Neoadjuvant systemic therapy refers to the use of systemic agent(s) for malignancy prior to surgical treatment and has recently emerged as an option for most breast cancer patients eligible for adjuvant systemic therapy. Consequently, treated breast carcinomas have become routine specimens in pathology practices. A standard protocol has not yet been universally adopted for the evaluation and reporting of these specimens. The American Joint Committee on Cancer staging system recognizes the challenges in staging breast carcinomas after neoadjuvant treatment and provides important data points but does not currently provide detailed guidance in estimating the residual tumor burden in the breast and lymph nodes. The Residual Cancer Burden system is the only Web-based system that quantifies treatment response as a continuous variable using residual tumor burden in the breast and the lymph nodes.To provide clarifications and guidance for evaluation and reporting of postneoadjuvant breast specimens, discuss issues with the current staging and reporting systems, and provide specific suggestions for future modifications to the American Joint Committee on Cancer system and the Residual Cancer Burden calculator.English-language literature on the subject and the data from the I-SPY 2, a multicenter, adaptive randomization phase 2 neoadjuvant platform trial for early-stage, high-risk breast cancer patients.This article highlights challenges in the pathologic evaluation and reporting of treated breast carcinomas and provides recommendations and clarifications for pathologists and clinicians. It also provides specific recommendations for staging and discusses future directions.
The primary origin of some ovarian mucinous tumors may be challenging to determine, because some metastases of extraovarian origin may exhibit gross, microscopic, and immunohistochemical features that are shared by some primary ovarian mucinous tumors. Metastases of primary colorectal, appendiceal, gastric, pancreatic, and endocervical adenocarcinomas may simulate primary ovarian mucinous cystadenoma, mucinous borderline tumor, or mucinous adenocarcinoma. Recently, immunohistochemical expression of SATB2, a transcriptional regulator involved in osteoblastic and neuronal differentiation, has been shown to be a highly sensitive marker of normal colorectal epithelium and of colorectal adenocarcinoma. SATB2 expression has not been reported in normal epithelium of the female reproductive tract. Therefore, we hypothesized that SATB2 may be of value in distinguishing ovarian metastases of colorectal adenocarcinoma from primary ovarian mucinous tumors and from primary ovarian endometrioid tumors. Among primary ovarian tumors, SATB2 staining was observed in 0/22 mucinous cystadenomas that lacked a component of mature teratoma, 4/12 mucinous cystadenomas with mature teratoma, 1/60 mucinous borderline tumors, 0/17 mucinous adenocarcinomas, 0/3 endometrioid borderline tumors, and 0/72 endometrioid adenocarcinomas. Among ovarian metastases, SATB2 staining was observed in 24/32 (75%) colorectal adenocarcinomas; 8/10 (80%) low-grade appendiceal mucinous neoplasms; and 4/4 (100%) high-grade appendiceal adenocarcinomas. No SATB2 staining was observed in any ovarian metastasis of pancreatic, gastric, gallbladder, or endocervical origin. Evaluation of primary extraovarian tumors showed the highest incidences of SATB2 staining among primary colorectal adenocarcinomas (71%), primary appendiceal low-grade mucinous neoplasms (100%), and primary appendiceal high-grade adenocarcinomas (100%). Similar to their metastatic counterparts, none of the primary pancreatic or gastric adenocarcinomas showed any SATB2 staining. In a subset of tumors for which CK7, CK20, and CDX2 were available, SATB2 was never positive in any tumor of any origin that was CK7+CK20-CDX2-. Among tumors that coexpressed all 3 markers (CK7+CK20+CDX2+), 6/7 SATB2 tumors were of colorectal or appendiceal origin, and 1/7 was a primary ovarian borderline tumor. We conclude that ovarian tumors with mucinous or endometrioid features that express SATB2 are unlikely to be of primary ovarian origin unless there is a component of mature teratoma in the ovary; instead, attention should be directed to a colorectal or appendiceal origin. SATB2 may be of particular value in ovarian mucinous tumors that are positive for all 3 markers (CK7+CK20+CDX2+), as SATB2 staining strongly implicates a colorectal or appendiceal origin.
Fallbericht einer Vena-Galeni-Malformation (VGM), einer seltenen, vital bedrohlichen Gefäßfehlbildungen mit meist letalem Ausgang. Wir berichten über ein kritisch krankes Neugeborenes (NG), dass shuntbedingt kardiopulmonal dekompensiert war und transarteriell embolisiert wurde.
