Xrcc4 and mre11 Roles and Transcriptional Response to Repair of Talen-Induced Double-Strand Dna Breaks

Double-strand breaks (DSB) are one of the most lethal forms of DNA damage that, if left unrepaired, can lead to genomic instability, cellular transformation, and cell death. In this work, we examined how repair of transcription activator-like effector nuclease (TALEN)-induced DNA damage was altered when knocking out, or inhibiting a function of, two DNA repair proteins, XRCC4 and MRE11, respectively. We developed a fluorescent reporter assay that uses TALENs to introduce DSB and detected repair by the presence of GFP fluorescence. We observed repair of TALEN-induced breaks in the XRCC4 knockout cells treated with mirin (a pharmacological inhibitor of MRE11 exonuclease activity), albeit with ~40% reduced efficiency compared to normal cells. Editing in the absence of XRCC4 or MRE11 exonuclease was robust, with little difference between the indel profiles amongst any of the groups. Reviewing the transcriptional profiles of the mirin-treated XRCC4 knockout cells showed 307 uniquely differentially expressed genes, a number far greater than for either of the other cell lines (the HeLa XRCC4 knockout sample had 83 genes, and the mirin-treated HeLa cells had 30 genes uniquely differentially expressed). Pathways unique to the XRCC4 knockout+mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that TALEN-induced DSBs are repaired, even when a key DSB repair protein or protein function is not operational, without a change in indel profiles. However, transcriptional profiles indicate the induction of unique cellular responses dependent upon the DNA repair protein(s) hampered

Validation of Induced Microglia-Like Cells (iMG Cells) for Future Studies of Brain Diseases

Microglia are the primary resident immune cells of the central nervous system that maintain physiological homeostasis in the brain and contribute to the pathogenesis of many psychiatric disorders and neurodegenerative diseases. Due to the lack of appropriate human cellular models, it is difficult to study the basic pathophysiological processes linking microglia to brain diseases. In this study, we adopted a microglia-like cellular model derived from peripheral blood monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-34 (IL-34). We characterized and validated this in vitro cellular model by morphology, immunocytochemistry, gene expression profiles, and functional study. Our results indicated that the iMG cells developed typical microglial ramified morphology, expressed microglial specific surface markers (P2RY12 and TMEM119), and possessed phagocytic activity. Principal component analyses and multidimensional scaling analyses of RNA-seq data showed that iMG cells were distinct from monocytes and induced macrophages (iMacs) but clustered closer to human microglia and hiPSC-induced microglia. Heatmap analyses also found that iMG cells, but not monocytes, were closely clustered with human primary microglia. Further pathway and relative expression analysis indicated that unique genes from iMG cells were involved in the regulation of the complement system, especially in the synapse and ion transport. Overall, our data demonstrated that the iMG model mimicked many features of the brain resident microglia, highlighting its utility in the study of microglial function in many brain diseases, such as schizophrenia and Alzheimer\u27s disease (AD)

Impact of viral factors on subcellular distribution and RNA export activity of HIV-1 rev in astrocytes 1321N1.

CNS associated cells are permissive to HIV-1 infection, but poor in virus production due to attenuated Rev activity. The temporal and the spatial distribution of Rev in human astrocyte 1321N1 and glioblastoma GO-G-CCM were monitored for explaining the reduced Rev activity and low viral production during HIV-1 infection. Rev remained localized to the nuclei of these cells upon infection, attenuating its export activity, as manifested by low copy number of RRE-containing viral-mRNA in the cytoplasm of these cells. In contrast to infection, when Rev alone was transiently expressed, it localized in the cytoplasm of 1321N1. The localization changed to the nucleus when Rev was expressed in the presence of other viral proteins through pro-viral DNA pNL4-3. This study, for the first time, revealed the impact of other HIV-1 proteins apart from host factors in regulating the subcellular localization of Rev in astrocytes and hence the fate of HIV-1 infection in these cells

qRT-PCR analysis of RRE-containing viral mRNA in the nuclear and the cytoplasmic fractions of different cell lines.

<p>Cells infected with NL4-3 virus were harvested 6 hours post infection. Nuclear and cytoplasmic fractions were separated for RNA extraction. qRT-PCR were performed using RRE specific probes for real time PCR which were FAM labeled. Cytoplasmic and nuclear viral RRE containing mRNA were plotted as percentage of total viral RRE containing mRNA in different cell types Experiments were done in triplicate and ± Standard Deviation (SD) values were determined. The significance is determined by student’s <i>t</i> test and p values are denoted.</p

Quantification of p24 equivalents of HIV-1 titers in different cell types.

<p>Cells infected with NL4-3 virus were scored for p24 levels from culture supernatant after 48 hours of infection. The viral p24 levels were normalized to the copy number of viral DNA incorporated into the host genome for each of the cell lines following HIV-1 infection. Experiments were done five times and ± SD values were determined. The significance is determined by student’s <i>t</i> test and p values are denoted. The bar indicates the groups compared.</p

Distribution of Rev 9 hours post infection.

