Wendy A. Burgers

DNA methyltransferase Dnmt1 associates with histone deacetylase activity

The DNA methyltransferase Dnmt1 is responsible for cytosine methylation in mammals and has a role in gene silencing. DNA methylation represses genes partly by recruitment of the methyl-CpG-binding protein MeCP2, which in turn recruits a histone deacetylase activity. Here we show that Dnmt1 is itself associated with histone deacetylase activity in vivo. Consistent with this association, we find that one of the known histone deacetylases, HDAC1, has the ability to bind Dnmt1 and can purify methyltransferase activity from nuclear extracts. We have identified a transcriptional repression domain in Dnmt1 that functions, at least partly, by recruiting histone deacetylase activity and shows homology to the repressor domain of the trithorax-related protein HRX (also known as MLL and ALL-1). Our data show a more direct connection between DNA methylation and histone deacetylation than was previously considered. We suggest that the process of DNA methylation, mediated by Dnmt1, may depend on or generate an altered chromatin state via histone deacetylase activity.

Dnmt3a binds deacetylases and is recruited by a sequence‐specific repressor to silence transcription

The Dnmt3a DNA methyltransferase is essential for mammalian development and is responsible for the generation of genomic methylation patterns, which lead to transcriptional silencing. Here, we show that Dnmt3a associates with RP58, a DNA‐binding transcriptional repressor protein found at transcriptionally silent heterochromatin. Dnmt3a acts as a co‐repressor for RP58 in a manner that does not require its de novo methyltransferase activity. Like other characterized co‐repressors, Dnmt3a associates with the histone deacetylase HDAC1 using its ATRX‐homology domain. This domain of Dnmt3a represents an independent transcriptional repressor domain whose silencing functions require HDAC activity. These results identify Dnmt3a as a co‐repressor protein carrying deacetylase activity and show that Dnmt3a can be targeted to specific regulatory foci via its association with DNA‐binding transcription factors.

Viral oncoproteins target the DNA methyltransferases

Small DNA tumour viruses have evolved a number of mechanisms to drive nondividing cells into S phase. Virally encoded oncoproteins such as adenovirus E1A and human papillomavirus (HPV) E7 can bind an array of cellular proteins to override proliferation arrest. The DNA methyltransferase Dnmt1 is the major mammalian enzyme responsible for maintaining CpG methylation patterns in the cell following replication. One of the hallmarks of tumour cells is disrupted DNA methylation patterns, highlighting the importance of the proper regulation of DNA methyltransferases in normal cell proliferation. Here, we show that adenovirus 5 E1A and HPV-16 E7 associate in vitro and in vivo with the DNA methyltransferase Dnmt1. Consistent with this interaction, we find that E1A and E7 can purify DNA methyltransferase activity from nuclear extracts. These associations are direct and mediated by the extreme N-terminus of E1A and the CR3 zinc-finger domain of E7. Furthermore, we find that a point mutant at leucine 20 of E1A, a residue known to be critical for its transformation functions, is unable to bind Dnmt1 and DNA methyltransferase activity. Finally, both E1A and E7 can stimulate the methyltransferase activity of Dnmt1 in vitro. Our results provide the first indication that viral oncoproteins bind and regulate Dnmt1 enzymatic activity. These observations open up the possibility that this association may be used to control cellular proliferation pathways and suggest a new mechanism by which small DNA tumour viruses can steer cells through the cell cycle.

DNA methyltransferases get connected to chromatin

The DNA methyltransferases (Dnmts) are responsible for the generation of genomic methylation patterns, which lead to transcriptional silencing. Recent studies have identified new protein partners for the Dnmts revealing novel mechanisms by which they can be targeted to specific genomic regions to silence genes. In particular, the links identified with histone deacetylases, histone methyltransferases and SNF-2-like ATPases are defining new requirements for the functioning of Dnmts. A picture is emerging whereby the Dnmts are intimately connected to the chromatin environment in which they are working.

Plasma cytokine levels during acute HIV-1 infection predict HIV disease progression.