Adipocytes are the predominant cell population in the normal breast and while recent attention has pointed to adipocyte-tumor cell crosstalk as a driver of breast cancer biology there have been few reports on the potential role of adipocytes in driving breast cancer initiation. Because normal breast tissue studies have invariably used reduction mammoplasty, benign biopsy or cancer-adjacent tissues, we studied random breast core biopsy samples donated by 145 healthy, parous, non-obese, white women (median age = 45, range 27-66 y) without any history of breast cancer. Using questionnaire data to calculate future breast cancer risk (Gail scores), we compared digitized microscopic breast tissue (H&E) images with whole genome transcriptome profiling (RNAseq) from FFPE-extracted RNA. We used unsupervised hierarchical clustering of 1487 genes (normalized, median centered, log2-scaled RSEM values) to identify 32% of normal samples with an "Active" (vs. 68% "Inactive") transcriptome phenotype previously associated with later-life risk of death from breast cancer. Despite slightly lower BMI values, donors with the Active transcriptome phenotype showed significantly higher Gail scores as well as higher mammary adipocyte nuclei counts (median 80% vs. 60%, p=2.3e-6). Tissue resident leukocytes were uncommon but Active transcriptome tissues expressed significantly altered immune modules enriched in TGFβ, interferon and macrophage gene signatures (including single gene increase in CD68) and depleted (relative to Inactive samples) of CD8+ T-cell and serum response/inflammation/wound healing signatures. Active samples were not enriched in cell senescence, SASP or DNA damage response gene signatures but were enriched in an autophagy-to-senescence-transition (AST) signature with increased CAV1 (caveolin-1, p=2.7e-12) and BNIP3 (Bcl2 interacting protein-3, p=4.7e-05) expression, genes that also regulate lipoprotein digestion/mobilization and adipocyte remodeling. Strongest among significant associations linking Active with adipocyte-enriched normal breast samples were increases in two adipokine growth factors, IGF-1 (p=2.2e-16) and FGF2 (p=3.0e-11), the adipokine (resistin) receptor CAP1 (p=0.04) recently linked to poor breast cancer outcomes, and a cAMP-dependent pro-lipolytic signature (p=0.01) known to drive breast cancer progression which, in these samples, correlated positively with average adipocyte area values. Altogether, the collective histologic and molecular features characterizing the normal breast tissue of >30% of healthy parous and non-obese women with increased predicted breast cancer risk seem to implicate a dysregulated mammary adipocyte microenvironment similar to but distinct from that associated with established breast tumors, that precedes microscopic and clinical evidence of breast tumorigenesis.Citation Format: Taekyu Kang, Christina Yau, Stephen Benz, Gregor Krings, Roman Camarda, Jill E. Henry, Mark Powell, Christopher C. Benz. Normal breast tissue at risk for cancer development: A breast cancer initiating role for mammary adipocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3314.
Abstract Purpose: Women treated with radiotherapy before 30 years of age have increased risk of developing breast cancer at an early age. Here, we sought to investigate mechanisms by which radiation promotes aggressive cancer. Experimental Design: The tumor microenvironment (TME) of breast cancers arising in women treated with radiotherapy for Hodgkin lymphoma was compared with that of sporadic breast cancers. To investigate radiation effects on carcinogenesis, we analyzed tumors arising from Trp53-null mammary transplants after irradiation of the target epithelium or host using immunocompetent and incompetent mice, some of which were treated with aspirin. Results: Compared with age-matched specimens of sporadic breast cancer, radiation-preceded breast cancers (RP-BC) were characterized by TME rich in TGFβ, cyclooxygenase 2, and myeloid cells, indicative of greater immunosuppression, even when matched for triple-negative status. The mechanism by which radiation impacts TME construction was investigated in carcinomas arising in mice bearing Trp53-null mammary transplants. Immunosuppressive TMEs (iTME) were recapitulated in mice irradiated before transplantation, which implicated systemic immune effects. In nu/nu mice lacking adaptive immunity irradiated before Trp53-null mammary transplantation, cancers also established an iTME, which pointed to a critical role for myeloid cells. Consistent with this, irradiated mammary glands contained more macrophages and human cells cocultured with polarized macrophages underwent dysplastic morphogenesis mediated by IFNγ. Treating mice with low-dose aspirin for 6 months postirradiation prevented establishment of an iTME and resulted in less aggressive tumors. Conclusions: These data show that radiation acts via nonmutational mechanisms to promote markedly immunosuppressive features of aggressive, RP-BCs.
