YY1 is indispensable for Lgr5+ intestinal stemcell renewalAnsu O. Perekatta, Michael J. Valdeza, Melanie Davilaa, A. Hoffmana, Edward M. Bonderb, Nan Gaob,and Michael P. Verzia,1

aDepartment of Genetics, Human Genetics Institute of New Jersey, Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, Piscataway, NJ08854; and bDepartment of Biological Sciences, Rutgers, The State University of New Jersey, Newark, NJ 07102

Edited by Brigid L. M. Hogan, Duke University Medical Center, Durham, NC, and approved April 17, 2014 (received for review January 4, 2014)

The intestinal stem cell fuels the highest rate of tissue turnover inthe body and has been implicated in intestinal disease and cancer;understanding the regulatory mechanisms controlling intestinalstem cell physiology is of great importance. Here, we provideevidence that the transcription factor YY1 is essential for intestinalstem cell renewal. We observe that YY1 loss skews normal homeo-static cell turnover, with an increase in proliferating crypt cells anda decrease in their differentiated villous progeny. Increased crypt cellnumbers come at the expense of Lgr5+ stem cells. On YY1 deletion,Lgr5+ cells accelerate their commitment to the differentiated popula-tion, exhibit increased levels of apoptosis, and fail to maintain stemcell renewal. Loss of Yy1 in the intestine is ultimately fatal. Mecha-nistically, YY1 seems to play a role in stem cell energy metabolism,with mitochondrial complex I genes bound directly by YY1 and theirtranscript levels decreasing on YY1 loss. These unappreciated YY1functions broaden our understanding of metabolic regulation in in-testinal stem cell homeostasis.

transcriptional regulation | mitochondria | crypt base columnar cell

The gut epithelium is the most proliferative tissue in the body,refreshing itself on a weekly basis. Epithelial turnover is

made possible by intestinal stem cells, which are located in epi-thelial pockets tucked into the intestinal wall called crypts ofLieberkühn. Intestinal stem cells give rise to all other intestinalepithelial lineages and maintain their own population indef-initely (1). A number of transgenic reporters has been used inlineage tracing assays to show stem cell activity arising from thebase of the crypt (2–8), and all have been reported to overlapwith crypt base columnar cells (9), which cooccupy the bottom ofcrypts with differentiated Paneth cells. Intestinal stem cellsmarked by leucine rich repeat containing G protein coupledreceptor 5 (Lgr5) expression have been the most extensivelycharacterized; these cells maintain their own population throughsymmetric divisions (10), and when they leave the niche, theygive rise to differentiated crypt cells, including a transit ampli-fying population that ultimately supplies differentiated cells ontoluminal projections called villi.Intestinal stem cells are of great importance to human health

and regenerative medicine. Mouse models of human colorectalcancer show that intestinal stem cells can function as cells oforigin for cancer (11, 12). There is a clear imperative to un-derstand the regulatory mechanisms governing intestinal stemcell function. Recent work has shown that intestinal stem cellsfrom both flies and humans are sensitive to the metabolic state ofthe organism and has implicated cellular metabolism as a criticalregulatory input of stem cell homeostasis (13–16). Intestinalstem cells were observed to exhibit higher levels of glycolysisthan oxidative phosphorylation compared with their differenti-ated progeny (17), and the oxidative state of intestinal stem cellsimpacts the ability of the cells to undergo transformation (18).Caloric restriction was recently shown to increase intestinal stemcell numbers (16). In Drosophila, intestinal stem cell expressionof the fly homolog to PGC-1α, a metabolic coregulator, is evencoupled to the organism’s lifespan (19). These exciting advances

highlight a great need to identify additional regulators of in-testinal stem cell metabolism.YY1 is a zinc finger transcription factor first discovered for its

function in viral gene expression (20, 21) and cloned during inves-tigations of viral (22), immunoglobulin (23), and ribosomal (24)gene expression. YY1 has since been implicated in a number ofprocesses, including development of muscle (25–30) and B cells(31–33), and stem cell regulation (34–37). In embryonic stem cells,YY1 is part of the Myc transcriptional network (36) but counter-intuitively, suppresses induced pluriopotent cell reprogramming(34). In the blood, YY1 overexpression promotes long-termhematopoietic stem cell maintenance (37). Conversely, YY1promotes satellite cell activation and differentiation duringmuscle regeneration (35). Thus, context-specific stem cell func-tions have been attributed to YY1, potentially owing to its abilityto operate in diverse protein complexes. The role of YY1 inintestinal stem cells has not been previously investigated.Here, we present YY1 as an essential transcription factor for

