Ku Haploinsuffiency Causes a Lymphoproliferative Disorder of Immature T-cell Precursors due to Ikaros Malfunction  

Zahide Ozer1,2 , Sanjive Qazi1,3 , Rita Ishkhanian1 , Paul Hasty4 , Hong Ma1 , Fatih M. Uckun1,5,6
1 Systems Immunobiology Laboratory and Developmental Therapeutics Program, Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA, 90027;
2 Molecular Oncology Program, Parker Hughes Institute, St. Paul, MN 55113;
3 Department of Biology and Bioinformatics Program, Gustavus Adolphus College, 800 W College Avenue, St. Peter, MN 56082;
4 Department of Molecular Medicine and Institute of Biotechnology, The University of Texas Health Science Center, San Antonio, Texas 78425;
5 Department of Pediatrics, University of Southern California Keck School of Medicine, Los Angeles, CA 90027, USA;
6 Developmental Therapeutics Program, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90089
Author    Correspondence author
International Journal of Molecular Medical Science, 2013, Vol. 3, No. 7   doi: 10.5376/ijmms.2013.03.0007
Received: 16 May, 2013    Accepted: 05 Jun., 2013    Published: 21 Jun., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Ikaros (IK) malfunction has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of IK activity is very important. Here we provide unique genetic and biochemical evidence that the Ku protein components Ku70 and Ku80 act as positive regulators of IK function via formation of IK-Ku70 and IK-Ku80 heterodimers with augmented sequence-specific DNA binding activity. siRNA-mediated depletion of Ku70 or Ku80 reduced the sequence-specific DNA binding activity of IK in EMSA as well as the RT-PCR measured IK target gene expression levels in human cells. The interaction of Ku components with IK likely contributes to the anti-leukemic effects of IK as a tumor suppressor, because Ku70 as well as Ku80 haploinsuffiency in mice caused development of a lymphoproliferative disorder (LPD) involving CD2+CD4+CD8+CD1+IL7R+ thymic T-cell precursors with functional IK deficiency.

Keywords
Immunity; Leukemia; Thymocyte; Lymphocyte; Tumor suppressor

1 Introduction
Ikaros (IK) is a zinc finger (ZF)-containing sequence-specific DNA-binding protein that plays an important role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny by both (a) gene transcriptional activation via efficient transcription initiation and elongation as well as (b) gene repression (Li et al., 2011; Dovat et al., 2011). Alternatively spliced transcripts of the IKFZ1 gene with selective inclusion of exons 4-7 encode at least eight zinc finger proteins with distinct DNA-binding capabilities and specificities (IK isoforms IK1 through IK8) in mice and humans (Li et al., 2011). The formation of homo- and heterodimers among the DNA binding IK isoforms increases their affinity for DNA (Li et al., 2011). Functional IK deficiency has been associated with development of T-cell precursor leukemias in mice and humans (Dovat et al., 2011; Sun et al., 1999).  Therefore, a stringent regulation of IK activity is considered of paramount importance.

NHEJ is the predominant DNA-double strand break (DSB) repair pathway in mammalian cells (Weterings and Chen, 2008). The heterodimeric Ku protein (Ku70/Ku80) functions as the versatile DNA-targeting regulatory subunit of the DNA-dependent protein kinase (DNA-PK) holoenzyme, a nuclear serine/threonine kinase that plays a critical role in the NHEJ pathway of DNA-DSB repair (Walker et al., 2001). Here we provide unique genetic and biochemical evidence that the Ku protein components Ku70 and Ku80 act as positive regulators of IK function both in vitro and in vivo. siRNA-mediated depletion of Ku70 or Ku80 reduced the sequence-specific DNA binding activity of IK in EMSA as well as the RTPCR measured IK target gene expression levels in human cells. The interaction of Ku components with IK likely contributes to the anti-leukemic effects of IK as a tumor suppressor, because Ku70 as well as Ku80 haploinsuffiency in mice caused development of a lymphoproliferative disorder (LPD) involving immature T-cell precursors with functional IK deficiency. Our study uniquely implicates Ku deficiency for the IK malfunction in T-cell precursors, which is a hallmark of pediatric high-risk T-lineage ALL.

2 Materials and methods
2.1 Standard biochemical, imaging, and transfection methods 
Confocal Laser Scanning Microscopy, co-immunoprecipitations, Western blot analyses, pull-down assays, and electrophoretic mobility shift assays (EMSA) were performed as per previously described standard procedures. 293T cells were transfected after reaching 70%~80% confluence using ONTARGETplus SMARTpool siRNA and DharmaFECT Transfection Reagent 4 (Catalog No. T-2004) (Thermo Scientific Dharmacon, Lafayette, CO, USA) (supplemental Methods). Reverse transcription (RT) and polymerase chain reaction (PCR) were used to evaluate the expression levels of IK target genes.

 
2.2 Bioinformatics and statistical analysis of gene expression profiles
The publically available archived GSE32311 database (Zhang et al., 2011)  was used to compare gene expression changes in CD4+CD8+ double-positive wild type (N=3; GSM800500, GSM800501, GSM800502) vs. IK null mouse thymocytes (N=3, GSM800503, GSM800504, GSM800505) from the same genetic background of (C57BL/6 x129S4/SvJae), as described (Uckun et al., 2012). Probe level RMA signal intensity values were obtained from the mouse 430_2.0 Genome Array. Up-regulated and downregulated transcripts in IKZF1/Ikaros knockout mice were identified by filtering changes greater than 2 fold and T-test P-values less than 0.05 (T-test, Unequal Variances, Excel formula). The GSE32311 database was also used to compare the gene expression profiles of normal thymocytes from 3 wildtype mice (GSM800500, GSM800501, GSM800502) to those of abnormal thymocytes of 8 preleukemic mice of the same genetic background of (C57BL/6x129S4/SvJae) with IKZF1/Ikaros null mutation (N=3, GSM800503, GSM800504, GSM800505) or expression of dominant negative IK isoforms (N=5, GSM800506, GSM800507, GSM802973, GSM802974, GSM802975).
 