<p>Supplementary Figure S3 shows all 8 patients with H3K36me3 IHC analysis. (A) Cohort A: Other Solid Tumor. (B) Cohort B: ccRCC. Images are at 40x power. Of note, the portal did not allow for uploading of a TIFF file alone as a "supplemental data" file, so the image included in this document is a TIFF file. We can easily provide the TIFF file if needed as well.</p>
Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor. https://doi.org/10.7554/eLife.10647.001 eLife digest Embryos develop from stem cells, which have the ability to mature into any type of cell in the body. The activity of proteins called transcription factors determines whether a stem cell will become a specialized cell type or remain in an immature "pluripotent" state that has the potential to become any cell type. These transcription factors bind to the cell's DNA to regulate the activity of target genes. SOX2 is a transcription factor that helps to maintain embryonic stem cells in a pluripotent state. In 2011, a group of researchers showed that a specific sugar molecule was added to SOX2 in mouse embryonic stem cells, in a process called O-GlcNAcylation. Now, Myers, Peddada et al. – including the researchers who performed the 2011 study – have studied the effects of this SOX2 modification in more detail. Transcription factors have two major activities – they bind to DNA and recruit other proteins that can turn target genes on or off. Myers, Peddada et al. found that, in pluripotent stem cells, a complex pattern of O-GlcNAcylation is present on SOX2 in a region that is responsible for recruiting other proteins. In addition, SOX2 O-GlcNAcylation decreases when stem cells are directed to become a new cell type. Further experiments investigated gene activity in stem cells that contained a mutant form of SOX2 that cannot be O-GlcNAc modified. In these cells, genes that help to maintain the cell in a pluripotent state were more active than in normal cells. The mutant form of SOX2 was altered in its ability to bind DNA and to associate with proteins that control gene activity. Myers, Peddada et al.'s findings raise several questions. Does O-GlcNAcylation control the activity of SOX2 in other cell types, such as neurons and cancer cells, in which this modification can be detected on SOX2? Why does a modification on the portion of the SOX2 that is thought to interact with other proteins affect SOX2 DNA binding activity? Finally, understanding how O-GlcNAcylation is employed to regulate SOX2 activity in response to developmental cues remains a major challenge. https://doi.org/10.7554/eLife.10647.002 Introduction SOX2 (sex determining region Y-box 2) is a transcription factor necessary for embryonic stem cell (ESC) self-renewal (Arnold et al., 2011; Masui et al., 2007). Precise control of SOX2 is critical for ESC maintenance, since increased or decreased expression of SOX2 interferes with self-renewal and pluripotency (Kopp et al., 2008; Masui et al., 2007). Post-translational modifications (PTMs) of SOX2 may play a role in its regulation, as SOX2 is reported to be phosphorylated, methylated, ubiquitinylated, SUMOylated, acetylated, and PARylated (Baltus et al., 2009; Brumbaugh et al., 2012; Fang et al., 2014; Gao et al., 2009; Lai et al., 2012; Swaney et al., 2009; Tahmasebi et al., 2013; Tsuruzoe et al., 2006; Van Hoof et al., 2009; Zhao et al., 2011). We have previously shown SOX2 is O-linked N-acetlyglucosamine (O-GlcNAc) modified in mouse ESCs (mESCs) (Myers et al., 2011). O-GlcNAcylation is the dynamic and regulatory mono-glycosylation of nucleocytosolic proteins catalyzed by a single O-GlcNAc transferase (OGT) and removed by a single hydrolase (OGA/MGEA5/NCOAT). O-GlcNAc signaling is essential for embryo viability (O'Donnell et al., 2004; Shafi et al., 2000; Yang et al., 2012) and mESC self-renewal (Jang et al., 2012). While OGT is critical for mESC maintenance, the protein- and site-specific functions of O-GlcNAcylation in mESCs have not been fully elucidated. Here, we show that O-GlcNAcylation of SOX2 at serine 248 (S248) is dynamically regulated in mESCs. Upon differentiation, O-GlcNAc occupancy is reduced and SOX2 is predominantly unmodified at this site. Replacement of wild type SOX2 (SOX2WT) with an O-GlcNAc-deficient mutant SOX2 (SOX2S248A) results in increased reprogramming efficiency. mESCs with SOX2S248A as their sole source of SOX2 have increased expression of genes associated with pluripotency and exhibit a decreased requirement for OCT4. SOX2S248A exhibits altered genomic occupancy and differential association with transcriptional