ORIGINAL ARTICLE

The Nude Mutant Gene Foxn1 Is a HOXC13 Regulatory Target during Hair Follicle and Nail Differentiation Christopher S. Potter1,2,7, Nathanael D. Pruett1,7, Michael J. Kern3, Mary Ann Baybo3, Alan R. Godwin4,5, Kathleen A. Potter1,2, Ron L. Peterson6, John P. Sundberg2 and Alexander Awgulewitsch1

Among the Hox genes, homeobox C13 (Hoxc13) has been shown to be essential for proper hair shaft differentiation, as Hoxc13 gene-targeted (Hoxc13tm1Mrc) mice completely lack external hair. Because of the remarkable overt phenotypic parallels to the Foxn1nu (nude) mutant mice, we sought to determine whether Hoxc13 and forkhead box N1 (Foxn1) might act in a common pathway of hair follicle (HF) differentiation. We show that the alopecia exhibited by both the Hoxc13tm1Mrc and Foxn1nu mice is because of strikingly similar defects in hair shaft differentiation and that both mutants suffer from a severe nail dystrophy. These phenotypic similarities are consistent with the extensive overlap between Hoxc13 and Foxn1 expression patterns in the HF and the nail matrix. Furthermore, DNA microarray analysis of skin from Hoxc13tm1Mrc mice identified Foxn1 as significantly downregulated along with numerous hair genes. This Foxn1 downregulation apparently reflects the loss of direct transcriptional control by HOXC13 as indicated by our results obtained through co-transfection and chromatin immunoprecipitation (ChIP) assays. As presented in the discussion, these data support a regulatory model of keratinocyte differentiation in which HOXC13-dependent activation of Foxn1 is part of a regulatory cascade controlling the expression of terminal differentiation markers. Journal of Investigative Dermatology (2011) 131, 828–837; doi:10.1038/jid.2010.391; published online 30 December 2010

INTRODUCTION Bieberich et al., 1991; Kanzler et al., 1994; Chuong and The HOX transcription factors are crucial regulators of Noveen, 1999; Reid and Gaunt, 2002). However, several Hox developmental processes (Duboule, 1992; Krumlauf, 1994; genes are expressed globally in all follicles (Awgulewitsch, Akin and Nazarali, 2005), including epidermal and hair 2003). This includes homeobox C13 (Hoxc13), which is follicle (HF) differentiation (Awgulewitsch, 2003). A primary expressed primarily in the upper bulb region of the HF of all role of Hox genes is defining regional identities in axial and hair types in mice (Godwin and Capecchi, 1998) and humans paraxial embryonic structures (Duboule, 1992; McGinnis (Jave-Suarez et al., 2002). Hoxc13 expression is initiated at the and Krumlauf, 1992; Capecchi, 1997), and certain Hox genes early stages of HF differentiation, both during HF morphogen- apparently establish topographical specificity in the skin esis and the progressive growth phase (anagen) in cycling hair, and its appendages, including HFs (Chuong et al., 1990; and it continues in the proximal-most region of catagen follicles during HF regression (Shang et al., 2002). In anagen follicles, 1Department of Medicine, Medical University of South Carolina, Charleston, the Hoxc13 expression domain encompasses distinct subpopu- South Carolina, USA; 2The Jackson Laboratory, Bar Harbor, Maine, USA; lations of cells primarily in the matrix and the three cylind- 3 Department of Regenerative Medicine and Cell Biology, Medical University rical layers of the differentiating hair shaft, including cuticle, of South Carolina, Charleston, South Carolina, USA; 4Department of Molecular and Integrative Physiology, University of Kansas Medical Center, cortex, and medulla (Jave-Suarez et al., 2002; Potter et al., Kansas City, Kansas, USA; 5Department of Microbiology, Immunology, and 2006). Functional studies revealed that both Hoxc13-null Pathology, Colorado State University, Fort Collins, Colorado, USA and tm1Mrc 6 (Hoxc13 )andHoxc13-overexpressing transgenic (FVB/ Integrative Expression Profiling, Novartis Institutes of Biomedical Research, NTac-Tg(Hoxc13)61B1Awg/J) mice exhibit severe hair growth Cambridge, Massachusetts, USA defects (Godwin and Capecchi, 1998; Tkatchenko et al., 2001), 7These authors contributed equally to this work. resulting in structurally defective hair shafts in both cases. In the Correspondence: Alexander Awgulewitsch, Department of Medicine/Division of Rheumatology and Immunology, Medical University of South Carolina, latter case, this manifests itself in the delayed formation of a CSB912, 96 Jonathan Lucas Street, Charleston, South Carolina, 29425, USA. thin and scruffy-looking hair coat during postnatal development E-mail: [email protected] and progressive alopecia during adulthood (Supplementary Abbreviations: BS, binding site; ChIP, chromatin immunoprecipitation Figure S1b online; Tkatchenko et al., 2001), whereas Hoxc13- FFL, feed-forward loop; Foxn1, forkhead box N1; Foxq1, forkhead box Q1; null mice fail to grow any external hair, thus resul- HF, hair follicle; Hoxc13, homeobox C13; KAP, keratin-associated ; p.n., post natum ting in a completely nude appearance (Supplementary Figure Received 16 February 2010; revised 24 October 2010; accepted 3 November S1d online; Godwin and Capecchi, 1998); in addition to 2010; published online 30 December 2010 alopecia, loss of Hoxc13 function results in defective nail