2.3 Characterization of thymocyte populations in Ku knockout mice 
We examined the surface antigen profiles of thymocytes from Ku knockout mice using flow cytometric immunophenotyping. The IK function of the thymocytes was examined by EMSA (supplemental Methods).
 
3 Results
3.1 Heterodimerization of ikaros with Ku70 or Ku80 augments its sequence-specific DNA binding function
In co-immunoprecipitation experiments using Triton X-100 whole cell lysates from EBV-transformed non-leukemic human B-cells, both Ku70 and Ku80 immune complexes contained both Ku70 and Ku80 consistent with a stable physical association between these two components of the Ku70/Ku80 heterodimer (Figure 1A1). Notably, both Ku70 and Ku80 immune complexes also contained IK indicating that IK constitutively exists in a stable physical association with Ku protein (Figure 1A2). This association was further confirmed by demonstrating that IK immune complexes contain Ku both Ku70 and Ku80 (Figure 1A3). There was more Ku70 in Ku70 immune complexes and more Ku80 in Ku80 immune complexes than in IK immune complexes. Likewise, there was more IK protein in IK immune complexes than in Ku70 or Ku 80 immune complexes. These results indicate that not all of the IK protein exists in a complex with Ku components. We next performed “pull-down” experiments with MBP-tagged recombinant IK proteins. DNA-binding isoforms of Ikaros, IK1, IK2, and IK3 each pulled down both Ku70 and Ku80 proteins (Figure 1B1 & Figure 1B2).
 
High affinity binding of the most abundant IK isoform IK1 to DNA requires its homo- or heterodimerization with other DNA-binding IK isoforms (Li et al., 2011). After demonstrating that native IK can form complexes with both Ku70 as well as Ku80, we next performed electrophoretic mobility shift assays (EMSA) in a cell-free platform devoid of other proteins to evaluate the ability of heterodimers of recombinant IK with recombinant Ku70 or Ku80 to bind the IK-Bs1 oligonucleotide probe containing a high-affinity IK1 binding site. The addition of increasing amounts of Ku70 or Ku80 protein to the MBP-tagged IK1 protein resulted in increased retardation of the IK-specific IK-Bs1 probe in a dose-dependent fashion, which indicates stronger sequence-specific DNA binding by IK1-Ku heterodimers vs. IK1 homodimers (Figure 1C1). By using anti-IK, anti-Ku70, anti-Ku80 antibodies in EMSA supershift assays, we experimentally confirmed the presence of both Ku70 or Ku80 along with IK1 in the IK-specific DNA binding molecular complexes (Figure 1C2).

 
Figure 1 Ku components Ku70 and Ku80 form heterodimers with Ikaros


3.2 siRNA mediated knockdown of Ku expression diminishes the constitutive transcription factor activity of Ikaros in human cells
We could not employ RNA interference to evaluate the role of native Ku components for IK function in human lymphoid cells as Ku70-specific siRNA  or Ku80-specific siRNA causes apoptosis. In order to experimentally document the significance of Ku components Ku70 and Ku80 to the function of IK, we therefore examined the effects of their depletion by RNA interference on IK-specific DNA binding activity in nuclear extracts from 293T cells using EMSAs with the end-labeled IK-BS1 oligonucleotide probe containing a high-affinity IK binding site (Figure 2). The selective depletion of Ku70 and Ku80 by specific siRNA was documented using Western blot analyses (Figure 2A). We recently published 293T cells as an IK+ human cell line that provides the opportunity to study post-translational regulation of native IK (Uckun et al., 2012). Nuclear extracts of 293T cells exhibited abundant IK activity in EMSAs performed using the IKBs1 oligonucleotide probe containing a high-affinity IK1 binding site (Figure 2B) and expressed transcripts of validated IK target genes ITGA4, KIF23, TNFAIP8L2, PREP, DNAJC6, and EIF4E3 (Figure 2C). IK-specific siRNA (but not scrambled control siRNA) abrogated or reduced the expression of each of these 6 IK target genes (Figure 2C). Notably siRNA-mediated depletion of Ku70 or Ku80 abolished the DNA binding activity of native IK (Figure 2B). To formally document the importance of the Ku components Ku70 and Ku80 for the ability of IK to activate the expression of its target genes, we also examined the effects of their depletion by RNA interference on IK target gene expression in human 293T cells using RT-PCR (Figure 2C). Notably, the expression levels of all IK target genes were reduced by siRNA-mediated depletion of Ku70 or Ku80, whereas treatment with scrambled siRNA (included as a negative control) had no such effect. The striking Ku-dependency of the IK target gene expression levels is consistent with the notion that Ku plays a critical role in regulation of the IK function.

