letter
`
`© 2001 Nature Publishing Group http://genetics.nature.com
`
`Disruption of a new forkhead/winged-helix protein,
`scurfin, results in the fatal lymphoproliferative disorder
`of the scurfy mouse
`
`Mary E. Brunkow1, Eric W. Jeffery1, Kathryn A. Hjerrild1, Bryan Paeper1, Lisa B. Clark1, Sue-Ann Yasayko1,
`J. Erby Wilkinson2, David Galas3, Steven F. Ziegler4 & Fred Ramsdell1
`
`Scurfy (sf) is an X-linked recessive mouse mutant resulting in
`lethality in hemizygous males 16–25 days after birth, and is char-
`acterized by overproliferation of CD4+CD8– T lymphocytes,
`extensive multiorgan infiltration and elevation of numerous
`cytokines1–4. Similar to animals that lack expression of either
`Ctla-4 (refs. 5,6) or Tgf-β (refs. 7,8), the pathology observed in sf
`mice seems to result from an inability to properly regulate
`CD4+CD8– T-cell activity3,9. Here we identify the gene defective
`in sf mice by combining high-resolution genetic and physical
`mapping with large-scale sequence analysis. The protein
`encoded by this gene (designated Foxp3) is a new member of
`the forkhead/winged-helix family of transcriptional regulators
`and is highly conserved in humans. In sf mice, a frameshift muta-
`tion results in a product lacking the forkhead domain. Genetic
`complementation demonstrates that the protein product of
`Foxp3, scurfin, is essential for normal immune homeostasis.
`
`The sf locus was mapped originally to a 1.7-cM interval
`between DXWas70 and Otc in the proximal region of the mouse
`X chromosome1,10. We used an intersubspecific backcross to fur-
`ther localize sf to a 0.3-cM interval (Fig. A, see http://genetics.
`nature.com/supplementary_info/) and, in parallel, constructed a
`sequence-ready BAC contig across the entire DXMit123–Otc
`interval. Probe content mapping of 11 overlapping BACs span-
`ning the DXCch1–DXCch2 region, however, indicated that the 4
`clones K50, K90, K60 and K70 defined the minimum tiling path
`(Fig. 1a); this region was estimated to be approximately 500 kb.
`By random shotgun sequencing, we identified 20 putative genes
`on the 4 BACs (Fig. 1a), which corresponded well with a recently
`published map in which transcripts were identified primarily
`through direct cDNA selection11. We discovered through computa-
`tional analysis an ORF with strong homology to the DNA-binding
`domain of the forkhead/HNF3/winged-helix family of proteins.
`
`a
`
`DXCch1
`
`DXMit55
`
`~ 500 kb
`
`scurfy
`(Foxp3)
`
`Cacna1f
`Syp Lmo6 Plp2
`DXMit26
`AA124124
`왘
`
`©2001 Nature Publishing Group http://genetics.nature.com
`
`AA273854
`
`AF229644
`왘
`
`왗
`
`K50 (196K10)
`100
`
`K90 (349I6)
`150
`
`왗
`
`←
`
`Cen
`
`b
`
`Hp
`
`Bs
`
`-12.5 kb
`
`왘
`
`왘
`
`왗
`K60 (8C22)
`180
`
`AA030924
`AF229636
`AF229635
`AF229645
`Ppia-ps
`왘 왘 왘 왘
`왗
`
`mT54
`
`왗
`
`Tcfe3
`DXCch4
`
`Kcnd1
`
`Smt3h2-ps
`왘
`
`왘
`
`AW342118
`왘
`
`왘
`
`Pim2
`(DXCch3)
`DXCch2
`AF229642
`→
`왘왘
`Tel
`
`K70 (284K10)
`200
`
`-2b
`
`-2a
`
`-1
`
`1
`
`2 3
`
`4 5
`
`6 7
`
`8
`
`9
`
`Bs
`
`10 11
`
`Bs
`
`Hp
`
`+18.3 kb
`
`wild type
`....GGACAAGAGCTC..
