Distinct signaling and transcriptional pathways regulate peri … · Distinct signaling and...

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Distinct signaling and transcriptional pathways regulate peri-weaning development and cold-induced recruitment of beige adipocytes Yixuan Wu a , Melissa A. Kinnebrew a,b , Vassily I. Kutyavin a , and Ajay Chawla a,c,d,1,2 a Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0795; b Department of Dermatology, University of California, San Francisco, CA 94115; c Department of Physiology, University of California, San Francisco, CA 94143-0795; and d Department of Medicine, University of California, San Francisco, CA 94143-0795 Edited by Christian Wolfrum, Swiss Federal Institute of Technology in Zurich, Zurich Schwerzenbach, Switzerland, and accepted by Editorial Board Member David J. Mangelsdorf February 6, 2020 (received for review November 20, 2019) Adipose tissue provides a defense against starvation and environ- mental cold. These dichotomous functions are performed by three distinct cell types: energy-storing white adipocytes, and thermogenic beige and brown adipocytes. Previous studies have demonstrated that exposure to environmental cold stimulates the recruitment of beige adipocytes in the white adipose tissue (WAT) of mice and humans, a process that has been extensively investigated. However, beige adipose tissue also develops during the peri-weaning period in mice, a developmental program that remains poorly understood. Here, we address this gap in our knowledge using genetic, imaging, physiologic, and genomic approaches. We find that, unlike cold- induced recruitment in adult animals, peri-weaning development of beige adipocytes occurs in a temperature- and sympathetic nerve- independent manner. Instead, the transcription factor B cell leukemia/lymphoma 6 (BCL6) acts in a cell-autonomous manner to regulate the commitment but not the maintenance phase of beige adipogenesis. Genome-wide RNA-sequencing (seq) studies reveal that BCL6 regulates a core set of genes involved in fatty acid oxidation and mitochondrial uncoupling, which are necessary for development of functional beige adipocytes. Together, our find- ings demonstrate that distinct transcriptional and signaling mech- anisms control peri-weaning development and cold-induced recruitment of beige adipocytes in mammals. BCL6 | sympathetic nervous system | thermogenesis | development | metabolism I n mammals, adipose tissue provides a defense against starva- tion and environmental cold (1). These distinct functions of adipose tissue are performed by specialized adipocytes that can store or dissipate energy. For example, white adipocytes, which contain a single large lipid droplet, are a primary site of energy storage in mammals, whereas multilocular beige and brown ad- ipocytes dissipate energy through uncoupled respiration (14). Consistent with their distinct functions in energy metabolism, loss of white adipocytes, as in lipodystrophy, leads to ectopic de- position of lipids in liver and other organs, whereas loss of beige and brown adipocytes compromises organismal defense against environmental cold (1). Beige and brown adipocytes are considered to be two distinct cell types based on their developmental origin, transcriptome, and epigenome (1, 4, 5). Myogenic precursors give rise to brown adipocytes during embryogenesis in a temperature-independent manner, whereas environmental cold is the primary stimulus for the recruitment of mesenchyme-derived beige adipocytes in the white adipose tissue (WAT) of adult animals. A focus on cold- induced recruitment of beige adipocytes has identified a large number of signaling and transcriptional pathways that control its biogenesis in adult animals (4). In nearly all of these cases, beige- promoting factors directly or indirectly modulate the adrenergic activity of sympathetic nerves, which densely innervate the in- guinal WAT (iWAT) of mice (6). Although less well appreciated, iWAT also undergoes beige adipogenesis during the peri-weaning period (712). However, the importance of sympathetic stimulation in acquisition of this phenotype remains controversial. For example, Contreras et al. demonstrated that denervation of iWAT impairs commitment to the beige adipocyte lineage, whereas Chabowska-Kita et al. reported presence of UCP1 + cells in the iWAT of thermoneutral mice that presumably lacked sympathetic stimulation (7, 8). These findings raise the possibility that a combination of cell autonomous and nonautonomous mechanisms might regulate beige adipocyte development during the peri-weaning period, a gap in our knowledge that we address here. Results and Discussion Beige Fat Develops during the Peri-Weaning Period in a Temperature- Independent Manner. To identify mechanisms that control beige adi- pogenesis during the peri-weaning period, we employed Adipo-Clear Significance Adipose tissue performs multiple functions in mammals, in- cluding insulation, mechanical tissue protection, and energy balance. These functions of adipose tissue are performed by three distinct cell types: white, beige, and brown adipocytes. While white adipocytes store energy, beige and brown adi- pocytes dissipate energy through mitochondrial respiration. Previous studies have elucidated how environmental cold stimulates recruitment of beige adipocytes in adult animals. However, beige adipogenesis also occurs during the peri- weaning period in mice, a process that is poorly understood. Here, we demonstrate that distinct mechanisms regulate de- velopment and recruitment of beige adipocytes. We find that B cell leukemia/lymphoma 6 (BCL6) controls development of beige adipocytes during the peri-weaning period, whereas sympathetic nerves regulate the recruitment of beige adipo- cytes in cold environments. Author contributions: Y.W., M.A.K., and A.C. designed research; Y.W., M.A.K., and V.I.K. performed research; V.I.K. contributed new reagents/analytic tools; Y.W., M.A.K., V.I.K., and A.C. analyzed data; and Y.W. and A.C. wrote the paper. The authors declare no competing interest. This article is a PNAS Direct Submission. C.W. is a guest editor invited by the Editorial Board. This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). Data deposition: All RNA-seq data are available at the Gene Expression Omnibus (GEO), https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE140259). 1 Present address: Discovery Biology, Merck Research Labs, South San Francisco, CA 94080. 2 To whom correspondence may be addressed. Email: [email protected]. This article contains supporting information online at https://www.pnas.org/lookup/suppl/ doi:10.1073/pnas.1920419117/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1920419117 PNAS Latest Articles | 1 of 7 PHYSIOLOGY Downloaded by guest on April 8, 2020

