcription remains poorly characterized. Here we identify specific CHD1-interacting proteins via H3K4me3 affinity purification, including numerous factors mediating postinitiation events. Conventional biochemical purification revealed a stable complex between CHD1 and components of the spliceosome. Depletion of CHD1 in extracts dramatically reduced splicing efficiency in vitro, indicating a functional link between CHD1 and the spliceosome. Knockdown of CHD1 and H3K4me3 levels by siRNA reduced association of U2 snRNP components with chromatin and, more importantly, altered the efficiency of pre-mRNA splicing on active genes in vivo. These findings suggest that methylated H3K4 serves to facilitate the competency of pre-mRNA maturation through the bridging of spliceosomal components to H3K4me3 via CHD1.
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http://dx.doi.org/10.1016/j.molcel.2007.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276655PMC
November 2007

Binding and activation of DNA topoisomerase III by the Rmi1 subunit.

J Biol Chem 2007 Sep 9;282(39):28971-9. Epub 2007 Aug 9.

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA.

Rmi1 is a conserved oligonucleotide and oligosaccharide binding-fold protein that is associated with RecQ DNA helicase complexes from humans (BLM-TOP3 alpha) and yeast (Sgs1-Top3). Although human RMI1 stimulates the dissolution activity of BLM-TOP3 alpha, its biochemical function is unknown. Here we examined the role of Rmi1 in the yeast complex. Consistent with the similarity of top3Delta and rmi1Delta phenotypes, we find that a stable Top3.Rmi1 complex can be isolated from yeast cells overexpressing these two subunits. Compared with Top3 alone, this complex displays increased superhelical relaxation activity. The isolated Rmi1 subunit also stimulates Top3 activity in reconstitution experiments. In both cases elevated temperatures are required for optimal relaxation unless the substrate contains a single-strand DNA (ssDNA) bubble. Interestingly, Rmi1 binds only weakly to ssDNA on its own, but it stimulates the ssDNA binding activity of Top3 5-fold. Top3 and Rmi1 also cooperate to bind the Sgs1 N terminus and promote its interaction with ssDNA. These results demonstrate that Top3-Rmi1 functions as a complex and suggest that Rmi1 stimulates Top3 by promoting its interaction with ssDNA.
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http://dx.doi.org/10.1074/jbc.M705427200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2804875PMC
September 2007

Molecular cloning and functional identification of invertase isozymes from green bamboo Bambusa oldhamii.

J Agric Food Chem 2006 Apr;54(8):3101-7

Institute of Microbiology and Biochemistry, National Taiwan University, Taipei 106, Taiwan.

Three Bo beta fruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Bo beta fruct1 and Bo beta fruct2 were determined to encode cell wall invertases, whereas Bo beta fruct3encodes a vacuolar invertase. The recombinant proteins encoded by Bo beta fruct2 and Bo beta fruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pI values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Bo beta fruct1 and Bo beta fruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Bo beta fruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Bo beta fruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement.
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http://dx.doi.org/10.1021/jf052711sDOI Listing
April 2006

Human but not yeast CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains.

J Biol Chem 2005 Dec 31;280(51):41789-92. Epub 2005 Oct 31.

Howard Hughes Medical Institute and Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

Defining the protein factors that directly recognize post-translational, covalent histone modifications is essential toward understanding the impact of these chromatin "marks" on gene regulation. In the current study, we identify human CHD1, an ATP-dependent chromatin remodeling protein, as a factor that directly and selectively recognizes histone H3 methylated on lysine 4. In vitro binding studies identified that CHD1 recognizes di- and trimethyl H3K4 with a dissociation constant (Kd) of approximately 5 microm, whereas monomethyl H3K4 binds CHD1 with a 3-fold lower affinity. Surprisingly, human CHD1 binds to methylated H3K4 in a manner that requires both of its tandem chromodomains. In vitro analyses demonstrate that unlike human CHD1, yeast Chd1 does not bind methylated H3K4. Our findings indicate that yeast and human CHD1 have diverged in their ability to discriminate covalently modified histones and link histone modification-recognition and non-covalent chromatin remodeling activities within a single human protein.
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http://dx.doi.org/10.1074/jbc.C500395200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1421377PMC
December 2005

Acute hemodynamic study of Tai-Ta left ventricular assist device in a canine model.

Artif Organs 2004 Dec;28(12):1095-101

Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan.

A revised Tai-Ta centrifugal impeller pump was designed to study the interaction of the left ventricular assist device (LVAD) with the cardiovascular system in a canine model. Six healthy dogs weighing 12-16 kg were used. Blood flows in the aortic arch, the pulmonary artery (PA), and the LVAD outlet were measured simultaneously with the arterial blood pressure (ABP), the pump outflow pressure (POP), and the electrocardiograph (ECG). Normally, the blood flows in the aorta and the PA started at the S-wave of the ECG. When the LVAD was operated at a higher rotational speed (increased from 2900 to 5400 rpm), the ABP, POP, the pump flow, and the maximum rate of change of PA flow increased. However, the fluctuating amplitudes of ABP, POP, and the pump flow decreased significantly. The cardiovascular hemodynamics change with the pump speeds. For a typical 1.1-1.5 L/min cardiac output in canine, the revised LVAD was able to deliver a flow bypass ratio from 15% up to 100%. The LVAD outflow appeared to be pulsatile and matched the cardiac cycle, showing that the centrifugal impeller pump could be used as a pediatric assist device when cardiac function was impeded.
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http://dx.doi.org/10.1111/j.1525-1594.2004.07314.xDOI Listing
December 2004