`....GGACAAAAGAGCTC... scurfy
`
`Fig. 1 Physical map of the sf region, genomic organization of Foxp3 and the sf mutation. a, The 500-kb sf candidate interval is limited by markers DXCch1 and
`DXCch2. BAC clones K50, K60, K70, K90 and library ID (in parentheses) are indicated. Computational analysis of this region identified 20 putative genes, including
`nine known genes (in bold): Tcfe3, Kcnd1, Pim2 and “human CMV-interacting protein”, as well as the mouse orthologs for SYP, LMO6, PLP2, T54 (ref. 15) and
`CACNA1F (encoding the calcium channel α1 subunit disrupted in congenital stationary night blindness30,31; MIM 310500); and two pseudogenes (on lowest level)
`related to mouse Smt-3B and Ppia. For the remaining new genes (underlined), corresponding to ESTs and/or GENSCAN predictions, RT–PCR experiments further
`extended and confirmed transcript structure. The transcriptional orientation of each transcript is indicated by a filled arrowhead. Polymorphic markers are
`shown in italics. b, Genomic organization of Foxp3 and the sf mutation. Coding exons are shown as filled black boxes, whereas noncoding regions are open
`boxes. Lines connecting exons indicate splicing events and the observed alternate polyadenylation signal is indicated by the dashed line. Note the two different
`5´ noncoding exons (-2a and -2b), separated by 640 bp on the chromosome, both of which splice to a second, common noncoding exon (-1). The putative 5´ end
`of the most distal non-coding exon, ‘-2b’, located 6.1 kb upstream from the first coding exon corresponds well to a promoter region predicted by GENSCAN. The
`sf mutation is the result of a 2-bp insertion in exon 8 (indicated below the schematic). Exon 8 was amplified and sequenced directly from genomic DNAs derived
`from the sf mutant, as well as four inbred (C57BL/6J, 101/Rl, C3Hf/Rl, 129/SvJ) and three wild-derived inbred (CAST/Ei, MOLF/Ei, SPRET/Ei) strains of mice. As
`expected, the 2-bp insertion was specific for sf mice. The HpaI sites (Hp) used to generate the 30.8-kb Foxp3 transgene, as well as the BstXI sites (Bs) used for
`Southern-blot analysis are shown. The transgene includes the entire gene as well as 12.5 kb and 2.8 kb of 5´ and 3´ flanking sequence, respectively.
`
`1Celltech Chiroscience, Inc., Bothell, Washington, USA. 2Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA. 3Keck Graduate Institute for
`Applied Life Sciences, Claremont, California, USA. 4Virginia Mason Research Center, Seattle, Washington, USA. Correspondence should be addressed to
`M.E.B. (e-mail: marybrunkow@chiroscience.com).
`
`68
`
`nature genetics • volume 27 • january 2001
`
`Page 1 of 6
`
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`
`letter
`
`NLC sf /Y sf /Y; Tg
`
`NLC
`L S T
`
`1292L 1292H
`L S T L S T
`
`2826
`2827
`2828
`L S T L S T L S T
`
`b
`
`c
`
`Bs
`
`9
`
`10 11
`
`Bs
`
`Bs
`
`Hp
`[
`30.8 kb
`
`3.1 kb
`
`4.8 kb
`
`2827
`
`NLC
`
`2828
`
`NLC
`
`2826
`
`1292H
`
`1292L
`
`NLC
`
`a
`
`Hp
`
`Bs
`
`[
`0 kb
`
`endogenous only
`transgene only
`endogenous
`3.1
`+ transgene
`
`Fig. 2 Analysis of Foxp3 transgenic mice. a, Southern-blot analysis was performed
`on genomic DNA prepared from tail tips, digested with BstXI (Bs) and probed with
`the fragment indicated by a heavy horizontal bar in the schematic diagram. At the
`endogenous Foxp3 locus, this fragment hybridizes to 3.1- and 4.8-kb fragments
`(shown in schematic, and on Southern blot); in the case of multiple copies of the
`30.8-kb HpaI (Hp) transgene integrated in a head-to-tail array, the probe
`hybridizes to the same internal 3.1-kb fragment as well as an unique 3.6-kb frag-
`ment (indicated on Southern blot). Representative samples from normal littermate controls (NLC) and transgene (Tg) lines 1292L, 1292H, 2826, 2827 and 2828 are
`shown. Using the endogenous 4.8-kb BstXI fragment as an internal control in each lane, transgene copy numbers were determined by analysis with a PhosphorImager
`445 SI. The copy numbers indicated in Table 1 are the result of analyzing multiple animals from each transgenic line. Additional higher molecular weight bands most
`apparent in the analyses of lines 1292H, 2827 and 2828 represent the unique single-copy BstXI fragments generated at the sites of transgene insertion. b, Lymph nodes
`from NLC, sf/Y mutant, and sf/Y; Tg animals were removed at 12 days of age. Lymphoid organs appeared normal in all sf/Y; Tg animals examined, irrespective of trans-
`gene copy number. c, Foxp3 transgene expression level in spleen (S) and thymus (T) roughly correlates with copy number, as determined by northern-blot analysis of
`total RNA prepared from animals at 10 days of age. A major transcript is seen at ∼2.4 kb and a minor species, at ∼3.7 kb, the difference in length being due to the use
`of alternative polyadenylation sites. As with endogenous Foxp3, Tg expression is undetectable in liver (L). We loaded 20 µg total RNA in each lane.
`
`~3.7 kb
`~2.4 kb
`
`This new transcript was not represented in the EST database, nor
`was it identified in the study mentioned above.
