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Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis

Nature Metabolism volume 6, pages 304–322 (2024)Cite this article

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Abstract

Skeletal muscle is dynamically controlled by the balance of protein synthesis and degradation. Here we discover an unexpected function for the transcriptional repressor B cell lymphoma 6 (BCL6) in muscle proteostasis and strength in mice. Skeletal muscle-specific Bcl6 ablation in utero or in adult mice results in over 30% decreased muscle mass and force production due to reduced protein synthesis and increased autophagy, while it promotes a shift to a slower myosin heavy chain fibre profile. Ribosome profiling reveals reduced overall translation efficiency in Bcl6-ablated muscles. Mechanistically, tandem chromatin immunoprecipitation, transcriptomic and translational analyses identify direct BCL6 repression of eukaryotic translation initiation factor 4E-binding protein 1 (Eif4ebp1) and activation of insulin-like growth factor 1 (Igf1) and androgen receptor (Ar). Together, these results uncover a bifunctional role for BCL6 in the transcriptional and translational control of muscle proteostasis.

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Fig. 1: Bcl6 controls the development of skeletal muscle mass.
Fig. 2: Rapid muscle loss following muscle Bcl6 deletion in adult mice.
Fig. 3: Bcl6 controls an oxidative to glycolytic shift in myofibres.
Fig. 4: Bcl6 enhances skeletal muscle force production.
Fig. 5: BCL6 directly controls the expression of anabolic regulators.
Fig. 6: Loss of Bcl6 reduces the rate of muscle protein synthesis.
Fig. 7: BCL6 controls translation in skeletal muscle.
Fig. 8: Model for BCL6-mediated control of skeletal muscle proteostasis.

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Data availability

All sequencing data generated in this study are publicly available in the GEO repository SuperSeries GSE190956. We also utilized publicly accessible RNA-seq and microarray data from GEO repository entries GSE65936, GSE112204, GSE107470 and GSE14710, as well as the mm10 reference assembly for the mouse genome. Source data are provided with this paper.

Code availability

No custom code was used.

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Acknowledgements

We thank N. Hay (University of Illinois Chicago), L. Goodyear (Joslin Diabetes Center, Harvard), C. He (Northwestern University), J. Bass (Northwestern University), D. Chakravarti (Northwestern University) and J. Madsen (University of Southern Denmark) for helpful advice and discussion. We thank Northwestern University’s NU-Seq Core, Comprehensive Metabolic Core and Mouse Histology and Phenotyping Laboratory (supported by NCI P30-CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center) for services. This research was supported by computational resources provided by the Quest high-performance computing facility at Northwestern University. Figures 1d, 2a, 6c and 8 were created with BioRender.com. This work was funded by Merit Review Award #I01BX004898 from the United States (U.S.) Department of Veterans Affairs (to G.D.B.), National Institutes of Health (NIH) grants R01DK108987 (to G.D.B.) and T32 GM008061 (to E.F.) and Pfizer Aspire award WI183795 (to G.D.B.). M.Q. is supported by NIH grants R56HL158531, R01HL166356-01, R03DK130908-01A1 and R01AG078174-01 and CCHMC grants RIP, CCRF Endowed Scholarship and HI Translational Funds. D.A. is supported by the Searle Scholar Program (grant no. SSP-2023-102). E.M.M. is supported by NIH grants R01HL061322 and R01NS047726. The article contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

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Authors and Affiliations

  1. Division of Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Krithika Ramachandran, Christopher R. Futtner, Meredith A. Sommars, Yasuhiro Omura, Ellen Fruzyna, Janice C. Wang, Nathan J. Waldeck, Madhavi D. Senagolage, Carmen G. Telles & Grant D. Barish

  2. Jesse Brown VA Medical Center, Chicago, IL, USA

    Christopher R. Futtner, Yasuhiro Omura & Grant D. Barish

  3. Center for Genetic Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Mattia Quattrocelli, Alexis R. Demonbreun & Elizabeth M. McNally

  4. Division of Molecular Cardiovascular Biology, Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Mattia Quattrocelli

  5. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Mattia Quattrocelli

  6. Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, USA

    Erin Prendergast & Ilya R. Bederman

  7. Department of Mechanical, Chemical, and Materials Engineering, University of Cagliari, Cagliari, Italy

    Nicola Lai

  8. Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Daniel Arango

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  1. Krithika Ramachandran
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Contributions

Experiments were designed by G.D.B., K.R., C.R.F., A.R.D., I.R.B. and E.M.M. Experiments were performed by K.R., C.R.F., M.A.S., M.Q., Y.O., E.F., J.C.W., M.D.S., C.G.T., E.P. and I.R.B. Data analysis was done by K.R., C.R.F., M.A.S., E.F., G.D.B., N.L., I.R.B., D.A., N.J.W. and M.Q. The paper was written by K.R., C.R.F., D.A. and G.D.B. with input from the other authors.