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Distinct signaling and transcriptional pathwaysregulate peri-weaning development and cold-inducedrecruitment of beige adipocytesYixuan Wua

, Melissa A. Kinnebrewa,b, Vassily I. Kutyavina, and Ajay Chawlaa,c,d,1,2

aCardiovascular Research Institute, University of California, San Francisco, CA 94143-0795; bDepartment of Dermatology, University of California, SanFrancisco, CA 94115; cDepartment of Physiology, University of California, San Francisco, CA 94143-0795; and dDepartment of Medicine, University ofCalifornia, San Francisco, CA 94143-0795

Edited by Christian Wolfrum, Swiss Federal Institute of Technology in Zurich, Zurich Schwerzenbach, Switzerland, and accepted by Editorial Board MemberDavid J. Mangelsdorf February 6, 2020 (received for review November 20, 2019)

Adipose tissue provides a defense against starvation and environ-mental cold. These dichotomous functions are performed by threedistinct cell types: energy-storing white adipocytes, and thermogenicbeige and brown adipocytes. Previous studies have demonstratedthat exposure to environmental cold stimulates the recruitment ofbeige adipocytes in the white adipose tissue (WAT) of mice andhumans, a process that has been extensively investigated. However,beige adipose tissue also develops during the peri-weaning period inmice, a developmental program that remains poorly understood.Here, we address this gap in our knowledge using genetic, imaging,physiologic, and genomic approaches. We find that, unlike cold-induced recruitment in adult animals, peri-weaning development ofbeige adipocytes occurs in a temperature- and sympathetic nerve-independent manner. Instead, the transcription factor B cellleukemia/lymphoma 6 (BCL6) acts in a cell-autonomous mannerto regulate the commitment but not the maintenance phase ofbeige adipogenesis. Genome-wide RNA-sequencing (seq) studiesreveal that BCL6 regulates a core set of genes involved in fatty acidoxidation and mitochondrial uncoupling, which are necessary fordevelopment of functional beige adipocytes. Together, our find-ings demonstrate that distinct transcriptional and signaling mech-anisms control peri-weaning development and cold-inducedrecruitment of beige adipocytes in mammals.

BCL6 | sympathetic nervous system | thermogenesis | development |metabolism

In mammals, adipose tissue provides a defense against starva-tion and environmental cold (1). These distinct functions of

adipose tissue are performed by specialized adipocytes that canstore or dissipate energy. For example, white adipocytes, whichcontain a single large lipid droplet, are a primary site of energystorage in mammals, whereas multilocular beige and brown ad-ipocytes dissipate energy through uncoupled respiration (1–4).Consistent with their distinct functions in energy metabolism,loss of white adipocytes, as in lipodystrophy, leads to ectopic de-position of lipids in liver and other organs, whereas loss of beigeand brown adipocytes compromises organismal defense againstenvironmental cold (1).Beige and brown adipocytes are considered to be two distinct

cell types based on their developmental origin, transcriptome,and epigenome (1, 4, 5). Myogenic precursors give rise to brownadipocytes during embryogenesis in a temperature-independentmanner, whereas environmental cold is the primary stimulus forthe recruitment of mesenchyme-derived beige adipocytes in thewhite adipose tissue (WAT) of adult animals. A focus on cold-induced recruitment of beige adipocytes has identified a largenumber of signaling and transcriptional pathways that control itsbiogenesis in adult animals (4). In nearly all of these cases, beige-promoting factors directly or indirectly modulate the adrenergicactivity of sympathetic nerves, which densely innervate the in-guinal WAT (iWAT) of mice (6).

Although less well appreciated, iWAT also undergoes beigeadipogenesis during the peri-weaning period (7–12). However,the importance of sympathetic stimulation in acquisition of thisphenotype remains controversial. For example, Contreras et al.demonstrated that denervation of iWAT impairs commitment tothe beige adipocyte lineage, whereas Chabowska-Kita et al.reported presence of UCP1+ cells in the iWAT of thermoneutralmice that presumably lacked sympathetic stimulation (7, 8).These findings raise the possibility that a combination of cellautonomous and nonautonomous mechanisms might regulatebeige adipocyte development during the peri-weaning period, agap in our knowledge that we address here.