intestinal stem cell renewal. YY1 loss leads to an imbalance inthe ratio of crypt to villus cell populations in the intestine, withexpanded crypt length and an increased zone of cell prolif-eration. Functional lineage tracing assays showed that intestinalstem cells lacking YY1 undergo an increased exit from theirniche, which corresponds to the expanded proliferative zone.Ultimately, depletion of the stem cell population was observed,which was indicated by transgenic markers, gene expression, andEM. To understand the functional mechanisms of YY1 in stemcell maintenance, expression profiling and ChIP were used toreveal that YY1 binds to mitochondrial complex I genes andis required for their expression. Our work shows that YY1 is

Significance

A subset of our body’s tissues is continuously renewed throughcell division. Tissue-specific stem cells support this tissue turn-over, and understanding the mechanisms that control the be-havior of these stem cells is important to understanding thehealth of the tissue. In this work, we identify a novel regulatorof the intestinal stem cells. We find that, when the transcrip-tion factor YY1 is inactivated, intestinal stem cells can no lon-ger renew themselves. We show that YY1 controls mitochondrialgene expression, and loss of YY1 results in loss of mitochondrialstructural integrity. This work, therefore, provides a link be-tween a mitochondrial regulator and stem cell function andbroadens our appreciation of metabolic regulation in tissue-specific stem cells.

Author contributions: A.O.P. and M.P.V. designed research; A.O.P., M.J.V., M.D., A.H.,E.M.B., and N.G. performed research; A.O.P., M.J.V., M.D., E.M.B., N.G., and M.P.V. ana-lyzed data; and A.O.P. and M.P.V. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE53503).1To whom correspondence should be addressed. E-mail: mverzi@gmail.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1400128111/-/DCSupplemental.

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required for intestinal stem cell renewal and links YY1 to cel-lular metabolism of the intestinal stem cell.

ResultsYY1 Is Required to Maintain Intestinal Homeostasis and Viability.Whether YY1 is expressed or contributes to the homeostaticrenewal of the adult intestinal epithelium is untested. We ob-served YY1 immunoreactivity throughout the length of the gutand along the crypt–villus axis. Notably, intestinal stem cells,identified by their crypt base columnar cell (CBC) morphology,showed strong YY1 immunoreactivity, whereas YY1 staining inPaneth and Goblet cells was lower by comparison (Fig. 1 A andF). To determine the function of YY1 in the intestinal epithe-lium, we inactivated a conditional Yy1 allele with a tamoxifen-inducible, epithelium-specific Cre driver, Yy1f/f;Vil-Cre-ERT2(38, 39). YY1 immunostain in the epithelium was specifically loston tamoxifen treatment in adult mice (Fig. 1 B and D), whereascells in the lamina propria remained YY1-positive (Fig. 1 B andD).Eleven days after the first tamoxifen injection, Yy1f/f;Vil-Cre-ERT2

mice lost weight (Fig. 1E) and became moribund, prompting us tohalt the experiment, and indicating that epithelial functions of YY1are essential for viability; 4 d after induced YY1 KO, both cryptsand villi were notably elongated, but by 10 d post-KO, villi becamesignificantly shorter than in controls, and crypts appeared furtherelongated and sinuous (Fig. S1), indicating that YY1 function isrequired for normal intestinal homeostasis. Interestingly, 10 d afterYY1 KO was induced, occasional hyperproliferative crypts wereobserved, characteristic of regenerative foci (Fig. 1G). The cellswithin these foci were positive for YY1 protein expression, sug-gesting that these foci originate from epithelial cells that had es-caped Yy1 deletion.

