Dynamic repression by BCL6 controls the genome-wide liver response to fasting and steatosis
- Northwestern University, United States
- Case Western Reserve University, United States
- Jesse Brown VA Medical Center, United States
Figures
Figure 1 with 2 supplements
BCL6 converges with PPARα at fasting-regulated lipid genes.
(A) MSigDB Pathway and Gene Ontology (GO) Cellular Compartment analysis of BCL6 ChIP-seq binding sites. (B) Motif enrichment analysis of BCL6-bound regions. (C) Gene expression as measured by reads …
Figure 1—figure supplement 1
BCL6 liver ChIP-seq reflects binding events specific to hepatocytes.
(A, B) MSigDB Pathway/Gene Ontology analysis (A) and motif densities (B) for BCL6 ChIP-seq peaks identified in wild-type livers, as determined by enrichment over input chromatin or over BCL6 …
Figure 1—figure supplement 2
BCL6 and PPARα reciprocally bind to shared regulatory regions near fasting genes.
(A) Fed and fasted BCL6 (top) and PPARα (bottom) ChIP-seq tag densities at all respective sites (left panel) or at shared sites (right panel). (B) Histogram of distances between BCL6 and PPARα peak …
Figure 2 with 1 supplement
BCL6 genome-wide DNA binding is independent of PPARα and PPARδ.
(A) BCL6 ChIP-seq tag densities in C57BL/6 and Ppara-/- livers at shared BCL6-PPARα peaks (left). PPARα tag density in Bcl6fl/fl and Bcl6LKO livers at shared BCL6-PPARα peaks (right). (B) BCL6 tag …
Figure 2—figure supplement 1
PPARα and PPARδ compete for binding at shared sites.
(A) qPCR gene expression of Ppard and Bcl6 genes in control (PpardWT) and PpardLKO mouse livers. N = 2 per group. (B) PPARδ tag densities in fed or fasted mouse livers at all PPARδ peaks. N = 3 per …
Figure 3 with 2 supplements
BCL6 complexes with SMRT/NCoR-HDAC3 to reduce H3K27ac and represses transcription.
(A) Volcano plots showing log2 fold change (LFC) in expression of fed Bcl6LKO over Bcl6fl/fl livers. Blue dots represent |LFC| greater than one with an adjusted p-value less than 0.05. Red dots …
Figure 3—figure supplement 1
Bcl6LKO mice exhibit efficient protein and mRNA reductions of BCL6.
(A) qPCR analysis of Bcl6 in Bcl6fl/fl and Bcl6LKO livers. N = 7 per group. (B) Western blots of BCL6 protein in Bcl6fl/fl and Bcl6LKO livers. Densitometric quantification is shown normalized to …
Figure 3—figure supplement 2
SMRT, NCoR, and HDAC3 are recruited to chromatin by BCL6 in liver.
(A) Enriched motifs for SMRT, NCoR and HDAC3 binding sites. For each corepressor, enriched motifs are shown in all peaks (all), peaks shared with BCL6 (BCL6 shared), and BCL6-exclusive peaks …
Figure 4 with 2 supplements
Bcl6 deletion de-represses a fasting liver gene program.
(A) Pathway enrichment analysis for genes differentially expressed (|LFC| greater than one and an adjusted p-value less than 0.05) with fasting or Bcl6 deletion in liver. (B) Volcano plots showing …
Figure 4—figure supplement 1
BCL6 deletion mimics the fasting gene program.
(A) Clustered heatmap of a distance matrix across fed and fasted Bcl6fl/fl and Bcl6LKO RNA-seq samples at genes differentially expressed with fasting. N = 4 per group. (B) Gene ontology analysis for …
Figure 4—figure supplement 2
The BCL6-PPARα regulatory module clusters along dynamic fasting genes.
(A) Distribution of fasting genes annotated with BCL6 and/or PPARα binding peaks. Differential fasting genes here include all significantly different genes with fasting (adjusted p-value<0.05). (B) …
Figure 5 with 2 supplements
Bcl6 ablation reduces HDAC3 activity and promotes PPARδ binding in Ppara-/- mice at rescued genes.
(A) Heatmap of RPKM values at fasting-regulated genes that are dysregulated in Ppara-/- mice. 209 genes have partially or completely restored expression upon fasting in Ppara-/-;Bcl6LKO mice. N = 4 …
Figure 5—figure supplement 1
BCL6 deletion in Ppara-/- mice restores fasting gene expression.
qPCR analysis of PPARα- and fasting-regulated transcripts in overnight fasted Bcl6fl/fl, Ppara-/-, and Ppara-/-;Bcl6LKO mouse livers, N = 5–6 per group. A one-way ANOVA and Holm-Sidak’s post-hoc …
Figure 5—figure supplement 2
Bcl6 deletion is linked to enhanced PPARδ binding in fasted Pparα-/- mice.