Background:Both T-cell activation during early HIV-1 infection and soluble markers of immune activation during chronic infection are predictive of HIV disease progression. Although the acute phase of HIV infection is associated with increased pro-inflammatory cytokine production, the relationship between cytokine concentrations and HIV pathogenesis is unknown. Objectives:To identify cytokine biomarkers measurable in plasma during acute HIV-1 infection that predict HIV disease progression. Design:Study including 40 South African women who became infected with HIV-1 and were followed longitudinally from the time of infection. Methods:The concentrations of 30 cytokines in plasma from women with acute HIV-1 infection were measured and associations between cytokine levels and both viral load set point 12 months postinfection and time taken for CD4 cell counts to fall below 350 cells/μl were determined using multivariate and Cox proportional hazards regression. Results: We found that the concentrations of five plasma cytokines, IL-12p40, IL-12p70, IFN-γ, IL-7 and IL-15 in women with acute infection predicted 66% of the variation in viral load set point 12 months postinfection. IL-12p40, IL-12p70 and IFN-γ were significantly associated with lower viral load, whereas IL-7 and IL-15 were associated with higher viral load. Plasma concentrations of IL-12p40 and granulocyte–macrophage colony-stimulating factor during acute infection were associated with maintenance of CD4 cell counts above 350 cells/μl, whereas IL-1α, eotaxin and IL-7 were associated with more rapid CD4 loss. Conclusion: A small panel of plasma cytokines during acute HIV-1 infection was predictive of long-term HIV disease prognosis in this group of South African women.

Association of HIV-Specific and Total CD8+ T Memory Phenotypes in Subtype C HIV-1 Infection with Viral Set Point

Understanding early immunological events during HIV-1 infection that may set the course of disease progression is important for identifying correlates of viral control. This study explores the association of differentiation profiles of HIV-specific and total memory CD8+ T cells with viral set point. A cohort of 47 HIV-1-infected individuals, with differing viral set points at 12 mo, were recruited during acute infection. We identified that the magnitude of IFN-γ+ T cell responses at 6 mo postinfection did not associate with viral set point at 12 mo. A subset of 16 individuals was further studied to characterize CD8+ T cells for expression patterns of markers for memory differentiation, survival (CD127), senescence (CD57), and negative regulation (programmed death-1). We show that viral control and the predicted tempo of HIV disease progression in the first year of infection was associated with a synchronous differentiation of HIV-specific and total CD8+ memory subpopulations. At 6–9 mo postinfection, those with low viral set points had a significantly higher proportion of early differentiated HIV-specific and total memory CD8+ cells of a central memory (CD45RO+CD27+CCR7+) and intermediate memory (CD45RO−CD27+CCR7−) phenotype. Those with high viral set points possessed significantly larger frequencies of effector memory (CD45RO+CD27−CCR7−) cells. The proportions of memory subsets significantly correlated with CD38+CD8+ T cells. Thus, it is likely that a high Ag burden resulting in generalized immune activation may drive differentiation of HIV-specific and total memory CD8+ T cells.

Impact of Mucosal Inflammation on Cervical Human Immunodeficiency Virus (HIV-1)-Specific CD8 T-Cell Responses in the Female Genital Tract during Chronic HIV Infection

ABSTRACT The female genital tract is the major route of heterosexual human immunodeficiency virus (HIV) acquisition and transmission. Here, we investigated whether HIV-specific CD8 T-cell-mediated immune responses could be detected in the genital mucosa of chronically HIV-infected women and whether these were associated with either local mucosal HIV shedding or local immune factors. We found that CD8+ T-cell gamma interferon responses to Gag were detectable at the cervix of HIV-infected women but that the magnitude of genital responses did not correlate with those similarly detected in blood. This indicates that ex vivo HIV responses in one compartment may not be predictive of those in the other. We found that increased genital tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) levels correlated significantly with levels of Gag-specific CD8+ T cells at the cervix. Women who were detectably shedding virus in the genital tract had significantly increased cervical levels of TNF-α, IL-1β, IL-6, and IL-8 compared to women who were not detectably shedding virus. We were, however, unable to detect any association between the magnitude of cervical HIV-specific responses and mucosal HIV shedding. Our results support the hypothesis that proinflammatory cytokines in the female genital tract may promote HIV replication and shedding. In addition, we further show that inflammatory cytokines are associated with increased levels of HIV-specific CD8 effector cells at the genital mucosa but that these were not able to control genital HIV shedding.

