Journal: bioRxiv

Article Title: A functional map of CDK-drug interactions at single amino acid resolution

doi: 10.1101/2025.11.02.685764

Figure Lengend Snippet: a) Scatterplot of median gRNA enrichment (log 2 fold-change) in the presence of 5,000 nM KI-CDK9d-32N relative to DMSO-treated control. Each dot represents a gRNA, filtered to exclude gRNAs below 10% sensor editing and with a base mean count ≤ 100. Hits labelled with FDR < 0.1 & LFC ≥ 2. ABE screen results shown in purple; CBE in blue. Regions shaded blue indicate ATP binding site residues. b) Same as (a), but for cells treated with KB-0742 (1,500 nM). c) Sanger sequencing of cells transduced with CDK7 L18F gRNA before and after selection with SY-5609. d) Same as (a), but for cells treated with SEL120 (4,000 nM). e) Same as (a), but for cells treated with Senexin B (15,000 nM). f) Top resistance variants in CDK8/19, as ranked by log 2 fold-change in SEL120 4,000 nM treatment condition. g) Sanger sequencing of CDK9 L156F/CDK7 L18F cells.

Article Snippet: KI-CDK9d-32 and KI-CDK9d-32N were provided by the Koehler lab. All other compounds were ordered from MedChemExpress (MCE) or SelleckChem (Selleck), with the following catalogue numbers: KB-0742 (MCE cat. no. HY-137478A), SY-5609 (MCE cat. no. HY-138293), Senexin B (MCE cat. no. HY-101800), SEL120 (Selleck cat. no. S8840), BSJ-4-116 (MCE cat. no. HY-139039), CDK12-IN-2, (MCE cat. no. HY-112626), HQ461 (MCE cat. no. HY-144981), Ribociclib (MCE cat. no. HY-15777), Palbociclib (MCE cat. no. HY-50767A), Abemaciclib (MCE cat. no. HY-16297), Atirmociclib (Selleck cat. no. E1495), Tagtociclib (MCE cat. no. HY-137894A), and INX-315 (MCE cat. no. HY-162001).

Techniques: Control, Binding Assay, Sequencing, Transduction, Selection

Journal: Molecular metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism.

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: Figure 3: Higher levels of H3K9Bu correlate with blunted stress- and diet-induced changes in gene expression. Twelve-week-old C57BL/6J mice were fed a fat-free (0 Kcal % fat) or high-fat (60 kcal % fat) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diets for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated and pooled for ChIPeSeq assay with anti-H3K9Bu or anti-H3K9Ac (n ¼ 1, pool of 3 hearts each) or the RNA was extracted for RNA-Seq (n ¼ 3 independent hearts each). AeF. The results were sorted according to mRNA of genes that were significantly (p 0.05) AeC. upregulated (2,221 genes) or DeF. downregulated (2,242 genes) during TAC and a 60 kcal % fat diet (Fat 60 diet). a. and d. Violin plots showing the median and quartiles of H3K9Bu and B. and E. H3K9Ac average values of reads at the promoter regions (1 kb to þ1 kb) after subtracting the input for each. c. and F. Violin plots of the median and quartiles of the shrunken log2 fold change (LFC) of the RNA-Seq for fat-free (Fat 0) TAC/Sham, Fat 60 TAC/Sham, Fat 60 TAC/Fat 0 TAC, and Fat 60 Sham/Fat 0 Sham. G. and H. MA plots of the DeSeq2 shrunken LFC (Y axis) vs mean of normalized counts (X axis) of the TAC/Sham RNA-Seq data with the different diets. I. A gene enrichment plot of GO term fatty acid b-oxidation generated from the heart tissue RNA-Seq data (n ¼ 3) from the mice maintained on the low-fat (10% fat) vs high-fat (60% fat) diet for 11 days. Heatmap of the differentially expressed genes is shown on the right. The red asterisks indicate p < 0.05. J. RNA-Seq reads for ACADS mRNA from the heart tissue of the mice on the indicated diets were averaged and plotted. Error bars represent S.E.M. The brackets encompass the values that were statistically compared for significance, with the p values listed above each. KeL. RNA-Seq results for K. Nppb, Ankrd1, and Acta1, or L. Tbx20, Rasl11b, and Grk5 were averaged (n ¼ 3) and plotted as bar graphs (see color keycode at top). * indicates p 0.05 vs Sham and # indicates p 0.05 vs TAC. MeN. H3K9Bu- and H3K9Ac-bound sequence fragments (Y axis) aligned to the chromosome coordinates (X axis) are shown for the genes in (KeL.). O. Gene enrichment plots of GO term respirasome generated from the Sham vs TAC heart tissue RNA-Seq data (n ¼ 3, each) from the mice maintained on a 0, 10, or 60 kcal % fat diets, with a heatmap of the differentially expressed genes shown on the right of each.