Results and DiscussionBeige Fat Develops during the Peri-Weaning Period in a Temperature-Independent Manner. To identify mechanisms that control beige adi-pogenesis during the peri-weaning period, we employed Adipo-Clear

Significance

Adipose tissue performs multiple functions in mammals, in-cluding insulation, mechanical tissue protection, and energybalance. These functions of adipose tissue are performed bythree distinct cell types: white, beige, and brown adipocytes.While white adipocytes store energy, beige and brown adi-pocytes dissipate energy through mitochondrial respiration.Previous studies have elucidated how environmental coldstimulates recruitment of beige adipocytes in adult animals.However, beige adipogenesis also occurs during the peri-weaning period in mice, a process that is poorly understood.Here, we demonstrate that distinct mechanisms regulate de-velopment and recruitment of beige adipocytes. We find that Bcell leukemia/lymphoma 6 (BCL6) controls development ofbeige adipocytes during the peri-weaning period, whereassympathetic nerves regulate the recruitment of beige adipo-cytes in cold environments.

Author contributions: Y.W., M.A.K., and A.C. designed research; Y.W., M.A.K., and V.I.K.performed research; V.I.K. contributed new reagents/analytic tools; Y.W., M.A.K., V.I.K.,and A.C. analyzed data; and Y.W. and A.C. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission. C.W. is a guest editor invited by theEditorial Board.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Data deposition: All RNA-seq data are available at the Gene Expression Omnibus (GEO),https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE140259).1Present address: Discovery Biology, Merck Research Labs, South San Francisco, CA 94080.2To whom correspondence may be addressed. Email: [email protected].

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1920419117/-/DCSupplemental.

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together with light sheet fluorescence microscopy to investigatehow the ambient temperature (Ta) regulates developmental ex-pression of UCP1 in the iWAT (13). In C57BL/6J mice that werebred at Ta = 22 °C, UCP1 expression began in the inguinal region(left) of iWAT as early as postnatal day (P)14, spread to thedorsolumbar region (right) by P21, peaked around P28, andbegan to regress by P35 (Fig. 1A, SI Appendix, Fig. S1 A and B,and Movie S1). To our surprise, this spatiotemporal pattern ofUCP1 expression was preserved in iWAT of C57BL/6J mice thatwere bred at thermoneutrality (Ta = 30 °C), suggesting that thecommitment to the beige adipocyte lineage occurs in a temperature-independent manner (Fig. 1B and Movie S2). In support of thishypothesis, quantitation of UCP1 protein expression during the peri-weaning period (P14–P35) revealed that while beige adipocyte de-velopment occurred in a temperature-independent manner, theamplitude of this response was gated by environmental temper-ature (Fig. 1C). For example, the UCP1+ volume was comparable

at P14 between mice bred at 22 °C and 30 °C but differed in older(P21–P35) animals (Fig. 1C). Hematoxylin and eosin staining oftissue sections independently confirmed the presence of multi-locular beige adipocytes in P28 iWAT of thermoneutral mice (Fig.1D). Taken together with previous observations that neonatalmice are poikilothermic (body temperature mirrors ambient tem-perature) and only develop full thermoregulatory capacity aroundP19 to P28 (14), our data suggest that beige adipogenesis is initiatedprior to acquisition of homeothermy in a temperature-independentmanner.We next asked whether the pattern of UCP1 expression in

iWAT of cold-exposed adult animals was similar to its develop-mental expression during the peri-weaning period. We foundthat cold-induced beige fat recruitment in adult animals followeda similar spatiotemporal pattern as young mice; i.e., it began inthe inguinal region and progressively spread to the dorsolumbarregion of iWAT, as previously reported (Fig. 1 A and E) (13).

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Fig. 1. Beige fat develops during the peri-weaning period in a temperature-independent manner. (A and B) Immunostaining for uncoupling protein 1 (UCP1)in iWAT of C57BL/6J mice at various postnatal ages that were bred and housed at 22 °C (A) or 30 °C (B). (C) Quantification of UCP1 in iWAT of mice housed at22 °C (A) or 30 °C (B) using Imaris. Data pooled from 2 to 4 litters per time point and housing temperature (n = 10–26 per group) and analyzed by Student’st test. (D) Representative hematoxylin and eosin staining of iWAT from P28 mice housed at 22 °C or 30 °C. (E) Immunostaining for UCP1 in iWAT of 14- to 15-wk-old C57BL/6J mice housed at 22 °C or 8 °C for 2–6 d. (F) Immunostaining for RFP in iWAT of 20-wk-old Ai14 x Ucp1Cre mice housed at 22 °C. (G) Comparisonof differentially expressed genes in iWAT of P28 mice housed at 22 °C with beige- and white-specific adipocyte signatures. (H) GO enrichment analysis andcorresponding P values of up-regulated genes in iWAT of P28 mice housed at 22 °C. (I) Heatmap of up-regulated genes in the “Lipid metabolic process” GOcategory in iWAT of P28 C57BL/6J mice housed at 22 °C (n = 4 per genotype). (J) GO enrichment analysis and corresponding P values of down-regulated genesin iWAT of P28 mice housed at 22 °C. (K) Heatmap of down-regulated genes in the “Muscle contraction” GO category in iWAT of P28 C57BL/6J mice housed at22 °C (n = 4 per genotype). Data are presented as mean ± SEM. (Scale bars: A, B, and F, 1 mm; D and E, 50 μm.)