`We evaluated candidates by direct sequencing of RT–PCR
`products obtained from mutant and normal animals. We
`observed a change in a protein-coding sequence in only the
`sequence of the new forkhead gene. The sequence derived from sf
`thymus RNA included a 2-bp insertion within the coding region
`(Fig. 1b), which was confirmed by direct sequencing of a PCR
`product obtained from sf genomic DNA. Likewise, the lack of this
`insertion in PCR products from genomic DNA of seven different
`mouse strains, as well as in the analogous cDNA from human,
`indicated that it was in fact unique to the sf mutant. The
`frameshift resulting from the insertion leads to a truncated gene
`product lacking the carboxy-terminal forkhead domain.
`We confirmed the identity of the sf gene by functional comple-
`mentation of the mutation in transgenic mice. A 30.8-kb
`genomic fragment containing the entire gene was used for
`microinjection of mouse ooctyes (Fig. 1b). We obtained 5 inde-
`pendent transgenic lines, with copy numbers ranging from 3 to
`approximately 70 (Fig. 2a). Transgenic (Tg) males were mated
`with sf Otcspf/++ carrier females, and the scurfy phenotype was
`analyzed in male progeny. As expected, 50% of the males carried
`the Otcspf (sparse-fur) allele; half of these animals also suc-
`cumbed to scurfy disease by 16–18 days of age. However,
`approximately 50% of the Otcspf males (also presumed to carry
`the sf mutation) survived to weaning; several of these animals
`were killed at approximately 4 weeks of age, and visual inspec-
`tion revealed normal lymph nodes (Fig. 2b), spleen and liver.
`Southern-blot analysis of these animals indicated that they car-
`ried the transgene. None of the Otcspf (and sf) males with scurfy
`disease were transgenic. We found complete rescue of the scurfy
`defect with all five transgenic lines (Table 1). Northern-blot
`
`analysis revealed elevated expression of the sf gene in spleen and
`thymus that roughly correlated with transgene copy number
`(Fig. 2c and Table 1). sf Otcspf/Y; Tg males show no other overt
`phenotype besides sparse-fur, and animals that are older than
`one year of age are fertile. Likewise, females carrying the trans-
`gene, with or without the sf mutation, also seem to be normal.
`The lymph nodes from transgenic animals, however, were often
`smaller than those of their littermate controls, primarily due to a
`decrease in total T-cell number (Fig. 3). This was particularly
`acute in animals with higher expression of the transgene, but the
`effect was less pronounced in the spleen. In contrast, the thymus
`of transgenic animals seemed to be normal in number of cells
`and phenotype. More detailed analyses of these transgenic mice
`will be presented elsewhere (manuscript in preparation).
`To rule out the possibility that the sf mutation results in a dom-
`inant-negative protein, we generated transgenic animals that
`overexpressed the sf form of the gene. In analysis of five indepen-
`dent lines, overexpression of the mutant gene did not result in
`any overt phenotype, nor did it rescue disease in scurfy mice
`(data not shown).
`We generated sequence of full-length sf cDNA by assembling
`overlapping RT–PCR, as well as 5´- and 3´-RACE products from a
`number of different tissues (embryo, thymus, spleen). Compari-
`son of cDNA with the BAC K60 sequence resulted in a gene struc-
`ture (Fig. 1b). Multiple forms of mature message are produced as
`a result of alternative noncoding first exons as well as polyadeny-
`lation sites. In keeping with the recent proposal to devise a stan-
`dard nomenclature for the rapidly growing family of forkhead
`genes12, the gene mutated in sf mice has been designated Foxp3,
`and we refer to the normal gene product as scurfin.
`We surveyed a large number of mouse tissues for expression of
`Foxp3 using real-time RT–PCR, and the gene Dad1 as an
`
`nature genetics • volume 27 • january 2001
`
`69
`
`©2001 Nature Publishing Group http://genetics.nature.com
`
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`
`© 2001 Nature Publishing Group http://genetics.nature.com
`
`(see also http://www.biology.pomona.edu/fox.html). A sequence
`alignment including a selection of members from these various
`subfamilies, along with mouse and human FOXP3, is available
`(Fig. B, see http://genetics.nature.com/supplementary_info/). As
`in other family members, the scurfin winged-helix domain con-
`tains several basic residues at either end, which may serve to
`direct the protein to the nucleus19,20 (Fig. 5).
`Analysis of the scurfin sequence using BLAST and Motifs
`(GCG Wisconsin Package) also revealed the presence of a single
`C2H2 zinc-finger domain 116 amino acids upstream from the
`winged-helix domain. Aside from these two well-described func-
`tional motifs, the scurfin sequence is unique. In particular, we did
`not find a region with obvious similarity to previously described
`transcriptional activation domains. The identification of such a
`domain awaits further functional characterization of the protein.
