YY1 is indispensable for Lgr5 + intestinal stem cell renewal

YY1 is indispensable for Lgr5+ intestinal stem cell renewal Ansu O. Perekatta, Michael J. Valdeza, Melanie Davilaa, A. Hoffmana, Edward M. Bonderb, Nan Gaob, and Michael P. Verzia,1 a Department of Genetics, Human Genetics Institute of New Jersey, Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, Piscataway, NJ 08854; and bDepartment of Biological Sciences, Rutgers, The State University of New Jersey, Newark, NJ 07102 The intestinal stem cell fuels the highest rate of tissue turnover in the body and has been implicated in intestinal disease and cancer; understanding the regulatory mechanisms controlling intestinal stem cell physiology is of great importance. Here, we provide evidence that the transcription factor YY1 is essential for intestinal stem cell renewal. We observe that YY1 loss skews normal homeostatic cell turnover, with an increase in proliferating crypt cells and a decrease in their differentiated villous progeny. Increased crypt cell numbers come at the expense of Lgr5+ stem cells. On YY1 deletion, Lgr5+ cells accelerate their commitment to the differentiated population, exhibit increased levels of apoptosis, and fail to maintain stem cell renewal. Loss of Yy1 in the intestine is ultimately fatal. Mechanistically, YY1 seems to play a role in stem cell energy metabolism, with mitochondrial complex I genes bound directly by YY1 and their transcript levels decreasing on YY1 loss. These unappreciated YY1 functions broaden our understanding of metabolic regulation in intestinal stem cell homeostasis. transcriptional regulation | mitochondria | crypt base columnar cell Downloaded by guest on June 12, 2020 T he 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 epithelial pockets tucked into the intestinal wall called crypts of Lieberkühn. Intestinal stem cells give rise to all other intestinal epithelial lineages and maintain their own population indefinitely (1). A number of transgenic reporters has been used in lineage tracing assays to show stem cell activity arising from the base of the crypt (2–8), and all have been reported to overlap with crypt base columnar cells (9), which cooccupy the bottom of crypts with differentiated Paneth cells. Intestinal stem cells marked by leucine rich repeat containing G protein coupled receptor 5 (Lgr5) expression have been the most extensively characterized; these cells maintain their own population through symmetric divisions (10), and when they leave the niche, they give rise to differentiated crypt cells, including a transit amplifying population that ultimately supplies differentiated cells onto luminal projections called villi. Intestinal stem cells are of great importance to human health and regenerative medicine. Mouse models of human colorectal cancer show that intestinal stem cells can function as cells of origin for cancer (11, 12). There is a clear imperative to understand the regulatory mechanisms governing intestinal stem cell function. Recent work has shown that intestinal stem cells from both flies and humans are sensitive to the metabolic state of the organism and has implicated cellular metabolism as a critical regulatory input of stem cell homeostasis (13–16). Intestinal stem cells were observed to exhibit higher levels of glycolysis than oxidative phosphorylation compared with their differentiated progeny (17), and the oxidative state of intestinal stem cells impacts the ability of the cells to undergo transformation (18). Caloric restriction was recently shown to increase intestinal stem cell numbers (16). In Drosophila, intestinal stem cell expression of the fly homolog to PGC-1α, a metabolic coregulator, is even coupled to the organism’s lifespan (19). These exciting advances www.pnas.org/cgi/doi/10.1073/pnas.1400128111 highlight a great need to identify additional regulators of intestinal stem cell metabolism. YY1 is a zinc finger transcription factor first discovered for its function in viral gene expression (20, 21) and cloned during investigations of viral (22), immunoglobulin (23), and ribosomal (24) gene expression. YY1 has since been implicated in a number of processes, 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 