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This suggested that cold-induced beige fat in adult animals mightresult from reactivation of dormant beige adipocytes (8, 12, 15).To test this hypothesis, we examined the iWAT of adult Ai14 ×Ucp1Cre mice, which allows for irreversible marking of all UCP1+

cells and their descendants with membrane-targeted tdTomatofluorescent protein. We found that the entire iWAT of 20-wk-oldmice that had not been exposed to environmental cold containedcells with a history of UCP1 protein expression (Fig. 1F andMovies S3 and S4), suggesting that commitment to the beige fatlineage in iWAT occurs during the peri-weaning period. Inagreement with these findings, a recent report demonstrated thatdepletion of UCP1+ cells during the peri-weaning period wassufficient to impair recruitment of beige fat in adult animals byβ3-adrenergic agonists or environmental cold (12).To examine similarities and differences between developmental

and cold-induced beige fat, we performed RNA-sequencing (seq)on P28 iWAT isolated from C57BL/6J mice bred at 22 °C or 30 °Cand compared it to signatures of adult beige and white adipocytesidentified by the Rosen laboratory (5). We found that genes up-regulated by cold in iWAT of P28 mice (22 °C vs. 30 °C) werepreferentially expressed by adult beige adipocytes, whereas genesthat were down-regulated in iWAT of P28 mice housed at 22 °Cwere enriched for genes found in adult white adipocytes (Fig. 1G).Gene ontology (GO) enrichment analysis of up-regulated genesrevealed that metabolic programs associated with catabolism offatty acids and carbohydrates, as well as biosynthesis of cholesteroland sterols, were enriched in P28 iWAT of mice housed at 22 °C(Fig. 1 H and I and SI Appendix, Fig. S1 C–E), processes that areknown to be important for development of beige adipocytes inadult animals (3, 16, 17). In contrast, GO enrichment analysis ofdown-regulated genes identified biological processes associatedwith muscle contraction and sarcomere organization (Fig. 1 J andK). Because myogenic precursors can contribute to glycolyticbeige adipocytes in the absence of adrenergic stimuli (18), ourfindings suggest environmental cold suppresses the developmentalexpression of this myogenic program in P28 iWAT.

Distinct Requirements for the SNS in Development and Recruitmentof Beige Fat. The sympathetic nervous system (SNS) regulatescold-induced recruitment of beige adipocytes in iWAT (19),prompting us to ask whether it might also be important forphysiologic development of beige adipocytes in young animals.To test this hypothesis, we generated mice in which TrkA, thehigh-affinity receptor for nerve growth factor (20), was selec-tively deleted in sympathetic nerves (designated Ntrk1f/fThCre)(21). We found that iWAT of Ntrk1f/fThCre mice lacked the densenetwork of sympathetic neurites that was present in iWAT ofcontrol (Ntrk1f/+ThCre) or C57BL/6J mice (Fig. 2 A and B andMovies S5–S7), as assessed by immunostaining for the SNSmarker tyrosine hydroxylase (TH). However, because Ntrk1f/f is ahypomorphic allele (20), the development of TH+ neurites wasalso reduced in iWAT of Ntrk1f/f mice (Fig. 2 A and B and MoviesS5–S7). Based on these observations, we used Ntrk1f/+ThCre orC57BL/6J mice as controls for our subsequent experiments.We next asked whether sympathetic innervation of iWAT was

necessary for the commitment to the beige fat lineage during theperi-weaning period. To our surprise, we found that both thespatiotemporal pattern and magnitude of UCP1 expression werepreserved in iWAT of Ntrk1f/f and Ntrk1f/fThCre mice housed at22 °C or 30 °C (Fig. 2 C–E and SI Appendix, Fig. S2 A and B),indicating that the SNS is dispensable for the development ofbeige adipocytes during the peri-weaning period. This was in-dependently confirmed by costaining for UCP1 and TH in P7iWAT of Ntrk1f/+ThCre and Ntrk1f/fThCre mice, which showedemergence of UCP1+ cells in regions lacking TH+ neurites (Fig.2F and Movies S8–S11). In agreement with these observations,histological examination revealed the presence of multilocular

beige adipocytes in iWAT of Ntrk1f/f and Ntrk1f/fThCre micehoused at 22 °C (SI Appendix, Fig. S2C).To understand the role of sympathetic nerves in regulation of

iWAT gene expression during the peri-weaning period, we per-formed RNA-seq on iWAT of P28 Ntrk1f/fThCre mice that werehoused at 22 °C. We found that only a small subset of genes wasaffected by the loss of sympathetic innervation in iWAT of P28Ntrk1f/fThCre mice, which did not correlate strongly with adultbeige and white adipocyte signatures (SI Appendix, Fig. S3A). GOenrichment analysis of differentially expressed genes revealed thatbiological processes associated with sterol, cholesterol, and fattyacid biosynthesis were decreased, whereas programs associatedwith inflammatory response, phagocytosis, apoptosis, and musclecontraction were increased in iWAT of P28 Ntrk1f/fThCre mice(Fig. 2 G and H and SI Appendix, Fig. S3 B–J). Although thecholesterol biosynthetic pathway has been implicated in cold-induced recruitment of beige adipocytes in adult animals (17),our results suggest that its induction is dispensable for biogenesisof beige adipocytes during the peri-weaning period.We next asked whether the SNS was required for recruitment