`Foxp3 encodes a new member of the forkhead family of tran-
`scriptional regulators and is required for normal T-cell function.
`The Foxp3sf allele results in truncation of the protein and an appar-
`ent lack of functional activity. A number of other transcription fac-
`tors are critical for various aspects of lymphocyte development or
`lineage commitment21. Previous studies have demonstrated a
`causal association between expression of specific factors and com-
`mitment to a specific T-cell lineage, such as Th1 versus Th2 (for
`example, c-Maf (ref. 22), GATA-3 (ref. 23) and T-bet (ref. 24)), or
`maintenance of a quiescent state in T cells (for example, LKLF; ref.
`25). Our data suggest that the scurfin putative transcription factor
`is critical for control of immune responses and potentially T-cell
`number, acting as an apparent rheostat for T-cell activation, and
`functions predominantly in peripheral T cells.
`Both the cellular and biochemical mechanisms by which scurfin
`controls T-cell responses are yet to be determined. Although similar
`in phenotype to Ctla-4 and Tgf-β deficient mice, sf animals express
`
`NLC
`
`1292H 1292L
`
`2826
`
`2827
`
`2828
`
`scurfy
`
`NLC
`
`1292H 1292L
`
`2826
`
`2827
`
`2828
`
`scurfy
`
`NLC
`
`1292H 1292L
`
`2826
`
`2827
`
`2828
`
`scurfy
`
`6.0
`
`5.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`7.0
`
`6.0
`
`5.0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`cell number (x106)
`
`ratio
`
`1.80
`1.60
`1.40
`1.20
`1.00
`0.80
`0.60
`0.40
`0.20
`0.00
`
`cells number (x106)
`
`
`
`a
`
`b
`
`c
`
`letter
`
`Table 1 • Scurfin transgene rescues scurfy disease
`
`Tg line
`
`1292L
`1292H
`2826
`2827
`2828
`
`Tg copy
`no.
`
`3
`9
`∼16
`∼70
`∼45
`Totals
`
`Genotype of males (no. with scurfy disease)
`wt
`wt
`sf
`non-Tg
`non-Tg
`Tg
`
`sf
`Tg
`
`1 (0)
`1 (0)
`0
`0
`1 (0)
`3 (0)
`
`2 (2)
`1 (1)
`1 (1)
`2 (2)
`0
`6 (6)
`
`2 (0)
`6 (0)
`4 (0)
`0
`8 (0)
`20 (0)
`
`2 (0)
`4 (0)
`1 (0)
`3 (0)
`4 (0)
`14 (0)
`
`For each of the five transgenic lines, the transgene was crossed onto the sf
`mutant background; resulting male progeny were genotyped with respect to
`sf mutation (wt or sf) and transgene (non-Tg or Tg) status. The number of ani-
`mals with scurfy disease within each genotypic class was ascertained (in paren-
`theses). The last column demonstrates that the presence of the transgene,
`from any of the five lines, prevents disease in genotypically sf animals. Tg,
`transgene; wt, wild type.
`
`endogenous reference13 (Fig. 4). We found the highest levels of
`Foxp3 expression in lymphoid organs such as thymus and
`spleen, consistent with transgene expression (Fig. 2c). Further
`analysis of subpopulations of purified lymphoid cells showed
`Foxp3 expression in Th1 and Th2 cells, and much lower (90%
`decreased) levels in CD4–CD8+ and B220+ cells.
`During the course of analyzing Foxp3, a highly conserved
`human sequence (JM2) was deposited in GenBank. The high
`degree of similarity with Foxp3, combined with the fact that JM2
`falls within a chromosomal region with the same gene organiza-
`tion as Foxp3 (refs. 11,14,15), indicates that JM2 represents the
`human ortholog. Because JM2 was generated through concep-
`tual translation of genomic sequence from Xp11.23, we isolated
`human FOXP3 cDNAs by RT–PCR from a variety of tissues and
`determined the complete coding sequence. There were a number
`of differences between our human FOXP3 sequence and JM2
`(Fig. 5). We have identified a single, noncoding exon located 6.1
`kb upstream from the first coding exon. The longest human
`FOXP3 transcript we have characterized so far includes a 5´ UTR
`of 188 bp, an ORF of 1,293 bp, and a 3´ UTR of 388 bp. Exon-
`intron boundaries are identical across the coding regions of the
`mouse and human genes. The complete mouse and human
`scurfin sequences are shown (Fig. 5).
