High risk HPV E6 protein promotes reprogramming of Fanconi Anemia patient cells through 1 ...

34 Pages·2014·2.89 MB·English

JVI Accepts, published online ahead of print on 16 July 2014
J. Virol. doi:10.1128/JVI.01533-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1 High risk HPV E6 protein promotes reprogramming of Fanconi Anemia patient cells through
2 repression of p53 but does not allow for sustained growth of iPSC
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4 Timothy M. Chlon1, Elizabeth E. Hoskins1, Christopher N. Mayhew2, Kathryn A. Wikenheiser-
5 Brokamp1, Stella M. Davies1, Parinda Mehta1, Kasiani C. Myers1, James M. Wells2, Susanne I.
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6 Wells1# o
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8 1Cincinnati Children’s Hospital Medical Center, Cancer and Blood Diseases Institute, Cincinnati, OH e
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9 2Cincinnati Children’s Hospital Medical Center, Division of Developmental Biology, Cincinnati, OH ro
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11 #Corresponding Author: //
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12 Cincinnati Children’s Hospital Medical Center s
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13 Cancer and Blood Diseases Institute o
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14 3333 Burnet Avenue, MLC-7013 o
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15 Cincinnati, OH 45229 p
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16 Phone: [phone]
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17 Fax: [phone]
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18 [email protected] y

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20 Running title: Use of the HPV oncogenes to study FA reprogramming
21 Abstract
22 DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming.
23 The Fanconi Anemia pathway, which promotes error-free repair of DNA double strand breaks, is
24 required for somatic cell reprogramming to iPSC. Thus, cells from Fanconi Anemia patients, which
25 lack this critical pathway, fail to reprogram to iPSC under standard conditions unless the defective FA
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26 gene is complemented. In this study, we utilized the oncogenes of high risk human papillomavirus o
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27 type 16 to overcome the resistance to reprogramming of FA patient cells. We found that E6, but not lo
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28 E7, recovers FA iPS colony formation, and furthermore, that p53 inhibition is necessary and sufficient e
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29 for this activity. The iPS colonies resulting from each of these approaches stained positive for o
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30 alkaline-phosphatase, NANOG, and Tra-1-60, indicating that they were fully reprogrammed into
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31 pluripotent cells. However, FA iPSC were incapable of outgrowth into stable iPSC lines regardless of //
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32 p53 suppression, whereas their FA-complemented counterparts grew efficiently. Thus, we conclude s
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33 that the FA pathway is required for the growth of iPSC beyond reprogramming, and that p53- o
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34 independent mechanisms are involved. o
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35 p
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36 Importance: A novel approach is described whereby HPV oncogenes are used as tools to uncover 3
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37 DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings 1
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38 indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a y

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39 previously unrecognized defect in FA iPSC proliferation independent of p53. e
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41 Introduction
42 Human papillomaviruses (HPVs) are pathogens that commonly infect basal stem and progenitor cells
43 in the epidermis and can control keratinocyte proliferation and differentiation as a means to
44 perpetuate the viral life cycle (1, 2). Two viral proteins, E6 and E7, have been extensively
45 characterized for their ability to bind and modulate cellular factors that regulate fundamental
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46 processes including proliferation, survival, transcription and histone modification (3, 4). In the adult o
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47 epidermis, E6/E7 proteins support the regenerating stem cell compartment while ensuring retention of lo
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48 full cellular differentiation capacity. The cellular processes affected by E6/E7 proteins all play key e
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49 roles during the reprogramming of somatic adult cells into induced pluripotent stem cells (iPSC). o
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51 Induced pluripotent stem cells are self-renewing, pluripotent cells derived by reprogramming of //
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52 somatic cells through exogenous expression of the embryonic stem cell transcription factors OCT-3/4, s
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53 SOX2, KLF4, and c-MYC (OSKM), termed the ‘Yamanaka factors’ (5). Complete conversion of a o
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54 somatic cell to a pluripotent stem cell requires drastic changes in proliferation rate, cell morphology, o
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55 metabolism, epigenetic modifications, and gene expression (6, 7). These changes occur over a 10- p
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56 20 day period during which the success of reprogramming in an individual cell depends stochastically 3
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57 on responses to various impediments (8). One such impediment is DNA damage that occurs during 1
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58 early reprogramming (9). The p53 tumor suppressor responds to this damage and can trigger cell y

