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1.  Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation 
Retrovirology  2014;11(1):101.
Background
Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses.
Results
The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations.
Conclusions
Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.
doi:10.1186/s12977-014-0101-0
PMCID: PMC4264250  PMID: 25407514
HIV-1; Fitness; Immune escape mutation; Compensatory mutation; Cytotoxic T lymphocytes; Transmitted/founder virus; Reversion
2.  Transposition-mediated DNA re-replication in maize 
eLife  null;3:e03724.
Every DNA segment in a eukaryotic genome normally replicates once and only once per cell cycle to maintain genome stability. We show here that this restriction can be bypassed through alternative transposition, a transposition reaction that utilizes the termini of two separate, nearby transposable elements (TEs). Our results suggest that alternative transposition during S phase can induce re-replication of the TEs and their flanking sequences. The DNA re-replication can spontaneously abort to generate double-strand breaks, which can be repaired to generate Composite Insertions composed of transposon termini flanking segmental duplications of various lengths. These results show how alternative transposition coupled with DNA replication and repair can significantly alter genome structure and may have contributed to rapid genome evolution in maize and possibly other eukaryotes.
DOI: http://dx.doi.org/10.7554/eLife.03724.001
eLife digest
To make accurate copies of its genome, a cell takes precautions to make sure each section of DNA is only duplicated once in every round of copying. However, there are some sections of DNA called transposons that can avoid these restrictions and be duplicated more often.
Transposons are mobile pieces of DNA: they can be ‘cut’ from one section of the genome and are able to ‘paste’ back in somewhere else. The amount of mobile DNA in a genome varies a great deal between species, and in the crop plant maize, it makes up nearly 85% of the genome.
Some transposons can move while the genome is being duplicated. If a transposon is cut out of a section of DNA that has already been copied and is pasted into a site that is yet to be copied, the transposon can be copied again. The transposon may now be present in two different places in the genome.
If two transposons are close together on a section of DNA, both transposons can move at the same time. As they move, they can carry along pieces of the genome, transferring them from one site to another. These transferred pieces can include sections of, or even entire, genes. This is called alternative transposition, but it is not clear whether this process can happen when the genome is actively being duplicated.
Here, Zhang et al. studied transposons in maize. The experiments found that alternative transposition can take place between a site that has already been copied and another site that is still waiting to be copied. Therefore, after the round of copying is completed, both transposons and the flanking DNA can be present in two places in the genome.
When single transposons move, or alternative transposition takes place, sections of the genome can be rearranged and genes can be deleted or new ones can be created. Therefore, transposons may have contributed to rapid evolution in maize and possibly other species.
DOI: http://dx.doi.org/10.7554/eLife.03724.002
doi:10.7554/eLife.03724
PMCID: PMC4270019  PMID: 25406063
transposition; DNA replication; duplication; maize; Ac/Ds; composite insertion; other
3.  Epitope Tags beside the N-Terminal Cytoplasmic Tail of Human BST-2 Alter Its Intracellular Trafficking and HIV-1 Restriction 
PLoS ONE  2014;9(10):e111422.
BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif “KRXK” in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2.
doi:10.1371/journal.pone.0111422
PMCID: PMC4210262  PMID: 25347789
4.  Protein Kinase A Activation Enhances β-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies 
PLoS ONE  2014;9(10):e109523.
The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling.
doi:10.1371/journal.pone.0109523
PMCID: PMC4192022  PMID: 25299576
5.  Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR 
PLoS ONE  2014;9(9):e106658.
Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 1013–1014 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations.
doi:10.1371/journal.pone.0106658
PMCID: PMC4161356  PMID: 25211143
6.  Identification of miRNAs and their target genes in developing maize ears by combined small RNA and degradome sequencing 
BMC Genomics  2014;15:25.
Background
In plants, microRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in many aspects of plant biology, including metabolism, hormone response, epigenetic control of transposable elements, and stress response. Extensive studies of miRNAs have been performed in model plants such as rice and Arabidopsis thaliana. In maize, most miRNAs and their target genes were analyzed and identified by clearly different treatments, such as response to low nitrate, salt and drought stress. However, little is known about miRNAs involved in maize ear development. The objective of this study is to identify conserved and novel miRNAs and their target genes by combined small RNA and degradome sequencing at four inflorescence developmental stages.