`We compared the amino acid sequences of mouse and human
`scurfin with other members of the forkhead/winged-helix/HNF3
`family of proteins. Of all these family members, the strongest
`similarity was to the partial protein sequence called glutamine-
`rich factor16, recently designated Foxp1. Based on these align-
`ments, we localized the winged-helix domain of scurfin to the
`region between amino acid residues 337 and 420, at the C termi-
`nus of the protein (Fig. 5). The scurfin forkhead domain contains
`many of the sequence features thought to be important for medi-
`ating protein-DNA contacts based on co-crystallization data
`using HNF3-γ (ref. 17) and another predicted structure16 (see
`also the accompanying report by Wildin et al.18). Recent phyloge-
`netic analysis of all available forkhead domains from chordates
`has resulted in the assignment of each into 1 of 17 subfamilies12
`
`Fig. 3 Diminished T-cell numbers in lymph nodes from transgenic mice. Lymph
`node cells from representative normal littermate controls (NLC), sf mice and
`the various transgene lines were examined for cell number and phenotype.
`Total cell number (a) from lymph nodes of transgenic mice are consistently less
`than that in NLC animals, and the extent of reduction is roughly parallel to
`transgene expression. The ratio of CD4+8– to CD4–8+ cells in the lymph nodes
`is altered in high-copy transgenic mice, due in part to a decrease in the per-
`centage of CD4–8+ cells (b). The total number of CD4+8– T cells in the lymph
`nodes of transgenic mice is less than that of NLC as well as sf animals (c). The
`alterations in cell numbers are consistent in at least 5 mice per transgenic line
`analyzed between 4 and 16 weeks of age, and the phenotype remains consis-
`tent on backcrossing to C57Bl/6 mice for at least six generations.
`
`70
`
`nature genetics • volume 27 • january 2001
`
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`
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`
`letter
`
`Fig. 4 Tissue distribution of Foxp3 expression. Levels of
`Foxp3 expression were determined using the standard
`curve method (separate tube reactions), in which Dad1
`served as endogenous reference, and the standard curve
`was generated from a dilution series of a standard cDNA
`sample. The standard curve was derived by plotting the
`threshold cycle (CT) versus starting quantity, in this case
`expressed in arbitrary units, and raw values for each of the
`two genes were then calculated from the CT of each test
`sample. The mean values from duplicate reactions for
`Dad1 (a) and Foxp3 (b) are shown. Normalized Foxp3 val-
`ues were then derived from the ratio of raw mean Foxp3
`to raw mean Dad1 value for each tissue sample (c). Mouse
`tissues analyzed were 18 days post coitum liver and thymus
`(lanes 1, 2); brain, heart, kidney, liver, lung, spleen and thy-
`mus from day 10 animals (lanes 3–9); brain, heart, kidney,
`lung, spleen and thymus adult tissues (lanes 10–15);
`CD4+CD8–, CD4–CD8+, B220+, Th1 and Th2 purified lym-
`phocytes (lanes 16–20); and no template control (lane 21).
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10 11 12 13 14 15 16 17 18 19 20 21
`
`tissue
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10 11 12 13 14 15 16 17 18 19 20 21
`
`tissue
`
`cDNA cloning and mutation detection. We extracted
`total RNA from thymus, spleen, brain and liver of 10-d
`normal and sf mice. We used total RNA (5 µg) to generate
`random-primed first-strand cDNA (SuperScript Pream-
`plification System, Gibco-BRL). For typical RT–PCR
`reactions, 1 µl cDNA (1/30th of total cDNA preparation)
`was used as template in a 25 µl reaction; typical amplifica-
`tion conditions on MJ Tetrad were 94 °C for 3 min, fol-
`lowed by 35 cycles of 94 °C for 60 s, 55 °C for 30 s, 72 °C
`for 60 s. The primers used for these experiments derived
`from all 18 transcription units located within the sf critical
`interval. In the case of known genes, we used published
`sequences to design primers spanning the entire cDNA.
`For new genes, primers were designed initially to span
`exons predicted by GENSCAN on partial genomic
`sequence contigs. RT–PCR products were either
`sequenced directly (using the same primers as were used
`for the initial amplification, as well as more internal
`primers as necessary) or cloned into the TA vector (Invit-
`rogen) and sequenced from vector-specific primers. The
`sf mutation was found by sequencing an ∼980-bp frag-
`ment resulting from amplification of spleen cDNA with
`the primers 5´–CTACCCACTGCTGGCAAATG–3´ and
`5´–GGTTGTGAGGGCTCTTTGAC–3´.
`For analysis of the sf mutation in genomic DNA,
`amplicons generated with primers 5´–CAGAGCCTG
`GTCTATACACTG–3´ and 5´–GAAGGAACTATTGC
`CATGGCTTC–3´ were sequenced directly using the same primers as in
`PCR. Cycling conditions were 94 °C for 5 min, followed by 35 cycles of 94
`°C for 45 s, 62 °C for 90 s, 72 °C for 90 s.
`
`Th1
`
`no te m pl.