Corresponding author

Correspondence to Grant D. Barish.

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Nature Metabolism thanks Stuart Phillips, Marco Sandri and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Bcl6 controls the development of skeletal muscle mass.

(a) qPCR expression of Bcl6 and myogenic markers in primary myoblasts and myotubes relative to the housekeeping gene Rn45s (n = 4 per group). Myoblast vs myotube P-value for Bcl6 = 0.0124, Mb = 0.0153, Mef2c = 0.0146 by unpaired, two-tailed t-test with Welch correction. (b) Western blot and densitometry of BCL6 and total (Memcode) protein in primary myoblasts and myotubes (n = 4/group). Myoblast vs myotube P = 0.0036 by unpaired, two-tailed t-test with Welch correction. (c) Western blot and densitometry of BCL6 and Actin protein in heart from Bcl6fl/fl and Bcl6MKO mice (n = 3/group). (d) Total body weight, fat, and lean mass in n = 7 Bcl6fl/fl and n = 10 Bcl6MKO female mice at 8 weeks of age. Bcl6fl/fl vs Bcl6MKO lean mass P = 0.0184 by unpaired, two-tailed t-test with Welch correction. (e) Tissue weights of quadriceps (quad), gastrocnemius (gastroc), tibialis anterior (TA), extensor digitorum longus (EDL) and soleus from female Bcl6fl/fl and Bcl6MKO mice at 16 weeks of age (n = 7/group). Unpaired, two-tailed t-test with Welch correction was performed and all p-values can be found in the source data file. All data are represented as mean + SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Source data

Extended Data Fig. 2 Inducible deletion of Bcl6 in adult mice results in muscle loss.

(a, b) Serial measurements of total body, lean, and fat mass in (a) n = 6 Cre+oil, n = 9 Cre-tmx and n = 9 Bcl6i-MKO male and (b) n = 6 Cre+oil, n = 8 Cre-tmx and n = 8 Bcl6i-MKO female mice starting at 8 weeks of age prior to (pre) and up to 12 weeks after treatment. Two-way ANOVA with Tukey’s multiple comparisons test was performed. (c, d) Weights of quadriceps (quad), gastrocnemius (gastroc), tibialis anterior (TA), extensor digitorum longus (EDL) and soleus from Cre+oil, Cre-tmx and Bcl6i-MKO (c) male and (d) female mice 12 weeks after treatment (n = same as (a,b)). One-way ANOVA with Dunnett’s multiple comparisons test was performed. (e) Tissue weights in n = 4 ad lib chow fed and n = 5 48-hour fasted C57BL/6 male mice. (f) qPCR of Bcl6 normalized to 36b4 in muscles from fed and 48-hour fasted mice (n = 3/group). (g) Western blot and densitometry of BCL6 and Actin in n = 4 fed and n = 5 from 48 hour fasted mouse quadriceps. Unpaired, two-tailed t-test with Welch correction was performed in (e-g). (h) Bcl6 levels in published cancer-associated cachexia syndrome (CACS) models. GEO accession numbers are listed. Control vs C26 colon cancer P = 0.0008 by unpaired, two-tailed t-test with Welch correction (n = 3/group). Boxplot shows min to max values with line at the mean. (i) Bcl6 expression in C2C12 myotubes exposed to 33% colon-26 carcinoma (C26) conditioned versus control media for 8 hours (n = 3/group; unpaired, two-tailed t-test with Welch correction was performed) and corresponding protein blots after 8 or 24 hours. (j) Bcl6 expression in C2C12 myotubes treated with cytokines (n = 3/group), growth hormone (n = 2/group) and glucocorticoids (n = 3/group) for 8 hours. Unpaired, two-tailed t-test with Welch correction and one-way ANOVA with Dunnett’s multiple comparisons were performed. Exact p-values can be found in the source data file. All data in (a-g) and (i-j) are represented as mean + SEM. δ p < 0.05, δδ p < 0.01 for Cre+oil vs Cre-tmx; # p < 0.05, ## p < 0.01, ### p < 0.001 for Cre+oil vs Bcl6i-MKO; * p < 0.05, ** p < 0.01, *** p < 0.001 for Cre-tmx vs Bcl6i-MKO or fed vs fasted or vehicle vs treated myotubes.

