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author:("Liu, bifan")
1.  Methylation of histone H3K23 blocks DNA damage in pericentric heterochromatin during meiosis 
eLife  2014;3:e02996.
Despite the well-established role of heterochromatin in protecting chromosomal integrity during meiosis and mitosis, the contribution and extent of heterochromatic histone posttranslational modifications (PTMs) remain poorly defined. Here, we gained novel functional insight about heterochromatic PTMs by analyzing histone H3 purified from the heterochromatic germline micronucleus of the model organism Tetrahymena thermophila. Mass spectrometric sequencing of micronuclear H3 identified H3K23 trimethylation (H3K23me3), a previously uncharacterized PTM. H3K23me3 became particularly enriched during meiotic leptotene and zygotene in germline chromatin of Tetrahymena and C. elegans. Loss of H3K23me3 in Tetrahymena through deletion of the methyltransferase Ezl3p caused mislocalization of meiosis-induced DNA double-strand breaks (DSBs) to heterochromatin, and a decrease in progeny viability. These results show that an evolutionarily conserved developmental pathway regulates H3K23me3 during meiosis, and our studies in Tetrahymena suggest this pathway may function to protect heterochromatin from DSBs.
eLife digest
Inside the nucleus of a cell, the DNA is wound around histone proteins. This forms a structure called chromatin that allows the long DNA strands to fit inside the cell. Variations in chromatin structure also help the cell to control the functional properties of DNA. For example, a large proportion of chromatin in the cell is in the form of heterochromatin, which is very densely packed, and is associated with many roles such as gene silencing and keeping DNA intact during reproduction.
Many animals and plants have two copies of each DNA molecule: one inherited from the mother, and one from the father of the organism. Reproductive cells undergo a process called recombination when they form, where the matching copies of each DNA molecule break in a number of places and rejoin to form a new ‘blend’ of their mother's and their father's DNA, which is passed on to their own offspring. In contrast, most heterochromatin is inherited without recombining, preserving it in an unaltered form. This is important since recombination in heterochromatin can create genetic abnormalities.
Adding small chemical modifications—such as methyl groups—to the histone proteins at the core of the chromatin can change how the DNA is packed. However, the histone modifications that yield different chromatin structures, and the effect of these modifications, are not very well understood.
Papazyan et al. have taken advantage of a distinct feature of the protozoan Tetrahymena thermophila: a single-celled organism that divides its chromatin into two different nuclei. The smaller micronuclei contain only heterochromatin, and Papazyan et al. discovered that the histone H3 protein in the micronuclei is modified by methyl groups at a specific site that had not been studied before. Furthermore, this protozoan makes more of these modifications when it reproduces. An enzyme called Ezl3p adds these methyl groups, and without this enzyme T. thermophila reproduces more slowly and has offspring that are less likely to survive and more likely to be infertile. Papazyan et al. provide evidence that these characteristics arise because the cells without the histone modification are unable to prevent DNA breaks from occurring in heterochromatin during recombination.
The same histone modification also occurs when the microscopic worm Caenorhabditis elegans reproduces, suggesting that this method of DNA protection has been conserved throughout evolution. Papazyan et al. propose that the histone modification may prevent another enzyme that induces DNA breaks from accessing the heterochromatin in reproductive cells; but more work is required to support this hypothesis.
These findings reveal the importance of a new histone modification during reproduction, and could provide new directions for infertility research.
PMCID: PMC4141274  PMID: 25161194
Tetrahymena thermophila; histones; chromatin; methylation; meiosis; DNA damage; C. elegans; other
2.  Quantification of histone modifications using 15N metabolic labeling 
Methods (San Diego, Calif.)  2013;61(3):236-243.
Mass spectrometry has made major contributions to recent discoveries in the field of epigenetics, particularly in the characterization of the myriad post-translational modifications (PTMs) of histones which are technically challenging to analyze. These new developments have further aroused great interest in development of robust, new mass spectrometric methods to quantitatively study the dynamics of histone modifications. This review covers quantitative analysis of histone PTMs and discuss an 15N metabolic labeling procedure for quantifying histone PTMs applied to the analysis of methyltransferase knockouts in the model organism, Tetrahymena thermophila.