`Th2
`
`6000
`
`5000
`
`4000
`
`3000
`
`2000
`
`1000
`
`0
`
`relative Dad1 mRNA
`
`a
`
`3000
`
`2500
`
`2000
`
`1500
`
`1000
`
`500
`
`0
`
`relativeFoxp3 mRNA
`
`b
`
`2
`1.8
`1.6
`1.4
`1.2
`1
`0.8
`0.6
`0.4
`0.2
`0
`
`(relative Foxp 3 / relative Dad1)
`
`c
`
`normalized mRNA levels
`
`©2001 Nature Publishing Group http://genetics.nature.com
`
`18 dpc thym us
`10 d brain
`C D4+C D8-
`10 d spleen
`ad brain
`10 d thym us
`ad thym us
`ad spleen
`10 d liver10 d lung
`
`18 dpc liver
`B220+
`10 d kidney
`C D4-C D8+
`ad lung
`10 d heart
`ad kidney
`ad heart
`
`both of these proteins. It remains to be determined which genes are
`regulated by scurfin activity, as does the mechanism by which
`scurfin itself is regulated. Nevertheless, the discovery that mutations
`in human FOXP3 lead to a syndrome very similar to scurfy (see
`Wildin et al.18 and Bennett et al.26, this issue) suggests that the
`scurfy mouse provides an invaluable model system through which
`to better understand, and ultimately to develop treatments for,
`severe autoimmune-related dysfunctions.
`
`Methods
`Animals. We conducted animal studies following PHS guidelines. Double-
`mutant sf Otcspf mice were maintained as described10. Carriers of the Otcspf
`mutation were identified by amplification of genomic DNA with the
`primers 5´–TCTGCTGGGAGGACACCC–3´ and 5´–GGCATTATCTAAG
`GAGAAGCATCA–3´, and subsequent digestion with the restriction
`endonuclease MseI (ref. 27). The original breeding stocks were obtained
`from Oak Ridge National Laboratory (ORNL), and Mus musculus casta-
`neous animals (CAST/Ei) were obtained from The Jackson Laboratory.
`
`Subcloning and sequencing the candidate region. We sequenced four BAC
`clones (CITB genomic library, Research Genetics) encompassing the sf crit-
`ical interval by the random shotgun method. All genomic sequences were
`subjected to BLAST (ref. 28) analysis against all publicly available GenBank
`databases, and exons were predicted using GENSCAN (ref. 29). All results
`of the sequence analyses were deposited in a viewer developed in-house (S.
`Bobick and G. Brickner, pers. comm.).
`
`Transgenic mice. We purified a 30.8-kb HpaI fragment from BAC K60 by
`treatment with GELase (Epicentre Technologies) after field-inversion gel elec-
`trophoresis (FIGE) through a 1% SeaPlaque GTG agarose (FMC BioProd-
`ucts) gel in 1×TAE. The HpaI fragment was ligated to a linearized, blunt-end-
`ed SuperCos I (Stratagene) vector overnight, then transformed into electro-
`competent DH10B. To prepare DNA for oocyte microinjection, the cosmid
`construct (20 µg) was digested with NotI and the ∼31-kb insert was isolated by
`FIGE on a 1.1% SeaPlaque GTG agarose gel in 1×TAE. The fragment was
`purified as above and dialyzed extensively against microinjection buffer (10
`mM Tris, 0.25 mM EDTA, pH 7.5). Microinjections of (C57BL/6×SJL)F2
`hybrid oocytes were carried out by DNX Transgenic Services.
`Lymph nodes from transgenic animals were removed, minced to create
`single-cell suspensions and analyzed for cell number and phenotype. Phe-
`notypic characterization was performed by staining of cells with fluores-
`cently labeled antibodies to CD4 and CD8 (Caltag) and analysis on a
`MoFlo flow cytometer (Cytomation).