(A–B) UCSC genome browser tracks showing (A) RNA-seq and H3K27ac ChIP-seq data in fasted control Bcl6fl/fl, Ppara-/-, and Ppara-/-;Bcl6LKO livers at Aldh3a2 and Acot4/3. (B) H3K27ac tags in Ppara-/-;…
Figure 6 with 1 supplement
Bcl6 deletion enhances fatty acid oxidation and ameliorates steatosis.
(A) Serum β-hydroxybutyrate levels were measured in mice over the course of a 48 hr fast. N = 8–12 per group. (B) Rates of 14C-palmitate oxidation in Bcl6fl/fl and Bcl6LKO liver homogenates measured …
Figure 6—figure supplement 1
Mice lacking hepatic Bcl6 have improved insulin sensitivity after high-fat diet.
(A–B) Overnight fasted serum (A) glucose and (B) insulin in Bcl6fl/fl and Bcl6LKO mice after 17 weeks of high-fat diet. N = 6–9 per group. (C) Western blots showing phosphorylated AKT (pAKT) and …
Author response image 1
Loss of BCL6 does not enhance STAT5 recruitment to shared BCL6-PPARα enhancers.
(A) STAT5 ChIP-seq tag density in Ppara-/- and Ppara-/-;Bcl6LKO livers at BCL6-PPARα peaks near rescued upregulated and downregulated fasting genes. ChIP was performed in biological triplicate. (B) …
Author response image 2
Rescued genes are enriched for nearby PPARδ binding and motifs for distinct transcription factors compared to non-rescued genes.
(A) PPARδ ChIP-seq tag density in Ppara-/- and Ppara-/-;Bcl6LKO livers at shared BCL6-PPARα peaks near up- and downregulated non-rescued fasting genes. (B) Log2 ratio of PPARδ ChIP-seq tag density …
Author response image 3
Overexpression of BCL6 downregulates fatty acid oxidation genes.
(A) qPCR analysis of Bcl6 in control and Bcl6LTG livers. N = 3-7 per group. (B) Western blot of BCL6 protein in control and Bcl6LTG livers. Densitometric quantification is shown normalized to actin …
Author response image 4
BCL6 suppress fatty acid oxidation genes while SMRT/NCoR and HDAC3 suppress lipogenic genes.
(A) Comparison of log fold change (LFC) at significant (p-value <0.05) differentially expressed genes in both Bcl6LKO and Ncor/Smrt double knockout (DKO) livers over control samples. (B) Pathway …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional Information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Bcl6fl/fl | PMID 30566857 | ||
| Genetic reagent (Mus musculus) | Albumin-cre | Jackson Laboratory | Stock #003574 | |
| Genetic reagent (Mus musculus) | Ppara-/- | Jackson Laboratory | Stock #008154 | |
| Genetic reagent (Mus musculus) | Ppardfl/fl | Jackson Laboratory | Stock #005897 | |
| Antibody | anti-BCL6 (guinea pig polyclonal) | PMID 30566857 | custom polyclonal 7.5 μg per IP | |
| Antibody | anti-PPARδ (guinea pig polyclonal) | PMID 28467934 | custom polyclonal 7.5 μg per IP | |
| Antibody | anti-SMRT (guinea pig polyclonal) | PMID 22465074 | custom polyclonal 7.5 μg per IP | |
| Antibody | anti-NCoR (guinea pig polyclonal) | PMID 22465074 | custom polyclonal 7.5 μg per IP | |
| Antibody | anti-HDAC3 (rabbit polyclonal) | Santa Cruz | Cat. #: sc-11417x | 5 μg per IP |
| Antibody | anti-H3K27ac (rabbit polyclonal) | Active Motif | Cat. #: 39133 | 5 μg per IP |
| Antibody | anti-pAKT (rabbit monoclonal) | Cell Signaling | Cat. #: 4060 s | (1:1000) |
| Antibody | anti-panAKT (rabbit monoclonal) | Cell Signaling | Cat. #: 4691 s | (1:1000) |
| Antibody | anti-BCL6 (mouse monoclonal) | Santa Cruz | Cat. #: sc7388 | (1:200) |