828 Journal of Investigative Dermatology (2011), Volume 131 & 2011 The Society for Investigative Dermatology CS Potter et al. HOX13 Regulation of Foxn1

development (Godwin and Capecchi, 1998). These hair and and broke off at the surface (Figure 1k), thus producing a nail defects are remarkably similar to the overtly same phenocopy of the Hoxc13-null hair defect in this region. phenotypic characteristics exhibited by nude mutant mice Anagen HFs of adult Hoxc13-null and nude mutant mice (Supplementary Figure S1d and S1c online; Mecklenburg et al., examined at X8 weeks showed essentially the same changes 2005) that are homozygous for the Foxn1nu mutated allele of as those seen at 5 days p.n. (data not shown). Foxn1 (forkhead box N1), a member of the forkhead domain Compared with C57BL/6J-Tyrc2J controls (Figure 2a–d), family of transcription factors (Nehls et al., 1994; Meier et al., the Hoxc13-null (homozygous B6.Cg-Tyrc2J Hoxc13tm1Mrc) 1999; Mecklenburg et al., 2001). Foxn1nu/Foxn1nu mice have mice had a normal-appearing proximal nail fold; however, primarily been studied for their immunodeficiency based on unlike the control mice where the stratum granulosum ended athymic aplasia (Mecklenburg et al., 2005). half way into the invagination, the stratum granulosum here The overt phenotypic parallels between nude and continued well into the matrix (Figure 2e–h). Instead of a Hoxc13-null mice compelled us to hypothesize that the regular translucent nail plate, these mice formed a structure two genes function in common pathways of keratinocyte that resembled a layer of cornified, stratified, squamous differentiation in both hair and nails. The striking similarities epithelium. Only near the proximal end of the nail bed a in the histopathological changes of hair and nail between structure reminiscent of a normal nail plate was formed. these mutants presented here strongly support this idea. The The nude mice exhibited similar, but not identical, changes downregulation of Foxn1 expression in the postnatal skin of (Figure 2i–l). The stratum granulosum extended from the Hoxc13-null mice combined with results from HOXC13/ proximal nail fold to the matrix (Figure 2j) as was seen for the Foxn1 transient co-transfection and chromatin immuno- Hoxc13-null mice. In contrast to the former, most nude mice precipitation (ChIP) assays suggest that Foxn1 is a direct had no evidence of a nail plate but rather a hyperplastic, regulatory target for HOXC13. Foxn1 is thus the second cornified, epidermis-like layer, although this was not consistent member of the forkhead transcription factor gene family between individuals (some mice had abortive nail plate-like regulated by HOXC13, as we have previously demon- structures). This finding differed from previous analyses of nude strated HOXC13-dependent transcriptional control of Foxq1 mice at 8–12 weeks of age in which a thinned nail plate was (forkhead box Q1) in the HF medulla (Potter et al., 2006). reported to form (Mecklenburg et al., 2004). This phenotypic Together, these results suggest that Hoxc13 is at the core variation may reflect age differences at the time of analysis (5 of a regulatory network essential for both HF and nail days vs. 8–12 weeks) and differences in strain backgrounds differentiation. (outbred Hsd-Foxn1nu/Foxn1nu used here vs. inbred NMRI- Foxn1nu/Foxn1nu mice in the Mecklenburg study). Similarly, the RESULTS thin nail plate-like structure observed with Hoxc13-null mice at Hair and nail defects of Hoxc13-null and Foxn1nu mice 5 days p.n. was replaced with a hyperplastic, cornified are strikingly similar epidermis at X8 weeks of age, which was similar to what Histopathological analysis of the skin from Hoxc13-null was seen in the young nude mice (data not shown). Depending (Hoxc13tm1Mrc/Hoxc13tm1Mrc) and nude (Hsd-Foxn1nu/Foxn1nu) upon environmental conditions and genetic background, the mice, as well as C57BL/6J mice (controls), was performed nail matrix either produces a cornified, hyperplastic epidermis at 5 days post natum (p.n.) and at X8 weeks of age. At 5 days or an abortive nail plate. p.n., all HFs in the dorsal skin of both mutants and the controls were near their final stage of morphogenesis Downregulation of Foxn1 in hair and nail of Hoxc13-null mice (Figure 1a, d, and h); at this stage, HFs are histologically DNA microarray analysis of the 5-day p.n. skin from identical to follicles in late anagen of cycling hair (Stenn and Hoxc13tm1Mrc/Hoxc13tm1Mrc mice versus wild-type littermates Paus, 2001). Bulb regions of HFs from Hoxc13-null mice revealed differential expression of 180 genes (qo0.05), includ- appeared superficially normal compared with controls, ing 31 upregulated and 149 downregulated genes (Supplemen- although the usually distinct columns of follicular keratino- tary Table S1 online; Gene Expression Omnibus series: cytes forming the different components of the hair shaft GSE23759). The most strongly dysregulated genes belonged to appeared grossly disorganized (Figure 1b and e). This loss of the gene ontology group / organization in the upper matrix was reflected in particular that included numerous and keratin-associated by the disruption of the normal septate pattern of the HF protein (KAP) genes. Table 1 lists differentially expressed genes medulla in more distal regions (Figure 1f). At the level of the knowntobeactiveinskinandhair(n ¼ 47), all of which were sebaceous gland, the hair shafts became twisted and distorted downregulated. The majority of these genes were keratin and and contained amorphous eosinophilic material (Figure 1g). KAP genes (n ¼ 37), and many were previously found to be These abnormal hair shafts apparently had insufficient differentially expressed in the 5-day p.n. skin of Hoxc13- structural rigidity to exit through the osteum like normal hair overexpressing mice (Tkatchenko et al., 2001; Potter et al., fibers (compare Figure 1c), a likely primary cause for the 2006), as well as in the dorsal skin of Dsg4lah/Dsg4lah mutant overall alopecic appearance of Hoxc13-null mice. The mice (Bazzi et al., 2009). Notably, Foxn1 was among the 13 follicular matrix of nude mice exhibited a similar loss of downregulated ‘‘non-keratin/KAP’’ genes belonging to diverse organization (Figure 1i), although medulla formation ap- gene ontology groups. peared to be normal (Figure 1j). However, at the level of the The Foxn1 expression differential in the skin (–2.3-fold; sebaceous gland, the hair twisted within the infundibulum Table 1) was validated by quantitative PCR indicating