counterintuitively, suppresses induced pluriopotent cell reprogramming (34). In the blood, YY1 overexpression promotes long-term hematopoietic stem cell maintenance (37). Conversely, YY1 promotes satellite cell activation and differentiation during muscle regeneration (35). Thus, context-specific stem cell functions have been attributed to YY1, potentially owing to its ability to operate in diverse protein complexes. The role of YY1 in intestinal 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 in the ratio of crypt to villus cell populations in the intestine, with expanded crypt length and an increased zone of cell proliferation. Functional lineage tracing assays showed that intestinal stem cells lacking YY1 undergo an increased exit from their niche, which corresponds to the expanded proliferative zone. Ultimately, depletion of the stem cell population was observed, which was indicated by transgenic markers, gene expression, and EM. To understand the functional mechanisms of YY1 in stem cell maintenance, expression profiling and ChIP were used to reveal that YY1 binds to mitochondrial complex I genes and is required for their expression. Our work shows that YY1 is Significance A subset of our body’s tissues is continuously renewed through cell division. Tissue-specific stem cells support this tissue turnover, and understanding the mechanisms that control the behavior of these stem cells is important to understanding the health of the tissue. In this work, we identify a novel regulator of the intestinal stem cells. We find that, when the transcription factor YY1 is inactivated, intestinal stem cells can no longer renew themselves. We show that YY1 controls mitochondrial gene expression, and loss of YY1 results in loss of mitochondrial structural integrity. This work, therefore, provides a link between a mitochondrial regulator and stem cell function and broadens our appreciation of metabolic regulation in tissuespecific 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. analyzed 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 Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE53503). 1 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1400128111/-/DCSupplemental. PNAS | May 27, 2014 | vol. 111 | no. 21 | 7695–7700 DEVELOPMENTAL BIOLOGY Edited by Brigid L. M. Hogan, Duke University Medical Center, Durham, NC, and approved April 17, 2014 (received for review January 4, 2014) C E Control YY1 IHC F D 130 110 90 70 WT YY1-KO ** ** n=8 50 YY1-KO YY1 IHC 0 1 2 3 4 5 6 7 8 9 Days After Tamoxifen Injection G Fig. 1. YY1 KO in the intestinal epithelium triggers weight loss and death. YY1 immunoreactivity (brown) is (A and C) seen throughout the intestinal epithelium 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 but preserved in the lamina propria. (E) Significant weight loss is observed after epithelial KO of Yy1 is induced. **Unpaired two-tailed t test, P < 0.01. (F) Relative YY1 immunoreactivity is stronger in cells with crypt base columnar morphology (black arrows) and weaker in adjacent Paneth cells (white arrows). (G) After 10 d of YY1 KO, large, YY1+ hyperplastic crypts are observed (arrows; Inset), presumably arising from epithelial cells escaping Cremediated recombination. IHC, immunohistochemistry. (Scale bars: 50 μm.) Lgr5+ and CBC Cells Are Lost on YY1 Deletion. The hyperplastic crypts observed in YY1 KO mice (Fig. 1G) are reminiscent of regenerative foci observed in epithelia recovering from loss of intestinal stem cell regulatory factors, such as Myc (40) or ASCL2 (41). The robust YY1 expression observed in CBCs (Fig. 1F) also suggested that YY1 could function as an essential regulator of the stem cell. To investigate the role of YY1 in stem cell function, we measured the relative transcript levels of proposed stem cell markers in YY1 KO and control mouse epithelia. YY1 KO mice showed a significant decrease in expression of several genes comprising an Lgr5+ stem cell signature (42), including Lgr5, Olfm4, and Smoc2. Conversely, expression of markers associated with a more quiescent reserve stem cell activity was not affected, including Bmi1, Hopx, Lrig1, and Tert (Fig. 2 A and B). These data suggest that