regulatory complexes. O-GlcNAcylation directly inhibits the SOX2 and PARP1 interaction, which plays a regulatory role in mESC pluripotency (Gao et al., 2009; Lai et al., 2012). This study implicates O-GlcNAc modification in coordinating genomic occupancy and protein-protein interactions of SOX2 in ESCs, and provides molecular insight into how this broadly expressed transcription factor is regulated to promote the pluripotency-specific expression program. Results SOX2 O-GlcNAcylation is regulated by differentiation cues Previously, we reported in mESCs SOX2 was O-GlcNAcylated in the transactivation domain (TAD) (Myers et al., 2011), a region described to possess several other PTMs (Brumbaugh et al., 2012; Swaney et al., 2009; Tahmasebi et al., 2013; Tsuruzoe et al., 2006; Van Hoof et al., 2009). To investigate whether PTMs in the TAD are subject to developmental regulation, we analyzed SOX2 during the initial stages of differentiation. Knock-in FLAG/HA tagged SOX2 mESCs (KI cells; Lai et al., 2012) were cultured in media containing MEK and GSK3b inhibitors (2i) and leukemia inhibitory factor (LIF), or were induced to differentiate by removing LIF and adding retinoic acid (RA). Liquid chromatography coupled with tandem mass spectrometric (LC-MS/MS) analysis of SOX2 in self-renewal conditions revealed four main populations of the TAD peptide containing serine 248 (hereinafter referred to as TAD peptide): unmodified, O-GlcNAcylated at one of two sites (S248 or T258), phosphorylated at S253, and doubly modified with O-GlcNAcylation at S248 and phosphorylation at S253, (Figure 1A—figure supplements 1–5). Removal of LIF and addition of RA for 48 hr resulted in a marked decrease in the O-GlcNAc occupancy of the TAD peptide with no change in the phosphorylation stoichiometry (Figure 1B–C). These data indicate O-GlcNAcylation of SOX2 S248 is responsive to differentiation cues. Figure 1 with 7 supplements see all Download asset Open asset SOX2 O-GlcNAc levels change during differentiation. (A) Diagram of SOX2 (bottom, with TAD and high mobility group DNA binding domain [HMG] indicated), the TAD peptide sequence (middle; amino acid numbering from the Uniprot accession number P48432), and the PTM isoforms identified on the TAD peptide (top, grey and white rectangles, g indicates O-GlcNAc and p indicates phosphate). Mass spectra can be seen in Figure 1—figure supplement 1. (B) and (C) Extracted ion chromatographs (XICs) of SOX2 TAD peptide PTM states from (B) undifferentiated KI SOX2 mESCs (2i+L) or (C) differentiated KI SOX2 mESCs (RA 48 hr). Traces for each PTM isoform are colored differently, key provided in the inset in (B). https://doi.org/10.7554/eLife.10647.003 In mESCs SOX2 heterodimerizes with OCT4, which is also reported to be O-GlcNAcylated in this cell type (Jang et al., 2012). Thus, it is possible that OGT targets both these transcription factors when they are complexed together, prompting us to query OCT4 O-GlcNAcylation. However, we were unable to detect OCT4 O-GlcNAcylation in mESCs (Figure 1—figure supplements 6–7). O-GlcNAc-deficient SOX2 increases somatic cell reprogramming efficiency To query whether O-GlcNAcylation at S248 is present in other contexts, we examined the PTM profile of the SOX2 TAD peptide during somatic cell reprogramming. We used four-factor retroviral reprogramming (Oct4, Sox2, Klf4 and Myc; OSKM) in which SOX2 contained a triple FLAG tag (OSFLAG-WTKM). LC-MS/MS analysis of purified SOX2 six days after transduction of mouse embryonic fibroblasts (MEFs) showed S248 is O-GlcNAcylated (Figure 2A). In addition, mutation of S248 to alanine (S248A) resulted in loss of O-GlcNAcylation without affecting the other PTMs of the TAD peptide (Figure 2B–C). These results demonstrate S248 is O-GlcNAc modified during somatic cell reprogramming and suggest a connection between this SOX2 PTM and pluripotency. Figure 2 with 2 supplements see all Download asset Open asset O-GlcNAc-deficient SOX2, SOX2S248A, increases somatic cell reprogramming efficiency. (A) Diagram of SOX2 and the PTMs identified from MEFs transduced with OSFLAG-WTKM, labeled as described in Figure 1A. Spectra can be found at tinyurl.com/iPSC-3xF-SOX2-ETD and tinyurl.com/iPSC-3xF-SOX2-HCD. (B) XICs of 3xF-SOX2WT TAD peptide PTM states from OSFLAG-WTKM-transduced MEFs. (C) XICs of 3xF-SOX2S248A TAD peptide PTM states from OSFLAG-S248AKM-transduced MEFs. Color key the same as in (B). (D) Number of GFP+ colonies from 1000 Nanog-Gfp MEFs transduced with OSWTKM or OSS248AKM and cultured on SNL feeders for 18 or 20 days (n=7 +/- S.E.M.). (E) Chimeric mouse derived from iPSCs obtained from transducing Nanog-Gfp MEFs with OSS248AKM and his black offspring, demonstrating germline transmission. (F) Western blots against FLAG, SOX2, OGT and TUBULIN