| Antibody | anti-β-Actin (mouse monoclonal) | Sigma-Aldrich | Cat. #: A1978 | 1:1000) |
| Antibody | anti-PPARα (rabbit polyclonal) | Santa Cruz | Cat. #: sc-9000x | 7.5 μg per IP; WB: (1:500) |
| Antibody | Peroxidase AffiniPure Goat Anti-Mouse IgG | Jackson ImmunoResearch | Cat. #: 115-035-174 | (1:20,000) |
| Antibody | Peroxidase IgG Fraction Monoclonal Mouse Anti-Rabbit IgG | Jackson ImmunoResearch | Cat. #: 211-032-171 | (1:20,000) |
| Antibody | Rabit Anti-guinea pig IgG H and L | Abcam | Cat. #: ab6698 | |
| Chemical compound, drug | DSG Crosslinker | ProteoChem | Cat. #: c1104 | |
| Chemical compound, drug | Formaldehyde, 16%, methanol-free, Ultra Pure | Polysciences, Inc | Cat. #: 18814–20 | |
| Chemical compound, drug | RNAlater Stabilization Solution | ThermoFisher Scientific | Cat. #: AM7020 | |
| Chemical compound, drug | TRIzol Reagent | ThermoFisher Scientific | Cat. #: 15596018 | |
| Chemical compound, drug | Poloxamer 407 | Sigma-Aldrich | Cat. #: 16758 | |
| Chemical compound, drug | cOmplete Ultra Tablets, EDTA-free | Sigma-Aldrich | Cat. #: 5892953001 | |
| Chemical compound, drug | Deuterium oxide | Sigma-Aldrich | Cat. #: 151882 | |
| Chemical compound, drug | Sodium Palmitate | Sigma-Aldrich | Cat. #: P9767 | |
| Chemical compound, drug | Palmitic Acid, [1–14C] | MP Biomedicals | Cat. #: 12195 | |
| Chemical compound, drug | BODIPY 500/510 C1, C12 | Invitrogen | Cat. #: D3823 | |
| Chemical compound, drug | Humulin R | Lilly | NDC 0002-8215-01 | |
| Commercial assay or kit | Dynabeads M-280 Sheep Anti-Rabbit IgG | Invitrogen | Cat. #: 11204D | |
| Commercial assay or kit | Dynabeads M-280 Tosylactivated | Invitrogen | Cat. #: 14204 | |
| Commercial assay or kit | Protein A Agarose/Salmon Sperm DNA | Millipore | Cat. #: 16–157 | |
| Commercial assay or kit | iScript cDNA Synthesis Kit | BioRad | Cat. #: 1708891 | |
| Commercial assay or kit | iTaq Universal SYBR Green | BioRad | Cat. #: 1725124 | |
| Commercial assay or kit | Infinity Triglyceride Assay Kit | ThermoFisher Scientific | Cat. #: TR22421 | |
| Commercial assay or kit | β-Hydroxybutyrate (Ketone Body) Colorimetric Assay Kit | Cayman Chemical | Cat. #: 700190 | |
| Commercial assay or kit | Ultra Sensitive Mouse Insulin ELISA Kit | Crystal Chem | Cat. #: 90080 | |
| Commercial assay or kit | Glucose Colorimetric /Fluorometric Assay Kit | BioVision | Cat. #: K606 | |
| Commercial assay or kit | Microvette CB 300 K2E | Sarstedt | Cat. #: 16.444 | |
| Commercial assay or kit | MemCode Reversible Protein Stain Kit | ThermoFisher Scientific | Cat. #: 24585 | |
| Commercial assay or kit | Whatman qualitative filter paper | Sigma-Aldrich | Cat. #: WHA1003055 | |
| Commercial assay or kit | KAPA Hyper Prep Library Prep Kit | Kapa Biosystems | Cat. #: KK8504 | |
| Commercial assay or kit | KAPA Stranded RNA-seq Kit with RiboErase | Kapa Biosystems | Cat. #: KK8483 | |
| Commercial assay or kit | NextSeq 500/550 High Output Kit v2.5 (75 cycles) | Illumina | Cat. #: 20024906 | |
| Other, research diet (45% kcal from fat) | HFD | Research Diets, Inc | Stock #D12451 |
Additional files
-
Supplementary file 1
Quantitative PCR primers for chromatin immunoprecipitation.
- https://doi.org/10.7554/eLife.43922.019
-
Supplementary file 2
Quantitative PCR primers for gene expression.
- https://doi.org/10.7554/eLife.43922.020
-
Transparent reporting form
- https://doi.org/10.7554/eLife.43922.021