YY1 is required specifically for an active Lgr5+ stem cell population. To further explore YY1 function in Lgr5+ stem cells, we generated mice in which we could both inactivate YY1 throughout the epithelium and monitor the Lgr5-expressing cell population with an Lgr5GFP knockin allele: Yy1f/f;Vil-creERT2; Lgr5-EGFP-IREScreERT2 (43). Mice treated with tamoxifen and monitored for 4, 5, or 7 d showed a decrease in GFP expression over time, with no detectible GFP+ cells remaining at 7 d after tamoxifen treatment A 1.5 Relative transcript levels B % of Original Weight A 1.25 WT YY1-KO ** ** ** Yy1 Lgr5 Olfm4 Smoc2 Msi1 0.5 0.25 0 Hprt Ascl2 Lgr5+ Cell Markers C HOPX IHC Bmi1 Hopx Tert Lrig1 “+4” Associated Markers YY1f/f; VilCreERT2;Lgr5-GFP Day 0 Control Results ** 0.75 B required for intestinal stem cell renewal and links YY1 to cellular metabolism of the intestinal stem cell. ** 1 Downloaded by guest on June 12, 2020 YY1 Is Required to Maintain Intestinal Homeostasis and Viability. 7696 | www.pnas.org/cgi/doi/10.1073/pnas.1400128111 YY1 KO Day 4 Day 5 D TEM Whether YY1 is expressed or contributes to the homeostatic renewal of the adult intestinal epithelium is untested. We observed YY1 immunoreactivity throughout the length of the gut and 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 in Paneth and Goblet cells was lower by comparison (Fig. 1 A and F). To determine the function of YY1 in the intestinal epithelium, we inactivated a conditional Yy1 allele with a tamoxifeninducible, epithelium-specific Cre driver, Yy1f/f;Vil-Cre-ERT2 (38, 39). YY1 immunostain in the epithelium was specifically lost on tamoxifen treatment in adult mice (Fig. 1 B and D), whereas cells in the lamina propria remained YY1-positive (Fig. 1 B and D). Eleven days after the first tamoxifen injection, Yy1f/f;Vil-Cre-ERT2 mice lost weight (Fig. 1E) and became moribund, prompting us to halt the experiment, and indicating that epithelial functions of YY1 are essential for viability; 4 d after induced YY1 KO, both crypts and villi were notably elongated, but by 10 d post-KO, villi became significantly shorter than in controls, and crypts appeared further elongated and sinuous (Fig. S1), indicating that YY1 function is required for normal intestinal homeostasis. Interestingly, 10 d after YY1 KO was induced, occasional hyperproliferative crypts were observed, characteristic of regenerative foci (Fig. 1G). The cells within these foci were positive for YY1 protein expression, suggesting that these foci originate from epithelial cells that had escaped Yy1 deletion. Control YY1 KO Day 7 Fig. 2. Lgr5+ and CBC cells are lost on deletion of Yy1 in the intestinal epithelium. (A) Relative transcript levels of Lgr5+ cell markers decrease in isolated YY1 KO crypts. Bars represent ± SE on four replicates. **P < 0.01, two-tailed t test. (B) HOPX immunoreactivity is preserved in the YY1 KO (circled cells are brown nuclei), consistent with preserved transcript levels (A). (C) An Lgr5-GFP knockin allele was integrated with Yy1f/f; Villin-creERT2 alleles to monitor GFP expression over 7 d after tamoxifen treatment to inactivate YY1. Green GFP+ cells diminish over time and are no longer detected at 7 d. Blue, DAPI counterstain. (Scale bar: 50 μm.) (D) Transmission EM (TEM) indicates reduction of cells with CBC morphology (outlined in white) in Yy1f/f; Villin- creERT2 after 4 d of tamoxifen treatment. Basement membrane is shown by the black dashed line. (Scale bar: 2 μm.) Perekatt et al. Downloaded by guest on June 12, 2020 YY1 Deletion Causes Lgr5+ Stem Cell Loss Primarily by Differentiation. Loss of Lgr5+ stem cells upon YY1 deletion could be attributed to stem cell differentiation, apoptosis, or both. Stripes of YY1-deficient Perekatt et al. C 120 GFP stripe height (# of cells) YY1f/f; Lgr5-CreERT2; ROSA-GFP B 5d pulse, 5d chase D 5d Tam pulse 1 day ROSA-GFP 2 day 100 Control KO 80 60 40 20 0 1 day 2 day 3 day 14 day YY1f/f; Lgr5-CreERT2-GFP YY1 IHC YY1 IHC Fig. 3. Lgr5+ cells lacking YY1 rapidly exit the niche and fail to renew. (A and C) Lineage tracing in Yy1f/f; Lgr5-EGFP-IRES-creERT2; Rosa26-EGFP stem cells shows increased exodus of GFP+ cells from the crypt base on tamoxifen treatment compared