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Figure 1. Homeobox C13 (Hoxc13)-null and nude mice exhibit similar hair defects. Histological comparison of hair follicles (HFs) in hematoxylin and eosin (H&E)-stained sections (10 mm) of dorsal skin from 5-day post natum (p.n.) mice of the following strains: (a–c) C57/BL6L-Tyrc2J (controls); (d–g) Hoxc13- null (homozygous B6.129-Hoxc13tm1Mrc/ Hoxc13tm1Mrc); and (h–k) nude (Hsd-Foxn1nu/ Foxn1nu). In all strains, HFs were uniformly in late anagen phase of the hair cycle. Note that in both Hoxc13-null and nude mice the hair shaft became twisted and distorted at the level of the sebaceous gland (sb) and failed to penetrate the epidermis (arrows in panels g and k). For further explanations, see text; m, medulla; mtx, matrix; ost, osteum; pctx, precortex. Scale bars ¼ 500 mm in panels a, d, and h, and 10 mm in the remaining panels.

2.4-fold downregulation (Figure 3k), and potential changes in In the differentiating nail epithelium of C57BL/6J-Tyrc2J the expression patterns of Hoxc13 and Foxn1 in HFs and nails control mice, Hoxc13 and Foxn1 expression patterns in the were cross-examined in Hoxc13-null (homozygous B6.Cg- upper matrix were essentially identical (Figure 3f and g), and Tyrc2J Hoxc13tm1Mrc) and nude mice at 5 days p.n. by in situ the Foxn1 pattern agreed with previously published data (Lee hybridization. The normal Hoxc13 and Foxn1 patterns in HFs et al., 1999). In Hoxc13-null mice, expression of the of C57BL/6J-Tyrc2J control mice showed extensive overlap truncated Hoxc13 transcript was no longer detectable, in several compartments, including cuticle and cortex of the probably because of the largely ablated and transformed differentiating hair shaft, and the matrix and inner root sheath matrix (Figure 3h). However, in the nail matrix of nude mice, (Figure 3a and b). In the hair of Hoxc13-null mice, expression residual Hoxc13 could still be observed (Figure 3i), whereas of the truncated Hoxc13 transcript was detected exclusively Foxn1 expression was not detectable in the truncated nail in the upper matrix (Figure 3c). In HFs of nude mice, Hoxc13 matrix of Hoxc13-null mice (Figure 3j). was expressed in a pattern resembling its pattern in the controls, including the typical proximal boundary in the HOXC13 regulates Foxn1 expression matrix at the mid-level of the dermal papilla (Figure 3d). In In silico analysis of the Foxn1 promoter region revealed contrast, expression of Foxn1 was no longer detectable in multiple matches to the HOXC13 consensus binding 0 A 0 Hoxc13-null HFs (Figure 3e), thus suggesting that Foxn1 sequence (5 -TT /TATNPuPu-3 ) (Figure 4a) known to interact expression requires Hoxc13 activity. with both mouse and human HOXC13 (Jave-Suarez et al.,

830 Journal of Investigative Dermatology (2011), Volume 131 CS Potter et al. HOX13 Regulation of Foxn1

Figure 2. Nails of homeobox C13 (Hoxc13)-null and nude mutant mice exhibit similar structural defects. Histological comparison of nails in hematoxylin and eosin (H&E)-stained sections (10 mm) of rear foot digits derived from the following mouse strains at 5 days post natum (p.n.): (a–d) C57/BL6J-Tyrc2J mice (controls); (e–h) Hoxc13-null (homozygous B6.Cg-Tyrc2JHoxc13tm1Mrc); (i–l) nude (Hsd-Foxn1nu/ Foxn1nu). Compared with control mice Hoxc13-null and nude mice had similar defects including reduced nail matrix (mtx), a defective nail plate (np), and an extended stratum granulosum (SG) with its distal limits (marked by arrows) reaching into the matrix as explained in the text; cu, cuticle; nb, nail bed; pnf, proximal nail fold. Scale bars ¼ 200 mm, panels a, e, and i, and 5 mmin the remaining panels; distal points to the right.

