The human telomeric proteome during telomere replication

Telomere shortening can cause detrimental diseases and contribute to aging. It occurs due to the end replication problem in cells lacking telomerase. Furthermore, recent studies revealed that telomere shortening can be attributed to difficulties of the semi-conservative DNA replication machinery to replicate the bulk of telomeric DNA repeats. To investigate telomere replication in a comprehensive manner, we develop QTIP-iPOND - Quantitative Telomeric chromatin Isolation Protocol followed by isolation of Proteins On Nascent DNA - which enables purification of proteins that associate with telomeres specifically during replication. In addition to the core replisome, we identify a large number of proteins that specifically associate with telomere replication forks. Depletion of several of these proteins induces telomere fragility validating their importance for telomere replication. We also find that at telomere replication forks the single strand telomere binding protein POT1 is depleted, whereas histone H1 is enriched. Our work reveals the dynamic changes of the telomeric proteome during replication, providing a valuable resource of telomere replication proteins. To our knowledge, this is the first study that examines the replisome at a specific region of the genome.     

HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.     

Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.     

Protection against Lethal Influenza with a Viral Mimic

Despite countermeasures against influenza virus that prevent (vaccines) and treat (antivirals) infection, this upper respiratory tract human pathogen remains a global health burden, causing both seasonal epidemics and occasional pandemics. More potent and safe new vaccine technologies would contribute significantly to the battle against influenza and other respiratory infections. Using plasmid-based reverse genetics techniques, we have developed a single-cycle infectious influenza virus (sciIV) with immunoprotective potential. In our sciIV approach, the fourth viral segment, which codes for the receptor-binding and fusion protein hemagglutinin (HA), has been removed. Thus, upon infection of normal cells, although no infectious progeny are produced, the expression of other viral proteins occurs and is immunogenic. Consequently, sciIV is protective against influenza homologous and heterologous viral challenges in a mouse model. Vaccination with sciIV protects in a dose- and replication-dependent manner, which is attributed to both humoral responses and T cells. Safety, immunogenicity, and protection conferred by sciIV vaccination were also demonstrated in ferrets, where this immunization additionally blocked direct and aerosol transmission events. All together, our studies suggest that sciIV may have potential as a broadly protective vaccine against influenza virus.     

Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion

HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env''s fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag''s ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells.     

Unique Role of the WD-40 Repeat Protein 5 (WDR5) Subunit within the Mixed Lineage Leukemia 3 (MLL3) Histone Methyltransferase Complex

The MLL3 (mixed lineage leukemia 3) protein is a member of the human SET1 family of histone H3 lysine 4 methyltransferases and contains the conserved WDR5 interaction (Win) motif and the catalytic suppressor of variegation, enhancer of zeste, trithorax (SET) domain. The human SET1 family includes MLL1–4 and SETd1A/B, which all interact with a conserved subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD) to form the minimal core complex required for full methyltransferase activity. However, recent evidence suggests that the WDR5 subunit may not be utilized in an identical manner within all SET1 family core complexes. Although the roles of WDR5 within the MLL1 core complex have been extensively studied, not much is known about the roles of WDR5 in other SET1 family core complexes. In this investigation, we set out to characterize the roles of the WDR5 subunit in the MLL3 core complex. We found that unlike MLL1, the MLL3 SET domain assembles with the RbBP5/Ash2L heterodimer independently of the Win motif-WDR5 interaction. Furthermore, we observed that WDR5 inhibits the monomethylation activity of the MLL3 core complex, which is dependent on the Win motif. We also found evidence suggesting that the WRAD subcomplex catalyzes weak H3K4 monomethylation within the context of the MLL3 core complex. Furthermore, solution structures of the MLL3 core complex assembled with and without WDR5 by small angle x-ray scattering show similar overall topologies. Together, this work demonstrates a unique role for WDR5 in modulating the enzymatic activity of the MLL3 core complex.     

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.     

Functional Characterization of Core Components of the Bacillus subtilis Cyclic-Di-GMP Signaling Pathway

Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that regulates adaptation processes, including biofilm formation, motility, and virulence in Gram-negative bacteria. In this study, we have characterized the core components of a c-di-GMP signaling pathway in the model Gram-positive bacterium Bacillus subtilis. Specifically, we have directly identified and characterized three active diguanylate cyclases, DgcP, DgcK, and DgcW (formerly YtrP, YhcK, and YkoW, respectively), one active c-di-GMP phosphodiesterase, PdeH (formerly YuxH), and a cyclic-diguanylate (c-di-GMP) receptor, DgrA (formerly YpfA). Furthermore, elevation of c-di-GMP levels in B. subtilis led to inhibition of swarming motility, whereas biofilm formation was unaffected. Our work establishes paradigms for Gram-positive c-di-GMP signaling, and we have shown that the concise signaling system identified in B. subtilis serves as a powerful heterologous host for the study of c-di-GMP enzymes from bacteria predicted to possess larger, more-complex signaling systems.