Results
We used deep-sequencing, miRNA microarray assays and computational methods to identify, profile, and describe conserved and non-conserved miRNAs at four ear developmental stages, which resulted in identification of 22 conserved and 21-maize-specific miRNA families together with their corresponding miRNA*. Comparison of miRNA expression in these developmental stages revealed 18 differentially expressed miRNA families. Finally, a total of 141 genes (251 transcripts) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs. Moreover, the differentially expressed miRNAs-mediated pathways that regulate the development of ears were discussed.
Conclusions
This study confirmed 22 conserved miRNA families and discovered 26 novel miRNAs in maize. Moreover, we identified 141 target genes of known and new miRNAs and ta-siRNAs. Of these, 72 genes (117 transcripts) targeted by 62 differentially expressed miRNAs may attribute to the development of maize ears. Identification and characterization of these important classes of regulatory genes in maize may improve our understanding of molecular mechanisms controlling ear development.
doi:10.1186/1471-2164-15-25
PMCID: PMC3901417  PMID: 24422852
7.  Interactions between HIV-1 Vif and human ElonginB-ElonginC are important for CBF-β binding to Vif 
Retrovirology  2013;10:94.
Background
The HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-β-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-β-EloB-EloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. EloB and EloC were initially reported as positive regulatory subunits of the Elongin (SIII) complex. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. CBF-β is a newly identified key regulator of Vif function, and more information is needed to further clarify its regulatory mechanism. Here, we comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-β-Cul5 E3 ligase complex.
Results
The results revealed that: (1) EloB (and EloC) positively affected the recruitment of CBF-β to Vif. Both knockdown of endogenous EloB and over-expression of its mutant with a 34-residue deletion in the COOH-terminal tail (EloBΔC34/EBΔC34) impaired the Vif-CBF-β interaction. (2) Introduction of both the Vif SLQ → AAA mutant (VifΔSLQ, which dramatically impairs Vif-EloB-EloC binding) and the Vif PPL → AAA mutant (VifΔPPL, which is thought to reduce Vif-EloB binding) could reduce CBF-β binding. (3) EloB-EloC but not CBF-β could greatly enhance the folding of full-length Vif in Escherichia coli. (4) The over-expression of EloB or the N-terminal ubiquitin-like (UbL) domain of EloB could significantly improve the stability of Vif/VifΔSLQ/VifΔPPL through the region between residues 9 and 14.
Conclusion
Our results indicate that the Vif interaction with EloB-EloC may contribute to recruitment of CBF-β to Vif, demonstrating that the EloB C-teminus may play a role in improving Vif function and that the over-expression of EloB results in Vif stabilization.
doi:10.1186/1742-4690-10-94
PMCID: PMC3765967  PMID: 23988114
CBF-β; Elongin BC complex; HIV-1; Protein binding; Ubiquitin-protein ligases; Vif
8.  Generation of Tandem Direct Duplications by Reversed-Ends Transposition of Maize Ac Elements 
PLoS Genetics  2013;9(8):e1003691.
Tandem direct duplications are a common feature of the genomes of eukaryotes ranging from yeast to human, where they comprise a significant fraction of copy number variations. The prevailing model for the formation of tandem direct duplications is non-allelic homologous recombination (NAHR). Here we report the isolation of a series of duplications and reciprocal deletions isolated de novo from a maize allele containing two Class II Ac/Ds transposons. The duplication/deletion structures suggest that they were generated by alternative transposition reactions involving the termini of two nearby transposable elements. The deletion/duplication breakpoint junctions contain 8 bp target site duplications characteristic of Ac/Ds transposition events, confirming their formation directly by an alternative transposition mechanism. Tandem direct duplications and reciprocal deletions were generated at a relatively high frequency (∼0.5 to 1%) in the materials examined here in which transposons are positioned nearby each other in appropriate orientation; frequencies would likely be much lower in other genotypes. To test whether this mechanism may have contributed to maize genome evolution, we analyzed sequences flanking Ac/Ds and other hAT family transposons and identified three small tandem direct duplications with the structural features predicted by the alternative transposition mechanism. Together these results show that some class II transposons are capable of directly inducing tandem sequence duplications, and that this activity has contributed to the evolution of the maize genome.