 
Figure 2 Depletion of native Ku70 or Ku80 abrogates sequence-specific DNA binding activity of ikaros in situ


3.3 Ku-Knockout mice develop a lymphoproliferative disorder of immature T-Cell with functional ikaros deficiency
Complete Ku deficiency is lethal to human cells (Wang et al., 2009) as well as C57Bl/6 mice, as the only mouse strain that mimics the human biology in regards to Kurequirements for viability and survival (Reliene et al., 2006). C57Bl/6 mice that are homozygous Ku knockouts are not viable past the early post-natal period (Reliene et al., 2006). Therefore, we sought to examine the biologic significance of partial Ku deficiency in C57Bl/6 mice that were heterozygous knockouts for Ku70 or Ku80 genes. These mice developed at 2~3 months of age a lymphoproliferative disorder (LPD) that was characterized by a 9.5 fold above baseline increase of the IL7R-positive immature T-precursor count in the thymus (29.3×106/thymus vs. 3.1×106/thymus, T-test p-value = 0.016) (Figure 3A). The composite immunophenotype of the thymocytes from Ku knockout mice was CD1+CD2+CD3+CD4+CD8+IL7R/CD127+FLT3/CD135+ consistent with the surface antigen profile of an immature cortico-thymocyte (Figure 3B; supplemental Figure 1). 

 
Figure 3 Heterozygous Ku-Knockout mice develop a PLD of IL7R+ immature thymic T-cell precursors


CD3 antigen is expressed on both very immature double-positive (CD4+CD8+) T-cell precursors and mature T-cells (Levelt et al., 1993; Koga et al., 1994), whereas CD1 antigen is expressed only on T-cell precursors at a corticothymocyte stage (Foon and Todd, 1986). Notably, IL7R gene is a transcriptional target for IK and expression of dysfunctional dominant negative IK isoforms or IKZF1 null mutation is associated with increased levels of IL7R gene expression in thymocytes during the preleukemic phase (Figure 4A). We therefore sought to determine the subcellular localization and DNA binding activity of IK in thymocytes of Ku-knockout mice. In sharp contrast to the nuclear localization of IK in all of the thymocytes from wildtype mice, IK was localized predominantly in the cytoplasm of all of the thymocytes from Kuknockout mice (Figure 4B & Figure 4C). The differences in IK localization between thymocytes from wildtype mice vs. thymocytes from the Ku80- and Ku80-knockout mice were statistically significant (Fisher’s exact test, 2-tailed, p<0.0001). Since the nuclear localization of IK is determined by its DNA binding activity, these results uniquely indicated that partial Ku deficiency adversely affects the DNA binding activity of native IK in murine thymocytes. IK has been shown to bind to repetitive sequences within PC-HC that contain consensus IK binding sites, and its localization to the PC-HC in the nucleus is directly related to its ability to bind to these sequences (Gurel et al., 2008; Popescu et al., 2009).  Therefore, we next performed EMSAs to directly examine the effect of partial Ku deficiency on the binding of native IK to the biotin labeled γ-satellite A probe derived from the centromeric γ-satellite repeat sequences. The γ-satellite A DNA probe contains two consensus IK binding sites in close proximity to each other and is a target for high affinity binding of wildtype IK and the binding affinity to this probe shows an excellent correlation with the homing of IK to PC-HC (Uckun et al., 2012; Gurel et al., 2008; Popescu et al., 2009). In agreement with their abnormal subcellular IK localization, thymocytes of Ku knockout mice showed no native IK activity as measured by binding of their nuclear extracts to the γ-satellite A probe (Figure 4D). The observed development of an LPD involving IL7R positive immature thymocytes with functional IK deficiency in Ku-heterozygous mice provided unique evidence for haploinsufficiency of Ku70/Ku80 genes and indicated that native Ku is physiologically important for normal IK function in mice.