Lgr5+ and CBC Cells Are Lost on YY1 Deletion. The hyperplasticcrypts observed in YY1 KO mice (Fig. 1G) are reminiscent ofregenerative foci observed in epithelia recovering from loss ofintestinal stem cell regulatory factors, such as Myc (40) orASCL2 (41). The robust YY1 expression observed in CBCs (Fig.1F) also suggested that YY1 could function as an essential reg-ulator of the stem cell. To investigate the role of YY1 in stemcell function, we measured the relative transcript levels of pro-posed stem cell markers in YY1 KO and control mouse epi-thelia. YY1 KO mice showed a significant decrease in expressionof several genes comprising an Lgr5+ stem cell signature (42),including Lgr5, Olfm4, and Smoc2. Conversely, expression ofmarkers associated with a more quiescent reserve stem cell ac-tivity was not affected, including Bmi1, Hopx, Lrig1, and Tert(Fig. 2 A and B). These data suggest that YY1 is required spe-cifically for an active Lgr5+ stem cell population. To furtherexplore YY1 function in Lgr5+ stem cells, we generated mice inwhich we could both inactivate YY1 throughout the epitheliumand monitor the Lgr5-expressing cell population with an Lgr5-GFP knockin allele: Yy1f/f;Vil-creERT2; Lgr5-EGFP-IRES-creERT2 (43). Mice treated with tamoxifen and monitored for 4,5, or 7 d showed a decrease in GFP expression over time, with nodetectible GFP+ cells remaining at 7 d after tamoxifen treatment

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Fig. 1. YY1 KO in the intestinal epithelium triggers weight loss and death.YY1 immunoreactivity (brown) is (A and C) seen throughout the intestinalepithelium but (B and D) lost on tamoxifen treatment of Yy1f/f; Villin-creERT2

mice. C and D show that immunoreactivity is lost in the epithelium butpreserved in the lamina propria. (E) Significant weight loss is observed afterepithelial KO of Yy1 is induced. **Unpaired two-tailed t test, P < 0.01. (F)Relative YY1 immunoreactivity is stronger in cells with crypt base columnarmorphology (black arrows) and weaker in adjacent Paneth cells (whitearrows). (G) After 10 d of YY1 KO, large, YY1+ hyperplastic crypts are ob-served (arrows; Inset), presumably arising from epithelial cells escaping Cre-mediated recombination. IHC, immunohistochemistry. (Scale bars: 50 μm.)

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alleles to monitor GFP expression over 7 d after tamoxifen treatment toinactivate YY1. Green GFP+ cells diminish over time and are no longerdetected at 7 d. Blue, DAPI counterstain. (Scale bar: 50 μm.) (D) TransmissionEM (TEM) indicates reduction of cells with CBC morphology (outlined inwhite) in Yy1f/f; Villin- creERT2 after 4 d of tamoxifen treatment. Basementmembrane is shown by the black dashed line. (Scale bar: 2 μm.)

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(Fig. 2C). Consistent with the loss of the Lgr5+ stem cell pop-ulation on Yy1 deletion, examination of crypt ultrastructure bytransmission EM confirmed the loss of cells with the CBC stemcell morphology (Fig. 2D); 90% of crypts observed in the controlcondition contained cells with CBC morphology, whereas lessthan 10% of crypts observed in the YY1 KO crypts containedcells with CBC morphology. Taken together, YY1 seems nec-essary for maintenance of the Lgr5+ stem cell population.