Source data

Extended Data Fig. 3 Bcl6 controls an oxidative to glycolytic shift in myofibres.

(a) Representative fluorescent antibody-stained images of four myosin heavy chain isoforms in quadriceps from Cre- tmx and Bcl6i-MKO mice (left). MYH7 (type 1 fiber) is blue, MYH2 (type 2a fiber) is green, MYH1 (type 2x fiber) is black, and MYH4 (type 2b fiber) is red. Quantification of fiber types (right) expressed as percentage of total (n = 3/group). Cre- tmx vs Bcl6i-MKO type 2a fiber P = 0.0374 by unpaired, two-tailed t-test with Welch correction. (b) qPCR DNA ratios of mitochondrial genes Cox1 and Cytb over nuclear genes Gcg and Hbb in quadriceps from Bcl6fl/fl and Bcl6MKO males (n = 7/group). (c) Western blot and densitometry of VDAC and total (Licor) protein in quadriceps from n = 6 Bcl6fl/fl and n = 7 Bcl6MKO males. (d) Protein levels of mitochondrial complex subunits and total (Memcode) protein in quadriceps from Bcl6fl/fl and Bcl6MKO mice. Western blot (left) and protein densitometry (right) are shown. (n = 4/group) Bcl6fl/fl vs Bcl6MKO complex II P = 0.0139 by unpaired, two-tailed t-test with Welch correction. (e, f) Cross-sectional areas of specific fiber types determined from fluorescent antibody-stained sections in quadriceps from (e) Bcl6fl/fl and Bcl6MKO mice (n = 3/group) and (f) Cre- tmx and Bcl6i-MKO mice (n = 3/group). Type 2b fiber in Bcl6fl/fl vs Bcl6MKO P = 0.003 and in Cre− tmx vs Bcl6i-MKO P = 0.0385 by unpaired, two-tailed t-test with Welch correction. All data are represented as mean + SEM. * p < 0.05, ** p < 0.01.

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Extended Data Fig. 4 Muscle loss due to Bcl6 deletion is associated with reduced muscle function.

(a, b) Absolute (left) and lean body mass normalized (right) forelimb grip strength measured in (a) 13-week old n = 7 Bcl6fl/fl and n = 10 Bcl6MKO female mice and (b) n = 6 Cre+ oil, n = 8 Cre- tmx and n = 8 Bcl6i-MKO female mice twelve weeks after tamoxifen or oil treatment. Whiskers in the boxplot show min to max values with line at the median and box representing the first to third quartile values. (c) Maximum tetanic force for the tibialis anterior (TA) muscle from 13-week old n = 3 Bcl6fl/fl and n = 2 Bcl6MKO female mice (P-value for maximum force = 0.0311 by unpaired, two-tailed t-test with Welch correction). (d, e) Distance and time run on a treadmill to exhaustion in (d) 13-week old n = 15 Bcl6fl/fl and n = 14 Bcl6MKO male mice and (e) Cre- tmx and Bcl6i-MKO female mice twelve weeks after tamoxifen treatment (n = 12/group). (f, g) Light phase, dark phase and total activity in (f) 13-week old Bcl6fl/fl and Bcl6MKO male mice (n = 5/group) and (g) Cre- tmx and Bcl6i-MKO male mice 1.5 weeks after tamoxifen treatment (n = 5/group). All data in (c-g) are represented as mean + SEM. * p< 0.05.

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Extended Data Fig. 5 BCL6 directly controls the expression of anabolic regulators.