Author Summary
The recent explosion of genome sequence data has greatly increased the need to understand the forces that shape eukaryotic genomes. A common feature of higher plant genomes is the presence of large numbers of duplications, often occurring as tandem repeats of thousands of base pairs. Despite the importance of gene duplications in evolution and disease, the precise mechanism(s) that generate tandem duplications are still unclear. In this study we identified nine new spontaneous duplications that arose flanking elements of the Ac transposon system. These duplications range in size from 8 kbp to >5,000 kbp, and all cases exhibit features characteristic of Ac transposition. Using similar criteria in a bioinformatics search, we identified three smaller duplications adjacent to other hAT family transposons in the maize B73 reference genome sequence. Our results show that transposable elements can directly generate tandem duplications via alternative transposition, and that this mechanism is responsible for at least some of the duplications present in the maize B73 genome. This work extends the significance of Barbara McClintock's discovery of transposable elements by demonstrating how they can act as agents of genome expansion.
doi:10.1371/journal.pgen.1003691
PMCID: PMC3744419  PMID: 23966872
9.  Small-Molecule Inhibition of Human Immunodeficiency Virus Type 1 Replication by Targeting the Interaction between Vif and ElonginC 
Journal of Virology  2012;86(10):5497-5507.
The HIV-1 viral infectivity factor (Vif) protein is essential for viral replication. Vif recruits cellular ElonginB/C-Cullin5 E3 ubiquitin ligase to target the host antiviral protein APOBEC3G (A3G) for proteasomal degradation. In the absence of Vif, A3G is packaged into budding HIV-1 virions and introduces multiple mutations in the newly synthesized minus-strand viral DNA to restrict virus replication. Thus, the A3G-Vif-E3 complex represents an attractive target for development of novel anti-HIV drugs. In this study, we identified a potent small molecular compound (VEC-5) by virtual screening and validated its anti-Vif activity through biochemical analysis. We show that VEC-5 inhibits virus replication only in A3G-positive cells. Treatment with VEC-5 increased cellular A3G levels when Vif was coexpressed and enhanced A3G incorporation into HIV-1 virions to reduce viral infectivity. Coimmunoprecipitation and computational analysis further attributed the anti-Vif activity of VEC-5 to the inhibition of Vif from direct binding to the ElonginC protein. These findings support the notion that suppressing Vif function can liberate A3G to carry out its antiviral activity and demonstrate that regulation of the Vif-ElonginC interaction is a novel target for small-molecule inhibitors of HIV-1.
doi:10.1128/JVI.06957-11
PMCID: PMC3347289  PMID: 22379088
10.  Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells 
Carcinogenesis  2011;32(6):812-821.
While tumor suppressor genes frequently undergo epigenetic silencing in cancer, how the instructions directing this transcriptional repression are transmitted in cancer cells remain largely unclear. Expression of cyclin-dependent kinase inhibitor 1C (CDKN1C), an imprinted gene on chromosomal band 11 p15.5, is reduced or lost in the majority of breast cancers. Here, we report that CDKN1C is suppressed by estrogen through epigenetic mechanisms involving the chromatin-interacting noncoding RNA KCNQ1OT1 and CCCTC-binding factor (CTCF). Activation of estrogen signaling reduced CDKN1C expression 3-fold (P < 0.001) and established repressive histone modifications at the 5′ regulatory region of the locus. These events were concomitant with induction of KCNQ1OT1 expression as well as increased recruitment of CTCF to both the distal KCNQ1OT1 promoter-associated imprinting control region (ICR) and the CDKN1C locus. Transient depletion of CTCF by small interfering RNA increased CDKN1C expression and significantly reduced the estrogen-mediated repression of CDKN1C. Further studies in breast cancer cell lines indicated that the epigenetic silencing of CDKN1C occurs in part as the result of genetic loss of the inactive methylated 11p15.5 ICR allele (R2 = 0.612, P < 0.001). We also found a novel cis-encoded antisense transcript, CDKN1C-AS, which is induced by estrogen signaling following pharmacologic inhibition of DNA methyltransferase and histone deacetylase activity. Forced expression of CDKN1C-AS was capable of repressing endogenous CDKN1C in vivo. Our findings suggest that in addition to promoter hypermethylation, epigenetic repression of tumor suppressor genes by CTCF and noncoding RNA transcripts could be more common and important than previously understood.
doi:10.1093/carcin/bgr017
PMCID: PMC3106431  PMID: 21304052
11.  Identification of a Cullin5-ElonginB-ElonginC E3 Complex in Degradation of Feline Immunodeficiency Virus Vif-Mediated Feline APOBEC3 Proteins▿ 
Journal of Virology  2011;85(23):12482-12491.
Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.
doi:10.1128/JVI.05218-11
PMCID: PMC3209414  PMID: 21957297
12.  Self-assembled mPEG–PCL-g–PEI micelles for simultaneous codelivery of chemotherapeutic drugs and DNA: synthesis and characterization in vitro 
Background
In this paper, a series of amphiphilic triblock copolymers based on polyethylene glycol–poly ɛ-caprolactone–polyethylenimine (mPEG–PCL-g–PEI) were successfully synthesized, and their application for codelivery of chemotherapeutic drugs and DNA simultaneously was investigated.