`To generate the ‘scurfy’ mutant transgene, the single EcoRV fragment
`(∼11 kb) from the wild-type cosmid construct was first subcloned into
`pBluescript SK+ (Stratagene). An ∼4-kb Asp718 fragment, including exon 8
`of Foxp3, was then subcloned into pBS SK+ to generate the template for
`site-directed mutagenesis. Mutagenic primers 5´–GCAGCAAGAGCTC
`TTTTGTCCATTGAGG–3´ and 5´–CCTCAATGGACAAAAGAGCTCTT
`GCTGC–3´ were used to introduce the ‘scurfy’ 2-bp insertion, using the
`
`nature genetics • volume 27 • january 2001
`
`71
`
`Page 4 of 6
`
`YEDA EXHIBIT NO. 2071
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`© 2001 Nature Publishing Group http://genetics.nature.com
`
`MPNPRPAKPMAPSLALGPSPGVLPSWKTAPKGSELLGTRGSGGPFQGRDL
`|||||| || ||||||||||| |||: ||| |:||| || || ||||||
`MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDL
`
`RSGAH.TSSSLNPLPPSQLQLPTVPLVMVAPSGARLGPSPHLQALLQDRP
`| ||| .||||||:|||||||||.|||||||||||||| |||||||||||
`RGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRP
`
`HFMHQLSTVDAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSLKARPGLP
`|||||||||||||.|||||| ||:.|||||| ||. ||||||||||||||
`HFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLP
`
`PGINVASLEWVSREPALLCTFPRSGTPRKDSNLLAAPQGSYPLLANGVCK
`|||||||||||||||||||||| ||||| | | || |||||||||||
`PGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCK
`
`WPGCEKVFEEPEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQQLELEK
`||||||||||||:|||||||||||||||:||||||||.|||||||| |||
`WPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEK
`*
`EKLGAMQAHLAGKMALAKAPSVASMDKSSCCIVATSTQGSVLPAWSAPRE
`||| |||||||||||| || |||| || |||||| .|| |.|||| |||
`EKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPRE
`
`50
`
`50
`
`99
`
`100
`
`149
`
`150
`
`199
`
`200
`
`249
`
`250
`
`299
`
`300
`
`Fig. 5 Mouse and human scurfin proteins contain
`a highly conserved forkhead domain. Needle-
`man-Wunsch algorithm was used to align the
`mouse and human sequences; the two proteins
`have an overall similarity index of 86%. The fork-
`head domain, with a similarity index of 94%, is
`shaded, the C2H2 zinc finger domain is boxed and
`potential nuclear localization signals are under-
`lined. Asterisk indicates position of 2-bp insertion
`in sf mutant. One difference between the hypo-
`thetical JM2 transcript and mouse sf cDNAs was
`an in-frame insertion of 180 bp in the forkhead
`domain of the human sequence (between coding
`exons 10 and 11). The arrow below the human
`FOXP3 sequence indicates the site of this inser-
`tion, which would encode an additional 60 aa.
`
`APDGGLFAVRRHLWGSHGNSSFPEFFHNMDYFKYHNMRPPFTYATLIRWA
`||| |||||||||||||||.|||| |||||||:||||||||||||||||
`APD.SLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWA
`
`349
`
`349
`
`ILEAPERQRTLNEIYHWFTRMFAYFRNHPATWKNAIRHNLSLHKCFVRVE
`||||||:||||||||||||||||:||||||||||||||||||||||||||
`ILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVE
`
`399
`
`399
`
`SEKGAVWTVDEFEFRKKRSQRPNKCSNPCP..
`||||||||||| ||||||||||.:|||| |
`SEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP
`
`429
`
`431
`
`5´–GGCATCCACCGTTGAGAGC–3´. In addi-
`tion, the Stratagene Human Universal cDNA
`Library (HUCL) was screened by hybridization
`with a 730-bp fragment corresponding to the 5´
`half of the gene, obtained by PCR with primers
`5´–ATGCCCAACCCCAGGCCTGGC–3´
`and
`5´–CTCCAGAGACTGTACCATCTC–3´. A sin-
`gle polyadenylated clone was identified. The
`complete cDNA sequence was ascertained by
`combining various fragments obtained by all methods. To confirm that the
`intron included in the hypothetical JM2 cDNA sequence is in-frame with the
`rest of the coding sequence, we isolated a FOXP3-containing BAC from the
`CITB_978_SKB genomic library (Research Genetics) and directly sequenced
`across the appropriate region.
`
`Quickchange kit (Stratagene). The cycling conditions were 95 °C for 45 s,
`followed by 16 cycles of 95 °C for 30 s, 55 °C for 60 s, 68 °C for 14 min, and
`a final elongation step of 72 °C for 5 min. After digestion with DpnI and
`transformation into Escherichia coli strain DH10B, a plasmid containing
`the appropriate sequence alteration was identified by sequencing. The
`mutated Asp718 fragment was then re-introduced into the cosmid con-
`struct by reversing the two original subcloning steps described above.
`Preparation of DNA for oocyte microinjection was as above.