PMCID: PMC3700598  PMID: 23454290
Histone; Post-translational modification; Mass spectrometry; 15N metabolic labeling
3.  Anion-activated, thermoreversible gelation system for the capture, release, and visual monitoring of CO2 
Scientific Reports  2014;4:4593.
Carbon dioxide (CO2) is an important green house gas. This is providing an incentive to develop new strategies to detect and capture CO2. Achieving both functions within a single molecular system represents an unmet challenge in terms of molecular design and could translate into enhanced ease of use. Here, we report an anion-activated chemosensor system, NAP-chol 1, that permits dissolved CO2 to be detected in organic media via simple color changes or through ratiometric differences in fluorescence intensity. NAP-chol 1 also acts as a super gelator for DMSO. The resulting gel is transformed into a homogeneous solution upon exposure to fluoride anions. Bubbling with CO2 regenerates the gel. Subsequent flushing with N2 or heating serves to release the CO2 and reform the sol form. This series of transformations is reversible and can be followed by easy-to-discern color changes. Thus, NAP-chol 1 allows for the capture and release of CO2 gas while acting as a three mode sensing system. In particular, it permits CO2 to be detected through reversible sol-gel transitions, simple changes in color, or ratiometric monitoring of the differences in the fluorescence features.
PMCID: PMC3975223  PMID: 24699626
5.  Gene Network Landscape of the Ciliate Tetrahymena thermophila 
PLoS ONE  2011;6(5):e20124.
Genome-wide expression data of gene microarrays can be used to infer gene networks. At a cellular level, a gene network provides a picture of the modules in which genes are densely connected, and of the hub genes, which are highly connected with other genes. A gene network is useful to identify the genes involved in the same pathway, in a protein complex or that are co-regulated. In this study, we used different methods to find gene networks in the ciliate Tetrahymena thermophila, and describe some important properties of this network, such as modules and hubs.
Methodology/Principal Findings
Using 67 single channel microarrays, we constructed the Tetrahymena gene network (TGN) using three methods: the Pearson correlation coefficient (PCC), the Spearman correlation coefficient (SCC) and the context likelihood of relatedness (CLR) algorithm. The accuracy and coverage of the three networks were evaluated using four conserved protein complexes in yeast. The CLR network with a Z-score threshold 3.49 was determined to be the most robust. The TGN was partitioned, and 55 modules were found. In addition, analysis of the arbitrarily determined 1200 hubs showed that these hubs could be sorted into six groups according to their expression profiles. We also investigated human disease orthologs in Tetrahymena that are missing in yeast and provide evidence indicating that some of these are involved in the same process in Tetrahymena as in human.
This study constructed a Tetrahymena gene network, provided new insights to the properties of this biological network, and presents an important resource to study Tetrahymena genes at the pathway level.
PMCID: PMC3102692  PMID: 21637855
6.  An Ash2L/RbBP5 Heterodimer Stimulates the MLL1 Methyltransferase Activity through Coordinated Substrate Interactions with the MLL1 SET Domain 
PLoS ONE  2010;5(11):e14102.