with controls. (B) YY1-negative stem cell contribution is not sustained long term, because GFP+ cells diminish by 14 d after tamoxifen treatment, indicating that YY1 loss in Lgr5+ cells is incompatible with their long-term renewal. (C) Quantification of average lineage-traced cell position in a number of cells to the top of GFP+ stripe. Replicate counts on one biological replicate. Bars ± SE. No GFP-expressing YY1-negative stripes were observed at 14 d after tamoxifen injection. Rare stripes observed were YY1+, and therefore, they were not included in the quantification. (D) YY1 immunostaining confirms that YY1-deficient stem cells give rise to YY1deficient progeny (outlined cells are blue nuclei) but are eventually replaced by YY1-positive cells within 5 d of tamoxifen withdrawal (5d chase; Lower). IHC, immunohistochemistry. stem cell progeny on tamoxifen treatment of Yy1f/f; Lgr5-EGFPIres-CreERT2 mice (Fig. 3D) suggested that YY1 deficiency might prompt stem cells to leave their niche and acquire a transit-amplifying (TA) cell identity. Because TA cells cycle about every 12 h (45), approximately two times as fast as Lgr5+ stem cells (46), we anticipated an increased number of proliferating cells in the YY1 KO because of an increased contribution of Lgr5+ cells to the TA population. Indeed, increased numbers of BrdU+ 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), further suggesting that YY1-deficient Lgr5+ stem cells exit the niche to acquire a TA cell fate. Alternatively, the increase in BrdU+ crypt cells could be explained by an increased mitotic index on YY1 loss in a TA cell-autonomous manner. However, because the BrdU+ cell increase in the TA zone was delayed and unsustained (Fig. 4 A and B), we favor the former possibility, because we note that the timing of TA zone expansion is consistent with the timing of stem cells loss from their niche (Figs. 2 and 3). Regardless of the cause of increased BrdU+ cells at 4 d after YY1 KO, the increase in proliferation indicates that YY1 is not generally required for intestinal cell division but that Lgr5+ cells require YY1 specifically to maintain stem cell renewal. We also investigated whether stem cell loss could be attributed to apoptosis. Immunohistochemistry for cleaved Caspase-3 showed that apoptosis does contribute to the loss of stem cells after YY1 KO (Fig. 4C), with increased apoptosis observed near the base of the crypt (Fig. 4D). However, the small fraction of crypts exhibiting an apoptotic event is incompatible with the number of stem cells PNAS | May 27, 2014 | vol. 111 | no. 21 | 7697 DEVELOPMENTAL BIOLOGY in the epithelium is necessary for stem cell renewal, the specific cells that require YY1 to maintain stem cell homeostasis were not clear; stem cells could require YY1 expression autonomously or YY1 function in neighboring cells to establish a supportive niche. To test for a stem cell autonomous function, we deleted YY1 specifically within Lgr5+ stem cells using the Lgr5-GFP-IRES-Cre driver (43) and followed the fate of these Yy1-deleted stem cells by lineage tracing. Use of a Cre-activated reporter allele (such as Cre-induced GFP expression from the Rosa-CAG-LSL-ZsGreen1-WPRE allele) combined with the Lgr5-EGFP-IRES-creERT2 Cre driver allows for sustained expression of GFP in Lgr5-Cre–expressing cells and all their descendants (43, 44). In control mice (Lgr5-EGFP-IREScreERT2; Rosa-CAG-LSL-ZsGreen1-WPRE), robust GFP expression from the ROSA locus was activated in Lgr5+ cells on tamoxifen treatment, and their GFP-expressing descendent cells could be visualized leaving the crypt base and migrating onto the villi over time, consistent with published reports (43). Two weeks after tamoxifen treatment, sustained stripes of GFP-expressing epithelium spanned the crypt–villus axis, indicating that labeled stem cells were competent to maintain a supply of GFP-marked stem cells and GFP-expressing progeny (Fig. 3 A, Left and B, Left). In mice with the same genotype but also harboring the conditional Yy1 alleles, tamoxifen treatment both inactivated Yy1 and activated GFP expression from the ROSA locus, specifically in the Lgr5+ stem cells. Interestingly, GFP-positive descendants of YY1-deficient