of beige adipocytes in adult animals. Consistent with a previousreport (19), we found that cold-induced recruitment of beigeadipocytes was severely impaired in adult Ntrk1f/f andNtrk1f/fThCre mice (Fig. 2 I and J and Movies S12–S14). Be-cause recruitment of beige adipocytes could be rescued by β3-adrenergic agonist CL-316,243 (Fig. 2K), our data suggest thatthe commitment to the beige adipocyte lineage occurs normally inNtrk1f/f and Ntrk1f/fThCre mice. Taken together, these results suggestthat sympathetic innervation is not necessary for the developmentof beige adipocytes during the peri-weaning period but is absolutelyrequired for their recall after cold exposure in adult animals.

BCL6 Is Required for Development of Beige Fat during the Peri-Weaning Period. We recently reported that BCL6 is required formaintenance of thermogenic competence in dormant brownadipocytes when adrenergic input is minimal (22), prompting usto ask whether it might function in a similar manner to regulatecommitment to the beige adipocyte lineage during the peri-weaning period. In support of this hypothesis, we observed thatBCL6 protein was expressed in iWAT during the peri-weaningperiod and its expression declined as animals progressed throughpuberty (Fig. 3A and SI Appendix, Fig. S4A). To investigatewhether BCL6 was required for the development of beige adi-pocytes, we generated Bcl6f/fPrx1Cre and Bcl6f/fAdipoqCre mice toselectively delete Bcl6 in subcutaneous white adipocytes of theiWAT (SI Appendix, Fig. S4B) or all adipocytes, respectively (2, 23,24). We found that iWAT of P28 Bcl6f/fPrx1Cre and Bcl6f/fAdipoqCre

mice had a near complete absence of multilocular UCP1+ beigeadipocytes (Fig. 3 B–F and Movies S15 and S16). In contrast,when Bcl6 was deleted after adipocytes expressed UCP1, as inBcl6f/fUcp1Cre mice, development and subsequent cold-inducedrecruitment of beige adipocytes were not significantly affected(SI Appendix, Fig. S4 C–E). These findings together suggest thatBCL6 is specifically required during the commitment but not themaintenance stage of beige adipogenesis.To determine whether loss of beige adipocytes impacted

thermogenic fitness, we subjected 4-wk-old (P28) Bcl6f/f andBcl6f/fPrx1Cre mice to an acute cold challenge at 4 °C. We foundthat core temperature declined at a faster rate in Bcl6f/fPrx1Cre

mice, which shortened their survival during the cold challenge(Fig. 3 G and H). Measurement of oxygen consumption revealedsignificant reduction in the respiratory rate of iWAT, but notBAT, of P28 Bcl6f/fPrx1Cre mice (Fig. 3 I and J), suggesting that adefect in beige fat thermogenesis likely compromised survival ofthese young animals during the cold challenge.We next performed RNA-seq on iWAT from P25 Bcl6f/f and

Bcl6f/fPrx1Cre mice to understand how BCL6 regulates develop-ment of beige adipocytes. Analysis of 207 down-regulated and

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174 up-regulated genes in iWAT of Bcl6f/fPrx1Cre mice revealedthat they largely clustered with signatures of beige and whiteadipocytes (SI Appendix, Fig. S4F). GOs associated with β-oxidationof fatty acids, response to cold, and brown fat cell differentiationwere enriched among the down-regulated genes (Fig. 3 K–M andSI Appendix, Fig. S4 G–I), whereas pathways associated with celladhesion, osteoblast differentiation, and mammary gland devel-opment were represented among the up-regulated genes iniWAT of Bcl6f/fPrx1Cre mice (SI Appendix, Fig. S4 J and K). Ofnote, we observed that loss of BCL6 shifted iWAT fromuncoupled to coupled respiration, as evidenced by reduction inUcp1 and concomitant increase in Atp5g1 expression (Fig. 3 Nand O), the latter encoding for a protein regulating coupledrespiration via ATP synthase (22). Together, these data dem-onstrate that dysregulation of only a subset of beige adipocytegenes (such as Ucp1, Elovl3, Crat, Hadhb, Echdc1, Slc25a20,Acadl, and Acadm) is sufficient to impair development of beigefat during the peri-weaning period, leading us to propose that

they might constitute a core set necessary for establishinguncoupled respiration in iWAT (SI Appendix, Table S1).