 
Figure 4 Functional ikaros deficiency of thymocytes in Ku-knockout mice


4 Discussion
Currently, our knowledge regarding the upstream regulators of IK function is relatively limited. IK function, stability, and subcellular localization are generally thought to be regulated by posttranslational modification and heterodimerization with other members of the IK family of DNA binding proteins (Li et al., 2011). Phosphorylation of IK by casein kinase II (CK2) inhibits its many functions and promotes its degradation via the ubiquitin/proteosome pathway (Uckun et al., 2012). Conversely, dephosphorylation of IK by protein phosphatase 1 is critical for its ability to bind to target DNA sequences, localize to PC-HC in the nucleus, and exert its regulatory functions (Popescu et al., 2009).  In a recent study, we identified the spleen tyrosine kinase (SYK) as a posttranslational regulator of IK and determined that SYK-induced activating phosphorylation of IK at unique C-terminal serine phosphorylation sites S358 and S361 is essential for its nuclear localization and optimal transcription factor function (Uckun et al., 2012). We now report direct evidence that Ku components Ku70 and Ku80 bind to IK thereby augmenting its nuclear localization and sequence-specific DNA binding activity. The present study provides experimental evidence that Ku colocalizes with IK in human cells and its heterodimerization amplifies the transcription factor function of IK. Ku is the only protein outside the IK family of ZF proteins shown to non-enzymatically improve the function of IK as a sequence-specific DNA binding protein. In human B-lymphocytes cells, IK was constitutively associated with both Ku70 and Ku80 components of the Ku heterodimer. Recombinant Ku70 and Ku80 formed stable complexes with recombinant IK in vitro and enhanced its DNA binding activity. Native IK exhibited a normal multifocal nuclear localization in wildtype thymocytes, but a predominantly cytoplasmic expression in thymocytes from heterozygous Ku knockout mice. These results uniquely indicated that native Ku protein forms a complex with IK, promotes its nuclear localization and enhances its sequence-specific DNA binding activity. siRNA-mediated depletion of Ku70 or Ku80 abolished the sequence-specific DNA binding activity of IK in EMSA and reduced the RTPCR measured IK target gene expression levels in human cells. We conclude that Ku is critical for the nuclear localization and optimal transcription factor function of IK. Pull-down experiments using recombinant IK isoforms demonstrated that the Cys2His2 zinc finger (ZF) motifs of IK near its N terminus are important for the IK-Ku70/Ku80 interactions. The very similar abilities of recombinant IK1, IK2 and IK3 proteins to pull down Ku70 and Ku80 demonstrate that the protein domains encoded by IK exons 3 (E3) (missing in IK2), or 5 (E5) and 6 (E6) (missing in IK3) are not required for IK-Ku interactions. By comparison, MBP-IK4 and MBP-IK5 pulled down much smaller amounts of Ku70 or Ku80 than the MBP-IK-1, MBP-IK2, and MBP-IK3 fusion proteins. In view of the pull-down efficiency of MBP-IK2 lacking the E3-domain and MBP-IK3 lacking the E5 domain, the diminished Ku-binding of MBP-IK4 cannot be explained by lack of E3 or E5 domains. The poor Ku-binding of MBP-IK5 indicates that E1, E2, E3, and E7 domains are not sufficient for optimal IK-Ku interactions. The most discriminating similarity between MBP-IK1, MBP-IK2, and MBP-IK3 vs. MBP-IK4 and MBP-IK5 is the number of their N-terminal zinc fingers (4 in IK1, 3 in IK2 and IK3 but only 2 in IK4 and 1 in IK5) indicating that the zinc fingers likely participate in the physical contact between IK and Ku proteins. The 3-D structure of IK has not been resolved and the exact roles of the 6 C2H2 ZFs of IK in its interactions with DNA and their relative contributions to its DNA binding affinity remain unknown (26). The future elucidation of the structural basis of IK activation by its interactions with Ku70 or Ku80 will require a 3-D structure determination of IK at atomic resolution using Xray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The interaction of Ku components with IK likely contributes to the anti-leukemic effects of IK as a tumor suppressor in vivo (Dovat et al., 2011), because Ku haploinsufficiency caused development of a lymphoproliferative disorder (LPD) in heterozygous Ku70 and Ku80 knockout mice involving IL7R positive immature T-cell precursors with functional IK deficiency. It is noteworthy that comprehensive bioinformatic studies on haploinsufficiency have indicated that the Ku80/XRCC5 gene is highly likely to be haploinsufficient (Huang et al., 2010). As Ku70 and Ku80 are not haploinsufficient for DNA double strand break (DSB) repair and even Ku-null cells have normal DNA repair activity due to hyperactive alternative NHEJ pathway (Fattah et al., 2010), the development of a T-cell precursor hyperplasia in haplodeficient mice cannot be explained by DNA repair deficits and provides compelling evidence that the interaction of Ku components with IK likely contributes to the antileukemic effects of IK as a tumor suppressor. Our study uniquely implicates Ku deficiency for the IK malfunction in T-cell precursors, which is a hallmark of pediatric high-risk T-lineage ALL.
 
Acknowledgments
F.M.U’s research program is funded in part by DHHS grants U01-CA-151837, R21-CA-164098, and R01CA-154471 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. F.M.U is also supported in part by the 2011 V-Foundation Translational Research Award. This work was supported by endowment funds of the Hughes Chair in Molecular Oncology at Parker Hughes Institute (FMU), Children’s Hospital Los Angeles Institutional Endowment and Special Funds (FMU), Couples Against Leukemia Foundation (FMU), Ronald McDonald House Charities of Southern California, Couples Against Leukemia Foundation (FMU), Nautica Triathalon and its producer Michael Epstein and a William Lawrence & Blanche Hughes Foundation grant (FMU). Ku70 and Ku80 cDNA were kindly provided by Dr.Stephen P. Jackson (Wellcome Trust/Cancer Research UK Gurdon Institute). We further thank all members of the Uckun lab, especially Anoush Shahidzadeh, Lisa Tuel-Ahlgren and Nancy Dvorak for their many invaluable technical assistance and contributions. We also thank Ernesto Barron of the USC Norris Comprehensive Cancer Center Cell and Tissue Imaging Core (supported by P30CA014089), and Mrs. Tsen-Yin Lin of the CHLA FACS Core for their assistance.
 
Authorship
Contribution: F.M.U designed and directed research; Z.O., R.I., S.Q., H.M., A.S performed research and analyzed data; P.H. provided Ku-deficient mice; Z.O., S.Q., and F.M.U wrote the paper; all authors reviewed and revised the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests Correspondence to: Fatih M. Uckun, M.D., Ph.D., Children’s Center for Cancer and Blood Diseases, Children’s Hospital Los Angeles, MS#160, Los Angeles, California 90027-0367.
 
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Supplemental information
1 Supplemental materials and methods
1.1 Ku-knockout mice and genotyping