Lgr5+ Stem Cells Require YY1 for Renewal.Although YY1 expressionin the epithelium is necessary for stem cell renewal, the specific cellsthat require YY1 to maintain stem cell homeostasis were not clear;stem cells could require YY1 expression autonomously or YY1function in neighboring cells to establish a supportive niche. To testfor a stem cell autonomous function, we deleted YY1 specificallywithin Lgr5+ stem cells using the Lgr5-GFP-IRES-Cre driver (43)and followed the fate of these Yy1-deleted stem cells by lineagetracing. Use of a Cre-activated reporter allele (such as Cre-inducedGFP expression from the Rosa-CAG-LSL-ZsGreen1-WPRE allele)combined with the Lgr5-EGFP-IRES-creERT2 Cre driver allowsfor sustained expression of GFP in Lgr5-Cre–expressing cells andall their descendants (43, 44). In control mice (Lgr5-EGFP-IRES-creERT2; Rosa-CAG-LSL-ZsGreen1-WPRE), robust GFP expres-sion from the ROSA locus was activated in Lgr5+ cells on tamoxifentreatment, and their GFP-expressing descendent cells could bevisualized leaving the crypt base and migrating onto the villi overtime, consistent with published reports (43). Two weeks aftertamoxifen treatment, sustained stripes of GFP-expressing epi-thelium spanned the crypt–villus axis, indicating that labeledstem cells were competent to maintain a supply of GFP-markedstem cells and GFP-expressing progeny (Fig. 3 A, Left and B,Left). In mice with the same genotype but also harboring theconditional Yy1 alleles, tamoxifen treatment both inactivated Yy1and activated GFP expression from the ROSA locus, specificallyin the Lgr5+ stem cells. Interestingly, GFP-positive descendantsof YY1-deficient stem cells showed an accelerated exodus fromthe crypt compartment relative to controls, indicating a morerobust contribution of stem cells to the differentiation streamon YY1 loss (Fig. 3 A, Right and C). However, this initial burstof GFP-expressing cells was not sustained; GFP-expressing, YY1-deficient cells were lost by 14 d after tamoxifen injection (Fig. 3B).The disappearance of YY1-deficient, GFP-positive cells over timeindicates that YY1-deficient stem cells are replaced by YY1-pro-ficient cells that were not targeted by Cre-recombinase. To cor-roborate this result, Yy1f/f; Lgr5-EGFP-Ires-CreERT2 mice weretreated for 5 consecutive days with tamoxifen to ablate Yy1 in Lgr5-expressing cells and then harvested immediately or after a 5-d chaseafter the tamoxifen treatment. Consistent with the reported mosaicexpression of the Lgr5-EGFP-Ires-CreERT2 allele, we saw a mosaicdistribution of YY1-postive (Fig. 3D, brown) and -negative (Fig. 3D,blue) epithelial stripes emanating from crypts of mice after 5 d oftamoxifen treatment; however, there were very few YY1-de-ficient cells remaining in the intestine after the 5-d chase (Fig.3D), further indicating that YY1-deficient stem cells are re-placed by YY1-proficient neighboring cells. These findings areconsistent with sustained expression of label-retaining reservestem cell markers on YY1 KO (Fig. 2A), although the exact originof the replacement cells was not defined. YY1−Lgr5+ stem cellreplacement by YY1+ cells also explains our observation that GFPexpression persists from the Lgr5 locus in tamoxifen-treated Yy1f/f;Lgr5-EGFP-Ires-CreERT2 mice (Fig. S2A). These mice do not ex-hibit a loss of GFP expression, which was observed when thepanepithelial Villin-CreERT2 driver was used to inactivate YY1throughout the epithelium (Fig. 2C). Taken together, these dataindicate that YY1 expression in stem cells is required for their long-term renewal, with stem cells lacking YY1 rapidly leaving theirniche and being replaced by YY1-expressing neighbors.

YY1 Deletion Causes Lgr5+ Stem Cell Loss Primarily by Differentiation.Loss of Lgr5+ stem cells upon YY1 deletion could be attributed tostem cell differentiation, apoptosis, or both. Stripes of YY1-deficient

stem cell progeny on tamoxifen treatment of Yy1f/f; Lgr5-EGFP-Ires-CreERT2 mice (Fig. 3D) suggested that YY1 deficiencymight prompt stem cells to leave their niche and acquire a tran-sit-amplifying (TA) cell identity. Because TA cells cycle aboutevery 12 h (45), approximately two times as fast as Lgr5+ stemcells (46), we anticipated an increased number of proliferatingcells in the YY1 KO because of an increased contribution ofLgr5+ cells to the TA population. Indeed, increased numbers ofBrdU+ crypt cells were observed when YY1 was inactivated (Fig.4 A and B), and YY1-deficient cells emanating from the Lgr5+

population were BrdU+ when in the TA zone (Fig. S2B), furthersuggesting that YY1-deficient Lgr5+ stem cells exit the niche toacquire a TA cell fate. Alternatively, the increase in BrdU+ cryptcells could be explained by an increased mitotic index on YY1loss in a TA cell-autonomous manner. However, because theBrdU+ cell increase in the TA zone was delayed and unsustained(Fig. 4 A and B), we favor the former possibility, because we notethat the timing of TA zone expansion is consistent with thetiming of stem cells loss from their niche (Figs. 2 and 3). Re-gardless of the cause of increased BrdU+ cells at 4 d after YY1KO, the increase in proliferation indicates that YY1 is notgenerally required for intestinal cell division but that Lgr5+ cellsrequire YY1 specifically to maintain stem cell renewal. We alsoinvestigated whether stem cell loss could be attributed to apo-ptosis. Immunohistochemistry for cleaved Caspase-3 showed thatapoptosis does contribute to the loss of stem cells after YY1 KO(Fig. 4C), with increased apoptosis observed near the base of thecrypt (Fig. 4D). However, the small fraction of crypts exhibitingan apoptotic event is incompatible with the number of stem cells