2002; Pruett et al., 2004; Potter et al., 2006). Transient proximal Foxn1 promoter region (Figure 4a) demonstrated co-transfection assays using C2C12 myofibroblasts were amplification of a Foxn1-specific DNA fragment encompass- performed with FLAG (F)-tagged HOXC13 expression plas- ing BS2 but not BS1 (Figure 4d). Interestingly, unlike BS1, BS2 mids and a Foxn1-luciferase reporter gene construct (Foxn1- was found to reside in a subregion of the Foxn1 promoter that luc) that included proximal Foxn1 promoter sequences is phylogenetically conserved between mouse, chimpanzee, extending 5 kb upstream of the Foxn1 transcriptional start and Rhesus monkey (Supplementary Figure S2 online). Control site (Waterston et al., 2002; Figure 4a). As demonstrated reactions were performed with precipitated chromatin and in Figure 4b, significant differences were seen in luciferase primers specific for a previously tested region of the Foxq1 activity between transfection groups (F(2, 33) ¼ 68.42, promoter (Foxq1-B) containing a functional HOXC13 binding Po0.001)). Specifically, co-transfection with Foxn1-luc and site (Potter et al., 2006) and a paired related homeobox 1 full-length HOXC13 expression vector (HOXC13-F) indicated (Prx1) promoter region not containing a HOXC13 consensus 6-fold upregulation of luciferase activity relative to control binding sequence; whereas a PCR amplification product was assays without HOXC13-F (Po0.001; Figure 4b). Notably, observed for Foxq1-B, none was detected for paired related co-transfection with a HOXC13 expression vector in which homeobox 1 (Figure 4d). Combined with the results from the the homeodomain had been deleted (HOXC13Dhd-F) failed co-transfection assays and the altered Foxn1 expression to demonstrate significant luciferase activation compared in vivo, these data demonstrate that HOXC13 mediates with the control (Foxn1-luc alone; Po0.05), thus suggesting transcriptional regulation of Foxn1; however, the specific homeodomain-dependent activation of Foxn1. Western blot in vivo contribution of the particular binding site BS2 will need analysis of C2C12 cells transfected with either HOXC13-F or to be determined through a systematic dissection of the Foxn1 HOXC13Dhd-F using FLAG-specific antibodies showed promoter region with further in vivo validation. roughly equal levels of protein expression for both the full- length and the truncated versions of the protein (Figure 4c). DISCUSSION In vivo occupancy of the Foxn1 promoter region by The 2.3-fold downregulation of Foxn1 in the 5-day p.n. skin HOXC13 was determined by ChIP of C2C12 cells transfected of Hoxc13-null mice was accompanied by a drastic down- with HOXC13-F. Testing of precipitated chromatin by PCR regulation of hair structural genes, primarily keratin and KAP amplification using two primer sets specific for putative genes (Table 1). Earlier reports of Foxn1-dependent dysregu- HOXC13 binding site (BS) 1 and 2, respectively, in the lation of some of the same genes, including Krt31, Krt32,

www.jidonline.org 831 CS Potter et al. HOX13 Regulation of Foxn1

Table 1. Subset of genes downregulated in the skin of Hoxc13-null mice Entrez clone ID Fold change Current symbol Gene name Previous/alternative gene symbol

71369 241.29 Krtap16-10 Keratin associated protein 16-10

170651 235.46 Krtap16-1 Keratin associated protein 16-1

71363 231.19 Krtap7-1 Keratin associated protein 7-1

170657 228.74 Krtap16-9 Keratin associated protein 16-9 71888 200.85 Krt33a Keratin 33A 68484 193.37 Krtap16-8 Keratin associated protein 16-8 77918 188.00 Krtap16-5 Keratin associated protein 16-5 16700 185.72 Krtap6-1 Keratin associated protein 6-1 16704 180.84 Krtap8-2 Keratin associated protein 8-2 23927 177.32 Krtap14 Keratin associated protein 14 mKAP13, Pmg1 16672 169.26 Krt34 Krt1-4, mHa4

76444 142.77 Krtap4-7 Keratin associated protein 4-7 KAP4.7

26560 135.74 Krtap15 Keratin associated protein 15 Pmg2

268905 133.33 Krtap13-1 Keratin associated protein 13-1 KAP13.1

16679 132.74 Krt86 Keratin 86 Krt2-10, Krt2-11, mHb6

53617 120.25 Krt35 Keratin 35 Krt1-24, mHa5 16701 98.67 Krtap6-2 Keratin associated protein 6-2

11571 94.56 Crisp1 Cysteine-rich secretory protein 1 Aeg1 170654 86.56 Krtap16-4 Keratin associated protein 16-4

16660 86.05 Krt31 Keratin 31 Krt1-1, Ha1 16671 69.95 Krt33b Keratin 33B Krt1-3, mHa3

57390 62.45 Psors1c2 Psoriasis susceptibility 1 candidate 2 (human)

77914 39.87 Krtap17-1 Keratin associated protein 17-1

114566 39.41 Krt82 Keratin 82 Krt2-20

69473 33.65 Krtap3-1 Keratin associated protein 3-1 KAP3.1 71623 33.43 Krtap5-2 Keratin associated protein 5-2