Trained personnel of the AAALAC-approved Saban Institute Animal Care Facility (ACF) at Children’s Hospital Los Angeles cared for animals. Mouse experiments were conducted with approval from the CHLA Animal Care and Use Committee (IACUC) (Protocol # 293-10, Approval Date: 06/24/2010) and all animal care procedures conformed to the Guide for the Care and Use of Laboratory Animals of the National Research Council (National Academy Press, Washington DC 1996). Ku knockout mice (genetic background: 129xC57BL/6 cross) were previously reported (Li et al., 2007). Standard rederivation protocols were used to obtain Ku knockout mice with a C57BL/6J genetic background by the surgical transfer of pre-implantation stage embryos to the reproductive tract of pseudo-pregnant recipient C57BL/6 females. Mice were electively sacrificed painlessly by standard CO2 asphyxiation, in agreement with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (AVMA) and following the standard operating procedures of the Saban ACF. For genotyping, tail DNA was isolated after elective sacrifice of mice using the QIAamp DNA Mini Kit (Catalog No. 51306) (Qiagen, Santa Clarita, CA, USA) following manufacturers instructions. 2 μL of isolated DNA was used in a 25 μl PCR reaction using AmpliTaq Gold PCR Master Mix (Catalog No. 4318739) (Life Technologies, Grand Island, NY, USA). The Ku70 and Ku80 wildtype and mutant primers were used as previously described (Li et al., 2007). The Ku80 wild-type allele was detected with the sense primer 5’-GAGAGTCTACGACAACTGTGC-3’ and the antisense primer 5’-AGAGGGACTGCAGCCATATTA-3’ located in sequences deleted in the mutant Ku80 allele described as xrc5M1 (Li et al., 2007). The mutant allele was detected with a sense primer (5’-GGTTGCCAGTCATGCTACGGT-3’), which anneals to intronic sequences upstream of the positive selection cassette, and an antisense primer (5’ CCAAAGGCCTACCCGCTTCCATT-3’), which anneals to the PGK promoter in the positive selection cassette. PCR reactions were preincubated at 94oC for 5 min and then 29 cycles of amplification at 94℃ for 30 sec, 59℃ for 1 min (with 1 min ramp), and 72oC for 30 sec in a PTC100 MJ Research thermal cycler. Likewise, the Ku70 wild-type allele was detected with the sense primer 5’-GGCTGGCTTTAGCACTGTCA-3’ and the antisense primer 5’-ACACGGCTTCCTTAATGTGA-3’) (3). The mutant Ku70 allele was detected with the sense primer 5’-GGCTGGCTTTAGCACTGTCA-3’ and the antisense primer 5’-ACGTAAACTCCTCTTCAGACCT-3’), as previously reported. PCR reactions were preincubated at 94℃ for 5 min and then 35 cycles of amplification at 94℃ for 45 sec, 56℃ for 45 sec and 72℃ for 1.5 min in a PTC100 MJ Research thermal cycler. PCR products were separated on a 1.5% TBE-agarose gel containing 0.5 μg/ml ethidium bromide and visualized using a UVP Epi Chemi II Darkroom Transilluminator. Surface antigen profiles of thymoytes from 3 months old wildtype (Median age: 83 days) and heterozygous Ku knockout mice (Median age: 106 days) were determined by direct immunofluoresecence and flow cytometry using a panel of monoclonal antibodies recognizing distinct T-lineage differentiation antigens using standard methods (Uckun et al., 2012). The labeled cells were analyzed using a The LSR II flow cytometer from Becton Dickinson (BD).

1.2 Reagents
The polyclonal antibodies for Ku70 (goat polyclonal, M-19, sc-1487), Ku80 (rabbit polyclonal, H-300, sc-9034) and mouse anti-goat IgG: HRPO (sc-2354) as well as mouse anti- Ku70 monoclonal antibody (MoAb) (sc-71470), and mouse anti-Ku80 MoAb (sc-53021) were purchased from Santa-Cruz Biotechnology, Inc. (Santa Cruz, CA). The rabbit polyclonal antibody for Ikaros (IK)1 (H-100, sc-13039) was purchased from Santa-Cruz Biotechnology, Inc. (Santa Cruz, CA) for Western blot analysis and fluorescence staining of IK using previously reported procedures.2 The mouse monoclonal anti-IK antibody was prepared in our laboratory. Goat anti-mouse IgG: Horseradish Peroxidase (HRPO) (M15345) and goat anti-rabbit IgG: HRPO (R14745) antibodies were purchased from Transduction Labs. Green-fluorescent Alexa Fluor 488 dye-labeled secondary antibody Alexa Fluor 488 goat anti-mouse IgG (A-11001) and red-fluorescent Alexa Fluor 568 dye-labeled secondary antibody Alexa Fluor 568 F(ab')2 fragment of goat anti-rabbit IgG (A-21069) for confocal microscopy were purchased from Invitrogen (Carlsbad, CA). UltraCruz™ Mounting Medium containing 1.5 μg/ml of 4', 6-diamidino-2-phenylindole (DAPI) was purchased from Santa-Cruz Biotechnology, Inc. (sc-24941). For immunophenotyping of the mouse thymocytes from wildtype and Ku knockout mice, we used the following fluorochrome-labeled rat anti-mouse monoclonal antibodies from BD Biosciences (SanJose, CA): IL7R/CD127-PE cat#552543, FLT3/CD135-APC cat#560718, c-KIT/CD117-FITC cat#, 553354, CD1D-FITC cat#553845, CD2-PE cat. # 553112, CD3-FITC cat#555274, CD4-PE cat# 561829, and CD8-FITC cat# 553030. Molecular Weight Markers was purchased from Amersham Pharmacia Biotech. All chemicals used were reagent grade or higher. pFastBacHT cloning vector, SF21 cells and lipofectamine were purchased from Invitrogen/Gibco (Carlsbad, CA). Protein A-Sepharose was purchased from Repligen Corp., Cambridge, MA). Restriction enzymes, proteinase inhibitors were purchased from Roche (Indianapolis, IN). Hi-Trap-Ni chelation column was purchased from Amersham Biosciences Piscataway, NJ). siRNA pools for (a) ku70 (Gene ID: 2547) (Catalog No. L-005084) including 5’-GAUCCAGGUUUGAUGCUCA-3’, 5’-UUAGUGAUGUCCAAUUCAA-3’, 5’-CAGGGUGGGAGUCAUAUUA-3’, and 5’-AUAAAGCUCUAUCGGGAAA-3’; (b) ku80 (Gene ID: 7520) (Catalog No. L-010491) including 5’-AAACUUCCGUGUUCUAGUG-3’, 5’-GAGCAGCGCUUUAACAACU-3’, 5’-CGAGUAACCAGCUCAUAAA-3’, and 5’-GCAUGGAUGUGAUUCAACA-3’; (c) btk (Gene ID: 695) (Catalog No. L-003107) including 5’-UGAGCAAUAUUCUAGAUGU-3’, 5’-CAACUCUGCAGGACUCAUA-3’, 5’-GGUGAUACGUCAUUAUGUU-3’, and 5’-GCGGAAGGGUGAUGAAUAU-3’; and (d) ikaros/ikzf1 (Gene ID: 10320) (Catalog No. L-019092) including 5’ -GCUCAUGGUUCACAAAAGA-3’, 5’-CAAGUAACGUCGCCAAACG-3’, 5’-GCGCAGCGGUCUCAUCUAC-3’, and 5’-GGACGCACUCCGUUGGUAA-3’; as well as ON-TARGETplus Non-Targeting siRNA scrambled control pool (Catalog No. D-001810) were purchased from Thermo Scientific Dharmacon, Lafayette, CO, USA.

