Journal: bioRxiv

Article Title: Deep transcriptome annotation suggests that small and large proteins encoded in the same genes often cooperate

doi: 10.1101/142992

Figure Lengend Snippet: ( a ) AltDDIT3 coding sequence is located in exons 1 and 2 or the DDIT3/CHOP/GADD153 gene and in the 5 UTR of the canonical mRNA (RefSeq NM_004083.5). +2 and +1 indicate reading frames. AltDDIT3 amino acid sequence is also shown. ( b ) Confocal microscopy analyses of HeLa cells co-transfected with altDDIT3 GFP (green channel) and DDIT3 mCherry (red channel). Scale bar, 10 μm. ( c, d ) Colocalization analysis of the images shown in ( b ) performed using the JACoP plugin (Just Another Co-localization Plugin) implemented in Image J software (two independent biological replicates). ( c ) Scatterplot representing 50 % of green and red pixel intensities showing that altDDIT3 GFP and DDIT3 mCherry signal highly correlate (with Pearson correlation coefficient of 0.92 ( p- value < 0.0001)). ( d ) Binary version of the image shown in ( b ) after Costes automatic threshold. White pixels represent colocalization events ( p- value < 0.001, based on 1000 rounds of Costes randomization colocalization analysis). The associated Manders Correlation Coefficient, M 1 and M 2 , are shown in the right upper corner. M 1 is the proportion of altDDIT3 GFP signal overlapping DDIT3 mCherry signal and M 2 is the proportion of DDIT3 mCherry signal overlapping altDDIT3 GFP . ( e ) Representative immunoblot of co-immunoprecipitation with GFP-Trap agarose beads performed on HeLa lysates co-expressing DDIT3 mcherry and altDDIT3 GFP or DDIT3 mcherry with pcDNA3.1 GFP empty vector (two independent experiments).

Article Snippet: Human altDDIT3 mCherry was cloned into pcDNA3.1 by Gibson assembly using coding sequence from transcript variant 1 (NM_004083.5) and mCherry coding sequence from pLenti-myc-GLUT4-mCherry (Addgene plasmid # 64049).

Techniques: Sequencing, Confocal Microscopy, Transfection, Software, Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice

doi: 10.1038/s41467-021-26277-w

Figure Lengend Snippet: a Immunofluorescence images of WAT after 26 weeks on HFD (SD controls are shown in Supplementary Fig. ). DAPI (blue) and neutral lipid staining by BODIPY-493 (green). Highlighted regions show recidual fat storage. Scale bar: 50 µm, enlarged view: 5 µm. b Violin-plot showing the MFI quantification of ( a ). Error bars indicate ±SEM. Statistical analysis was performed by one-way ANOVA followed by Kruskal–Wallis test, * p = 0.04, **** p = 0.0001. n = 3 biological replicates. Dotted lines inside the violin plots show the quartiles and dashed lines depict the medians. Dotted horizontal line marks the zero. c Violin-plot showing total quantification of adipocytes per section ( n = 3, 2–3 sections per animal). Normality was scored as in ( b ). Statistical analysis using Mann–Whitney test showed no significance. Dotted lines show the quartiles and dashed lines depict the median. d Barplot showing enrichment test for target GO terms and DEG in WAT. The black box represents differentially regulated genes in WAT of Mof +/− animals on SD. Statistical test was performed using the absolute numbers of genes and p values were scored by the two-sided Fisher’s exact test. The red bar highlights the only significantly enriched pathway. See also: Supplementary Fig. . e Left: Representative immunofluorescence images showing GLUT4 (red) expression in WAT of animals on HFD. Scale bar: 50 µm. Right: MFI quantification of GLUT4 signal. Statistical analysis was performed by two-sided Mann–Whitney test, * p = 0.048. n = 3 biological replicates. f Representative immunofluorescence images showing neutral lipids (BODIPY-493; green) in adipocytes differentiated from mesenchymal stromal cells (MSC). Scale bar: 20 µm. g Boxplot showing the quantification of overall lipid droplet area per droplet (µm 2 ) in logarithmic scale (right). Statistical analysis was performed by two-sided ANOVA followed by Tukey post test, * p = 0.03, *** p = 0.0007. n = 3 biological replicates. h Graphical scheme illustrating the pre-adipocyte (iAdipo) in vitro differentiation and induction of Mof knockout ( Mof -iKO) by treatment with 4-hydroxy-tamoxifen (4-OHT). See also: Supplementary Fig. . i RT-qPCR analyses of iAdipo cells with (+) or without (−) insulin/glucose challenge. Violin plot shows average Glut4 mRNA expression relative to Hprt of biological replicates ( n = 5). Statistical analysis was performed by two-way ANOVA followed by Holm–Sidak’s comparison test, * p = 0.05. Dotted lines show the quartiles and dashed lines depict the medians. j Representative histogram showing glucose uptake capacity after insulin/glucose challenge (left). Gray histogram represents the untreated cells, control (black), and Mof -iKO (purple). Floating plots showing the 2-NBD ratio uptake (treated MFI/untreated MFI). “+” indicates the treatment employed and the dashed horizontal line marks the basal glucose uptake. Statistical analysis was performed by one-way ANOVA followed by Tukey’s comparison test, * p = 0.013, *** p = 0.0001. n = 5 biological replicates. k Left panel: Line-plots showing the percentage of extracellular acidification rate (%ECAR) of control (black) or Mof -iKO (purple) iAdipo cells without (open circles) or upon insulin challenge (filled circles). Arrows indicate the addition of inhibitors. Statistical analysis was performed by two-sided two-way ANOVA followed by Holm–Sidak’s comparison test. Right panel: Violin plot showing quantification overall glycolysis. “+” indicates insulin treatment. Biological replicates ( n = 3). Statistical analysis was performed by two-sided two-way ANOVA followed by Holm–Sidak’s comparison test, ** p = 0.01, ns = not significant. Dotted lines show the quartiles and dashed lines depict the medians. See also: Supplementary Fig. .