69533 32.53 Krtap26-1 Keratin associated protein 26-1

16670 21.89 Krt32 Keratin 32 Krt1-2, mHa2

66708 17.04 Krtap3-2 Keratin associated protein 3-2 KAP3.2

114666 15.78 Krtap5-5 Keratin associated protein 5-5

16673 12.70 Krt36 Keratin 36 Krt1-22, Krt1-5

17752 11.40 Mt4 Metallothionein 4 MT-IV

16694 8.24 Krtap12-1 Keratin associated protein 12-1 D10Jhu14e

50775 7.89 Krtap5-4 Keratin associated protein 5-4

20216 7.36 Acsm3 Acyl-CoA synthetase medium-chain family member 3

60594 7.36 Capn12 calpain 12

13506 5.72 Dsc2 Desmocollin 2 Dsc2a, Dsc2b

56370 4.89 Tagln3 Transgelin 3 Np25

105866 4.61 Krt72 Keratin 72

16680 4.30 Krt84 Keratin 84 Krt2-16, Krt2-3, mHb4

237987 4.18 Otop2 Otopetrin 2

320864 3.73 Krt26 Keratin 26

72685 3.37 Dnajc6 DnaJ (Hsp40) homolog, subfamily C, member 6

16846 2.71 Lep Leptin

15218 2.32 Foxn1 Forkhead box N1 Whn, Hfh11

12163 2.19 Bmp8a Bone morphogenetic protein 8a Bmp7r1, OP2

20652 2.02 Soat1 Sterol O-acyltransferase 1 ACAT-1 Subset of differentially expressed genes in the dorsal skin of 5-day post natum (p.n.) homeobox C13 (Hoxc13)-null mice that are known to be active in skin and hair as determined by querying the Mouse Genome Informatics (MGI) database (http://www.informatics.jax.org/). Genes are listed under current gene nomenclature symbols; as the nomenclature, particularly of keratin genes, was substantially revised recently (Schweizer et al., 2006), common previously used gene symbols are listed in the last column. For genes shown in bold, evidence for direct regulation by FOXN1 has previously been demonstrated as discussed in the text; shading indicates genes downregulated in the skin of Desmoglein4 (Dsg4)-null mice (Bazzi et al., 2009); underlining denotes HOXC13 target genes validated by DNA-binding studies (see text).

832 Journal of Investigative Dermatology (2011), Volume 131 CS Potter et al. HOX13 Regulation of Foxn1

1.5

1.0

0.5

0.0

mRNA level change - Foxn1 fold Relative Wild type Hoxc13 null

Figure 3. Disruption of forkhead box N1 (Foxn1) expression pattern in the hair follicles (HFs) and nails of homeobox C13 (Hoxc13)-null mice. In situ hybridization (ISH) analysis of Hoxc13 and Foxn1 expression patterns in the hair and nails from C57BL/6J-Tyrc2J (control), Hoxc13-null (homozygous B6. Cg-Tyrc2JHoxc13tm1Mrc), and nude mice. (a, b) Hoxc13 and Foxn1 expression (bluish stain) overlapped in cortical/precortical regions of control HFs (yellow arrows). (c) Reduced Hoxc13 expression in HF matrix of Hoxc13-null mice. (d) HFs of nude mice showed characteristic aspects of the Hoxc13 pattern, whereas (e) Foxn1 expression was undetectable in HFs of Hoxc13-null mice; note twisted hair shafts in Hoxc13-null and nude mice (arrows in c, d). (f) Hoxc13 expression in nail matrix (mtx) of control mice (g) mirrored the Foxn1 pattern. (h) Hoxc13 expression was undetectable in the nail matrix of Hoxc13-null mice, but (i) weakly present (orange arrow) in the nails of nude mice, and (j) Foxn1 expression was undetectable in Hoxc13-null nail matrix. (k) Bar graph showing the relative 2.4-fold change in Foxn1 expression in Hoxc13-null versus control (wild-type) mice as measured by reverse transcriptase PCR (RT-PCR). Scale bars ¼ 50 mmina–e, and 100 mminf–j.

Krt33b, Krt34, Krt35, Krt84, Krt86, and Krtap5-4 (Schlake expression does not exclusively result from the loss of et al., 2000; Schorpp et al., 2000; Schlake, 2001; Schlake and HOXC13 as a putative direct regulator, but may reflect the Boehm, 2001), combined with the similar histopathological collapse of a regulatory cascade involving Foxn1 (and possibly changes in hair and nail of Hoxc13-null and nude mice Hoxc12; see Shang et al., 2002) as a potential amplifier of observed here, suggest that Hoxc13 and Foxn1 act in some of HOXC13 activity in Krtap16 regulation (Figure 5). the same differentiation pathways. The data from HOXC13/ This proposed co-regulation of Krtap16 and other KAP and Foxn1 co-transfection and ChIP analysis (Figure 5) suggest keratin genes by HOXC13 and FOXN1 is consistent with that HOXC13 acts as a transcriptional regulator of Foxn1. findings demonstrating strong activation of Krt33b (previously The complexity of the Hoxc13 expression pattern in Krt1-3, mHa3) and Krt85 (previously Krt2-18, mHb5) upon different HF compartments suggests that Hoxc13 is involved tetracycline-induced FOXN1 expression in HeLa cells that in controlling the differentiation of various distinct trichocyte was dependent on the presence of the FOXN1 N-terminal lineages, each expressing a specific set of terminal differ- DNA-binding domain (Schlake et al., 2000). Accordingly, entiation markers. Studies involving electrophoretic mobility one may conceptualize a regulatory network of hair shaft shift assays and co-transfection assays suggested that differentiation that involves, in addition to a hierarchical HOXC13 controls the expression of human keratin genes HOXC13-controlled FOXN1 pathway, parallel pathways in KRT32 (previously KRTHA2), specifically expressed in the which keratin genes are regulated directly by HOXC13 and hair shaft cuticle, and KRT35 (KRTHA5), expressed in both FOXN1, as well as other transcription factors known to be cuticle and cortex (Jave-Suarez et al., 2002). Consistent with involved in hair shaft differentiation, such as lymphoid these data, the mouse versions of both genes (Krt32 and enhancer binding factor 1/ (cadherin associated Krt35) are among the hair keratin genes strongly down- protein) beta 1 (LEF1/CTNNB1); homeobox msh-like 2 regulated in the skin of Hoxc13-null mice (Table 1). (MSX1), and MAD homolog 4 (SMAD4) (Fuchs and Ragha- Furthermore, we previously showed sequence-specific inter- van, 2002; Millar, 2002; Qiao et al., 2006; Duverger and action of HOXC13 with cognate binding sites abundantly Morasso, 2008; Owens et al., 2008; Cai et al., 2009). present in the Krtap16 KAP gene complex (Pruett et al., A shortcoming of this rather simple network model is that 2004). Most members of this gene complex (Krtap16-1, -4, -5, it fails to take likely HOXC13 co-factor requirements into -8, -9, and -10) are expressed in the differentiating cortex, and account because pertinent information is presently limited. they are among the most strongly downregulated genes (up to As a member of the AbdB-type HOX , HOXC13 was 240-fold) in the skin of Hoxc13-null mice (Table 1). It is predicted to interact most likely with MEIS- and PREP-type tempting to speculate that this massive reduction in Krtap16 members of the TALE (three amino-acid loop extension)