for the first six days of reprogramming with either OSFLAG-WTKM or OSFLAG-S248AKM. "Endo" refers to the apparent molecular weight at which the endogenous SOX2 would be expected, "3xF" refers the the FLAG tagged version from the viral transduction. https://doi.org/10.7554/eLife.10647.011 To determine whether the S248A mutation impacted induced pluripotent stem cell (iPSC) colony formation, we used somatic cell reprogramming of Nanog-Gfp reporter MEFs (Takahashi and Yamanaka, 2006). Nanog-Gfp MEFs transduced with OSS248AKM produced significantly more GFP+ iPSC colonies compared to OSWTKM (Figure 2D). iPSCs generated with OSS248AKM exhibited standard colony morphology and contributed to chimeric mice capable of germ line transmission (Figure 2E), indicating these OSS248AKM iPSCs exhibit the features of normal iPSCs. By Western blot and immunostaining of MEFs transduced with OSFLAG-WTKM or OSFLAG-S248AKM showed equal levels of exogenous SOX2 for the first six days ofof reprogramming (Figure 2F and Figure 2—figure supplement 1), indicating comparable expression of WT and S248A triple FLAG tagged SOX2. OGT levels were also similar for the first six days of reprogramming between OSFLAG-WTKM and OSFLAG-S248AKM transduced MEFs (Figure 2F). These results indicate that SOX2S248A is more efficient than wild type SOX2 at inducing pluripotency and suggest O-GlcNAcylation at S248 inhibits SOX2 activity. The homologous SOX2 residue has been reported to be phosphorylated in human ESCs (Swaney et al., 2009). While our lab and others were unable to detect this phosphorylation in mESCs (Brumbaugh et al., 2012) or during murine reprogramming, the S248A mutation could potentially remove a regulatory phosphorylation site. Therefore, we performed reprogramming experiments using the phospho-mimetic SOX2 mutant, S248D. This mutation also increased reprogramming efficiency (Figure 2—figure supplement 2), suggesting it is the removal of an O-GlcNAcylation site, and not of a phosphorylation site, which mediates the effect on SOX2 activity. SOX2S248A can replace wild type SOX2 in mESCs Since the reprogramming results suggested the S248A mutation increased SOX2 activity, we examined whether this mutant SOX2 supported mESC self-renewal. We generated mESC lines that express either a FLAG-tagged wild-type Sox2 transgene (fSOX2-Tg cells) or an S248A transgene (fS248A-Tg cells) (Figure 3A). We introduced the transgenes into 2TS22C mESCs, in which endogenous Sox2 is removed and a doxycycline repressible SOX2 cDNA transgene supports self-renewal (Masui et al., 2007)(Figure 3—figure supplement 1). Under doxycycline repression, the sole source of SOX2 in these transgenic lines is the FLAG-tagged wild-type or S248A mutant SOX2 (Figure 3B). SOX2 levels in fSOX2-Tg and fS248A-Tg mESCs are comparable to SOX2 levels in the 2TS22C parental cell line and nucleo-cytoplasmic distribution was not altered by the mutation (Figure 3C). OCT4 and NANOG abundance and distribution were comparable between fSOX2-Tg and fS248A-Tg mESCs (Figure 3C), arguing that there is no gross effect on these pluripotency transcription factors. Figure 3 with 3 supplements see all Download asset Open asset SOX2S248A can replace wild type SOX2 in mESCs. (A) Characterization of fSOX2-Tg and fS248A-Tg mESCs. fSOX2-Tg and fS248A-Tg mESCs exhibit AP staining, a marker of pluripotency, similar to parental 2TS22C cells. (B) Western blot analysis of SOX2 and FLAG in 2TS22C, fSOX2-Tg and fS248A-Tg mESCs. TUBULIN (TUB) is used as a loading control. "3xFLAG" and "untagged" refer to expected molecular weights of SOX2 with the 3xFLAG tag or no tag, respectively. (C) Immunofluorescence staining for NANOG, SOX2, FLAG and OCT4 in wild type E14, parental 2TS22C, fSOX2-Tg, and fS248A-Tg mESCs. Antibody staining is green, nuclear stain with DAPI is blue. (D) and (E) XICs of the TAD peptides of SOX2 immunopurified from fSOX2-Tg (D) and fS248A-Tg (E) mESCs. Insets: pie charts showing the mean percentage of each PTM form to total TAD peptide signal (n=3). The doubly phosphorylated TAD peptide is below the limit of quantitation for both cell lines. https://doi.org/10.7554/eLife.10647.014 LC-MS/MS analysis of immunopurified SOX2 from fSOX2-Tg mESCs identified nine PTM forms of the SOX2 TAD peptide (Figure 3—figure supplement 2). LC-MS analysis of the TAD peptide precursor masses from fSOX2-Tg mESCs showed unmodified and singly O-GlcNAcylated were the most abundant forms of the SOX2 TAD peptide (33.2 and 44.1% of total TAD, respectively) (Figure 3D). LC-MS analysis confirmed the loss of S248 O-GlcNAcylation in fS248A-Tg mESCs (Figure 3E). Analysis of synthetic SOX2 TAD peptides showed chromatographic separation of PTM or mutant isoforms, and lack of electrospray ionization suppression, validating our label