Journal: Communications Biology

Article Title: IDLV-HIV-1 Env vaccination in non-human primates induces affinity maturation of antigen-specific memory B cells

doi: 10.1038/s42003-018-0131-6

Figure Lengend Snippet: NHP immunization schedule and binding antibody responses. a Six Indian rhesus macaques were primed intramuscularly with 3 × 10 8 transducing units (TUs) of IDLV-1086.C and boosted twice, at 1-year intervals, first with the same vector IDLV-1086.C and then with IDLV expressing a different envelope 1176.C in an attempt to garner neutralization breadth. Anti-vector immunity in the repeated IDLV immunizations was minimized using two different VSV serotypes for the first and second immunizations. b Anti C.1086 gp140 and c anti 1176.C gp140 protein binding Abs induced by IDLV-Env immunization. The magnitude and durability of anti-Env IgG were measured in 6 animals in plasma. No binding response was detected in plasma samples collected before immunization (week 0). Lines indicate mean values at each time point. Each sample was analyzed in duplicate and the data shown are representative of at least three experiments

Article Snippet: Macaque memory B cells were stained with both AlexaFluor 647 (AF647) and Brilliant Violet (BV421)-tagged HIV-1 1086.C gp140 and 1176.C gp140 and sorted using a BD FACSAria II (BD Biosciences, San Jose, CA).

Techniques: Binding Assay, Plasmid Preparation, Expressing, Neutralization, Protein Binding

Journal: Communications Biology

Article Title: IDLV-HIV-1 Env vaccination in non-human primates induces affinity maturation of antigen-specific memory B cells

doi: 10.1038/s42003-018-0131-6

Figure Lengend Snippet: Cross-reactivity of memory B cell-derived antibodies from longitudinal samples against 1086.C and 1176.C gp140 Envs. a Flow plots of 1086.C and 1176.C gp140 staining of Env-reactive memory B cells in the indicated animals at 0, 57, and 109 weeks post immunization. Memory B cells which bound both BV421 ( x -axis) and AF647 ( y- axis) labeled Envs were defined as double positive (DP) Env-reactive memory B cells. Sorting of DP Env-reactive B cells was performed to ensure sort accuracy. No pre-existing cross-reacting memory B cells were present before vaccination (week 0). b Env reactivity of each individual sorted memory B cell was confirmed in ELISA after in vitro expansion and differentiation into antibody-secreting cells as described in the Methods. The Venn diagrams show the percentage of Env-specific supernatants from Ig-secreting cultured memory B cells that bound to 1086.C gp140 (yellow) and/or 1176.C gp140 (blue) Env. The number of Env-specific memory B-cell supernatants screened for each animal at each time point is indicated below each diagram

Article Snippet: Macaque memory B cells were stained with both AlexaFluor 647 (AF647) and Brilliant Violet (BV421)-tagged HIV-1 1086.C gp140 and 1176.C gp140 and sorted using a BD FACSAria II (BD Biosciences, San Jose, CA).

Techniques: Derivative Assay, Staining, Labeling, Enzyme-linked Immunosorbent Assay, In Vitro, Cell Culture

Journal: Communications Biology

Article Title: IDLV-HIV-1 Env vaccination in non-human primates induces affinity maturation of antigen-specific memory B cells

doi: 10.1038/s42003-018-0131-6

Figure Lengend Snippet: Persistence of antigen-specific circulating memory B cells following IDLV vaccination. a To assess the persistence of the 1086.C-specific circulating memory B cell pool, longitudinal flow cytometry analysis was performed on PBMCs from two vaccinated animals. b Flow plots of gp140 and gp120 (1086, CH505 or RSC3) staining of Env-reactive memory B cells in animal 4430 at 57 weeks post immunization. Memory B cells which bound both BV421 ( x -axis) and AF647 ( y -axis) labeled Envs were defined as double positive (DP) Env-reactive memory B cells. Sorting of DP Env-reactive B cells was performed to ensure sort accuracy. Memory B cells which bound to the wild-type form of gp120 but not to the d371 mutated Env were defined as differential binders (putative CD4bs Abs)

Article Snippet: Macaque memory B cells were stained with both AlexaFluor 647 (AF647) and Brilliant Violet (BV421)-tagged HIV-1 1086.C gp140 and 1176.C gp140 and sorted using a BD FACSAria II (BD Biosciences, San Jose, CA).

Techniques: Flow Cytometry, Staining, Labeling

Journal: bioRxiv

Article Title: Epigenetic Interaction between UTX and DNMT1 Regulates Diet-Induced Myogenic Remodeling in Brown Fat

doi: 10.1101/2020.08.05.238923

Figure Lengend Snippet: MyoD1 mediates the effect of DNMT1 deficiency on brown fat myogenesis. (A) RRBS profiling of DNA methylation level at MyoD1 promoter in iBAT of D1KO and fl/fl mice. (B)-(C) Ucp1 (B) and Myod1 (C) expression in iBAT of mice during late embryonic and postnatal development (n=4/group). (D)-(E) Ucp1 (D) and Myod1 (E) expression in iBAT of mice during cold exposure (n=4/group). (F) Ucp1 , Prdm16 and myogenic marker gene expression in iBAT and gastrocnemius (GAS) muscle (n=4/group). (G) Pyrosequencing analysis of DNA methylation level at Myod1 promoter in iBAT and GAS muscle (n=4/group). (H) ChIP assay of DNMT1 binding to Myod1 promoter in undifferentiated BAT1 preadipocytes and differentiated BAT1 brown adipocytes (n=3/group). (I) ChIP assay of DNMT1 binding to the Myod1 promoter in iBAT from HFD- or LFD-fed mice (n=4/group). (J) Pyrosequencing analysis of DNA methylation levels at Myod1 promoter in BAT1 brown adipocytes transfected with scramble or Dnmt1 siRNA (n=6/group). (K) Quantitative RT-PCR analysis of myogenic marker gene and BAT gene expression in BAT1 brown adipocytes transfected with scramble, Dnmt1 , Myod1 , or Dnmt1 + Myod1 siRNA (n=4/group). Different lowercase letters represent significant differences at p<0.05. For (J) and (K), BAT1 cells were differentiated into brown adipocytes as described under Methods and scramble and targeting siRNAs were transfected into day 4 differentiated BAT1 cells using Amaxa Nucleofector II Electroporator with an Amaxa cell line nucleofector kit L. Cells were harvested 2 days after for pyrosequencing (J) or gene expression (K) analysis. All data are expressed as mean ± SEM. *p<0.05 by Student’s t-test or ANOVA with Bonferroni post-hoc analysis.