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Fig. 3. Lgr5+ cells lacking YY1 rapidly exit the niche and fail to renew. (Aand C) Lineage tracing in Yy1f/f; Lgr5-EGFP-IRES-creERT2; Rosa26-EGFP stemcells shows increased exodus of GFP+ cells from the crypt base on tamoxifentreatment compared with controls. (B) YY1-negative stem cell contribution isnot sustained long term, because GFP+ cells diminish by 14 d after tamoxifentreatment, indicating that YY1 loss in Lgr5+ cells is incompatible with theirlong-term renewal. (C) Quantification of average lineage-traced cell posi-tion in a number of cells to the top of GFP+ stripe. Replicate counts on onebiological replicate. Bars ± SE. No GFP-expressing YY1-negative stripes wereobserved at 14 d after tamoxifen injection. Rare stripes observed were YY1+,and therefore, they were not included in the quantification. (D) YY1immunostaining confirms that YY1-deficient stem cells give rise to YY1-deficient progeny (outlined cells are blue nuclei) but are eventually replacedby YY1-positive cells within 5 d of tamoxifen withdrawal (5d chase; Lower).IHC, immunohistochemistry.

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being depleted. We, therefore, favor the conclusion that Lgr5+

stem cell loss upon Yy1 deletion is primarily because of accel-erated stem cell exit from the niche, with minor contribution ofcell loss through apoptosis.

YY1 Binds to and Activates Mitochondrial Complex I Genes. To de-termine the mechanism underlying the dependence of stem cellon YY1, we performed microarray analysis on crypt epitheliaisolated from YY1 KO and littermate controls 4 d after tamox-ifen treatment. Of 39,000 transcripts on the microarray, 1,019were elevated, and 792 were reduced on Yy1 loss (P < 0.1 andfold change > 1.25). Gene ontology term analysis revealed cellcycle and mitotic functions prominent among the up-regulatedtranscripts (Fig. 5 A and B), consistent with the observed in-crease in proliferation on Yy1 loss (Fig. 4A). More revealing wasgene ontology categories enriched among down-regulated tran-scripts, including mitochondrial proteins (Fig. 5 A and B andDataset S1). Specifically, Yy1 deletion resulted in decreased ex-pression of several nuclear genes encoding mitochondrial com-plex I components (Fig. 5A). To determine whether YY1 wasdirectly regulating mitochondrial complex I genes, we used ChIPwith YY1-specific antibodies and deep sequencing of the immu-noprecipitated DNA (YY1 ChIP-seq). ChIP-seq analysis identified

4,261 YY1 binding sites (Model-based Analysis of ChIP-Seq P value< 10−4). Consistent with bona fide YY1 binding sites, YY1 ChIP-seq regions were enriched in the YY1 DNA binding motif (Fig.S3A), and binding regions were conserved across multiple verte-brate species (Fig. S3B). Genes nearby YY1 binding sites wereenriched for functions associated with RNA processing and mi-tochondria (Fig. 5D), echoing the results of Yy1 KO gene ex-pression analysis (Fig. 5B). Robust YY1 binding to mitochondrialcomplex components was observed at Ndufb5, Ndufs2, and Ndufa13,consistent with a direct role of YY1 in regulating mitochondrialcomplex I genes (Fig. 5C and Fig. S3 C and D). Together, thesedata suggest that YY1 influences the metabolic state of the cellthrough direct control of mitochondrial function.