stem cells showed an accelerated exodus from the crypt compartment relative to controls, indicating a more robust contribution of stem cells to the differentiation stream on YY1 loss (Fig. 3 A, Right and C). However, this initial burst of GFP-expressing cells was not sustained; GFP-expressing, YY1deficient cells were lost by 14 d after tamoxifen injection (Fig. 3B). The disappearance of YY1-deficient, GFP-positive cells over time indicates that YY1-deficient stem cells are replaced by YY1-proficient cells that were not targeted by Cre-recombinase. To corroborate this result, Yy1f/f; Lgr5-EGFP-Ires-CreERT2 mice were treated for 5 consecutive days with tamoxifen to ablate Yy1 in Lgr5expressing cells and then harvested immediately or after a 5-d chase after the tamoxifen treatment. Consistent with the reported mosaic expression of the Lgr5-EGFP-Ires-CreERT2 allele, we saw a mosaic distribution of YY1-postive (Fig. 3D, brown) and -negative (Fig. 3D, blue) epithelial stripes emanating from crypts of mice after 5 d of tamoxifen treatment; however, there were very few YY1-deficient cells remaining in the intestine after the 5-d chase (Fig. 3D), further indicating that YY1-deficient stem cells are replaced by YY1-proficient neighboring cells. These findings are consistent with sustained expression of label-retaining reserve stem cell markers on YY1 KO (Fig. 2A), although the exact origin of the replacement cells was not defined. YY1−Lgr5+ stem cell replacement by YY1+ cells also explains our observation that GFP expression persists from the Lgr5 locus in tamoxifen-treated Yy1f/f; Lgr5-EGFP-Ires-CreERT2 mice (Fig. S2A). These mice do not exhibit a loss of GFP expression, which was observed when the panepithelial Villin-CreERT2 driver was used to inactivate YY1 throughout the epithelium (Fig. 2C). Taken together, these data indicate that YY1 expression in stem cells is required for their longterm renewal, with stem cells lacking YY1 rapidly leaving their niche and being replaced by YY1-expressing neighbors. T2; 3 day Lgr5+ Stem Cells Require YY1 for Renewal. Although YY1 expression A Lgr5-CreER 14 day (Fig. 2C). Consistent with the loss of the Lgr5+ stem cell population on Yy1 deletion, examination of crypt ultrastructure by transmission EM confirmed the loss of cells with the CBC stem cell morphology (Fig. 2D); 90% of crypts observed in the control condition contained cells with CBC morphology, whereas less than 10% of crypts observed in the YY1 KO crypts contained cells with CBC morphology. Taken together, YY1 seems necessary for maintenance of the Lgr5+ stem cell population. B 1.4 ** 1.3 1.2 BrdU Fold Change in BrdU+ Cells A 1.1 1 0.9 0.8 Control D 4 Day KO 10 Day KO 30 % of crypts with apoptotic cells Cl. Caspase 3 C 2 3 4 10 Days Post YY1 KO Control YY1 KO 20 10 0 Cell position from crypt base Fig. 4. Stem cell loss in YY1 mutants results from apoptosis and accelerated differentiation. (A) Increased numbers of BrdU+ cells are observed in YY1 KO crypts, peaking at 4 d posttamoxifen treatment. Bars indicate ± SE. **P < 0.01, unpaired two-tailed t test. (B) BrdU immunostaining shows increased proliferation on 4 d of tamoxifen administration to Yy1f/f; Villin-creERT2 mice, coinciding with increased stem cells exiting their niche. (Scale bar: 10 μm.) (C and D) Cleaved Caspase-3 immunostain indicates that increased apoptosis contributes to stem cell loss, but the small percentage of crypts with an apoptotic event fails to explain the amount of stem cells lost. YY1-deficient cells are observed higher up the villus (Fig. 3D), indicating that most cells escape apoptosis. Error bars represent ± SE on three biological replicates. (Scale bar: 25 μm.) being depleted. We, therefore, favor the conclusion that Lgr5+ stem cell loss upon Yy1 deletion is primarily because of accelerated stem cell exit from the niche, with minor contribution of cell loss through apoptosis. YY1 Binds to and Activates Mitochondrial Complex I Genes. To de- termine the mechanism underlying the dependence of stem cell on YY1, we performed microarray analysis on crypt epithelia isolated from YY1 KO and littermate controls 4 d after tamoxifen treatment. Of 39,000 transcripts on the microarray, 