(a) Principal component analysis of RNA-seq from n = 5 Bcl6i-MKO and n = 4 tamoxifen-treated controls (Cre- tmx) or n = 4 corn oil-treated controls (Cre+ oil). (b) Venn diagram depicting overlap between differentially expressed (DE) genes in muscle Stat5 knockout (Stat5MKO) mice (GEO series GSE14710) compared to muscle Bcl6i-MKO mice. (c) Quadrant plot of log2 fold change in gene expression in Stat5MKO/controls versus Bcl6i-MKO/Cre- tmx controls. Spearman correlation coefficient rs = 0.03. (d) Scatterplot (left) of normalized H3K27ac tag counts and boxplot (right) showing quantification of H3K27ac tag densities within 1kb of BCL6 peaks in Cre- tmx and Bcl6i-MKO quadriceps (n = 3/group; P = 3.5e−10 by unpaired, two-tailed Mann Whitney test). Whiskers in the boxplot show min to max values with line at the median and box representing the first to third quartile values. (e) Receiver operator curve (ROC) of the association between differential expression in Bcl6-ablated muscle and BCL6 binding peaks. (f) UCSC browser tracks of RNAseq in quadriceps from Cre+ oil, Cre- tmx and Bcl6i-MKO mice, H3K27ac ChIP-seq in quadriceps from Cre- tmx and Bcl6i-MKO mice 1 week after oil or tamoxifen treatment, nascent RNA-seq in quadriceps from mice treated with DMSO or FX1 for 3 hours, and BCL6 ChIP-seq track in quadriceps from C57BL/6 animals along the Mstn, Ar, and Smox genes. (g) qPCR expression of Bcl6, Akt1, Eif4ebp1, Igf1 and Ar in quadriceps from males (n = 5/group) and females (n = 4 Bcl6fl/fl and n = 5 Bcl6MKO mice). Two-way ANOVA followed by Tukey’s multiple comparisons test was performed and all individual p-values can be found in the source data file. (h, i) Western blot and densitometry of (h) BCL6 and actin (i) 4EBP1 and total protein (licor) in quadriceps from male and female n = 3 Bcl6fl/fl and n = 4 Bcl6MKO mice. For BCL6, P(male Bcl6fl/fl vs Bcl6MKO) = 0.005, P(male Bcl6fl/fl vs female Bcl6MKO) = 0.005, P(male Bcl6MKO vs female Bcl6fl/fl) = 0.0299, P(female Bcl6fl/fl vs Bcl6MKO) = 0.0305 and for 4EBP1, P(male Bcl6fl/fl vs Bcl6MKO) = 0.0034, P(male Bcl6fl/fl vs female Bcl6MKO) = 0.0199 by two-way ANOVA followed by Tukey’s multiple comparisons. All data in (g-i) are represented as mean + SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001.

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Extended Data Fig. 6 Loss of Bcl6 reduces the rate of muscle protein synthesis.

(a) Heatmap of ribosome protected fragment (RPF) sizes in sequencing libraries of tamoxifen-treated Bcl6i-MKOmRiboTag and mRiboTag mice. (b) Pie chart distribution of mapped RPF reads. (c) Empirical cumulative distribution frequency (Ecdf) plots of the translation efficiencies (natural logarithm of TE) in quadriceps for all genes. Bcl6i-MKO mRiboTag vs mRiboTag P = 2.2e-16 by two-sided Kolmogorov-Smirnov testing (d) Metagene distribution plot showing normalized ribo-seq RPF reads from the TSS to TTS for all genes (left) and genes with increased TE (right). (e) Western blot and densitometry of ubiquitin protein and actin in quadriceps from Cre- tmx and Bcl6i-MKO males 1.5 weeks after tamoxifen treatment (n = 4/group). (f) EIF4E co-immunoprecipitation and western blots for EIF4G (top) and 4EBP1 (bottom) in gastrocnemius from Cre- tmx and Bcl6i-MKO males 1 week after tamoxifen treatment (n = 2/group). (g) Western blot and densitometry of p4EBP1, Non-phospho 4EBP1, Total 4EBP1, and total (Memcode) protein in quadriceps from 10 week old Bcl6fl/fl and Bcl6MKO males (n = 4/group). P(Bcl6fl/fl vs Bcl6MKO) for p4EBP1 = 0.0009, total 4EBP1 = 0.0004, and nonP 4EBP1 = 0.0002 by unpaired, two-tailed t-test with Welch correction. (h) qPCR of protein synthesis and degradation regulators in n = 4 Cre+ oil, n = 4 Cre- tmx and n = 5 Bcl6i-MKO male mice 7 days after treatment. One-way ANOVA with Dunnett’s multiple comparisons test showed that P(Cre+ oil vs Bcl6i-MKO) for Eif4ebp1 = 0.0086, Mstn = 0.0018, Igf1 = 0.0332 and Ar = 0.0420; P(Cre- tmx vs Bcl6i-MKO) for Eif4ebp1 = 0.0163, Mstn = 0.0011, Igf1 = 0.0140 and Ar = 0.0330. (i, j) Analysis of Eif4ebp1 knockdown efficiency in muscles (n = 3 mice/group for scramble and Eif4ebp1 shRNA1 and n = 2 mice/group for Eif4ebp1 shRNA2). (i) qPCR of Eif4ebp1 and (j) western blot and densitometry of 4EBP1 in Cre- tmx and Bcl6i-MKO male mice transduced with scramble (black border), Eif4ebp1 shRNA1 (green border), or Eif4ebp1 shRNA2 (yellow border) and treated with tamoxifen one week after viral infection. Tissues were analyzed eight weeks later. All bar graph data in (e, g-j) are represented as mean + SEM. # p < 0.05, ## p < 0.01 for Cre+oil vs Bcl6i-MKO and * p < 0.05, ** p < 0.01, *** p < 0.001 for Cre-tmx vs Bcl6i-MKO and Bcl6fl/fl vs Bcl6MKO.