<p>Cells were fixed with 3% paraformaldehyde 9 hours post infection followed by detection of Rev with anti-Rev antibody and FITC labeled secondary antibody. DAPI (blue) stains nuclei, FITC (green) stains Rev and merged images of blue and green channels indicates the subcellular localization of Rev protein. The distribution of Rev in a representative cell is plotted using Huygens Essential software twin slicer tool and shown as distribution plot. Ratios of mean intensities of green channel inside and outside the nucleus (blue) were calculated for at least 10–15 cells per field, with minimum of three fields per cell types. The ratios of nucleus to cytoplasmic levels of Rev are given at the end of each panel. Panel A: SUP-T1; Panel B: THP-1; Panel C: 1321N1 and Panel D: GO-G-CCM. The experiments were done at the least in triplicate and representative pictures are shown here.</p

Comparison of Rev distribution in infected and transfected astrocyte 1321N1.

<p>Panel A: 1321N1 cells infected with NL4-3 virus; Panel B: 1321N1 cells transfected with Rev-EGFP plasmid; Panel C: 1321N1 cells transfected with pro-viral DNA pNL4-3; Panel D: HEK293T cells transfected with Rev-EGFP plasmid; Panel E: HEK293T cells transfected with pro-viral DNA pNL4-3. DAPI (blue) stains nuclei, FITC (green) stains Rev and Merged images of blue and green channels indicates the subcellular localization of Rev protein. The distribution of Rev in a representative cell is plotted using Huygens Essential software twin slicer tool and shown as distribution plot. All the experiments were done thrice and representative pictures are shown here.</p

Earliest time point for detection of Rev expression in different cell lines.

<p>Cell lines infected with NL4-3 virus were fixed at 1 hour post infection with 3% paraformaldehyde followed by detection with mouse anti HIV-Rev primary antibody, goat anti mouse-FITC (green) secondary antibody and mounted on slides with fluoroshield containing DAPI (blue) for nucleus staining. Uninfected cells were treated similarly and used as control experiments. Panel A: SUP-T1 (A1–A2) uninfected cells; (A3–A4) infected cell line, Panel B: THP-1 (B1–B2) uninfected cells; (B3–B4) infected cell line, Panel C: 1321N1 (C1–C2) uninfected cells; (C3–C4) infected cell line, Panel D: GO-G-CCM (D1–D2) uninfected cells; (D3–D4) infected cell line. DAPI (blue) stains nuclei and FITC (green) shows Rev expression in infected cells. The experiments were done at the least in triplicate and representative pictures are shown here.</p

Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134, a Novel Positive Regulator of HIV-1 LTR Activity and Viral Propagation

<div><p>Background</p><p>Concurrent occurrence of HIV and Tuberculosis (TB) infections influence the cellular environment of the host for synergistic existence. An elementary approach to understand such coalition at the molecular level is to understand the interactions of the host and the viral factors that subsequently effect viral replication. Long terminal repeats (LTR) of HIV genome serve as a template for binding trans-acting viral and cellular factors that regulate its transcriptional activity, thereby, deciding the fate of HIV pathogenesis, making it an ideal system to explore the interplay between HIV and the host.</p><p>Methodology/Principal Findings</p><p>In this study, using biotinylated full length HIV-1 LTR sequence as bait followed by MALDI analyses, we identified and further characterized human-Zinc-finger-protein-134 (hZNF-134) as a novel positive regulator of HIV-1 that promoted LTR-driven transcription and viral production. Over-expression of hZNF-134 promoted LTR driven luciferase activity and viral transcripts, resulting in increased virus production while siRNA mediated knockdown reduced both the viral transcripts and the viral titers, establishing hZNF-134 as a positive effector of HIV-1. HIV, <i>Mycobacteria</i> and HIV-TB co-infections increased hZNF-134 expressions in PBMCs, the impact being highest by mycobacteria. Corroborating these observations, primary TB patients (n = 22) recorded extraordinarily high transcript levels of hZNF-134 as compared to healthy controls (n = 16).</p><p>Conclusions/Significance</p><p>With these observations, it was concluded that hZNF-134, which promoted HIV-1 LTR activity acted as a positive regulator of HIV propagation in human host. High titers of hZNF-134 transcripts in TB patients suggest that up-regulation of such positive effectors of HIV-1 upon mycobacterial infection can be yet another mechanism by which mycobacteria assists HIV-1 propagation during HIV-TB co-infections. hZNF-134, an uncharacterized host protein, thus assumes a novel regulatory role during HIV-host interactions. Our study provides new insights into the emerging role of zinc finger proteins in HIV-1 pathogenesis.</p></div

hZNF-134-GFP is localized in the nucleus of HEK293T and Astrocytoma 1321N1 cells.

<p>HEK293T and 1321N1 cells were transfected with expression plasmids pEGFP-C3 or ZNF-134-GFP-C3. The cells were fixed after 48 hours and visualized under confocal microscopy. Panels show Trans, DAPI, GFP, and Merged images. hZNF-134-GFP protein was localized in the nucleus of HEK293T and 1321N1 cells whereas GFP control showed both nuclear and cytoplasmic localization. Localization was confirmed by 3 independent experiments.</p