1.3 siRNA transfections
293T cells were transfected with siRNA after reaching 70-80% confluence using ON-TARGETplus SMARTpool siRNA and DharmaFECT Transfection Reagent 4 (Catalog No. T-2004) (Thermo Scientific Dharmacon, Lafayette, CO, USA) following the manufacturer’s instructions, as previously reported (Uckun et al., 2012). 50 nM of ON-TARGETplus SMARTpool siRNA and transfection reagent were mixed in antibiotic-free complete media and added to adherent cells. In experiments aimed at evaluating the effects of Ku70 or Ku80 knockdown on native IK function, cells were incubated with the respective siRNA for 72 h at 37℃ in a humidified 5% CO2 atmosphere before they were examined for the subcellular localization andfunction of IK.

1.4 Confocal laser scanning microscopy
Subcellular co-localization studies using immunofluorescence and confocal microscopy were performed as previously described (Uckun et al, 2012). During confocal imaging, slides were imaged using the PerkinElmer Ultraviewer Confocal Dual Spinning Disc Scanner (Shelton, CT). Images were analyzed and processed using the Velocity ver 5.4 imaging visualization software.

1.5 Expression of recombinant human Ku in Sf21 insect ovary cells
The 2.0 kb Ku70 and 2.2 kb Ku80 cDNA fragments including their protein coding segments5 were individually cloned into the NcoI/KpnI site of the 4.9 kb pFastBacHT (PFBH) donor vector (Life Technologies) containing a 6x-histidine (6xHis) tag to construct recombinant PFBH-Ku70 and PFBH-Ku80 plasmids. PFBH-ku70 and PFBH-ku80 were used to generate recombinant baculoviruses by site-specific transposition in Escherichia (E.) coli DH10Bac competent cells (Life Technologies), which harbor a baculovirus shuttle vector (bacmid), bMON14272 with a mini-attTn7 target site for site-specific transposition using previously reported procedures (Uckun et al., 2010a; Uckun et al., 2010b; Uckun et al., 2010c).3,6,7 The bacterial colonies containing recombinant bacmids were identified by disruption of the lacZa gene. High molecular weight miniprep DNA was prepared from selected E.coli clones containing recombinant bacmid and transfected into Sf21 cells using the Cellfectin reagent (Life Technologies) as previously described (Uckun et al., 2010a; Uckun et al., 2010c; Mahajan et al., 2001). Sf21 cells were infected with both recombinant baculoviruses to produce Ku70/Ku80 heterodimer. Cultures were incubated at 28℃ and stirred at 80-100 rpm using a magnetic stirrer (Bellco Glass, Inc., Vineland, NJ) for 48 h. Infected cells were harvested by gentle centrifugation in a Beckman GS-6 centrifuge at 500 x g for 7 min at room temperature. Cells from 1-liter cultures were flash-frozen at -80℃ and stored at -80℃ until purification of recombinant Ku70 and Ku86 proteins. Frozen Sf21 insect cells were lysed in 1x Triton X-100 extraction buffer (1% Triton X-100, 10 mM Tris, 130 mM NaCl, 10 mM NaF, 10 mM sodium phosphate, pH 7.5) (1 mL lysis buffer per 20×106 cells). One pellet of CompleteTM protease inhibitors (Roche Molecular Biochemicals) was added for each 25 mL of the lysate and the mixture was rotated for 2 h at 4℃. The cell pellets were centrifuged at 45 000 rpm × 1 h a Beckman Optima LE-80K ultracentrifuge using a 45 Ti rotor. Following centrifugation, the clarified supernatant was filtered through a 0.22 μm membrane filter (S100) and dialyzed for 4 h into buffer A (20 mM sodium phosphate, 10% glycerol, pH 7.2). For purification of Ku70 and Ku80 proteins as well as Ku70/Ku80 heterodimer, dialyzed supernatants were applied to a Nickel-chelation column (Pharmacia) that was equilibrated with buffer B (20 mM sodium phosphate, 500 mM NaCl, 0.5 M imidazole, 10% glycerol, pH 7.2). Ku70 or Ku80 enriched fractions were dialyzed against buffer C (20 mM Tris, pH 8.0 with 1 M DTT, 10% glycerol) overnight to remove imidazole and then applied to a Sepharose Q HP26/10 ion exchange column (column volume 50 ml; Amersham Pharmacia Biotech) followed by size exclusion chromatography on a Superdex 200 HR 10/30 column (Pharmacia) for further purification.