free quantitation approach (Figure 3—figure supplement 3). In addition, the TAD peptide in fS248A-Tg mESCs showed increased phosphorylation at S253, from 10.9 to 18.7% of total TAD, suggesting cross talk between phosphorylation and O-GlcNAcylation. SOX2S248A alters gene expression in mESCs To determine if the S248A mutation altered global transcript levels we used microarrays to compare the gene expression profiles of fSOX2-Tg and fS248A-Tg mESCs. Significant changes in mRNA levels were observed, with 320 genes up regulated and 344 genes down regulated in fS248A-Tg cells (Figure 4A) and gene set enrichment analysis of differentially expressed genes did not show significant enrichment of any pathways. Several genes that promote pluripotency and self-renewal were upregulated, while several genes associated with differentiation were down-regulated in fS248A-Tg cells compared to WT. RT-qPCR confirmed the differential expression of these pluripotency or differentiation genes (Figure 4B). These data suggest the S248A mutation alters the balance between self-renewal and differentiation gene expression in mESCs. Figure 4 with 1 supplement see all Download asset Open asset fS248A-Tg mESCs show altered gene expression and decreased dependence on OCT4. (A) Volcano plot of global changes in gene expression between fSOX2-Tg and fS248A-Tg cells. Red indicates genes with increased or decreased expression (fold change cutoff 1.5 and paired t-test p<0.05) (Supplementary file 1a). (B) RT-qPCR of select genes differentially expressed between fSOX2-Tg and fS248A-Tg cells (* indicates p<0.05, n=3, +/- S.E.M.). (C) fSOX2-Tg or fS248A-Tg cells were depleted of OCT4 using siRNA pools (esiRNAs) and Western blot analysis of OCT4 and TUBULIN were performed. (D) and (E), (D) AP staining and (E) quantitation of fold change in AP staining three days after OCT4 or GFP depletion in fSOX2-Tg and fS248A-Tg cells. Additional example fields of view for relative quantitation can be seen in Figure 4—figure supplement 1. F, RT-qPCR analysis of Oct4 and Nanog mRNA levels in fSOX2-Tg or fS248A-Tg cells depleted of OCT4 compared to the control knockdown of GFP. https://doi.org/10.7554/eLife.10647.018 The altered gene expression profile of fS248A-Tg cells suggested this mutation may promote self-renewal at the expense of differentiation. Therefore, we examined the effects of OCT4 depletion, which causes mESCs to differentiate (Figure 4C) (Hough et al., 2006). While fSOX2-Tg mESCs exhibited altered cell and colony morphology (Figure 4—figure supplement 1), decreased AP staining (Figure 4D), and decreased expression of Nanog (Figure 4E), fS248A-Tg mESCs were relatively unaffected by OCT4 depletion. These data indicate that fS248A-Tg mESCs can maintain key features of pluripotency when OCT4 levels are reduced, and are consistent with a role for the O-GlcNAc modification inhibiting SOX2 activity. SOX2S248A exhibits altered genomic occupancy To examine whether the altered gene expression associated with the S248A mutation was accompanied by changes in SOX2 genomic occupancy, we performed FLAG chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) to compare SOX2 genomic distribution in fSOX2-Tg and fS248A-Tg mESCs (Figure 5A). SOX2 distribution exhibited considerable overlap, with 4,191 sites bound in both lines (Figure 5B). The mutant form of SOX2 occupied 1000 sites not bound by the wild type form (Figure 5A). De novo motif analysis identified the SOX2 binding motif in fS248A-Tg specific peaks (Figure 5C). In mESCs, SOX2 and OCT4 heterodimerize and co-occupy a substantial portion of their target regulatory sequences (Boyer et al., 2005). De novo motif analysis of SOX2 peaks shared between fSOX2-Tg and fS248A-Tg mESCs identified the OCT4:SOX2 motif (Figure 5D), which was present in 2335 of the shared peaks. The OCT4:SOX2 motif was not identified in any of the fS248A-Tg-specific peaks (Figure 5E). These data indicate the S248A mutation alters SOX2 genomic distribution, increasing its ability to associate with SOX2 binding sites that would not ordinarily be bound by wild type SOX2 in mESCs. Figure 5 Download asset Open asset S248A mutation alters genome-wide distribution of SOX2. (A) Representative UCSC genome browser tracks of FLAG ChIP-seq in fSOX2-Tg (blue) and fS248A-Tg (red) cells. Examples of fS248A-Tg specific peaks (Pou5f1, Esrrb) and shared peaks (Abca4, Sox2) are shown for 2 biological replicates (2 technical replicates were performed for each biological replicate, Spearman correlations for technical replicates are 1, for biological replicates 0.45 for fSOX2-Tg and 0.55 for fS248A-Tg). Each track is 15 kb. Green arrows indicate fS248A-Tg specific peaks. For Sox2 track, the region shown is not encompassed in the deletion removing endogenous