Methods and results
These copolymers could self-assemble into micelles with positive charges. The size and zeta potential of the micelles was measured, and the results indicate that temperature had a large effect on the micelles obtained. In vitro gene transfection evaluation in cancer cells indicated that the self-assembled micelles could serve as potential gene delivery vectors. In addition, hydrophobic drug entrapment efficiency and codelivery with the gene was also studied in vitro. The self-assembled micelles could load doxorubicin efficiently and increase cellular uptake in vitro, while maintaining high gene transfection efficiency.
Conclusion
The triblock copolymer mPEG–PCL-g–PEI could be a novel vector for codelivery of drug and gene therapy.
doi:10.2147/IJN.S28932
PMCID: PMC3356179  PMID: 22619525
self-assembly; triblock copolymer; DNA; drug codelivery; gene transfection
13.  Epigenetic Silencing Mediated Through Activated PI3K/AKT Signaling in Breast Cancer 
Cancer research  2011;71(5):1752-1762.
Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we demonstrate that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future.
doi:10.1158/0008-5472.CAN-10-3573
PMCID: PMC3048165  PMID: 21216892
Epigenetic silencing; H3K27me3; DNA methylation; PI3K/AKT signaling; breast cancer
14.  Epigenetic influences of low-dose bisphenol A in primary human breast epithelial cells 
Toxicology and applied pharmacology  2010;248(2):111-121.
Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.
doi:10.1016/j.taap.2010.07.014
PMCID: PMC2946518  PMID: 20678512
Bisphenol A; Estrogen; DNA methylation; Epigenetics; Breast cancer
15.  Polarity Changes in the Transmembrane Domain Core of HIV-1 Vpu Inhibits Its Anti-Tetherin Activity 
PLoS ONE  2011;6(6):e20890.
Tetherin (BST-2/CD317) is an interferon-inducible antiviral protein that restricts the release of enveloped viruses from infected cells. The HIV-1 accessory protein Vpu can efficiently antagonize this restriction. In this study, we analyzed mutations of the transmembrane (TM) domain of Vpu, including deletions and substitutions, to delineate amino acids important for HIV-1 viral particle release and in interactions with tetherin. The mutants had similar subcellular localization patterns with that of wild-type Vpu and were functional with respect to CD4 downregulation. We showed that the hydrophobic binding surface for tetherin lies in the core of the Vpu TM domain. Three consecutive hydrophobic isoleucine residues in the middle region of the Vpu TM domain, I15, I16 and I17, were important for stabilizing the tetherin binding interface and determining its sensitivity to tetherin. Changing the polarity of the amino acids at these positions resulted in severe impairment of Vpu-induced tetherin targeting and antagonism. Taken together, these data reveal a model of specific hydrophobic interactions between Vpu and tetherin, which can be potentially targeted in the development of novel anti-HIV-1 drugs.
doi:10.1371/journal.pone.0020890
PMCID: PMC3107245  PMID: 21674066
16.  Somatic Single-hits Inactivate the X-linked Tumor Suppressor FOXP3 in the Prostate 
Cancer cell  2009;16(4):336-346.
Despite clear epidemiological and genetic evidence for X-linked prostate cancer risk, all prostate cancer genes identified are autosomal. Here we report somatic inactivating mutations and deletion of the X-linked FOXP3 gene residing at Xp11.23 in human prostate cancer. Lineage-specific ablation of FoxP3 in the mouse prostate epithelial cells leads to prostate hyperplasia and prostate intraepithelial neoplasia. In both normal and malignant prostate tissues, FOXP3 is both necessary and sufficient to transcriptionally repress cMYC, the most commonly over-expressed oncogene in prostate cancer as well as among the aggregates of other cancers. FOXP3 is an X-linked prostate tumor suppressor in the male. Since the male has only one X chromosome, our data represents a paradigm of “single-genetic-hit” inactivation-mediated carcinogenesis.
doi:10.1016/j.ccr.2009.08.016
PMCID: PMC2758294  PMID: 19800578
17.  Methods in DNA methylation profiling 
Epigenomics  2009;1(2):331-345.