www.jidonline.org 833 CS Potter et al. HOX13 Regulation of Foxn1

Foxn1 5′ UTR Δhd-F Fwd primer Rev primer

HOXC13-F HOXC13 BS2 BS1 Intron 14.5 kb Anti-FLAG Anti-β- Ex 1 Ex 2 1.80 ⏐ 1 kb = HOXC13 BS (TT A/T ATNPuPu) 1.60 1.40 80,000 1.20 1.00 70,000 0.80 60,000 0.60 0.40 50,000 0.20 Relative expression levels expression Relative 0.00 40,000

30,000 Δhd-F HOXC13-F 20,000 HOXC13

Normalized luciferase activity Normalized luciferase 10,000 I FNW 0 Foxn1 BS1 -Luc -Luc -Luc Foxn1 BS2 Δhd-F Foxn1 Foxn1 Foxn1 Foxq1-B + HOXC13-F + HOXC13 Prx1

Figure 4. Homeobox C13 (HOXC13) directly regulates forkhead box N1 (Foxn1) expression. (a) HOXC13 consensus binding sequences (TTA/TATNPuPu, black bars) upstream of Foxn1 transcription start (angled arrow); putative binding sites (BS) 1 and 2 examined by chromatin immunoprecipitation (ChIP) and locations of PCR primers used for isolating the 5 kb region included in Foxn1-luc are indicated; boxed region: transcribed sequences. UTR, untranslated region. (b) Normalized luciferase activities resulting from co-transfection of C2C12 cells with Foxn1-luc and HOXC13 expression vectors as indicated; co-transfection with HOXC13Dhd-F, in which the homeodomain was deleted, resulted in expression levels similar to baseline levels. (c) Western blot analysis (top) using anti-FLAG antibodies confirmed expression of HOXC13-F and HOXC13Dhd-F in transfected C2C12 cells at approximately equal levels (n ¼ 3) as determined by densitometric quantification of chemiluminescent signals (see bar diagram below). (d) ChIP assays of C2C12 cells transfected with HOXC13-FLAG; PCR analysis of immunoprecipitated chromatin (F) indicated amplification of HOXC13-bound sequences specific for BS2 but not BS1 after 42 cycles; unprecipitated input DNA was used as positive control (I), whereas ChIP DNA from untransfected cells (N) and distilled water (W) were used as negative controls; additionally, control reactions using primers specific for Foxq1-B yielded a specific PCR product, whereas reactions with primers specific for Prx1 sequences containing no HOXC13 consensus binding sequences failed to yield PCR products with the same batch of immunoprecipitated DNA. Prx1, paired related homeobox 1.

M Distal

C T X D (1) (2) (3) C i U f f /KAPs Dsg4 Dsc2 I R e r S (1) ? (3) e O (2) n R ? S t Foxn1 Foxn1 Foxq1 i M a G t a L t i Hoxc13 Hoxc13 Hoxc13 r DP o i n x GMC Proximal

Figure 5. Models of homeobox C13 (HOXC13)-controlled network motifs involved in regulating hair follicle (HF) differentiation. (Left) Schematics of three HOXC13-controlled feed-forward loop (FFL)-type network motifs (1), (2), and (3) as explained in the text; a HOXC13-negative feedback loop (Tkatchenko et al., 2001) is included in all three circuits but requires validation. (Right) Schematic of bisected lower portion of anagen HF indicating the medulla (M), cortex (CTX), and cuticle (CU) of the hair shaft, and the internal root sheath (IRS) and outer root sheath (ORS) that transitions into the germinal layer (GL) surrounding the dermal papilla (DP); the germinal compartment in the lower matrix (GMC) contains proliferating, transit-amplifying cells (Langbein and Schweizer, 2005); the topography of regulatory network motifs shown on the left is indicated by the numbers. Dsc2, desmocollin 2; Dsg4, desmoglein 4; KAP, keratin-associated protein.