BCL6 Regulates the Core Program Necessary for Development andRecruitment of Beige Fat. We next investigated the requirementof BCL6 in cold-induced recruitment of beige fat in adult ani-mals. Compared to Bcl6f/f mice, cold-induced recruitment ofUCP1+ adipocytes was nearly absent in iWAT of 14-wk-oldBcl6f/fPrx1Cre mice (Fig. 4 A and B and Movies S17 and S18).As a consequence, ex vivo oxygen consumption was reduced iniWAT, but not BAT, of Bcl6f/fPrx1Cre mice (Fig. 4 C and D).Because immunostaining for TH demonstrated similar density ofsympathetic nerves in iWAT of Bcl6f/f and Bcl6f/fPrx1Cre mice(Fig. 4E and Movies S19 and S20), it suggests that BCL6 acts in acell autonomous and SNS-independent manner to support thecommitment of precursor cells to the beige adipocyte lineage. Insupport of this hypothesis, cold-induced recruitment of beigeadipocytes was unaffected in Bcl6f/fUcp1Cre mice (Fig. 4 F and

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Fig. 2. Distinct requirements for the SNS in development and recruitment of beige fat. (A) Immunostaining for tyrosine hydroxylase (TH) in iWAT of P28Ntrk1f/+ThCre, Ntrk1f/f, and Ntrk1f/fThCre mice housed at 22 °C. (B) Higher-magnification images of TH immunostained iWAT of P28 C57BL/6J, Ntrk1f/f, andNtrk1f/fThCre mice housed at 22 °C. (C and D) Immunostaining for UCP1 in iWAT of Ntrk1f/f (C) or Ntrk1f/fThCre (D) mice at various postnatal ages that werebred and housed at 22 °C. (E) Quantification of UCP1 in iWAT of C57BL/6J (same as in Fig. 1C), Ntrk1f/f and Ntrk1f/fThCre mice using Imaris. Data pooled from1 to 4 litters per time point and genotype (n = 8–26 samples per group) and analyzed by Student’s t test. (F) Immunostaining for TH and UCP1 in iWAT of P7Ntrk1f/+ThCre and Ntrk1f/fThCre mice housed at 22 °C. (G and H) Heatmaps of down-regulated genes in the “Cholesterol biosynthetic process” (G) and “Lipidmetabolic process” (H) GO categories in iWAT of P28 Ntrk1f/fThCre mice housed at 22 °C (n = 4 per genotype). (I) Immunostaining for TH in iWAT of 8- to 10-wk-old Ntrk1f/+ThCre, Ntrk1f/f, and Ntrk1f/fThCre mice housed at 22 °C. (J) Immunostaining for UCP1 in iWAT of 14- to 15-wk-old C57BL/6J, Ntrk1f/f, andNtrk1f/fThCre mice housed at 10 °C for 11 d. (K) Immunostaining for UCP1 in iWAT of 10- to 11-wk-old Ntrk1f/+ThCre, Ntrk1f/f, and Ntrk1f/fThCre mice treateddaily with vehicle or CL-316,243 (1 mg/kg intraperitoneally) for 7 d at 22 °C. The mesh-like structures in the Veh-22 °C- Ntrk1f/f image are autofluorescentmammary ducts. Data are presented as mean ± SEM. (Scale bars: A, C, D, and I–K, 1 mm; B, 200 μm; F, 500 μm.)

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G), which had normal development of beige adipocytes duringthe peri-weaning period (SI Appendix, Fig. S4 C–E). These datatogether suggest a model in which BCL6 acts primarily at thecommitment phase of beige adipogenesis, whereas cold exposureleads to the recall of committed cells in a SNS-dependent andBCL6-independent manner.To gain insights into how signals downstream of environ-

mental cold interact with BCL6 to regulate recruitment of beigeadipocytes in adult animals, we performed RNA-seq on iWAT ofBcl6f/f and Bcl6f/fPrx1Cre mice that were subjected to a coldchallenge at 4 °C for 2 d. We found that 493 genes were down-regulated and 902 genes were up-regulated in iWAT of 9-wk-oldBcl6f/fPrx1Cre mice after exposure to environmental cold, whichwere enriched in the signature genes for beige and white adipo-cytes, respectively (SI Appendix, Fig. S5A). GO terms associatedwith lipid metabolic process, fatty acid β-oxidation, and heat gen-eration were enriched in the down-regulated genes (Fig. 4 H and I

and SI Appendix, Fig. S5 B and C), whereas processes associatedwith cell adhesion, inflammatory response, and angiogenesis werefound among the up-regulated genes in Bcl6f/fPrx1Cre mice (SIAppendix, Fig. S5 D–F).We next compared these BCL6-dependent genes with those

regulated by environmental cold in WT C57BL/6J mice. Wefound that BCL6-dependent genes only partially overlapped withthose regulated by cold challenge at 4 °C in C57BL/6J adultmice. For example, among the cold-induced genes in C57BL/6Jmice, only 122 genes (∼21%) exhibited dependence on BCL6(Fig. 4J). In contrast, cold exposure led to down-regulation of780 genes in iWAT of C57BL/6J mice, but only ∼17% (135 genes)required BCL6 for their suppression (Fig. 4K). This suggests thatthe interactome between BCL6 and environmental cold repre-sents a core set of genes necessary for the recruitment of beigeadipocytes in adult animals. Indeed, comparison of cold- andBCL6-dependent differentially expressed genes with the signature