Article Snippet: Construction of GFP fusion ACADS and NLS mutant Plasmids harboring cDNAs of turbo-GFP (tGFP) and mouse ACADS (NM_007383) were purchased from Origene.

Techniques: Gene Expression, Isolation, RNA Sequencing, Generated, Sequencing

Journal: Molecular metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism.

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: Figure 5: ACADS is a mitochondrial and nuclear protein and its deficiency is associated with a specific increase in H3K9Bu. AeB. Cardiac myocytes were isolated and cultured for 24 h before they were supplemented with Ad-tGFP, Ad-ACADS-tGFP, or Ad-NLS-mutant ACADS (Ad-mt-ACADS) for an additional 24 h before the cells were harvested. CeD. Twelve-week-old Balc/cJ and Balb/cByJ (ACADS-deficient) mice were maintained on a high-fat (60 Kcal %) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diet for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated. A. and C. Protein from isolated cardiac myocytes or heart tissue was fractionated into cytosol (Cyto) membranes, including mitochondria (Mem/Mito), nucleoplasm (Nuc), and chromatin (Chrom). The protein was analyzed by Western blotting with antibodies for the molecules listed on the right of the panels or A. with anti-tGFP (top panel) or anti-ACADS (second from the top panel). The first and last lanes show protein standards, and their sizes are listed on the left of each panel. B. and D. Western blotting signals were quantitated, and cytosolic proteins were normalized to AKT1, membrane/mitochondrial protein to VDAC1, nucleoplasm proteins to Pol II, and H3K9Bu to H3 or Pol II (n ¼ 4e6 each). D. The results were plotted relative to a cJ-Sham signal adjusted to 1. B. Signals for tGFP, ACADS-tGFP, and mt-ACADS-tGFP in the Cyto and Nuc fractions were plotted each relative to its corresponding Mem/Mito signal adjusted to 1. Error bars represent S.E.M. The brackets encompass the values that were statistically compared for significance, with the p values listed above each.

Article Snippet: Construction of GFP fusion ACADS and NLS mutant Plasmids harboring cDNAs of turbo-GFP (tGFP) and mouse ACADS (NM_007383) were purchased from Origene.

Techniques: Isolation, Cell Culture, Mutagenesis, Western Blot, Membrane

Journal: Molecular metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism.

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: Figure 6: Deletion of ACADS reverses stress-induced downregulation of H3K9Bu in the heart. Twelve-week-old Balc/cJ and Balb/cByJ (ACADS-deficient) mice were maintained on a low-fat (10% of calories) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diet for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated and pooled (n ¼ 3, each). Chromatin was extracted from the heart tissue and subjected to anti-H3K9Bu ChIPeSeq (n ¼ 1, a pool of 3 hearts each). AeB. The average signal (Avg Signal) of the chromatin-bound H3K9Bu reads assembled at all of the gene promotors (2 Kb to þ2 Kb from the TSS) were plotted for each condition (see color keycode at top) CeD. or all sequence reads were represented by a heatmap across the same region. EeF. H3K9Bu-bound sequence fragments (Y axis) aligned to chromosome coordinates (X axis) are shown for Clasp1, Arpc4,Usp1, and Myl2 genes. g. Violin plots showing the median and quartiles of H3K9Bu average values of sequence reads within gene bodies in 3,242 genes (þ1 Kb from TSS to gene end). HeI. The average signal (Avg Signal) of the chromatin-bound H3K9Ac reads assembled at all of the gene promotors (2 Kb to þ2 Kb from the TSS) graphed for each condition (see color keycode at top) or JeI. all reads were represented by heatmaps across the same region. The input was generated from a pool of Balb/cJ and Balb/cByJ, Sham, and TAC heart chromatin.