`
`Full-length mouse and human cDNAs. cDNA libraries prepared from nor-
`mal 10-d mouse thymus, adult mouse spleen (M.A. Gayle, pers. comm.) and
`mouse 15-d embryo (5´-STRETCH cDNA library, Clontech) were screened
`by nested PCR to obtain additional sequences 5´ and 3´ of the 980-bp Foxp3
`fragment described above. In these PCRs, vector-specific primers were paired
`with Foxp3-specific primers. For all three libraries, we used the following
`λgt10 vector primers: 5´–CGAGCTGCTCTATAGACTGCTGGGTAGTC–3´
`nested with 5´–CCTTTTGAGCAAGTTCAGCCTGG–3´; and 5´–TTGCAT
`ATCGCCTCCATCAACAAAC–3´ nested with 5´–GGTGGCTTATGAGTAT
`TTCTTCC–3´. Foxp3-specific primers used for isolating 5´ end sequences
`were as follows: 5´–CCAGGCCACTTGCAGA–3´ nested with 5´–CAT
`TTGCCAGCAGTGGGTAG–3´, and 5´–GCAGCTGGGATGGTGGCAG–3´
`nested with 5´–GCCCCACTTCGCAGGTCC–3´. For the 3´ end, a number of
`different primers were used, such as 5´–CATGGACTACTTCAAGTAC
`CAC–3´, and 5´–GTCAGAACACGCTCGTGTGCAC–3´, nested with
`5´–GTACACACATGCAGCCCCTCC–3´. We amplified the concentrated
`phage library stock (1 µl) with outer primers (94 °C for 3 min, followed by 35
`cycles of 94 °C for 45 s, 62 °C for 90 s, 72 °C for 90 s). This primary reaction (1
`µl) was then used as template in a second round of PCR using the appropriate
`nested primers (same cycling conditions). We also obtained 5´ sequences by
`applying the RACE technique on normal thymus and spleen RNAs, following
`the manufacturer’s instructions (Gibco-BRL 5´-RACE System v. 2.0). Full-
`length cDNA sequences of the alternative forms were ascertained by combin-
`ing all overlapping sequences; RT–PCR using primers from the extreme 5´
`and 3´ ends of Foxp3 cDNAs confirmed the structures as shown.
`We originally isolated human FOXP3 cDNA as two overlapping PCR prod-
`ucts from prostate cDNA (Marathon, Clontech). Primers were designed
`based on the sequence of JM2 and its similarity to the mouse gene:
`5´–CACACTGCCCCTAGTCATGG–3´ and 5´–GCATGGCACTCAGCTTC
`TC–3´ to amplify the 5´ region; 5´–GATGGTACAGTCTCTGGAG–3´ and
`5´–GCAAGACAGTGGAAACCTCAC–3´ for the 3´ region. To obtain addi-
`tional 5´ and 3´ sequences, including the UTRs, cDNA libraries derived from
`peripheral blood lymphocytes (λgt10 vector, M.A. Gayle, pers. comm.) and
`testis (λgt11 vector, Clontech) were screened with vector-specific/gene-spe-
`cific primer pairs. The longest 5´ extension was obtained from the testis
`λgt11
`library
`using
`vector-specific
`primer
`5´–CGGTTTCCAT
`ATGGGGATTGGTGGCGAC–3´ paired with the FOXP3 anti-sense primer
`
`Foxp3 expression. Northern-blot analysis was performed on RNA extract-
`ed from normal and transgenic tissues using reagents from Ambion (Total-
`ly RNA and NorthernMax-Gly kits) and overnight transfer to HyBond N
`filters (Amersham). A 360-bp fragment of Foxp3 amplified from thymus
`cDNA with primers 5´–CAGCTGCCTACAGTGCCCCTAG–3´ and 5´–CAT
`TTGCCAGCAGTGGGTAG–3´ was labeled with 32P-—dCTP by random
`hexamer incorporation (MegaPrime kit, Amersham) and used to probe the
`filters. After washing at high stringency, the filters were exposed to Hyper-
`film MP (Amersham Life Science).
`We also examined Foxp3 expression by real-time RT–PCR using an ABI
`Prism 7700 instrument. Random-primed first-strand cDNA (SuperScript
`Preamplification System, Gibco-BRL) was used as template in separate-tube
`amplification reactions. All samples were run in duplicate in each experiment.
`Primers were custom designed and obtained from PE Biosystems. Foxp3
`primers were 5´–GGCCCTTCTCCAGGACAGA–3´ and 5´–GCTGATCATG
`GCTGGGTTGT–3´, and the internal TaqMan probe was 5´–6FAM-AGCTTC
`ATCCTAGCGGTTTGCCTGAGAATAC-TAMRA–3´; Dad1 primers were
`5´–CCTCTCTGGCTTCATCTCTTGTGT–3´ and 5´–CCGGAGAGATGC
`CTTGGAA–3´, with TaqMan probe 5´–6FAM-AGCTTCATCCTAGCG
`GTTTGCCTGAGAATAC-TAMRA–3´. The TaqMan Universal Master Mix
`(PE Applied Biosystems) was used for all the reaction components, except
`primers, probe and template. The final primer concentrations were 300 nM
`for Foxp3 and 50 nM for Dad1. The final probe concentration for both Foxp3
`and Dad1 was 100 nM. Cycling conditions were 50 °C for 2 min; 95 °C for 10
`min; and 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The data was collected
`and analyzed by the ABI Prism 7700 Sequence Detection System Software,
`