Histone H3 lysine 4 (K4) methylation is a prevalent mark associated with transcription activation and is mainly catalyzed by the MLL/SET1 family histone methyltransferases. A common feature of the mammalian MLL/SET1 complexes is the presence of three core components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. Unlike most other histone lysine methyltransferases, all four proteins are required for efficient H3 K4 methylation. Despite extensive efforts, mechanisms for how three core components regulate MLL/SET1 methyltransferase activity remain elusive. Here we show that a heterodimer of Ash2L and RbBP5 has intrinsic histone methyltransferase activity. This activity requires the highly conserved SPRY domain of Ash2L and a short peptide of RbBP5. We demonstrate that both Ash2L and the MLL1 SET domain are capable of binding to S-adenosyl-L- [methyl-3H] methionine in the MLL1 core complex. Mutations in the MLL1 SET domain that fail to support overall H3 K4 methylation also compromise SAM binding by Ash2L. Taken together, our results show that the Ash2L/RbBP5 heterodimer plays a critical role in the overall catalysis of MLL1 mediated H3 K4 methylation. The results we describe here provide mechanistic insights for unique regulation of the MLL1 methyltransferase activity. It suggests that both Ash2L/RbBP5 and the MLL1 SET domain make direct contacts with the substrates and contribute to the formation of a joint catalytic center. Given the shared core configuration among all MLL/SET1 family HMTs, it will be interesting to test whether the mechanism we describe here can be generalized to other MLL/SET1 family members in the future.
PMCID: PMC2990719  PMID: 21124902
7.  Microarray Analyses of Gene Expression during the Tetrahymena thermophila Life Cycle 
PLoS ONE  2009;4(2):e4429.
The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression.
Methodology/Principal Findings
A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation.
Of the ∼27,000 predicted open reading frames, transcripts homologous to only ∼5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5× corrected background and 95 genes are expressed at >250× corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.
PMCID: PMC2636879  PMID: 19204800
8.  Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure 
BMC Genomics  2008;9:562.
Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.
We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.
We report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.
PMCID: PMC2612030  PMID: 19036158
9.  Deposition and Function of Histone H3 Variants in Tetrahymena thermophila▿ †  
Molecular and Cellular Biology  2006;26(20):7719-7730.
In Tetrahymena, HHT1 and HHT2 genes encode the same major histone H3; HHT3 and HHT4 encode similar minor H3 variants (H3s), H3.3 and H3.4. Green fluorescent protein (GFP)-tagged H3 is deposited onto chromatin through a DNA replication-coupled (RC) pathway. GFP-tagged H3.3 and H3.4 can be deposited both by a transcription-associated, replication-independent (RI) pathway and also weakly by an RC pathway. Although both types of H3s can be deposited by the RC pathway, DNA repair synthesis associated with meiotic recombination utilizes H3 specifically. The regions distinguishing H3 and H3.3 for their deposition pathways were identified. RC major H3 is not essential. Cells can grow without major H3 if the minor H3s are expressed at high levels. Surprisingly, cells lacking RI H3s are also viable and maintain normal nucleosome density at a highly transcribed region. The RC H3 is not detectably deposited by the RI pathway, even when there are no RI H3s available, indicating that transcription-associated RI H3 deposition is not essential for transcription. Minor H3s are also required to produce viable sexual progeny and play an unexpected role in the germ line micronuclei late in conjugation that is unrelated to transcription.
PMCID: PMC1636873  PMID: 16908532
10.  The H1 Phosphorylation State Regulates Expression of CDC2 and Other Genes in Response to Starvation in Tetrahymena thermophila 
Molecular and Cellular Biology  2005;25(10):3914-3922.
In Tetrahymena thermophila, highly phosphorylated histone H1 of growing cells becomes partially dephosphorylated when cells are starved in preparation for conjugation. To determine the effects of H1 phosphorylation on gene expression, PCR-based subtractive hybridization was used to clone cDNAs that were differentially expressed during starvation in two otherwise-isogenic strains differing only in their H1s. H1 in A5 mutant cells lacked phosphorylation, and H1 in E5 cells mimicked constitutive H1 phosphorylation. Sequences enriched in A5 cells included genes encoding proteases. Sequences enriched in E5 cells included genes encoding cdc2 kinase and a Ser/Thr kinase. These results indicate that H1 phosphorylation plays an important role in regulating the pattern of gene expression during the starvation response and that its role in transcription regulation can be either positive or negative. Treatment of starved cells with a phosphatase inhibitor caused CDC2 gene overexpression. Expression of the E5 version of H1 in starved cells containing endogenous, wild-type H1 caused the wild-type H1 to remain highly phosphorylated. These results argue that Cdc2p is the kinase that phosphorylates Tetrahymena H1, establish a positive feedback mechanism between H1 phosphorylation and CDC2 expression, and indicate that CDC2 gene expression is regulated by an H1 phosphatase.