Mitochondrial Ultrastructure Is Compromised and Oxidative DNADamage Occurs on YY1 Loss. We next investigated whether YY1loss compromised mitochondrial ultrastructure using trans-mission EM. Four days after induced YY1 KO, mitochondriaexhibited a range of structural defects, including a distendedintermembrane space and fragmented cristae (Fig. 6 A and B).Mitochondrial dysfunction has been shown to increase genera-tion of reactive oxygen species (ROS) and DNA damage (47–51). Indeed, immunohistochemistry revealed elevated levels of8-hydroxyguanosine, an antigen formed on DNA oxidation (Fig.6C), and γ-H2AX (a marker of ROS-induced DNA damage)(Fig. S4A) (52–56). Immunoreactivity for both markers wasspecifically enriched in YY1-deficient crypt bottoms, consistentwith disrupted mitochondrial function. To test our hypothesisthat ROS generation was contributing to YY1-deficient stemcell loss, we tested the ability of antioxidants to slow or reversethe phenotype. Intestinal organoids were derived from Yy1f/f;Vil-CreERT2 mice and after 3 d of culture, YY1 KO induced withtamoxifen and organoids scored for survival in the presenceor absence of α-tocopherol, an antioxidant that localizes to themitochondrial membranes and promotes mitochondrial integrity(57). On loss of YY1, organoids deteriorated over time; how-ever, in the presence of α-tocopherol, the organoids persisted inthe absence of YY1 over a 5-d time course, significantly longerthan vehicle-treated controls and with increased Lgr5 transcriptlevels (Fig. 6 D and E and Fig. S4B). These findings suggest thata major component of the YY1 KO phenotype is attributed tomitochondrial dysfunction and ROS generation.

DiscussionIn this report, we identify YY1 as previously unappreciatedregulator of intestinal stem cell homeostasis. Conditional abla-tion of Yy1 in the intestinal epithelium leads to short-term ex-pansion of proliferative cells in the crypt, driven in part byaccelerated contribution of Lgr5+ stem cells to the TA cell pop-ulation. However, increased proliferation is not sustained, becausethe stem cell population is ultimately exhausted, and loss of Yy1 isultimately fatal for the mouse. Lineage tracing analysis shows thatLgr5+ stem cells depend on YY1 for long-term self-renewal andthat YY1-deficient stem cells are replaced by YY1-expressing,

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neighboring epithelial cells. Mechanistically, we observe that mi-tochondrial complex I genes are under direct regulation of YY1 byChIP-seq and expression profiling analysis and that compromisedmitochondrial gene expression corresponds to disrupted mito-chondrial ultrastructure and oxidative damage. Together, theseworks implicate YY1 in intestinal stem cell homoeostasis andprovide a previously unidentified mechanism coupling stem cellmetabolism to stem cell renewal.YY1 has previously been linked to mitochondrial function in

skeletal muscle through the mechanistic target of rapamycin(mTOR) signaling pathway by the coregulator PGC-1α (26, 28,58), raising the possibility that the intestinal stem cell may usea similar mechanism to couple metabolic state and the renewalvs. differentiation decision. Indeed, recent work has implicatedthe mTOR pathway in homoestatic control of Lgr5+ stem cellnumbers based on nutrient availability (16). PGC-1α is alsonecessary for intestinal stem cell (ISC) renewal in Drosophila(19), suggesting that the process is conserved across species.Within mammalian species, oxidative stress also compromiseshematopoietic stem cell self-renewal (59). It will be interesting todetermine whether oxidative state is a common mechanism tomediate the renewal vs. differentiation decision of many stemcell types.Although stem cell renewal is compromised on YY1 loss,

proliferation of TA cells is decidedly unaffected, with increasednumbers of BrdU+ cells observed on YY1 loss. It is possible thatstem cells, which operate under a metabolism favoring glycolysis(17), are exquisitely sensitive to the mitochondrial dysfunctioninduced by YY1 loss, whereas transit-amplifying progeny aremore tolerant of ROS generated by a compromised mitochon-drial complex I. Alternatively, Lgr5+ stem cells may be less tol-erant to the DNA damage observed on YY1 loss, and DNAdamage could serve as a trigger for the differentiation decision.However, recent investigations report that Lgr5+ cells are rela-tively radiation resistant compared with their differentiatedprogeny (60). Although YY1 could certainly play multiple rolesin ISCs, because α-tocopherol only partially rescued YY1-deficient organoid growth, we favor compromised mitochondrialmetabolism as a primary explanation for failure of stem cellrenewal in the absence of YY1.Finally, we report intriguing and potentially related observa-

tions regarding stem cell turnover, metabolism, and the dis-crepancy between active and reserve stem cell marker transcriptlevels. Reserve stem cell populations are believed to be com-mitted progenitor cells that retain the ability to revert to the stemcell state on appropriate physiological conditions (61, 62). OnYY1 loss, reserve stem cells markers are unaffected, whereasactive stem cell markers are lost (Fig. 2 A and B). Underlying thisobservation may be the coupling between stem cell metabolism