L

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Fig. 3. BCL6 is required for development of beige fat during the peri-weaning period. (A) Immunoblotting for BCL6 in iWAT of C57BL/6J male mice housed at22 °C. (B) Representative hematoxylin and eosin staining of iWAT from P28 Bcl6f/f and Bcl6f/fPrx1Cre mice housed at 22 °C. (C and D) Immunostaining for UCP1in iWAT of P28 Bcl6f/f, Bcl6f/fPrx1Cre (C) and Bcl6f/fAdipoqCre (D) mice housed at 22 °C. (E and F) Quantification of UCP1 in iWAT of P28 Bcl6f/f, Bcl6f/fPrx1Cre (E),and Bcl6f/fAdipoqCre (F) mice housed at 22 °C using Imaris. Data analyzed by Student’s t test (n = 12–20 per genotype). (G and H) Core temperature mea-surements (G) and survival curves (H) of P28 Bcl6f/f and Bcl6f/fPrx1Cre mice housed at 4 °C for up to 6 h (n = 6–7 per genotype). Kaplan–Meier survival curveanalyzed by Log-Rank (Mantel–Cox) test; humane endpoint = 28 °C. (I and J) Oxygen consumption rate of iWAT (I) and BAT (J) from P28 Bcl6f/f andBcl6f/fPrx1Cre mice housed at 22 °C. Data analyzed by Student’s t test (n = 8 per genotype). (K) GO enrichment analysis and corresponding P values of down-regulated genes in iWAT of P25 Bcl6f/fPrx1Cre mice housed at 22 °C. (L and M) Heatmaps of down-regulated genes in the “Fatty acid beta oxidation” and“Response to cold & brown fat cell differentiation” GO categories in iWAT of P25 Bcl6f/fPrx1Cre mice housed at 22 °C (n = 7–8 per genotype; M, male; F,female). (N and O) Normalized counts of Ucp1 and Atp5g1 transcripts in iWAT of P25 Bcl6f/f and Bcl6f/fPrx1Cre mice housed at 22 °C. Data analyzed by Student’st test (n = 7–8 per genotype). (Scale bars: B, 50 μm; C and D, 1 mm.)

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genes of beige and white adipocytes revealed that the inducedgenes were highly enriched in beige adipocytes, whereas therepressed genes were abundant in white adipocytes (Fig. 4L andSI Appendix, Table S2). GO enrichment analysis confirmed thatlipid and fatty oxidation processes were highly represented inthe induced genes, whereas processes of skeletal muscle de-velopment, cell adhesion, and wound healing were enrichedamong the repressed genes (Fig. 4 M and N).

DiscussionWe present here evidence that BCL6 and the SNS differentiallyregulate biogenesis and recruitment, respectively, of beige adi-pocytes in the iWAT. While previous studies had suggested thatcontinuous sympathetic stimulation was required for commit-ment to the beige adipocyte lineage (8), we found that genetic

absence of sympathetic nerves (as in Ntrk1f/fThCre mice) did notsignificantly impact development of beige adipocytes during theperi-weaning period. In contrast, we observed that cold-inducedrecruitment of beige adipocytes was completely dependent on thesympathetic nerves. Because this recruitment defect in Ntrk1f/fThCre

mice could be rescued by administration of the β3-selective ad-renergic agonist CL-316,243, it suggests that cell-autonomousmechanisms, rather than adrenergic signaling, regulate commit-ment of mesenchyme-derived precursor cells to the beige adipocytelineage.We investigated the cell-autonomous requirement of BCL6 in

beige adipocyte development for two reasons. First, we had re-cently reported that BCL6 was required for maintenance ofthermogenic fitness in dormant brown adipocytes (22), an adi-pocyte cell type that shares molecular and functional features

A B D FC E

G H I J

K L

M

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Fig. 4. BCL6 regulates the core program necessary for development and recruitment of beige fat. (A) Immunostaining for UCP1 in iWAT of 14-wk-old Bcl6f/f

and Bcl6f/fPrx1Cre mice housed at 4 °C for 2 d. (B) Quantification of UCP1 immunostaining in A using Imaris (n = 10–14 per genotype). Data analyzed byStudent’s t test. (C and D) Oxygen consumption rate of iWAT (C) and BAT (D) from 9- to 10-wk-old Bcl6f/f and Bcl6f/fPrx1Cre mice housed at 4 °C for 2 d (n = 11–12per genotype). Data analyzed by Student’s t test. (E) Immunostaining for TH in iWAT of 8-wk-old Bcl6f/f and Bcl6f/fPrx1Cre mice housed at 22 °C. (F) Immu-nostaining for UCP1 in iWAT from 9-wk-old Bcl6f/f and Bcl6f/fUcp1Cre mice housed at 4 °C for 2 d. (G) Quantification of UCP1 in iWAT of Bcl6f/f and Bcl6f/fUcp1Cre