PMCID: PMC1087734  PMID: 15870266
11.  Elimination of Foreign DNA during Somatic Differentiation in Tetrahymena thermophila Shows Position Effect and Is Dosage Dependent†  
Eukaryotic Cell  2005;4(2):421-431.
In the ciliate Tetrahymena thermophila, approximately 15% of the germ line micronuclear DNA sequences are eliminated during formation of the somatic macronucleus. The vast majority of the internal eliminated sequences (IESs) are repeated in the micronuclear genome, and several of them resemble transposable elements. Thus, it has been suggested that DNA elimination evolved as a means for removing invading DNAs. In the present study, bacterial neo genes introduced into the germ line micronuclei were eliminated from the somatic genome. The efficiency of elimination from two different loci increased dramatically with the copy number of the neo genes in the micronuclei. The timing of neo elimination is similar to that of endogenous IESs, and they both produce bidirectional transcripts of the eliminated element, suggesting that the deletion of neo occurred by the same mechanism as elimination of endogenous IESs. These results indicate that repetition of an element in the micronucleus enhances the efficiency of its elimination from the newly formed somatic genome of Tetrahymena thermophila. The implications of these data in relation to the function and mechanism of IES elimination are discussed.
PMCID: PMC549336  PMID: 15701804
12.  HEPA/Vaccine Plan for Indoor Anthrax Remediation 
Emerging Infectious Diseases  2005;11(1):69-76.
A mathematical model suggests that a HEPA/vaccine approach is viable for most buildings after a large-scale anthrax attack.
We developed a mathematical model to compare 2 indoor remediation strategies in the aftermath of an outdoor release of 1.5 kg of anthrax spores in lower Manhattan. The 2 strategies are the fumigation approach used after the 2001 postal anthrax attack and a HEPA/vaccine plan, which relies on HEPA vacuuming, HEPA air cleaners, and vaccination of reoccupants. The HEPA/vaccine approach leads to few anthrax cases among reoccupants if applied to all but the most heavily contaminated buildings, and recovery is much faster than under the decades-long fumigation plan. Only modest environmental sampling is needed. A surge capacity of 10,000 to 20,000 Hazmat workers is required to perform remediation within 6 to 12 months and to avoid permanent mass relocation. Because of the possibility of a campaign of terrorist attacks, serious consideration should be given to allowing or encouraging voluntary self-service cleaning of lightly contaminated rooms by age-appropriate, vaccinated, partially protected (through masks or hoods) reoccupants or owners.
PMCID: PMC3294362  PMID: 15705325
research; HEPA filter; anthrax; mathematical model; bioterrorism; remediation; vaccine
13.  Phosphorylation and an ATP-dependent process increase the dynamic exchange of H1 in chromatin 
The Journal of Cell Biology  2002;158(7):1161-1170.
In Tetrahymena cells, phosphorylation of linker histone H1 regulates transcription of specific genes. Phosphorylation acts by creating a localized negative charge patch and phenocopies the loss of H1 from chromatin, suggesting that it affects transcription by regulating the dissociation of H1 from chromatin. To test this hypothesis, we used FRAP of GFP-tagged H1 to analyze the effects of mutations that either eliminate or mimic phosphorylation on the binding of H1 to chromatin both in vivo and in vitro. We demonstrate that phosphorylation can increase the rate of dissociation of H1 from chromatin, providing a mechanism by which it can affect H1 function in vivo. We also demonstrate a previously undescribed ATP-dependent process that has a global effect on the dynamic binding of linker histone to chromatin.
PMCID: PMC2173238  PMID: 12356861
H1; phosphorylation; ATP remodeling; FRAP; Tetrahymena

Results 1-13 (13)