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superclass of homeodomain proteins (Jave-Suarez and shaft differentiation within the HOXC13 expression domain Schweizer, 2006). However, failure to demonstrate nuclear may be expected to require different combinations of co-localization of candidate MEIS and PREP proteins with accessory transcription factors and some of these are likely HOXC13 in KRT32- and KRT35-expressing cells seems to to be induced by HOXC13. argue against this idea, thus providing a rationale for In summary, the data demonstrating HOXC13-dependent considering alternative co-factors. Both FOXN1 expressed activation of Foxn1 reported in this study and of Foxq1 shown in the cortex, cuticle, and internal root sheath, and FOXQ1 in previously (Potter et al., 2006), combined with the apparent the medulla might be potential candidates, particularly in control of downstream effecter genes (Dsg4,Bazziet al., 2009; view of data indicating that other members of the FOX family Crisp1 (cysteine-rich secretory protein 1), Peterson et al., 2005; act as HOX co-factors in different systems. Specifically, hair keratin and KAP genes, Jave-Suarez et al., 2002; Pruett FOXP1 was shown to determine spinal motor neuron et al., 2004), collectively provide a framework for outlining a columnar fate in cooperation with various HOX proteins HOXC13-controlled regulatory network of hair shaft differ- (Dasen et al., 2008); this FOXP1 expression requires HOX entiation. Furthermore, mutated alleles of the HOXC13 target activity, an apparent regulatory parallel to Drosophila, where genes FOXN1 and DSG4 in humans have been linked to an the Hox protein Scr directly activates expression of forkhead inherited immunodeficiency with baldness (Frank et al., 1999) (Ryoo and Mann, 1999). Furthermore, HOXA11 interfaces and localized autosomal recessive hypotrichosis (Kljuic et al., with FOXO1A in regulating prolactin (prl) gene expression in 2003), respectively. The emerging Hoxc13-regulated pathways endometrial stromal cells (Lynch et al., 2009). and network motifs presented and discussed in this study Accordingly, our data are consistent with the idea that provide critical entry points for further studies promising more HOXC13 and FOXN1 operate in a regulatory network motif detailed insight into the molecular control mechanisms of HF known as coherent feed-forward loop (FFL) (Mangan and differentiation. This information will be a prerequisite for Alon, 2003). This type of FFL is one of the most abundantly understanding pertinent disease processes and designing occurring three-gene regulatory circuits found in biological effective therapeutic approaches. systems in which transcription factor X regulates transcription factor Y and both regulate gene Z in such a way that the signs MATERIALS AND METHODS (activation or repression) of the direct and indirect paths of Mice and histological analysis of tissues gene Z regulation are the same. Although requiring further The mouse strains used were B6.129-Hoxc13tm1Mrc (Godwin and validation, the proposed HOXC13-FOXN1-keratin/KAP gene Capecchi, 1998), C57BL/6J þ / þ ,C57BL/6J-Tyrc2J, and Hsd-Foxn1nu/ regulatory circuit in the HF cortex (Figure 5) fits the model of Foxn1nu;B6.129-Hoxc13tm1Mrc were crossed with C57BL/6J-Tyrc2J a coherent FFL and, in the case of the Krtap16 gene cluster mice to generate albino B6.Cg-Tyrc2JHoxc13tm1Mrc.Micewere (Pruett et al., 2004), is supported by the presence of 430 maintained in a temperature and light/dark (12/12 hours) cycle- copies of the core motif of the Forkhead domain-binding controlled vivarium. All studies were done using protocols approved sequence (TAAACA; Furuyama et al., 2000; Yan et al., 2006) by the institutional animal care and use committee. Dorsal skin and within 23 kb of DNA, more than 6 times its random statistical digits (front and rear feet) were collected from euthanized (CO2 frequency (data not shown). Common functions of coherent asphyxiation) mice at 5 days and X8 weeks p.n., fixed in Fekete’s FFLs are modulation of the amplitude and timing (delay) of acid alcohol/formalin solution, and transferred to 70% ethanol effecter gene activation or repression (Mangan and Alon, (Seymour et al., 2004) before paraffin embedding. Sections 2003), which are both critical for orchestrating differentiation (6–10 mm) were stained with hematoxylin and eosin. processes such as hair shaft formation. The regulation of Foxq1 by HOXC13 in the medulla (Potter In situ hybridization et al., 2006) might be part of an FFL that potentially involves Dissected tissues (scapular skin and rear feet) from homozygous Hsd- desmocollin 2 (Dsc2) as a common target; this is supported by Foxn1nu,B6.Cg-Tyrc2J,Hoxc13tm1Mrc, and C57BL/6J-Tyrc2 mice at in silico analysis of the Dsc2 promoter region that identified 5 days p.n. were fixed and processed for cryo-sectioning (10 mm) as clusters of bona fide binding sites for both HOXC13 and previously described (Potter et al., 2006). Hoxc13-specific probe was Forkhead domain transcription factors (data not shown). prepared by using pC13rev (Tkatchenko et al., 2001). Foxn1 probe Recent data suggesting transcriptional regulation of another template was generated by PCR using complementary DNA from the desmosomal cadherin gene, desmoglein 4 (Dsg4), by HOXC13 skin of 5 day p.n. FVB/NTac mice and primers 50-TCCCAGCCTCT and FOXN1 in the differentiating follicular cortex (Bazzi et al., GCACCCAAT-30 and 50-TGCATGTCTCCCAGAGCACC-30;PCR 2009) are consistent with the concept of a HOXC13-controlled products were cloned into pCRII-TOPO vector (Invitrogen, Carlsbad, FFL-type network motif as well (Figure 5). CA) for the generation of digoxigenin (Roche Applied Science, Multifactorial regulation of Hox target genes has been Indianapolis, IN)-labeled antisense and sense (control) RNA probes studied extensively in Drosophila. A good example is the (Pruett et al., 2004). Hybridization and colorimetric signal detection activation of the cell death gene reaper (rpr) by the Hox gene was performed as described. Deformed (Dfd) in a distinct subregion of the Dfd expression domain during Drosophila head development that requires Transient co-transfection assays interaction with at least eight other transcription factors Transfection assays using C2C12 myofibroblasts were done as on a minimal enhancer (Sto¨be et al., 2009). Based on this described (Potter et al., 2006). The reporter plasmid included 5 kb paradigm, target gene specificity in distinct lineages of hair of the Foxn1 promoter region isolated from mouse genomic DNA