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Extended Data Fig. 7 Chronic Bcl6 loss evokes compensatory signaling to maintain muscle mass.

(a) SUnSET analysis of puromycin incorporation into quadriceps (top) in Cre- tmx and Bcl6i-MKO mice 12 weeks after tamoxifen treatment. Puromycin labeling relative to total protein (Licor) by densitometry (bottom) (n = 3/group; P = 0.0017 by unpaired, two-tailed t-test with Welch correction). (b) Western blot and densitometry in quadriceps from Cre- tmx and Bcl6i-MKO males 15 weeks after tamoxifen treatment for LC3, 3 days after colchicine or vehicle treatment. (n = 3/group; Two-way ANOVA followed by Tukey’s multiple comparisons test was performed. P (Veh-Cre- tmx vs Col-Cre- tmx) = 0.0174; P (Veh-Cre- tmx vs Col-Bcl6i-MKO) = 0.0005; P (Veh-Bcl6i-MKO vs Col-Bcl6i-MKO) = 0.0083.) Flux is the difference in average LC3-II levels in colchicine minus vehicle (bottom right). (c) Western blot and densitometry of ubiquitin and total (Licor) protein in quadriceps from Cre- tmx and Bcl6i-MKO males 12 weeks after tamoxifen treatment (n = 4/group). (d) Quadrant plot of log2 fold change in gene expression in Bcl6i-MKO/Cre- tmx 12 weeks after tamoxifen treatment (chronic) versus Bcl6i-MKO/Cre- tmx 1 week after tamoxifen treatment (acute). (e) Ontology analysis of differential genes unique in chronic Bcl6 deletion. Cumulative hypergeometric statistical testing was used. (f) Log p-value versus Pearson correlation plotted for causal transcription factors identified by IMAGE that were unique to either acute or chronic Bcl6 deletion. Pink = high, green = medium, and gray = low confidence factors. Factors in acute dataset are shown as circles and factors in chronic are squares. (g) Causal transcription factors with opposing Pearson correlations to gene expression in acute versus chronic Bcl6 deletion. Acute = green circle, chronic = orange square. (h-l) Western blot and densitometry of (h) pSMAD1/5/8, Total SMAD1, and total (Licor) protein, (i) pFOXO1, Total FOXO1, and Actin, (j) p4EBP1, Non-phospho 4EBP1, Total 4EBP1, and Actin, (k) pAKT, Total AKT, and Actin and (l) pS6, Total S6, and Actin in quadriceps from Cre- tmx and Bcl6i-MKO males 12 week after tamoxifen treatment (n = 4/group). Unpaired, two-tailed t-test with Welch correction and all individual p-values can be found in the source data file. All bar graph data in (a-c, h-l) are represented as mean + SEM. * p < 0.05 and ** p < 0.01.

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Supplementary information

Supplementary Information

Supplementary Table 1. List of qPCR primers.

Reporting Summary

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Ramachandran, K., Futtner, C.R., Sommars, M.A. et al. Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis. Nat Metab 6, 304–322 (2024). https://doi.org/10.1038/s42255-024-00983-3

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