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59 cycle arrest, senescence, or apoptosis, depending on the severity of the damage and the ability of the e
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60 cell to repair it. Thus, p53 activity represses reprogramming at this early stage (10, 11). Repression
61 of p53 increases reprogramming frequency and anti-p53 shRNA is now often introduced alongside
62 the Yamanaka factors to improve efficiency (10-13). The acquisition of the high proliferation rate
63 characteristic of pluripotent cells can also be difficult to achieve in reprogramming somatic cells, and
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64 thus increasing the proliferation rate by targeting cell cycle regulators, such as the Retinoblastoma
65 protein (RB), has been demonstrated to increase reprogramming efficiency (14).
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67 IPSC approximate embryonic stem cells (ESC), a cell type that exists only in the inner cell mass of
68 the blastocyst and ultimately give rise to the entire embryo proper. These cells possess the unique
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69 responsibility to prevent genomic mutations that would be passed on to the cells of the entire o
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70 organism, including the germline. It is likely for this reason that ESC have evolved to maintain a lo
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71 significantly lower mutation frequency compared to somatic cells (15). They accomplish this by both e
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72 increasing the use of error-free DNA repair pathways at the expense of error-prone pathways, and by o
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73 undergoing rapid apoptosis in response to elevated DNA damage levels (16-21).
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74 //
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75 Fanconi Anemia (FA) is a genetic disease characterized by bone marrow failure (BMF) and extreme s
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76 cancer incidence (22). It is caused by mutations in genes that participate in the FA DNA repair o
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77 pathway, which is required for error-free repair of DNA interstrand crosslinks by homologous o
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78 recombination (HR) and is also involved in promoting HR at DNA double strand breaks (23). The FA p
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79 pathway comprises a core complex of FA proteins, including FANCA, FANCB, FANCC, FANCE, 3
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80 FANCF, FANCG, FANCL, and FANCM and other associated proteins, which is assembled in the 1
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81 presence of interstrand crosslink DNA damage and functions to promote activating ubiquitination of y

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82 FANCD2 and FANCI, which then dimerize and localize to the site of damage where they recruit e
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83 nucleases, the helicase FANCJ, and the HR machinery to repair the damaged DNA (24). Consistent
84 with the essential role of the FA pathway in repair of ICLs, cells deficient for the FA pathway are
85 hypersensitive to ICL-inducing agents such as Mitomycin C (MMC), which causes G2/M arrest and
86 the formation of radial chromosomes in FA cells. Recent studies have revealed that cells from FA
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87 patients are resistant to somatic cell reprogramming and that complementation of the defective FA
88 gene restores normal reprogramming efficiency to these cells (25, 26). Thus, a functional FA
89 pathway is required for efficient somatic cell reprogramming. However, the mechanism by which FA
90 cells fail to reprogram is not yet fully understood. A recent study by Muller et al showed that mouse
91 FA cells experience even higher levels of reprogramming-induced DNA damage and senescence
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92 than control cells and suggested that the senescence was caused by inability to repair the damage o
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93 (26). By culturing the reprogramming cells in hypoxia, which is known to increase reprogramming lo
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94 efficiency in normal cells, they were able to reprogram mouse and human FA cells and grow iPS lines e
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95 (26). However, the mechanism by which hypoxia promoted FA reprogramming and iPSC cell growth o
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96 remains elusive. Importantly, these human FA iPS lines were not characterized under normoxic
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97 conditions. In a separate study, Gonzalez et al reported that mouse FA and HR-deficient cells //
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98 experience higher rates of apoptosis and reduced cell proliferation during reprogramming than control s
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99 cells and also that the reprogramming efficiency could be increased by inhibition of p53 (27). o
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100 However, there was no reported attempt to derive iPS lines from the reprogrammed colonies in this o
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101 study. A final study attempted to reprogram FA patient cells and reported the derivation of lines at low p
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102 frequency (28). However, these lines failed to produce teratomas, suggesting that they are not fully 3
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103 pluripotent. Thus, it remains unknown whether iPS lines from FA patients can be grown at normoxic 1
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104 conditions. y