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by PCR using primers 50-TCAGTCCGTCAGCCTGATTG-30 and Awgulewitsch A (2003) Hox in hair growth and development. Naturwis- 50-ACTTATGGCAATGCTCCTGC-30 (Figure 4a) and inserted into senschaften 90:193–211 Photinus pyralis luciferase-containing pGL2-Basic (Promega, Bazzi H, Demehri S, Potter CS et al. (2009) Desmoglein 4 is regulated by Madison, WI). Homeodomain-deleted HOXC13 expression vector transcription factors implicated in hair shaft differentiation. Differentia- tion 78:292–300 HOXC13Dhd-F was generated by removal of an EcoN1–XhoI Bieberich CJ, Ruddle FH, Stenn KS (1991) Differential expression of the Hox B 0 fragment of 550 bp containing the Hoxc13 homeobox and 3 3.1 gene in adult mouse skin. Ann NY Acad Sci 642:346–53 untranslated region sequences from HOXC13-F (also known as Cai J, Lee J, Kopan R et al. 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Normalized data were averaged ( SE) developing feather buds. Development (Cambridge, England) and analyzed by analysis of variance with Bonferroni post hoc 110:1021–30 analyses performed as applicable. Significance was set at P40.05. Dasen JS, De Camilli A, Wang B et al. (2008) Hox repertoires for motor neuron diversity and connectivity gated by a single accessory factor, ChIP and western blot analysis FoxP1. Cell 134:304–16 ChIP assays (Ren and Dynlacht, 2004) with C2C12 cells transfected Duboule D (1992) The vertebrate limb: a model system to study the Hox/ HOM gene network during development and evolution. Bioessays with FLAG-tagged HOXC13 expression vector were performed as 14:375–84 described (Potter et al., 2006). Precipitated Foxn1 DNA was detected Duverger O, Morasso MI (2008) Role of homeobox genes in the patterning, by PCR using Foxn1-specific primer sets for BS1 and BS2 (Figure 4); specification, and differentiation of ectodermal appendages in mammals. 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DNA microarray and quantitative PCR analysis Genes Dev 12:11–20 tm1Mrc Scapular skin from three 5-day p.n. male Hoxc13 / Jave-Suarez LF, Schweizer J (2006) The HOXC13-controlled expression of Hoxc13tm1Mrc mice and three age- and sex-matched wild-type early hair keratin genes in the human hair follicle does not involve TALE littermates was used for RNA extraction, complementary DNA proteins MEIS and PREP as cofactors. Arch Dermatol Res 297:372–6 synthesis, and probe preparation for DNA microarray analysis using Jave-Suarez LF, Winter H, Langbein L et al. (2002) HOXC13 is involved in the MOE430v2.0 GeneChip arrays (Affymetrix, Santa Clara, CA) and for regulation of human hair keratin gene expression. J Biol Chem 277:3718–26 quantitative PCR analysis as described in detail (Supplementary Kanzler B, Viallet JP, Le Mouellic H et al. (1994) Differential expression of Material online). two different homeobox gene families during mouse tegument morpho- genesis. Int J Dev Biol 38:633–40 CONFLICT OF INTEREST Kljuic A, Bazzi H, Sundberg JP et al. (2003) Desmoglein 4 in hair follicle Dr Sundberg has a research contract with Procter and Gamble on an differentiation and epidermal adhesion: evidence from inherited unrelated topic. All other authors state no conflict of interest. hypotrichosis and acquired pemphigus vulgaris. Cell 113:249–60 Krumlauf R (1994) Hox genes in vertebrate development. Cell 78:191–201 ACKNOWLEDGMENTS Langbein L, Schweizer J (2005) Keratins of the human hair follicle. Int Rev We acknowledge the technical assistance of Margaret Romano with Cytol 243:1–78 processing tissue samples and Donna Jacob’s help with managing mouse strains. We thank Tim Stearns and Kathleen Silva from the Jackson Laboratory Lee D, Prowse DM, Brissette JL (1999) Association between mouse nude gene for help with DNA microarray data analysis and for providing tissue sam- expression and the initiation of epithelial terminal differentiation. 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