1.6 Standard biochemical assays
Immunoprecipitations (50 x 106 cells/sample) and immunoblotting (10 x 106 cells/sample) using the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech) were performed, as described in detail in previous publications (Li et al., 2007; Uckun et al., 2012; Uckun et al., 2010a; Uckun et al., 2010b; Mahajan et al., 2001).

1.7 Pull-down experiments using MBP-Ikaros fusion proteins
The cDNAs encoding each of 5 distinct isoforms of IK (IK1, IK2, IK3, IK4, IK5) were individually cloned into the E-coli expression vector pMAL-C2 with the isopropyl-1-thio-β-galactopyranoside-inducible Ptac promoter to create an in frame fusion between the particular IK isoform coding sequence and the 3’end of the Ecoli maIE gene, which codes for maltose-binding protein (MBP). E-coli strain DH5a was transformed with the generated recombinant plasmid and single transformants were expanded in 5 ml of LB medium (1% tryptone, 1% NaCl, 0.5% yeast extract) containing ampicillin (1 000 μg/ml) by overnight culture at 37℃. Expression of the MBP-IK fusion proteins was induced with 10 mM isopropyl-1-thio-β-galactopyranoside. The cells were harvested by centrifugation at 4500 xg in a Sorvall RC5B centrifuge for 10 min at 4℃, lysed in sucrose-lysozyme buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 10% sucrose, 1 mM EDTA, 20 mM lysozyme), and further disrupted by sonication. After removal of the cell pellets by centrifugation at 35 000 xg for 1 h at 4℃, each MBP-IK fusion protein was purified from the respective culture supernatant by amylose affinity chromatography, as previously described (Uckun et al., 2010c; Mahajan et al., 2001). MBP-IK proteins were noncovalently bound to amylose beads under conditions of saturating protein, as reported (Uckun et al., 2010a; Mahajan et al., 2001). Nonidet P-40 lysates of the EBV-transformed lymphoblastoid cell line BCL-1 were prepared and 500 μg of the lysates was incubated with 50 μl of MBP-IK fusion protein coupled to amylose beads for 2 h on ice. The fusion protein adsorbates were washed with ice-cold 1% Nonidet P-40 buffer and resuspended in reducing SDS sample buffer. Samples were boiled for 5 min and then fractionated on SDS-PAGE. Proteins were transferred to Immobilon-P (Millipore Corp) membranes, and membranes were immunoblotted with anti-Ku antibodies.

1.8 Electrophoretic mobility shift assays (EMSAs)
EMSAs were performed on purified recombinant proteins as well as nuclear extracts from 293T cells, as previously described (Uckun et al., 2012;  Mahajan et al., 2001). Preparation of nuclear extracts was carried out using CHEMICON’s Nuclear Extraction Kit (Catalog No. 2900) (Millipore, Billerica, MA, USA) with some modifications. 5 million cells were spun down and washed with 1X phosphate buffered saline (PBS). Cells were resuspended in cytoplasmic buffer containing 0.5 mM DTT and a 1/1000 dilution of Protease Inhibitor Cocktail by gentle inversion and were then incubated on ice for 15 minutes. The cell suspension was centrifuged at 250 xg for 5 minutes at 4℃. The cell pellet was resuspended in ice-cold cytoplasmic lysis buffer and disrupted using a 27-gauge needle. The disrupted cell suspension was centrifuged at 8 000 x g for 20 minutes at 4℃. The supernatant containing the cytosolic portion of the cell lysate was removed. The remaining pellet containing the nuclear portion of the cell lysate was resuspended in ice-cold nuclear extraction buffer containing 0.5 mM DTT and 1/1000 dilution of Protease Inhibitor Cocktail. The nuclear suspension was gently agitated using an orbital shaker at 4℃ for 1 hour and then centrifuged at 16 000 x g for 5 minutes at 4℃ to isolate the extracted nuclear protein fraction. The protein concentration of extracts was determined by using the Quick StartTM Bradford Protein Assay Kit (Catalog No. 500-0202) (Bio-Rad, Hercules, CA, USA). Oligonucleotide probes for EMSA were purchased from Integrated DNA Technologies (IDT, San Diego, CA, USA) and included IK-BS1 (5’-TCAGCTTTTGGGAATACCCTGTCA-3’) and IK-BS5 (5’-TCAGCTTTTGAGAATACCCTGTCA-3’) (Uckun et al., 2012). The IK-BS1 oligonucleotide probe containing a high-affinity IK1 binding site was endlabeled with [γ-32P]ATP (3 000 Ci/mmol) using T4 polynucleotide kinase and purified using a Nuctrap probe purification column (Stratagene). ~3 μg samples of the nuclear extracts and 1 ng of labeled IK-BS1 probe (1×105 cpm/ng) were used in the DNA binding reaction. For competition reactions, a 60-fold excess of unlabeled IK-BS1 probe was added prior to the addition of the labeled IK-BS1 probe. The IK-BS5 oligonucleotide has a single base pair (G>A) substitution at position 3 within the core consensus and does not bind IK (4). In addition, we used the gamma (γ)-satellite probe A (5’-TATGGC GAGGAAAACTGAAAAAGGTGGAAAATTTAGAAATGT-3’ and 5’-ACATTTCTAAATTTTCCACCTTTTTCAGTTTTCCTCGCCATA-3’) derived from the centromeric γ-satellite repeat sequences (Uckun et al., 2012). The γ-satellite A DNA probe contains two consensus IK binding sites in close proximity to each other and is a target for high affinity binding of wildtype IK and the binding affinity to this probe shows an excellent correlation with the homing of IK to PC-HC. EMSAs were also performed using the Thermo Scientific LightShift Chemiluminescent EMSA Kit (Catalog No. 20148) (Pierce, Rockford, IL, USA) following the manufacturer’s protocol (Uckun et al., 2012). In these experiments, single stranded IK-BS1 and IK-BS5 oligonucleotides were biotin-labeled using the Biotin 3’ End Labeling Kit (Catalog No. 89818) (Pierce, Rockford, IL, USA). 100 nmols of unlabeled oligonucleotides were incubated for 30 minutes at 37℃ in a reaction mixture containing 1X TdT Reaction Buffer (500mM cacodylic acid, 10mM CoCl2, 1 mM DTT, pH 7.2), 0.5 μM Biotin-11-UTP and 0.2 Units of TdT. 0.2 M EDTA was added to stop the reaction and biotin-labeled oligonucleotides were extracted using chloroform: isoamyl alcohol (24:1). Singlestranded biotinylated oligonucleotides were duplexed by mixing together equal amounts and incubating for 1 hour at room temperature. Each binding reaction for EMSA included 10X Binding Buffer (100 mM Tris, 500 mM KCl, 10 mM DTT), 2.5% Glycerol, 5 mM MgCl2, 50 ng Poly (dI∗dC), 0.05% NP-40, ~0.4 (1x) or ~4 μg (10x) nuclear protein extract (NE), and 20 fmols of the biotin-labeled duplexed probe in a total volume of 20 μl. Binding reactions were performed at room temperature for 20 minutes. A 6% non-denaturing polyacrylamide gel was pre-run during the 20 min incubation time at 200 V in pre-chilled 0.5X TBE buffer (89 mM Tris base, 89 mM boric acid, 1 mM EDTA, pH ~8.0). 5X Loading Buffer was added to each reaction sample and samples were loaded onto a polyacrylamide gel. Samples were electrophoresed at 100V and transferred at 380 mA (~50 V) for 30 minutes to a Biodyne B Nylon Membrane (Catalog No.77016) (Thermo Scientific, Rockford, IL, USA) soaked in 0.5X TBE buffer. When the transfer was complete, biotin-labeled DNA was cross-linked to the membrane at 120 mJ/cm2 using a Spectrolinker XL-1000 UV cross-linker with 254 nm UV light bulbs. The biotin-labeled DNA was detected using a stabilized streptavidin-horseradish peroxidase (HRP) conjugate and a highly sensitive chemiluminescent substrate according to the manufacturer’s instructions (4). The membrane was exposed to X-ray film and developed with a film processor.