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105 e
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106 In this study, we sought to overcome the resistance to reprogramming in FA patient cells, and study
107 associated molecular mechanisms through expression of the oncogenes encoded by the high risk
108 human papillomaviruses (HPVs). High risk HPV E6 and E7 target various cell cycle and genome
109 stability regulators to drive proliferation of their target cell. E6 targets p53 for degradation through
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110 interaction with the E6AP ubiquitin ligase (29). It can also activate c-Myc and telomerase (30-32). E7
111 targets the Rb family pocket proteins for degradation, allowing constitutive activation of the E2F
112 transcription factors (33), and also binds and inhibits p21, which controls the G1/S transition
113 downstream of p53 (34, 35). Thus, these proteins systematically regulate the cell cycle checkpoints
114 and the DNA damage response, two regulatory processes that are critical to somatic cell
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115 reprogramming. We tested the effect of these oncogenes by themselves and in combination on o
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116 reprogramming efficiency in FA patient cells and found that E6, but not E7, overcomes the block to lo
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117 reprogramming. We then utilized mutants of E6 to establish that it requires the ability to degrade p53 e
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118 in order to promote reprogramming, establishing a role for p53 in failed reprogramming of human FA o
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119 cells. Lastly, we attempted to grow both FA-complemented and non-complemented FA iPSC lines
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120 from the iPS colonies, but only FA-complemented cells produced lines in normal culture conditions //
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121 despite continued repression of p53. Together, our studies reveal that repression of p53 overcomes s
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122 the barrier to reprogramming in FA patient cells but that resulting iPSC remain sensitive to deficiency o
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123 of the FA pathway and fail to self-renew. This observation suggests a broader role and versatile o
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124 activities for FA and the HR machinery in ESC and iPSC biology. p
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125 3
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126 Methods 1
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127 Cell Culture and Reprogramming y

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128 Patient-derived keratinocytes were obtained under a Cincinnati Children’s Hospital IRB approved e
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129 protocol, and cultured from fresh skin punch biopsies on irradiated J2-3T3 feeder cells as described
130 previously (36). After the second passage, keratinocytes were transduced with empty retroviral LXSN
131 vector or LXSN encoding wild-type or mutant human papillomavirus type 16 E6. LXSN-E6 vectors
132 were generous gifts from Elliot Androphy (Indiana University) and Saleem Khan (University of
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133 Pittsburgh). Keratinocytes were transduced with anti-TP53 shRNA or non-specific shRNA control by
134 retroviral infection with pRS vectors (reference). All transductions used supernatants produced from
135 293T cells in the presence of 8μg/mL polybrene. The transduced keratinocytes were selected with
136 200μg/mL G418 or 10μg/mL Hygromycin-B for 3 days. The resulting keratinocyte populations were
137 then transduced with either empty or FANCA-expressing MIEG retrovirus, which also expresses GFP.
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138 GFP-positive cells were sorted 3 days post-transduction on a BD FACS Aria cell sorter, and replated. o
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139 These cells were then transduced with a polycistronic lentivirus expressing OCT-4, SOX2, KLF4, and lo
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140 C-MYC (OSKM) as well as RFP (37). RFP-expression was used to confirm equal transduction. At 4 e
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141 days post transduction with OSKM, the cells were trypsinized and plated on irradiated CF-1 MEFs o
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142 (GlobalStem) in hESC media as described previously (38). The media was changed daily for 19 days.
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143 To establish iPS lines, colonies with characteristic iPS morphology were picked and transferred to //
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144 dishes coated with Matrigel (BD Biosciences) in mTeSR1 media (STEMCELL Technologies). s
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145 mTeSR1 media was changed daily. Cells were passaged by treatment with 1mg/mL Dispase until o
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146 colony edges were rounded, washed 3 times with 1:1 DMEM/F12 (Invitrogen), scraped off the dish o
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147 with a cell scraper in mTeSR1, gently triturated, and then replated. p
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148 3
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149 Staining and Immunofluorescence 1
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150 Reprogrammed cultures were stained on day19 post-plating using the Alkaline Phosphatase y

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151 Detection Kit (Millipore) following the manufacturer’s instructions. Parallel cultures were stained for e
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152 TRA-1-60 using a Biotin-conjugated TRA-1-60 antibody (eBiosciences), streptavidin-conjugated HRP
153 (BioLegend), and the DAB substrate kit (Vector Labs). Briefly, cells were fixed on the culture dish in
154 4% paraformaldehyde (PFA), blocked with 10% goat serum in 0.5% PBS-Tween for 10min, incubated
155 with primary antibody in blocking buffer overnight, washed with PBS, incubated with Streptavidin HRP
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156 in blocking buffer for 1hr, washed with PBS, and then treated with DAB substrate following
157 manufacturer’s instructions. For OCT-4 and NANOG immunofluorescence (IF), cells were fixed on the
158 culture dish with 4% PFA, washed with PBS, permeabilized with 0.5% PBS-Triton X-100 for 10min,
159 blocked with 10% donkey serum in 0.5% PBS-Triton X-100 for 30min, incubated with primary
160 antibodies in blocking buffer overnight, washed with PBS, incubated with appropriate fluorescent
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161 secondary antibodies (Jackson Laboratories) with DAPI, washed and overlaid in PBS, and then o
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162 imaged. Antibodies included OCT-3/4 (Santa Cruz Biotechnologies sc-5279) and NANOG (Abcam, lo
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163 ab109250). e
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164 o
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165 Gene Expression Analysis
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166 iPS cultures were treated with 1mg/mL Dispase until the colony edges were rounded, then the plates //
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167 were washed with 1:1 DMEM/F12, and the colonies were scraped off the dish. The colonies were s
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168 pelleted by gentle centrifugation, and then the RNA was extracted from the cell pellet using the o
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169 RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. 1μg of total RNA was used for cDNA o
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170 synthesis using the QuantiTect Reverse Transcription Kit (Qiagen). The resulting cDNA solution was p
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171 diluted 1:25 and used for real-time PCR analysis using Sybr green and gene-specific primers. 3
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172 Relative gene-expression values were determined using the ΔΔCT method with GAPDH as the 1
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173 housekeeping gene. Error bars indicate the standard deviation, and significant differences were y