Innate Lymphoid Cells Are Depleted Irreversibly during Acute HIV-1 Infection in the Absence of Viral Suppression

Innate lymphoid cells (ILCs) play a central role in the response to infection by secreting cytokines crucial for immune regulation, tissue homeostasis, and repair. Although dysregulation of these systems is central to pathology, the impact of HIV-1 on ILCs remains unknown. We found that human blood ILCs were severely depleted during acute viremic HIV-1 infection and that ILC numbers did not recover after resolution of peak viremia. ILC numbers were preserved by antiretroviral therapy (ART), but only if initiated during acute infection. Transcriptional profiling during the acute phase revealed upregulation of genes associated with cell death, temporally linked with a strong IFN acute-phase response and evidence of gut barrier breakdown. We found no evidence of tissue redistribution in chronic disease and remaining circulating ILCs were activated but not apoptotic. These data provide a potential mechanistic link between acute HIV-1 infection, lymphoid tissue breakdown, and persistent immune dysfunction.

Impact of human immunodeficiency virus 1 infection and inflammation on the composition and yield of cervical mononuclear cells in the female genital tract.

Cervical cytobrush sampling is a relatively non‐invasive method for obtaining mucosal cells from the female genital tract. To define mucosal immune cells sampled by cervical cytobrushing and to validate this approach for local immunity studies, we investigated the impact of human immunodeficiency virus (HIV) status and inflammation on the yield and composition of cervical cytobrush specimens. Cervical cytobrush samples were obtained from 89 chronically HIV‐infected and 46 HIV‐negative women. The HIV‐infected women had significantly higher yields of CD3+, CD45+, CD19+, CD14+, Langerin+ and CD24+ cells than the uninfected women. While cytobrush‐derived T cells from uninfected women were predominantly CD4+ (4·2 CD4 : 1 CD8), CD8+ T cells were predominant in HIV‐infected women (0·6 CD4 : 1 CD8). The majority of CD4+ and CD8+ T cells from HIV‐infected and uninfected women were of the effector memory (CD45RA− CCR7− CD27−) phenotype. HIV‐infected women had significantly elevated levels of interleukin (IL)‐1β, IL‐6 and IL‐8 in cervical supernatants compared with uninfected women. We observed a significant positive correlation between T‐cell counts and IL‐1β, tumour necrosis factor (TNF)‐α and IL‐12 concentrations. Neutrophil counts correlated significantly with cervical concentrations of IL‐1β, TNF‐α, IL‐8, IL‐6 and IL‐10. Antigen‐presenting cell numbers correlated significantly with TNF‐α and IL‐12 concentrations. HIV‐infected women on antiretroviral therapy had similar levels of cervical lymphocyte infiltration and inflammation to women naïve to therapy. In conclusion, we suggest that inflammation at the cervix and HIV infection are likely to be key determinants in the absolute number of mucosal immune cells recovered by cervical cytobrushing.

Construction, Characterization, and Immunogenicity of a Multigene Modified Vaccinia Ankara (MVA) Vaccine Based on HIV Type 1 Subtype C

Candidate vaccines composed of a DNA construct to prime the immune system, followed by modified vaccinia Ankara (MVA) containing matching genes as a booster vaccination, have produced encouraging immune responses in human volunteers. This study presents the detailed construction and characterization of a recombinant MVA that will be tested in combination with a DNA vaccine in Phase I clinical trials in South Africa and the United States. To match recently transmitted viruses in the southern African region and to maximize epitope coverage, the vaccines were constructed to contain five HIV-1 subtype C genes, namely gag, reverse transcriptase, tat, and nef (grttn), expressed as a polyprotein, and a truncated env (gp150). An initial recombinant MVA construct containing wild-type env was found to be genetically unstable, and thus a human codon-optimized gene was used. Grttn and gp150 were inserted into two different sites in MVA yielding a double recombinant, SAAVI MVA-C. The recombinant MVA was shown to be genetically stable and high level expression of the transgenes was observed. Env retained infectivity in a functional infectivity assay despite a point mutation that arose during virus generation. Mice inoculated with SAAVI MVA-C at various doses developed high levels of Gag, RT, and Env-specific CD8(+) and CD4(+) T cells, and some of these responses could be boosted by a second inoculation. An accompanying paper describes the immunogenicity of SAAVI MVA-C when given in combination with SAAVI DNA-C.