mice that were housed at 4 °C for 2 d (n = 8–9 per genotype). Data analyzed by Student’s t test. (H) GO enrichment analysis and corresponding P values of down-regulated genes in iWAT of 9-wk-old Bcl6f/fPrx1Cre mice housed at 4 °C for 2 d (n = 4 per genotype). (I) Heatmap of down-regulated genes in the “Fatty acid betaoxidation” GO category in iWAT of 9-wk-old Bcl6f/fPrx1Cre mice housed at 4 °C for 2 d (n = 4 per genotype). (J) Venn diagram showing overlap of genes down-regulated in iWAT of 9-wk-old Bcl6f/fPrx1Cre mice housed at 4 °C for 2 d (versus Bcl6f/f) and genes up-regulated in 9-wk-old C57BL/6J mice housed at 4 °C for 2 d(versus 22 °C). Fold change > 1.5, adjusted P value <0.05. (K) Venn diagram showing overlap of genes up-regulated in iWAT of 9-wk-old Bcl6f/fPrx1Cre micehoused at 4 °C for 2 d (versus Bcl6f/f) and genes down-regulated in 9-wk-old C57BL/6J mice housed at 4 °C for 2 d (versus 22 °C). Fold change > 1.5, adjusted Pvalue < 0.05. (L) Comparison of cold- and BCL6-dependent genes in J and K with beige and white adipocyte signatures. (M and N) GO enrichment analysis andcorresponding P values of cold- and BCL6-induced (M), and cold- and BCL6-repressed (N) genes in iWAT of 9-wk-old mice. (Scale bars: A, E, and F, 1 mm.)

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with beige adipocytes. Second, we and others have demonstratedthat BCL6 reinforces a cell’s identity by repressing alternativecell fate programs, including in immune cells and brown adipo-cytes (22, 25, 26). This observation led us to ask whether BCL6might function as a molecular switch between beige and whiteadipocytes that reside in the iWAT. Indeed, using three dif-ferent knockout mouse lines (Bcl6f/fPrx1Cre, Bcl6f/fAdipoqCre,and Bcl6f/fUcp1Cre), we found that BCL6 was specifically re-quired at the commitment, but not the maintenance, phase ofbeige adipogenesis. For example, in Bcl6f/fPrx1Cre mice, we foundthat expression of genes involved in β-oxidation of fatty acids(Acaa2, Acadm, Acadl, Acadvl, Acaa2b, Decr1, Slc27a2, Hadha,Hadhb, and Ehhadh), thermogenesis (Ucp1, Elovl3, Cox7a1,Cox8b, and Cidea), and the beige adipocyte markers Tbx1 andTmem26 was reduced in the iWAT (SI Appendix, Tables S1 andS2). Furthermore, the near absence of UCP1 protein in iWAT ofBcl6f/fPrx1Cre mice was accompanied by induction of Atp5g1,which switches mitochondria from uncoupled to coupled respira-tion (22). Together, these data suggest that BCL6 regulates a coreset of genes that are required for full expression of the beige adi-pocyte phenotype during the peri-weaning period of development.The peri-weaning period is an important transition point in

mammalian physiology, as neonates switch from mother’s milk toadult food. During this critical period, a number of physiologicalsystems undergo maturation and remodeling. For example, theabsorptive intestinal epithelium remodels from its neonatal toadult state to support nutrient absorption, a process regulated bythe transcriptional repressor B lymphocyte-induced maturationprotein 1 (Blimp1). Deletion of Blimp1 in the intestinal epithe-lial cells compromises nutrient intake in neonates, resulting ingrowth retardation and mortality (27, 28). In a similar manner,the microbiome, which is seeded at birth, remodels during the

peri-weaning period. This stabilization of the microbiome in-duces a weaning reaction in the immune system, which is nec-essary for induction of protective immunity in the gut (29). Ourdata extends this paradigm by demonstrating that BCL6 is re-quired for peri-weaning remodeling of iWAT into thermogenicbeige adipose tissue. Thus, in the future, it will be important todetermine how these tissue remodeling responses are coordi-nated during the peri-weaning period to enable physiologicaladaptations in the adult animal.

Materials and MethodsAll experiments involving mice were conducted under an approved In-stitutional Animal Care and Use Committee (IACUC) protocol at University ofCalifornia, San Francisco (UCSF). Animal, tissue clearing and immunostaining,three-dimensional imaging, image processing and quantification, histology,immunoblotting, tissue oxygen consumption, RNA isolation and qRT-PCR,sequencing library preparation, next generation sequencing, RNA-seq, andstatistical analyses for this study are described in detail in SI Appendix,Materials and Methods.

Data Availability. Data generated or analyzed during this study are includedin this published article and its SI Appendix files. The RNA-seq data has beendeposited at the Gene Expression Omnibus (GEO), https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE140259).

ACKNOWLEDGMENTS.We thank members of the A.C. laboratory and A. Lohfor comments on the manuscript, and X. Cui for assistance with mousehusbandry. This work was supported by NIH Grants DK094641 and DK101064(to A.C.); Agency for Science, Technology, and Research (A*STAR, Singapore)National Science Scholarship (PhD to Y.W.); Dermatology Foundation Derma-tologist Investigator Research Fellowship (to M.A.K.); and American Heart As-sociation (AHA) Predoctoral Fellowships 18PRE34080250 and 16PRE26960008(to V.I.K.).

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