Dynamic repression by BCL6 controls the genome-wide liver response to fasting and steatosis

  1. Meredith A Sommars
  2. Krithika Ramachandran
  3. Madhavi D Senagolage
  4. Christopher R Futtner
  5. Derrik M Germain
  6. Amanda L Allred
  7. Yasuhiro Omura
  8. Ilya R Bederman
  9. Grant D Barish  Is a corresponding author
  1. Northwestern University, United States
  2. Case Western Reserve University, United States
  3. Jesse Brown VA Medical Center, United States
10 figures, 1 table and 3 additional files

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 …

https://doi.org/10.7554/eLife.43922.003
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 …

https://doi.org/10.7554/eLife.43922.004
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 …

https://doi.org/10.7554/eLife.43922.005
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 …

https://doi.org/10.7554/eLife.43922.006
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 …

https://doi.org/10.7554/eLife.43922.007
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 …

https://doi.org/10.7554/eLife.43922.008
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 …

https://doi.org/10.7554/eLife.43922.009
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 …

https://doi.org/10.7554/eLife.43922.010
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 …

https://doi.org/10.7554/eLife.43922.011
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 …

https://doi.org/10.7554/eLife.43922.012
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) …

https://doi.org/10.7554/eLife.43922.013
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 …

https://doi.org/10.7554/eLife.43922.014
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 …

https://doi.org/10.7554/eLife.43922.015
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-/-;…

https://doi.org/10.7554/eLife.43922.016
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 …

https://doi.org/10.7554/eLife.43922.017
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 …

https://doi.org/10.7554/eLife.43922.018
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) …

https://doi.org/10.7554/eLife.43922.022
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 …

https://doi.org/10.7554/eLife.43922.023
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 …

https://doi.org/10.7554/eLife.43922.024
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 …

https://doi.org/10.7554/eLife.43922.025

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
Information
Genetic reagent
(Mus musculus)
Bcl6fl/flPMID 30566857
Genetic reagent
(Mus musculus)
Albumin-creJackson LaboratoryStock #003574
Genetic reagent
(Mus musculus)
Ppara-/-Jackson LaboratoryStock #008154
Genetic reagent
(Mus musculus)
Ppardfl/flJackson LaboratoryStock #005897
Antibodyanti-BCL6 (guinea
pig polyclonal)
PMID 30566857custom polyclonal
7.5 μg per IP
Antibodyanti-PPARδ (guinea
pig polyclonal)
PMID 28467934custom polyclonal
7.5 μg per IP
Antibodyanti-SMRT (guinea
pig polyclonal)
PMID 22465074custom polyclonal
7.5 μg per IP
Antibodyanti-NCoR (guinea
pig polyclonal)
PMID 22465074custom polyclonal
7.5 μg per IP
Antibodyanti-HDAC3
(rabbit polyclonal)
Santa CruzCat. #: sc-11417x5 μg per IP
Antibodyanti-H3K27ac
(rabbit polyclonal)
Active MotifCat. #: 391335 μg per IP
Antibodyanti-pAKT
(rabbit monoclonal)
Cell SignalingCat. #: 4060 s(1:1000)
Antibodyanti-panAKT
(rabbit monoclonal)
Cell SignalingCat. #: 4691 s(1:1000)
Antibodyanti-BCL6
(mouse monoclonal)
Santa CruzCat. #: sc7388(1:200)
Antibodyanti-β-Actin
(mouse monoclonal)
Sigma-AldrichCat. #: A19781:1000)
Antibodyanti-PPARα
(rabbit polyclonal)
Santa CruzCat. #: sc-9000x7.5 μg per IP; WB: (1:500)
AntibodyPeroxidase AffiniPure
Goat Anti-Mouse IgG
Jackson ImmunoResearchCat. #: 115-035-174(1:20,000)
AntibodyPeroxidase IgG Fraction
Monoclonal Mouse
Anti-Rabbit IgG
Jackson ImmunoResearchCat. #: 211-032-171(1:20,000)
AntibodyRabit Anti-guinea
pig IgG H and L
AbcamCat. #: ab6698
Chemical compound, drugDSG CrosslinkerProteoChemCat. #: c1104
Chemical compound, drugFormaldehyde, 16%,
methanol-free, Ultra Pure
Polysciences, IncCat. #: 18814–20
Chemical compound, drugRNAlater Stabilization SolutionThermoFisher ScientificCat. #: AM7020
Chemical compound, drugTRIzol ReagentThermoFisher ScientificCat. #: 15596018
Chemical compound, drugPoloxamer 407Sigma-AldrichCat. #: 16758
Chemical compound, drugcOmplete Ultra Tablets,
EDTA-free
Sigma-AldrichCat. #: 5892953001
Chemical compound, drugDeuterium oxideSigma-AldrichCat. #: 151882
Chemical compound, drugSodium PalmitateSigma-AldrichCat. #: P9767
Chemical compound, drugPalmitic Acid, [1–14C]MP BiomedicalsCat. #: 12195
Chemical compound, drugBODIPY 500/510 C1, C12InvitrogenCat. #: D3823
Chemical compound, drugHumulin RLillyNDC 0002-8215-01
Commercial assay or kitDynabeads M-280
Sheep Anti-Rabbit IgG
InvitrogenCat. #: 11204D
Commercial assay or kitDynabeads M-280
Tosylactivated
InvitrogenCat. #: 14204
Commercial assay or kitProtein A Agarose/Salmon
Sperm DNA
MilliporeCat. #: 16–157
Commercial assay or kitiScript cDNA Synthesis KitBioRadCat. #: 1708891
Commercial assay or kitiTaq Universal SYBR GreenBioRadCat. #: 1725124
Commercial assay or kitInfinity Triglyceride Assay KitThermoFisher ScientificCat. #: TR22421
Commercial assay or kitβ-Hydroxybutyrate
(Ketone Body) Colorimetric
Assay Kit
Cayman ChemicalCat. #: 700190
Commercial assay or kitUltra Sensitive Mouse
Insulin ELISA Kit
Crystal ChemCat. #: 90080
Commercial assay or kitGlucose Colorimetric
/Fluorometric Assay Kit
BioVisionCat. #: K606
Commercial assay or kitMicrovette CB 300 K2ESarstedtCat. #: 16.444
Commercial assay or kitMemCode Reversible
Protein Stain Kit
ThermoFisher ScientificCat. #: 24585
Commercial assay or kitWhatman qualitative filter paperSigma-AldrichCat. #: WHA1003055
Commercial assay or kitKAPA Hyper Prep Library Prep KitKapa BiosystemsCat. #: KK8504
Commercial assay or kitKAPA Stranded
RNA-seq Kit with RiboErase
Kapa BiosystemsCat. #: KK8483
Commercial assay or kitNextSeq 500/550 High
Output Kit v2.5 (75 cycles)
IlluminaCat. #: 20024906
Other, research diet
(45% kcal from fat)
HFDResearch Diets, IncStock #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

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