Sox2. (B) Overlap (purple) in called peaks from anti-FLAG ChIP-seq in fSOX2-Tg (blue) and fS248A-Tg (red) mESCs. (C) De novo SOX2 motif identified in shared ChIP-seq peaks between fSOX2-Tg and fS248A-Tg cells (top) compared to the canonical SOX2 motif [Jaspar M01271] (bottom). (D) OCT4:SOX2 motif identified in peaks shared between fSOX2-Tg and fS248A-Tg cells using de novo motif analysis (top) compared to the canonical OCT4:SOX2 motif [Jaspar MA0142.1] (bottom). (E) Proportion of peaks containing a motif matching the OCT4:SOX2 de novo motif in shared peaks (left) and fS248A-Tg specific peaks (right). https://doi.org/10.7554/eLife.10647.020 O-GlcNAc alters SOX2 protein-protein interactions S248 lies in the TAD of SOX2, a region responsible for interactions with transcriptional regulatory machinery (Ambrosetti et al., 2000; Nowling et al., 2000; Yuan et al., 1995). Therefore, we tested whether the S248A mutation altered SOX2 centered protein-protein interactions (PPIs). We performed affinity purifications against FLAG from nuclear extracts of fSOX2-Tg, fS248A-Tg or an equivalent mESC line expressing transgenic HA-tagged SOX2 (haSOX2-Tg, Figure 6—figure supplement 1) and used quantitative LC-MS to identify proteins that co-purified with FLAG in each cell type. We identified 329 proteins enriched in both fSOX2-Tg and fS248A-Tg, but not haSOX2-Tg FLAG IPs. Many of these interactors exist in complexes involved in histone modification, DNA damage repair, or nucleosome remodeling (Figure 6A). Several SOX2 interactors have been previously described (Cox et al., 2013; Engelen et al., 2011; Gao et al., 2012), indicating fSOX2-Tg and fS248A-Tg cells recapitulate some known SOX2 interactions (Supplementary file 1b). Figure 6 with 2 supplements see all Download asset Open asset O-GlcNAcylation of SOX2 at S248 alters protein-protein interactions. (A) Interaction diagram of a subset of SOX2 interactors that exhibit differential association with 3xF-SOX2S248A relative to 3xF- SOX2WT. Color of circles indicates with which SOX2 proteoform a protein preferentially interacts. Interaction diagram based on high confidence, experimental interactions identified by STRING. (B), Anti-FLAG IP-WB for SOX2, PARP1, GATAD2B, and SMARCA4 in fSOX2-Tg and fS248A-Tg cells. (C) Heatmap of median enrichment values of NuRD subunits that preferentially associate with 3xF-SOX2WT or 3xF-SOX2S248A as determined by quantitative mass spectrometry (n=3). (D) Western blot analysis of in vitro interaction between SOX2 +/- O-GlcNAcylation and PARP1. Bio-SOX2 and His-OGT were incubated with and without UDP-GlcNAc, Bio-SOX2 purified away from OGT and UDP-GlcNAc using streptavidin beads and incubated with GST-PARP1. Western blots examine proteins associated with streptavidin beads. Comparable amounts of input and pull down were loaded for all blots, except O-GlcNAc, in which more material was loaded in the pull down lanes. WB, Western blot; GST, glutathione S-transferase tag; Bio, biotinylated Bio tag; His, polyhistidine tag. (E) Flow chart outlining scheme for D. https://doi.org/10.7554/eLife.10647.021 We next examined whether any co-purifying proteins were enriched in either the fSOX2-Tg or fS248A-Tg co-IPs, by plotting the enrichment ratios between S248A and wild type. 22 of the interacting proteins were enriched at least four-fold in the co-IP with fSOX2-Tg (z-score > 1.5) and 60 were enriched in the fS248A-Tg co-IP (Supplementary file 1b). Co-IP followed by Western blotting corroborated the IP-MS data, showing preferential enrichment of PARP1 and GATAD2B with mutant and wild type SOX2, respectively, while SMARCA4 was associated equally with both forms of SOX2 (Figure 6B). Examination of the protein complexes enriched by either wild type or S248A SOX2 showed a subset of components behaved discordantly with the rest of the complex subunits. For example, MBD3 and MTA3, both of which can be a part of the NuRD complex, were consistently enriched in fS248A-Tg co-IPs while other NuRD components were enriched with fSOX2-Tg (Figure 6B). To more thoroughly investigate the subunit distribution of a subset of the NuRD complex, we used a targeted proteomic approach based on interacting proteins from an MBD3 co-IP experiment. We performed anti-FLAG affinity purifications in FLAG tagged MBD3 mESCs (Yildirim et al., 2011) followed by LC-MS to generate a representative set of NuRD complex peptides. The top two, best scoring, unique peptides for each NuRD component were used to determine the relative enrichment of these proteins from fSOX2-Tg and fS248A-Tg co-IPs (Supplementary file 1c). Targeted analysis showed the majority of the NuRD complex preferentially associated with SOX2WT, while MBD3 and MTA3 components prefer SOX2S248A (Figure 6C). These results suggest that the S248A