1.9 RT-PCR analysis of ikaros target genes
Reverse transcription (RT) and polymerase chain reaction (PCR) were used to evaluate the expression levels of 6 IK target genes, as previously reported (Uckun et al., 2012). Total cellular RNA was extracted from cells using the QIAamp RNA Blood Mini Kit (Catalog No. 52304) (Qiagen, Santa Clarita, CA, USA) following manufacturers instructions. Oligonucleotide primers were ordered from Integrated DNA Technologies (IDT, San Diego, CA,USA) to amplify a 244 bp region of the ITGA4 transcript (Gene ID 3676: 5’-GAGTGCAATGCAGACCTTGA-3’ and 5’-TGGATTTGGCTCTGGAAAAC-3’), a 236 bp region of the KIF23 transcript (Gene ID 9493: 5’-CGGAAACCTACCGTGAAAAA-3’ and 5’-AGTTCCTTCTGGGTGGTGTG-3’), a 168 bp region of the TNFAIP8L2 transcript (Gene ID79626: 5’-GGCACTTAGCTTTGGTGAGG-3’ and 5’-AGCAGACCTGGGTCAGAGAA-3’), a 245 bp region of the PREP transcript (Gene ID 5550: 5’-TGAGCAGTGTCCCATCAGAG-3’ and 5’- CATCTTCGCTGAACGCATAA-3’), a 232 bp region of the DNAJC6 transcript (Gene ID9829: 5’-GTCCTTCGCCCACAGTACAT-3’ and 5’-TTGCTGGCAAAGGAAGAACT-3’), and a 209 bp region of the EIF4E3 transcript (Gene ID 317649: 5’-CCGCAGCAGATGATGAAGTA-3’ and 5’- GTGTTTTCCACGTCCACCTT-3’). QIAGEN One-Step RT-PCR Kit (Catalog No.210212) (Qiagen, Santa Clarita, CA, USA) was used following manufacturer’s instructions tamplify the target PCR products. The conditions were 1 cycle x (30 min 50 ℃, 15 min 95℃) and 35 cycles x (45 sec 94℃, 1 min 60℃, 1 min 72℃). PCR products were separated on a 1.2% agarose gel and visualized after ethidium bromide staining using UVP Epi Chemi II Darkroom
Transilluminator.

2 Supplemental data
Supplemental Figure 1. Composite immunophenotypes of thymic T-cell precursors from Ku- knockout mice. The surface antigen profiles of thymocytes from heterozygous Kuknockout mice were determined using multi-parameter flow cytometry using fluorochromelabeled rat anti-mouse monoclonal antibodies and their isotype matched controls. Depicted are the results from M-63 thymocytes (Ku80 K/O), M-98 thymocytes (Ku70 K/O), and M-100 thymocytes (Ku70 K/O). The majority of the cells had a composite immunophenotype of a double-positive CD1+CD4+CD8+ corticothymocyte with elevated IL7R expression level: CD1+CD2+CD3+CD4+CD8+CD117+CD127+CD135+.
 
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