1,019 were elevated, and 792 were reduced on Yy1 loss (P < 0.1 and fold change > 1.25). Gene ontology term analysis revealed cell cycle and mitotic functions prominent among the up-regulated transcripts (Fig. 5 A and B), consistent with the observed increase in proliferation on Yy1 loss (Fig. 4A). More revealing was gene ontology categories enriched among down-regulated transcripts, including mitochondrial proteins (Fig. 5 A and B and Dataset S1). Specifically, Yy1 deletion resulted in decreased expression of several nuclear genes encoding mitochondrial complex I components (Fig. 5A). To determine whether YY1 was directly regulating mitochondrial complex I genes, we used ChIP with YY1-specific antibodies and deep sequencing of the immunoprecipitated DNA (YY1 ChIP-seq). ChIP-seq analysis identified Catalase Ndufc1 Ppargc1a Naprt Fmo4 YY1 ChIP-Seq Tags Upregulated Ndufb8 Ndufs2 Ndufb4 Ndufs7 Ndufa3 Ndufb11 YY1-KO Genes RNA processing cell cycle mitotic cell cycle translation 0 40 generation of precursor metabolites oxidation reduction cofactor metabolic process 0 4 - log10 p-value 8 loss compromised mitochondrial ultrastructure using transmission EM. Four days after induced YY1 KO, mitochondria exhibited a range of structural defects, including a distended intermembrane space and fragmented cristae (Fig. 6 A and B). Mitochondrial dysfunction has been shown to increase generation of reactive oxygen species (ROS) and DNA damage (47– 51). Indeed, immunohistochemistry revealed elevated levels of 8-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 was specifically enriched in YY1-deficient crypt bottoms, consistent with disrupted mitochondrial function. To test our hypothesis that ROS generation was contributing to YY1-deficient stem cell loss, we tested the ability of antioxidants to slow or reverse the phenotype. Intestinal organoids were derived from Yy1f/f; Vil-CreERT2 mice and after 3 d of culture, YY1 KO induced with tamoxifen and organoids scored for survival in the presence or absence of α-tocopherol, an antioxidant that localizes to the mitochondrial membranes and promotes mitochondrial integrity (57). On loss of YY1, organoids deteriorated over time; however, in the presence of α-tocopherol, the organoids persisted in the absence of YY1 over a 5-d time course, significantly longer than vehicle-treated controls and with increased Lgr5 transcript levels (Fig. 6 D and E and Fig. S4B). These findings suggest that a major component of the YY1 KO phenotype is attributed to mitochondrial dysfunction and ROS generation. Discussion In this report, we identify YY1 as previously unappreciated regulator of intestinal stem cell homeostasis. Conditional ablation of Yy1 in the intestinal epithelium leads to short-term expansion of proliferative cells in the crypt, driven in part by accelerated contribution of Lgr5+ stem cells to the TA cell population. However, increased proliferation is not sustained, because the stem cell population is ultimately exhausted, and loss of Yy1 is ultimately fatal for the mouse. Lineage tracing analysis shows that Lgr5+ stem cells depend on YY1 for long-term self-renewal and that YY1-deficient stem cells are replaced by YY1-expressing, 5kb Ndufs2 D 7698 | www.pnas.org/cgi/doi/10.1073/pnas.1400128111 Mitochondrial Ultrastructure Is Compromised and Oxidative DNA Damage Occurs on YY1 Loss. We next investigated whether YY1 YY1 ChIP-seq 3 20 mitochondrion Downregulated Ndufb5 Downloaded by guest on June 12, 2020 C 10 B GO Terms Enriched among ChIP-seq Target Genes GO Term Enrichment A 4,261 YY1 binding sites (Model-based Analysis of ChIP-Seq P value < 10−4). Consistent with bona fide YY1 binding sites, YY1 ChIPseq regions were enriched in the YY1 DNA binding motif (Fig. S3A), and binding regions were conserved across multiple vertebrate species (Fig. S3B). Genes nearby YY1 binding sites were enriched for functions associated with RNA processing and mitochondria (Fig. 5D), echoing the results of Yy1 KO gene expression analysis (Fig. 5B). Robust YY1 binding to mitochondrial complex components was observed at Ndufb5, Ndufs2, and Ndufa13, consistent with a direct role of YY1 in regulating mitochondrial complex I genes (Fig. 5C and Fig. S3 