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174 determined by t-test with p<0.05. e
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176 Antibodies
177 Antibodies for western blot analysis included p53 (Calbiochem, DO-1), p21 (Calbiochem, EA10), actin
178 (Seven Hills Bioresearch,C4), vinculin (Sigma, V9131), FANCA (Cascade Biosciences), and FANCD2
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179 (Novus).
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181 Teratoma Formation
182 Three confluent 35mm culture dishes of iPSC were harvested by Dispase followed by scraping, as
183 above. The cells were pelleted and gently resuspended to retain cell clumps in 70μL cold 1:1
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184 DMEM:F12. Immediately before injection, 35μL of Matrigel was added, and then loaded into a o
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185 syringe. Cell clumps were injected into the flanks of NSG mice with a 30-gauge needle. The mice lo
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186 were monitored for tumor formation for at least 16 weeks, and were then sacrificed. The tumors were e
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187 paraffin embedded, sectioned, and stained by H&E. Slides were evaluated by a clinical pathologist for o
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188 the presence of structures derived from all three germ layers.
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189 //
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190 Statistics s
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191 All pairwise comparisons were examined for statistical significance by Student’s t-test with p<0.05. o
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192 Error bars indicate the standard deviation of the mean. o
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194 Results 3
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195 HPV E6 oncogene expression rescues reprogramming in FA patient cells. 1
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196 Recent studies have established that cells from both mice and humans with defects in the FA y

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197 pathway undergo somatic cell reprogramming at dramatically reduced efficiency (25-28, 39). We e
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198 have previously immortalized skin keratinocytes from individuals with FA using the high risk HPV E6
199 and E7 oncogenes (36). Since immortalized somatic cells are known to reprogram with increased
200 efficiency (40), we hypothesized that these cells could be reprogrammed despite being deficient for
201 the FA pathway. Keratinocytes from patients within the FANCA complementation group were
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202 immortalized with HPV16 E6 and E7 and then either control transduced or transduced with a
203 retroviral FANCA expression vector. Resulting isogenic FA-deficient and –proficient keratinocytes
204 were then subjected to reprogramming by transduction with an Oct3/4-Sox2-Klf4-Myc polycistronic
205 lentiviral vector (OSKM). After 19 days in ES-cell culture conditions, both non-complemented and
206 complemented (FANCA) cells formed large AP+ colonies with ES-like morphology, suggesting that
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207 immortalization by HPV overcomes the resistance to reprogramming in FA cells. (Figure 1A). Next, o
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208 we sought to determine which oncogene was sufficient for reprogramming or if E6 and E7 were lo
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209 required. To do this, cells that had been immortalized with E6 and E7, either individually or together, e
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210 were compared for their relative reprogramming efficiencies (Figure 1B and C). Remarkably, E6 o
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211 immortalized cells produced as many AP-positive colonies as E6 and E7 in combination, while E7
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212 immortalized cells produced no colonies. This result suggested that E6 possesses an activity that //
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213 overcomes the resistance to reprogramming in FA patient cells. s
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214 o
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215 The above immortalized FA keratinocytes were passaged extensively during immortalization, and we o
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216 therefore sought primary FA patient samples. Transductions with control or HPV oncogene- p
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217 expressing vectors, control or FANCA complementation vector, and the OSKM reprogramming vector 3
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218 were carried out consecutively such that primary control cell populations proliferated actively at the 1
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219 point of reprogramming (Figure 2A). We obtained fresh skin biopsies from 4 patients in the FANCA y

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220 complementation group (FA-A) and one with unknown complementation group (FA-U), and cultured e
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221 keratinocytes from each. As expected, transduction with a FANCA-expressing retrovirus
222 complemented the FA pathway in FA-A cells, as shown by the ability to mono-ubiquitinate FANCD2 in
223 response to treatment with hydroxyurea (Figure 2B). The ability of E6 and E7 to promote
224 reprogramming was then analyzed in both complemented (FANCA) and control-transduced cells from
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