Metastable and somatically heritable patterns of DNA methylation provide an important level of genomic regulation. In this article, we review methods for analyzing these genome-wide epigenetic patterns and offer a perspective on the ever-expanding literature, which we hope will be useful for investigators who are new to this area. The historical aspects that we cover will be helpful in interpreting this literature and we hope that our discussion of the newest analytical methods will stimulate future progress. We emphasize that no single approach can provide a complete view of the overall methylome, and that combinations of several modalities applied to the same sample set will give the clearest picture. Given the unexpected epigenomic patterns and new biological principles, as well as new disease markers, that have been uncovered in recent studies, it is likely that important discoveries will continue to be made using genome-wide DNA methylation profiling.
doi:10.2217/epi.09.31
PMCID: PMC2880494  PMID: 20526417
bisulfite sequencing; CpG island; differential methylation hybridization; DNA methylation; methylation profiling; methylated CpG island recovery assay; methylated DNA immunoprecipitation; methylation-sensitive SNP chip analysis; next-generation DNA sequencing
18.  Xenoestrogen-Induced Epigenetic Repression of microRNA-9-3 in Breast Epithelial Cells 
Cancer research  2009;69(14):5936-5945.
Early exposure to xenoestrogens may predispose to breast cancer risk later in adult life. It is likely that long-lived, self-regenerating epithelial progenitor cells are more susceptible to these exposure injuries over time and transmit the injured memory through epigenetic mechanisms to their differentiated progeny. Here, we used progenitor-containing mammospheres as an in vitro exposure model to study this epigenetic effect. Expression profiling identified that, relative to control cells, 9.1% of microRNAs (82 of 898 loci) were altered in epithelial progeny derived from mammospheres exposed to a synthetic estrogen, diethylstilbestrol. Repressive chromatin marks, trimethyl Lys27 of histone H3 (H3K27me3) and dimethyl Lys9 of histone H3 (H3K9me2), were found at a down-regulated locus, miR-9-3, in epithelial cells preexposed to diethylstilbestrol. This was accompanied by recruitment of DNA methyltransferase 1 that caused an aberrant increase in DNA methylation of its promoter CpG island in mammosphere-derived epithelial cells on diethylstilbestrol preexposure. Functional analyses suggest that miR-9-3 plays a role in the p53-related apoptotic pathway. Epigenetic silencing of this gene, therefore, reduces this cellular function and promotes the proliferation of breast cancer cells. Promoter hypermethylation of this microRNA may be a hallmark for early breast cancer development, and restoration of its expression by epigenetic and microRNA-based therapies is another viable option for future treatment of this disease.
doi:10.1158/0008-5472.CAN-08-4914
PMCID: PMC2855843  PMID: 19549897
19.  Seed in soil, with an epigenetic view 
Biochimica et biophysica acta  2008;1790(9):920-924.
It is becoming increasingly evident that discrete genetic alterations in neoplastic cells alone cannot explain multistep carcinogenesis whereby tumor cells are able to express diverse phenotypes during the complex phases of tumor development and progression. The epigenetic model posits that the host microenvironment exerts an initial, inhibitory constraint on tumor growth that is followed by acceleration of tumor progression through complex cell–matrix interactions. This review emphasizes the epigenetic aspects of breast cancer development in light of such interactions between epithelial cells (“seed”) and the tumor microenvironment (“soil”). Our recent research findings suggest that epigenetic perturbations induced by the tumor microenvironment may play a causal role in promoting breast cancer development. It is believed that abrogation of these initiators could offer a promising therapeutic strategy.
doi:10.1016/j.bbagen.2008.12.004
PMCID: PMC2845923  PMID: 19162126
Breast cancer; Tumor microenvironment; Stromal fibroblast; Epigenetics; DNA methylation; Chromatin remodeling
20.  Breast Cancer-associated Fibroblasts Confer AKT1-mediated Epigenetic Silencing of Cystatin M in Epithelial Cells 
Cancer research  2008;68(24):10257.
The interplay between histone modifications and promoter hypermethylation provides a causative explanation for epigenetic gene silencing in cancer. Less is known about the upstream initiators that direct this process. Here, we report that the Cystatin M (CST6) tumor suppressor gene is concurrently down-regulated with other loci in breast epithelial cells co-cultured with cancer-associated fibroblasts (CAFs). Promoter hypermethylation of CST6 is associated with aberrant AKT1 activation in epithelial cells, as well as the disabled INNP4B regulator resulted from the suppression by CAFs. Repressive chromatin, marked by trimethyl-H3K27 and dimethyl-H3K9, and de novo DNA methylation is established at the promoter. The findings suggest that microenvironmental stimuli are triggers in this epigenetic cascade, leading to the long-term silencing of CST6 in breast tumors. Our present findings implicate a causal mechanism defining how tumor stromal fibroblasts support neoplastic progression by manipulating the epigenome of mammary epithelial cells. The result also highlights the importance of direct cell-cell contract between epithelial cells and the surrounding fibroblasts that confer this epigenetic perturbation. Since this two-way interaction is anticipated, the described co-culture system can be used to determine the effect of epithelial factors on fibroblasts in future studies.
doi:10.1158/0008-5472.CAN-08-0288
PMCID: PMC2821873  PMID: 19074894
21.  FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene 
Cell  2007;129(7):1275-1286.