C and D). Together, these data suggest that YY1 influences the metabolic state of the cell through direct control of mitochondrial function. ribonucleoprotein complex intracellular organelle lumen mitochondrian ribosome chromatin organization 0 5 - log10 p-value 10 Fig. 5. YY1 binds and regulates mitochondrial complex I genes. (A and B) Mitochondrial complex I genes, among other metabolic and oxidative related gene ontology (GO) categories, were enriched among transcripts down-regulated in Yy1f/f; Villin-creERT2 crypt epithelia 4 d after tamoxifen treatment. Cell cycle and mitotic functions were among the most up-regulated GO categories, consistent with the proliferative burst observed on YY1 loss (Fig. 4A). (C) Representative YY1 binding event at the mitochondrial complex I gene Ndufs2. (D) GO terms enriched among genes nearby YY1 binding sites include ribosomal and mitochondrial functions. Perekatt et al. Fig. 6. YY1 loss leads to compromised miWT KO KO 140 tochondrial structure and oxidative DNA 120 damage. (A) Transmission EM of controls and Control 100 YY1 KOs reveals compromised mitochondrial Tam + 80 structure 4 d after YY1 loss in crypt base 500 μM αT columnar cells, including outer and inner 60 Tam + 100μM αT 100 membrane deterioration (arrowheads) and 40 CT Tam + 80 dilation of the intermembrane space (arrows). 10 μM αT KO 20 60 (Scale bar: 200 nm.) (B) Quantification of Tam only 40 0 the histopathology of >100 mitochondria 0 1 2 3 4 5 20 from each genotype revealed defects in Days post tamoxifen induced knockout 0 the majority of YY1 KO mitochondria. (C ) Grade 0 Grade 1 Grade 2 Grade 3 Control KO KO+100μM αT Consistent with mitochondrial dysfunction, KO WT 8-hydroxyguanosine (8-OH-dG) immunostaining shows increased levels of oxidized DNA in YY1 KO cells. (D) Intestinal organoids derived from YY1 conditional KO mice were induced with tamoxifen (Tam) and monitored 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; SE bars. (E ) Representative images of organoids from the experiment detailed in D. Mitochondrial Histopathology (%) B % of surviving organoids D A E Downloaded by guest on June 12, 2020 neighboring epithelial cells. Mechanistically, we observe that mitochondrial complex I genes are under direct regulation of YY1 by ChIP-seq and expression profiling analysis and that compromised mitochondrial gene expression corresponds to disrupted mitochondrial ultrastructure and oxidative damage. Together, these works implicate YY1 in intestinal stem cell homoeostasis and provide a previously unidentified mechanism coupling stem cell metabolism 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 use a similar mechanism to couple metabolic state and the renewal vs. differentiation decision. Indeed, recent work has implicated the mTOR pathway in homoestatic control of Lgr5+ stem cell numbers based on nutrient availability (16). PGC-1α is also necessary for intestinal stem cell (ISC) renewal in Drosophila (19), suggesting that the process is conserved across species. Within mammalian species, oxidative stress also compromises hematopoietic stem cell self-renewal (59). It will be interesting to determine whether oxidative state is a common mechanism to mediate the renewal vs. differentiation decision of many stem cell types. Although stem cell renewal is compromised on YY1 loss, proliferation of TA cells is decidedly unaffected, with increased numbers of BrdU+ cells observed on YY1 loss. It is possible that stem cells, which operate under a metabolism favoring glycolysis (17), are exquisitely sensitive to the mitochondrial dysfunction induced by YY1 loss, whereas transit-amplifying progeny are more tolerant of ROS generated by a compromised mitochondrial complex I. Alternatively, Lgr5+ stem cells may be less tolerant to the DNA damage observed on YY1 loss, and DNA damage could serve as a trigger for the differentiation decision. However, recent investigations report that Lgr5+ cells are relatively radiation resistant compared with their differentiated progeny (60). Although YY1 could certainly play multiple roles in ISCs, because α-tocopherol only partially rescued YY1deficient organoid growth, we favor compromised mitochondrial metabolism as a