mutation can affect the stoichiometry of subunits in complexes that associate with SOX2. The altered PPIs with SOX2S248A may occur as a direct result of the lack of O-GlcNAcylation of S248. To test whether the O-GlcNAcylation of SOX2 was directly responsible for alterations in a PPI, we used recombinant proteins to assess the effect of this PTM on the SOX2-PARP1 interaction. O-GlcNAcylation of Bio-tagged, recombinant human SOX2 (Bio-SOX2, 96% identical to mouse) by recombinant human OGT (His-OGT, 99% identical to mouse), which depended on the sugar donor UDP-GlcNAc, was detected by Western blotting (Figure 6D) and specificity confirmed by mass spec (Figure 6—figure supplement 2). Bio-SOX2 was bound to streptavidin magnetic beads to remove OGT and UDP-GlcNAc. Beads bound by O-GlcNAcylated or unmodified SOX2 were incubated with GST-tagged, recombinant human PARP1 (GST-PARP1, 91% identical to mouse) (Figure 6E). Pull down efficiency of Bio-SOX2 was not affected by O-GlcNAcylation and His-OGT was not detected in pull downs, indicating any potential SOX2:OGT interaction was not stable under our wash conditions. Unmodified Bio-SOX2 pulled down GST-PARP1, indicating the interaction between mouse SOX2 and PARP1 can be recapitulated by their conserved human homologues. Pulldown efficiency of GST-PARP1 by glycosylated Bio-SOX2 was diminished compared to that of unmodified Bio-SOX2 (Figure 6D). Together, these data demonstrate SOX2 O-GlcNAcylation directly alters its interaction with a transcriptional regulatory protein involved in maintaining the balance of self-renewal and differentiation (Figure 7). Figure 7 Download asset Open asset Model for the role of O-GlcNAcylation in regulation of SOX2 in mESCs. (A) O-GlcNAc (sugar moiety) affects the affinity of SOX2 (red) for interacting proteins (ovals). Some proteins (blue shapes) exhibit greater affinity for unmodified SOX2, while others exhibit lower affinity (orange shapes). In addition, O-GlcNAcylation affects SOX2 binding to a subset of target DNA sequences. (B) As a result of altered genomic distribution and protein-protein interactions when SOX2 cannot be O-GlcNAcylated (SOX2S248A), pluripotency gene expression is promoted at the expense of differentiation. https://doi.org/10.7554/eLife.10647.024 Discussion Depletion of OGT, the sole enzyme that mediates intracellular O-GlcNAcylation, disrupts mESC self-renewal (O'Donnell et al., 2004; Shafi et al., 2000), prompting us to identify OGT targets to elucidate link between O-GlcNAc and self-renewal. Using an unbiased strategy for enrichment of native O-GlcNAcylated nuclear peptides, we previously identified SOX2 S248 as an OGT substrate (Myers et al., 2011). Here, we find that S248 is O-GlcNAcylated during somatic cell reprogramming and that mutation of this residue to alanine increases reprogramming efficiency. We also find mESCs expressing SOX2S248A exhibit changes in transcription consistent with increased expression of pluripotency promoting genes at the expense of differentiation promoting genes. Together, these analyses from both mESCs and during somatic cell reprogramming reveal the S248A mutation promotes SOX2 activity, which suggests SOX2 O-GlcNAcylation is inhibitory during maintenance and establishment of pluripotency. Our data indicate that S248 O-GlcNAcylation is regulated by developmental signaling molecules, since removing LIF and adding RA to trigger differentiation resulted in a substantial decrease in this PTM. This decrease in S248 O-GlcNAcylation appears contradictory to the finding that the S248A mutation, which eliminates O-GlcNAcylation, promotes mESC self-renewal. However, the effects of this, or any, PTM are likely to be context specific, and determined by the transcription factors and signaling molecules present in each cell type. GlcNAc-S248 may inhibit SOX2 activity in mESCs, where the decrease in this PTM may alter SOX2 activity upon differentiation, such that SOX2 functions appropriately for the changing cellular context. As this work shows, use of methods that allow analysis of SOX2 PTM-specific PPIs and genomic occupancy may be crucial to understand how combinations of PTMs are used to regulate SOX2 activity in response to developmental cues. In addition to changes in gene expression, the S248A mutation altered SOX2 genomic distribution. As well as occupying the same sites as SOX2WT, SOX2S248A was found at an additional 1000 sites. The majority of these sites contained a predicted SOX2 binding motif, indicating the mutation allows SOX2S248A to occupy sites that SOX2WT is unable to access in mESCs. This result suggests O-GlcNAcylation can regulate the affinity of SOX2 for its target sites. Since the mutation lies in the TAD, but affects the activity of the high mobility group DNA binding r