Article Snippet: The Glut4 coding sequence was sub-cloned from Addgene plasmid number 52872 and Mof coding sequence was cloned into a pcDNATM5/pCMV vector.

Techniques: Immunofluorescence, Staining, MANN-WHITNEY, Expressing, In Vitro, Knock-Out, Quantitative RT-PCR, Comparison, Control

Journal: Nature Communications

Article Title: Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice

doi: 10.1038/s41467-021-26277-w

Figure Lengend Snippet: a , b , d , f Left: representative images of neutral lipid staining (BODIPY-493; green). Scale bars: 5 µm. Right: Boxplots showing quantification of overall lipid droplet area per droplet (µm 2 ) in logarithmic scale with the boxes depicting interquartile range. Number of biological replicates per condition: n = 3. Total number of droplets used for quantification are indicated in the figure. Dashed horizontal lines mark the mean area of controls. a Control, Mof -iAdipo or Mg149-treated (50 nM) control cells at baseline (“-”, upper panel) or after 15 min of insulin/glucose challenge (“+”, lower panel). Statistical analysis was performed using the raw data and one-way ANOVA followed by Kruskal–Wallis comparison test, **** p = 10 −16 . b Control and Mof -iAdipo cells treated with Ex-527 (200 nM) after insulin/glucose challenge. Statistical analysis was performed using the raw data and one-way ANOVA followed by Kruskal–Wallis comparison test, *** p = 0.006. See Supplementary Fig. for baseline images. c Scatter-plot depicting the H4K16ac MFI in control and Mof- iKO iAdipo with (open circles) or without (filled circles) insulin and glucose challenge. Each dot represents a single iAdipo. Statistical analysis was performed using the raw data and two-sided one-way ANOVA followed by Kruskal–Wallis comparison test, ** p = 0.014, **** p = 10 −16 . Number of biological replicates n = 4. d Control and Mof -iAdipo cells at baseline, following overexpression of Glut4 , or following ectopic expression of wild-type Mof (wt- Mof ) or Mof catalytic mutant ( Mof -E350Q) challenged with insulin/glucose (+Ins/Gluc). Statistical analysis was performed using the raw data and two-sided one-way ANOVA followed by Dunn’s multiple comparison test, *** p = 0.001. Note that the data for untreated wild type (n = 1299) and Mof -iKO (n = 4644) is identical to the data depicted in ( a ). e Heatmap showing the H4K16ac levels from wild type and Mof -iKO upon ectopic expression of Glut4 , wt- Mof or Mof -E350Q with or without glucose and insulin treatment. Statistical analysis was performed using the raw data and two-sided two-way ANOVA followed by Sidak multiple comparison post test, **** p = 10 −16 . f Control and Mof -iAdipo cells treated with chloroquine at baseline or upon insulin/glucose challenge. Statistical analysis was performed using the raw data and two-sided one-way ANOVA followed by Dunn’s multiple comparison test, *** p = 0.0001, **** p = 10 −16 . g RT-qPCR analyses of visceral WAT. Violin plots show the mRNA expression of Glut4 , Pparg, Pcg1α and Mef2c as an average of biological replicates ( Mof +/+ n = 5; Mof +/− n = 5 for Glut4 , Pparg, Pcg1α and Mof +/+ n = 3; Mof +/− n = 3 for Mef2c ). Statistical analysis was performed by two-sided Mann–Whitney test, * p = 0.026. Dotted lines show the quartiles and solid lines depict the medians. h ChIP-qPCR analyses of MOF at promoters of Pparg , Glut4 , Pcg1α and Mef2c promoters in visceral WAT. The data are expressed as fold change over H3. Violin plots show the average of biological replicates ( Mof +/+ n = 6 and Mof +/− n = 5). Statistical analysis was performed by two-sided Mann–Whitney test, * p = 0.015. Dotted lines show the quartiles and dashed lines depict the medians. i ChIP-qPCR analyses of MOF (left) and H4K16ac (right) at promoters of Pparg in the presence or absence of Glu/Ins in wild-type and Mof -iKO adipocytes. Dotted lines inside the violin plots show the quartiles and dashed lines depict the medians.