Article Snippet: Construction of GFP fusion ACADS and NLS mutant Plasmids harboring cDNAs of turbo-GFP (tGFP) and mouse ACADS (NM_007383) were purchased from Origene.

Techniques: Isolation, Sequencing, Generated

Journal: Molecular metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism.

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: Figure 7: Deletion of ACADS blunts stress-induced changes in gene expression. Twelve-week-old Balc/cJ and Balb/cByJ (ACADS-deficient) mice were maintained on a low- fat (10% of calories) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diet for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated and pooled (n ¼ 3 each) for ChIPeSeq with anti-H3K9Bu or anti-H3K9Ac (n ¼ 1, a pool of 3 hearts each) or the RNA was extracted for RNA-Seq (n ¼ 3 independent hearts each). AeF. The results were sorted according to mRNA of genes that were significantly (p 0.05) AeC. upregulated (1,272 genes) during TAC in the Balb/cJ mice (cJ) or DeF. downregulated (1,016) during TAC in the cJ mice. A. and D. Violin plots showing the median and quartiles of H3K9Bu and B. and E. H3K9Ac average values of sequence reads at the promoter regions (1,000 to þ1,000) after subtracting the input for each. C. and F. Violin plots of the median and quartiles of the shrunken log2 fold change (LFC) of the RNA-Seq for cJ-TAC/Sham, cByJ-TAC/Sham, cJ-TAC/cByJ-TAC, and cJ-Sham/cByJ-Sham. G. and H. MA plots of the DeSeq2 shrunken LFC (Y axis) vs mean of normalized counts (X axis) of the TAC/Sham RNA-Seq data for Balb/cJ and Balb/cByJ. IeJ. Enrichment plots for GO terms generated from the Sham and TAC and heart RNA-Seq results of the Balb/cJ (cJ) and Balb/cByJ (cByJ) mice.

Article Snippet: Construction of GFP fusion ACADS and NLS mutant Plasmids harboring cDNAs of turbo-GFP (tGFP) and mouse ACADS (NM_007383) were purchased from Origene.

Techniques: Gene Expression, Isolation, RNA Sequencing, Sequencing, Generated

Journal: Molecular metabolism

Article Title: Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism.

doi: 10.1016/j.molmet.2021.101249

Figure Lengend Snippet: Figure 5: ACADS is a mitochondrial and nuclear protein and its deficiency is associated with a specific increase in H3K9Bu. AeB. Cardiac myocytes were isolated and cultured for 24 h before they were supplemented with Ad-tGFP, Ad-ACADS-tGFP, or Ad-NLS-mutant ACADS (Ad-mt-ACADS) for an additional 24 h before the cells were harvested. CeD. Twelve-week-old Balc/cJ and Balb/cByJ (ACADS-deficient) mice were maintained on a high-fat (60 Kcal %) diet for 4 days before subjecting them to transverse aortic constriction (TAC) or sham surgeries. They were then maintained on the same diet for 7 days before they were assessed by echocardiography, sacrificed, and the hearts isolated. A. and C. Protein from isolated cardiac myocytes or heart tissue was fractionated into cytosol (Cyto) membranes, including mitochondria (Mem/Mito), nucleoplasm (Nuc), and chromatin (Chrom). The protein was analyzed by Western blotting with antibodies for the molecules listed on the right of the panels or A. with anti-tGFP (top panel) or anti-ACADS (second from the top panel). The first and last lanes show protein standards, and their sizes are listed on the left of each panel. B. and D. Western blotting signals were quantitated, and cytosolic proteins were normalized to AKT1, membrane/mitochondrial protein to VDAC1, nucleoplasm proteins to Pol II, and H3K9Bu to H3 or Pol II (n ¼ 4e6 each). D. The results were plotted relative to a cJ-Sham signal adjusted to 1. B. Signals for tGFP, ACADS-tGFP, and mt-ACADS-tGFP in the Cyto and Nuc fractions were plotted each relative to its corresponding Mem/Mito signal adjusted to 1. Error bars represent S.E.M. The brackets encompass the values that were statistically compared for significance, with the p values listed above each.

Article Snippet: Construction of GFP fusion ACADS and NLS mutant Plasmids harboring cDNAs of turbo-GFP (tGFP) and mouse ACADS (NM_007383) were purchased from Origene.

Techniques: Isolation, Cell Culture, Mutagenesis, Western Blot, Membrane