Article Snippet: For sub-cloning of Prdm16 and Dnmt1 fragments for co-immunoprecipitation experiments, Fragments of Prdm16 (1–223, 224–454, 455–680, 681–880, 881–1038, and 1039–1176) were PCR-amplified using the full-length Prdm16 plasmids (Addgene #15503) and sub-cloned into XbaI/EcoRI sites of Flag-HA-pcDNA3.1 vector (Addgene #52535).

Techniques: DNA Methylation Assay, Expressing, Marker, Gene Expression, Binding Assay, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Epigenetic Interaction between UTX and DNMT1 Regulates Diet-Induced Myogenic Remodeling in Brown Fat

doi: 10.1101/2020.08.05.238923

Figure Lengend Snippet: DNMT1 silences Myod1 expression via interacting with PRDM16. (A) Quantitative RT-PCR analysis of Prdm16 mRNA in iBAT of LFD- or HFD-fed mice (n=8/Group). (B)-(C) ChIP assay of UTX binding to Prdm16 promoter (B) and ChIP assay of H3K27me3 levels at Prdm16 promoter (C) in iBAT of LFD- or HFD-fed mice (n=4/Group). (D) ChIP assay of H3K27me3 levels at Prdm16 promoter in control or Utx knockdown BAT1 brown adipocytes treated with isoproterenol (n=4/Group). (E) Pyrosequencing analysis of DNA methylation at Myod1 promoter in BAT1 brown adipocytes transfected with scramble or Utx siRNA (n=6/Group). (F) Pyrosequencing analysis of DNA methylation at Myod1 promoter in BAT1 brown adipocytes transfected with scramble or Prdm16 siRNA (n=4/group). (G) Pyrosequencing analysis of DNA methylation at Myod1 promoter in BAT1 brown adipocytes transfected with pSport6 or pSport6 encoding Prdm16 overexpressing plasmids (n=4/group). (H) ChIP assay of DNMT1 binding to Myod1 promoter in control or Prdm16 knockdown BAT1 brown adipocytes treated with or without isoproterenol (n=4/group). Different lowercase letters represent significant differences at p<0.05. (I) Co-IP of DNMT1 and FLAG-PRDM16 in HEK293T cells. (J) Co-IP of DNMT1 and various fragments of PRDM16. HA-tagged fragments of PRDM16 were expressed along with full-length DNMT1 in HEK293T cells. Cell lysates were immunoprecipitated with anti-DNMT1 antibodies followed by immunoblotting with HA or DNMT1 antibodies. Color-coded domain architecture of PRDM16 shows a PR/SET domain (PR), an N-terminal zinc-finger domain containing seven C2H2 zinc finger motifs (ZF1), a proline rich domain (PRR), a repression domain (RD), a second C-terminal zinc-finger domain containing three C2H2 zinc finger motifs (ZF2), and an acidic activation domain (AD). (K) Co-IP of PRDM16 and various fragments of DNMT1. HA-tagged fragments of DNMT1 were expressed along with full-length PRDM16 in HEK293T cells. Cell lysates were immunoprecipitated with anti-HA antibodies followed by immunoblotting with HA or PRDM16 antibodies. Color-coded domain architecture of DNMT1 shows the N-terminal independently folded domain (NTD), replication foci-targeting sequence (RFTS) domain, a Zn-finger like CXXC motif, two bromo adjacent homology (BAH1 and BAH2) domains, and the catalytic domain. (L) Schematic illustration of the interaction between UTX-regulated PRDM16 and DNMT1 in the maintenance of brown fat identity and suppression of myogenic remodeling in mature brown adipocytes. In brief, in mature brown adipocytes, UTX maintains the persistent demethylation of the repressive mark H3K27me3 at Prdm16 promoter, leading to high expression of Prdm16 ; PRDM16 then recruits the DNA methyltransferase DNMT1 to Myod1 promoter, causing Myod1 promoter hypermethylation, and suppressing Myod1 expression. The interaction between PRDM16 and DNMT1 coordinately serves to maintain brown adipocyte identity while repressing myogenic remodeling in mature brown adipocytes, thus promoting their active brown adipocyte thermogenic function. Suppressing this interaction by HFD feeding induces brown adipocyte-to-myocyte remodeling, which limits brown adipocyte thermogenic capacity and compromises diet-induced thermogenesis, leading to the development of obesity.

Article Snippet: For sub-cloning of Prdm16 and Dnmt1 fragments for co-immunoprecipitation experiments, Fragments of Prdm16 (1–223, 224–454, 455–680, 681–880, 881–1038, and 1039–1176) were PCR-amplified using the full-length Prdm16 plasmids (Addgene #15503) and sub-cloned into XbaI/EcoRI sites of Flag-HA-pcDNA3.1 vector (Addgene #52535).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Control, Knockdown, DNA Methylation Assay, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Activation Assay, Sequencing