Article Snippet: The Glut4 coding sequence was sub-cloned from Addgene plasmid number 52872 and Mof coding sequence was cloned into a pcDNATM5/pCMV vector.

Techniques: Staining, Control, Comparison, Over Expression, Expressing, Mutagenesis, Quantitative RT-PCR, MANN-WHITNEY, ChIP-qPCR

Journal: Nature Communications

Article Title: Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice

doi: 10.1038/s41467-021-26277-w

Figure Lengend Snippet: a Left: representative images of neutral lipid staining (BODIPY-493; green). Control or Mof -iAdipo treated with thiazolidinedione (TZD)(10 −4 mol/L) at baseline (upper panel) or upon insulin/glucose challenge (bottom panel). Scale bar: 5 µm. Right: boxplot showing quantification of overall lipid droplet area per droplet (µm 2 ) in logarithmic scale with boxes showing the interquartile range. Number of biological replicates per condition: n = 3. Total number of droplets used for quantification are indicated on the figure. Dashed horizontal lines mark the mean area of controls. Statistical analysis was performed using the raw data and one-way ANOVA followed by Kruskal–Wallis comparison test, * p = 0.039. b , c RT-qPCR analyses of control or Mof -iAdipo cells treated with TZD at baseline or upon insulin/glucose challenge. Violin plots show the average Pgc1α ( b ) and Glut4 ( c ) mRNA expression of biological replicates ( n = 3). Dotted lines show the quartiles and solid lines depict the medians. d Heatmap showing H4K16ac levels after TZD treatment at different conditions. Statistical analysis was performed using the raw data and two-sided two-way ANOVA followed by Sidak multiple comparison post test, **** p = 10 −16 . e Representative images of neutral lipid staining (BODIPY-493; green). Control or Mof -iAdipo treated with Glut4 siRNA in the presence or absence of thiazolidinedione (TZD)(10 −4 mol/L) at baseline or upon insulin/glucose challenge (+Ins/Gluc). Scale bar: 5 µm. f – h Boxplots showing quantification of overall lipid droplet area per droplet (µm 2 ) in logarithmic scale. Dashed horizontal lines mark the mean area of controls. Number of biological replicates per condition: n = 3. The “Control siRNA ” data is identical in ( f ), ( g ) and ( h ). ( i ) Schematic representation of proposed MOF-mediated regulation of the Glut4 transcription network. See also: Supplementary Fig. .

Article Snippet: The Glut4 coding sequence was sub-cloned from Addgene plasmid number 52872 and Mof coding sequence was cloned into a pcDNATM5/pCMV vector.

Techniques: Staining, Control, Comparison, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice

doi: 10.1038/s41467-021-26277-w

Figure Lengend Snippet: Schematic overview describing the proposed mechanism by which chronic reduction of MOF results in global remodeling of metabolism toward diabetic predisposition. In wild-type cells, insulin binds to the insulin receptor, which will trigger a cascade of phosphorylation events culminating in the phosphorylation of GLUT4 inside storage vesicles. These will be transported to the cytoplasmic membrane to regulate glucose import. In parallel, insulin triggers the transcription of Glut4 predominantly through the transcriptional network downstream of PPARγ and PGC1α. In healthy adipose tissue and skeletal muscle (SKM), MOF mediates glucose uptake by positively regulating gene expression of the nuclear hormone receptor Pparg , thereby triggering the downstream transcriptional network resulting in increased levels of the major insulin-dependent glucose transporter GLUT4. On the other hand, Mof haploinsufficient animals show impaired insulin secretion upon glucose challenge, leading to severe destablization of glucose homeostasis. Whilst insulin signaling and GLUT4 vesicle transport in Mof- depleted cells are functional, the transcriptional network regulating Glut4 expression is impaired, resulting in reduced glucose import. Limitation of intracellular glucose is responsible for impaired neutral lipid storage in Mof +/− adipocytes. This study identifies the epigenetic regulator MOF as the first histone acetyltransferase to regulate the onset of diet-induced obesity.

Article Snippet: The Glut4 coding sequence was sub-cloned from Addgene plasmid number 52872 and Mof coding sequence was cloned into a pcDNATM5/pCMV vector.

Techniques: Phospho-proteomics, Membrane, Gene Expression, Functional Assay, Expressing