primary explanation for failure of stem cell renewal in the absence of YY1. Finally, we report intriguing and potentially related observations regarding stem cell turnover, metabolism, and the discrepancy between active and reserve stem cell marker transcript levels. Reserve stem cell populations are believed to be committed progenitor cells that retain the ability to revert to the stem cell state on appropriate physiological conditions (61, 62). On YY1 loss, reserve stem cells markers are unaffected, whereas active stem cell markers are lost (Fig. 2 A and B). Underlying this observation may be the coupling between stem cell metabolism Perekatt et al. and homeostasis, which was observed in a model in which cells marked by high GFP expression from an Sox9-EGFP BAC transgene represent a differentiated lineage with the potential to act as reserve stem cells (8). Sox9-EGFP high reserve cells undergo a shift in mitochondrial gene expression consistent with a shift to reduced oxidative metabolism when they acquire stemness after radiation-induced injury. It is possible that the reverse process triggers Lgr5+ stem cells to differentiate on mitochondrial dysfunction triggered by deletion of Yy1. How metabolic state leads to regulation of stem cell-specific genes (and vice versa) is unclear, but it is likely an indirect consequence of YY1 loss, because no direct binding of YY1 at stem cell genes was observed. Curiously, whereas all Lgr5+ stem cell markers tested were reduced on YY1 loss, ASCL2, the stem cell transcription factor (41), was unchanged. We speculate that reserve cells attempting to restore the Lgr5+ population are transcribing ASCL2. Better appreciation of metabolic regulation of intestinal stem cells will continue to be of great interest, particularly with expectations that regulation of normal stem cell homeostasis will be applicable to understanding mechanisms of intestinal regeneration and oncogenesis. We suggest that YY1 may function as part of a critical bottleneck in tuning stem cell metabolism to niche-dependent regulatory signals. Materials and Methods Compound mouse genotypes were established by breeding Villin-CreER(T2) transgenic mice (39), Lgr5-EGFP-Ires-CreERT2 knockin mice (43), Rosa-CAGLSL-ZsGreen1-WPRE mice (63), and Yy1f/f mice (38). Yy1f/f; Villin-CreER(T2) were injected with tamoxifen for 4 consecutive days and harvested the following day for microarray and IHC analysis unless otherwise stated; 0.05 mg tamoxifen per gram body weight was injected i.p., except for in Rosa-EGFP lineage tracing, where a single dose of tamoxifen (0.1 mg per gram body weight) was administered to Yy1f/f; Lgr5-EGFP-Ires-CreERT2; Rosa-CAG-LSLZsGreen1-WPRE (experimental) and Lgr5-EGFP-Ires-CreERT2; Rosa-CAG-LSLZsGreen1-WPRE (control) mice. Experiments were conducted according to protocol 11–017 approved by the Institutional Animal Care and Use Committee of Rutgers University. All tissues were collected between 12:00 and 14:00 h to avoid circadian variability. Additional experimental details on tissue preparation, histology, microscopy, and genomics can be found in SI Materials and Methods. All omics data can be accessed in Gene Expression Omnibus (accession no. GSE53503). ACKNOWLEDGMENTS. The authors acknowledge David Axelrod for critical comments on the manuscript and Chen X. Chen, C. S. Yang, Josh Thackray, Noriko Goldsmith, and Lourdes Serrano for technical advice on immunostaining and imaging. This work was supported by a postdoctoral fellowship (to A.O.P.) and graduate fellowship (to A.H.) from the New Jersey Commission on Cancer Research, an undergraduate research fellowship from Rutgers (to M.J.V.), a startup grant from the Human Genetics Institute of New Jersey, and National Institutes of Health Grants K01DK085194 (to N.G.), R03DK099251 (to M.P.V.), and K01DK08886 (to M.P.V.). PNAS | May 27, 2014 | vol. 111 | no. 21 | 7699 DEVELOPMENTAL BIOLOGY 8-OH-dG C Downloaded by guest on June 12, 2020 1. Clevers H (2013) The intestinal crypt, a prototype stem cell compartment. Cell 154(2): 274–284. 2. Sangiorgi E, Capecchi MR (2008) Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet 40(7):915–920. 3. 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