and homeostasis, which was observed in a model in which cellsmarked by high GFP expression from an Sox9-EGFP BACtransgene represent a differentiated lineage with the potential toact as reserve stem cells (8). Sox9-EGFP high reserve cells un-dergo a shift in mitochondrial gene expression consistent witha shift to reduced oxidative metabolism when they acquirestemness after radiation-induced injury. It is possible that thereverse process triggers Lgr5+ stem cells to differentiate on mi-tochondrial dysfunction triggered by deletion of Yy1. How met-abolic state leads to regulation of stem cell-specific genes (andvice versa) is unclear, but it is likely an indirect consequence ofYY1 loss, because no direct binding of YY1 at stem cell geneswas observed. Curiously, whereas all Lgr5+ stem cell markerstested were reduced on YY1 loss, ASCL2, the stem cell tran-scription factor (41), was unchanged. We speculate that reservecells attempting to restore the Lgr5+ population are transcribingASCL2. Better appreciation of metabolic regulation of intestinalstem cells will continue to be of great interest, particularly withexpectations that regulation of normal stem cell homeostasis willbe applicable to understanding mechanisms of intestinal regen-eration and oncogenesis. We suggest that YY1 may function aspart of a critical bottleneck in tuning stem cell metabolism toniche-dependent regulatory signals.

Materials and MethodsCompound mouse genotypes were established by breeding Villin-CreER(T2)

transgenic mice (39), Lgr5-EGFP-Ires-CreERT2 knockin mice (43), Rosa-CAG-LSL-ZsGreen1-WPRE mice (63), and Yy1f/f mice (38). Yy1f/f; Villin-CreER(T2)

were injected with tamoxifen for 4 consecutive days and harvested thefollowing day for microarray and IHC analysis unless otherwise stated; 0.05mg tamoxifen per gram body weight was injected i.p., except for in Rosa-EGFPlineage tracing, where a single dose of tamoxifen (0.1 mg per gram bodyweight) was administered to Yy1f/f; Lgr5-EGFP-Ires-CreERT2; Rosa-CAG-LSL-ZsGreen1-WPRE (experimental) and Lgr5-EGFP-Ires-CreERT2; Rosa-CAG-LSL-ZsGreen1-WPRE (control) mice. Experiments were conducted according toprotocol 11–017 approved by the Institutional Animal Care and Use Commit-tee of Rutgers University. All tissues were collected between 12:00 and 14:00 hto avoid circadian variability.

Additional experimental details on tissue preparation, histology, micros-copy, and genomics can be found in SI Materials and Methods. All omics datacan be accessed in Gene Expression Omnibus (accession no. GSE53503).

ACKNOWLEDGMENTS. The authors acknowledge David Axelrod for criticalcomments on the manuscript and Chen X. Chen, C. S. Yang, Josh Thackray,Noriko Goldsmith, and Lourdes Serrano for technical advice on immunos-taining and imaging. This work was supported by a postdoctoral fellowship(to A.O.P.) and graduate fellowship (to A.H.) from the New JerseyCommission on Cancer Research, an undergraduate research fellowshipfrom Rutgers (to M.J.V.), a startup grant from the Human Genetics Instituteof New Jersey, and National Institutes of Health Grants K01DK085194 (toN.G.), R03DK099251 (to M.P.V.), and K01DK08886 (to M.P.V.).

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Fig. 6. YY1 loss leads to compromised mi-tochondrial structure and oxidative DNAdamage. (A) Transmission EM of controls andYY1 KOs reveals compromised mitochondrialstructure 4 d after YY1 loss in crypt basecolumnar cells, including outer and innermembrane deterioration (arrowheads) anddilation of the intermembrane space (arrows).(Scale bar: 200 nm.) (B) Quantification ofthe histopathology of >100 mitochondriafrom each genotype revealed defects inthe majority of YY1 KO mitochondria. (C )Consistent with mitochondrial dysfunction,8-hydroxyguanosine (8-OH-dG) immuno-staining shows increased levels of oxidizedDNA in YY1 KO cells. (D) Intestinal organoidsderived from YY1 conditional KO mice wereinduced with tamoxifen (Tam) and moni-tored for viability. In the absence of YY1,organoids fail to expand and survive, but survival is prolonged with addition of the antioxidant α-tocopherol (αT). n = 3 biological replicates; SEbars. (E ) Representative images of organoids from the experiment detailed in D.

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