The X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germ-line mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that Foxp3sf/+ heterozygous mice developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and ErbB2 was over-expressed. Foxp3 bound and repressed the ErbB2 promoter. Deletion, functionally significant somatic mutations and down-regulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2 over-expression, regardless of the status of HER-2 amplification. In toto, the data demonstrate that FOXP3 is an X-linked breast cancer suppressor gene and an important regulator of the HER-2/ErbB2 oncogene.
doi:10.1016/j.cell.2007.04.034
PMCID: PMC1974845  PMID: 17570480
22.  FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2  
The Journal of Clinical Investigation  2007;117(12):3765-3773.
S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex. Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpress ERBB2/HER2, additional FOXP3 targets may be involved in its tumor suppressor activity. Here, we show that mammary carcinomas from mice heterozygous for a Foxp3 mutation exhibited increased Skp2 expression. Ectopic expression of FOXP3 in mouse mammary cancer cells repressed SKP2 expression with a corresponding increase in p27 and polyploidy. Conversely, siRNA silencing of the FOXP3 gene in human mammary epithelial cells increased SKP2 expression. We also show that Foxp3 directly interacted with and repressed the Skp2 promoter. Moreover, the analysis of over 200 primary breast cancer samples revealed an inverse correlation between FOXP3 and SKP2 levels. Finally, we demonstrated that downregulation of SKP2 was critical for FOXP3-mediated growth inhibition in breast cancer cells that do not overexpress ERBB2/HER2. Our data provide genetic, biochemical, and functional evidence that FOXP3 is a novel transcriptional repressor for the oncogene SKP2.
doi:10.1172/JCI32538
PMCID: PMC2075479  PMID: 18008005
23.  The Scurfy mutation of FoxP3 in the thymus stroma leads to defective thymopoiesis 
The Journal of Experimental Medicine  2005;202(8):1141-1151.
The Scurfy mutation of the FoxP3 gene (FoxP3sf) in the mouse and analogous mutations in human result in lethal autoimmunity. The mutation of FoxP3 in the hematopoietic cells impairs the development of regulatory T cells. In addition, development of the Scurfy disease also may require mutation of the gene in nonhematopoietic cells. The T cell–extrinsic function of FoxP3 has not been characterized. Here we show that the FoxP3sf mutation leads to defective thymopoiesis, which is caused by inactivation of FoxP3 in the thymic stromal cells. FoxP3 mutation also results in overexpression of ErbB2 in the thymic stroma, which may be involved in defective thymopoiesis. Our data reveal a novel T cell–extrinsic function of FoxP3. In combination, the T cell–intrinsic and –extrinsic defects provide plausible explanation for the severity of the autoimmune diseases in the scurfy mice and in patients who have immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome.
doi:10.1084/jem.20050157
PMCID: PMC2213221  PMID: 16230479
24.  B7DC/PDL2 Promotes Tumor Immunity by a PD-1–independent Mechanism 
The Journal of Experimental Medicine  2003;197(12):1721-1730.
B7H1 (PDL1) and B7DC (PDL2) are two new members of the B7 family that can interact with PD-1, a putative negative regulator for immune function. Recent studies have provided evidence for inhibitory functions of both members via PD-1. Meanwhile, compelling evidence exists for costimulatory function of both members. Here we demonstrate that expression of B7DC on the tumor cells promotes CD8 T cell–mediated rejection of tumor cells, at both the induction and effector phase of antitumor immunity. Moreover, B7DC binds to PD-1(−/−) cells and enhances T cell killing in a PD-1–independent mechanism. Our results demonstrate a novel pathway for B7DC to promote tumor immunity and may reconcile the apparently contradictory findings on the function of B7DC.
doi:10.1084/jem.20022089
PMCID: PMC2193953  PMID: 12810690
tumor immunity; costimulatory molecules; cytolytic T lymphocytes

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