Characterization of the Promoter Regions of Two Sheep Keratin-Associated Protein Genes for Hair Cortex-Specific Expression

The keratin-associated proteins (KAPs) are the structural proteins of hair fibers and are thought to play an important role in determining the physical properties of hair fibers. These proteins are activated in a striking sequential and spatial pattern in the keratinocytes of hair fibers. Thus, it is important to elucidate the mechanism that underlies the specific transcriptional activity of these genes. In this study, sheep KRTAP 3–3 and KRTAP11-1 genes were found to be highly expressed in wool follicles in a tissue-specific manner. Subsequently, the promoter regions of the two genes that contained the 5′ flanking/5′ untranslated regions and the coding regions were cloned. Using an in vivo transgenic approach, we found that the promoter regions from the two genes exhibited transcriptional activity in hair fibers. A much stronger and more uniformly expressed green fluorescent signal was observed in the KRTAP11-1-ZsGreen1 transgenic mice. In situ hybridization revealed the symmetrical expression of sheep KRTAP11-1 in the entire wool cortex. Consistently, immunohistochemical analysis demonstrated that the pattern of ZsGreen1 expression in the hair cortex of transgenic mice matches that of the endogenous KRTAP11-1 gene, indicating that the cloned promoter region contains elements that are sufficient to govern the wool cortex-specific transcription of KRTAP11-1. Furthermore, regulatory regions in the 5′ upstream sequence of the sheep KRTAP11-1 gene that may regulate the observed hair keratinocyte specificity were identified using in vivo reporter assays.


Introduction
Due to the favorable properties of wool for use in textiles, the improvement of wool quality (such as the fiber diameter, length, strength and elasticity) is important for the sheep industry. The wool/hair fiber that is produced by the wool/hair follicle bulb is composed of the cuticle, cortex and sometimes a central medulla. The primary structural proteins of hair fibers are the hair keratins and the keratin-associated proteins (KAPs). Hair keratins produce the keratin murine UHS KRTAP that could direct the appropriate expression of a reporter gene construct in transgenic mice [17]. In addition, a recent report revealed that a 5 0 upstream region containing an NF-kappa-B binding site may activate sheep KRTAP6.1 expression in fetal fibroblast cells [18]. However, due to the restricted expression in the three structural compartments of hair fibers and the lack of suitable cell lines, the number of KRTAP genes with defined regulatory elements is small. Therefore, the objectives of this study were to isolate KRTAP genes that are highly expressed in the sheep wool follicle, characterize the associated cDNA sequences, detect the active promoter sequences of the isolated KRTAP genes using transgenic mice and then investigate the regulatory regions and factors involved in the observed tempo-spatial expression patterns.

Animal materials and sample collection
The animal experiments and procedures were carried out in strict accordance with regulations of the Standing Committee of Hubei People's Congress and approved by the Biological Studies Animal Care Committee of Hubei Province, P.R. China, and the Ethics Committee of Huazhong Agricultural University, P. R. China.
In order to investigate the regional expression patterns of the KRTAP genes in the three wool fiber structural compartments (cuticle, cortex and medulla), Tibetan sheep whose wool fiber contains all the three parts were used as the experiment animals. Three healthy (one male and two female), 1-year-old Tibetan sheep were obtained from the Agriculture and Animal Husbandry College of Tibet, China. All sheep were raised according to the sheep feeding standard of China. Animals were slaughtered with a rapid intravenous infusion of T-61 euthanasia solution. Fifteen tissue samples were collected. From those samples, fourteen tissues (skin, heart, liver, spleen, lung, kidney, rumen, abomasums, duodenum, rectum, muscle, lymph, testis and ovary) were immediately frozen in liquid nitrogen and then stored in a -80°C freezer for total RNA extraction. However, the wool follicle bulb tissue samples were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA) immediately after collection and then centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was preserved and stored at -80°C.

Analysis of gene expression profiles
Total RNA was isolated from each tissue using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer 0 s instructions. The PrimeScript™ RT reagent kit (Takara Bio Inc., Dalian, China), which contains the oligo dT and random primer mix, was used for the first-strand cDNA synthesis. The mRNA expression levels of KRTAP3-3 and KRTAP11-1 in fifteen tissue samples from Tibetan sheep were evaluated via RT-PCR, and the mRNA expression levels of those genes in wool follicles and skin were further measured using qRT-PCR. The employed primers (KRTAP3-3 F/KRTAP3-3 R and KRTAP11-1 F/KRTAP11-1 R) (S1 Table) were designed based on conserved regions between the bovine KRTAP3-3 (Gen-Bank: BC114204.1) and ovine KRTAP11-1 (GenBank: HQ595347.1) sequences, respectively. The qRT-PCR assay was performed using the SYBR Green All-in-One QPCR Mix (Genecopoeia, Rockville, MD, USA). The relative gene expression was analyzed using the 2 −ΔΔCT method [19]. The ovine 18S ribosomal RNA gene (S1 Table) was used as an internal control.
Molecular cloning of the KRTAP3-3 and KRTAP11-1 cDNAs The total RNA extracted from the skin was used to clone the KRTAP3-3 and KRTAP11-1 cDNAs. The CDS sequences of the bovine KRTAP3-3 gene and the ovine KRTAP11-1 gene were BLAST searched against the ovine genome to obtain the 5 0 and 3 0 untranslated regions of the sheep KRTAP3-3 and KRTAP11-1 genes, respectively. The primers (S2 Table) were designed based on the 5 0 and 3 0 untranslated region sequences. The purified PCR products were sequenced by TSINGKE Biological Technology Co., Ltd. (Beijing, China).

Construction of expression vectors and generation of transgenic mice
Genomic DNA was extracted from Tibetan sheep skin using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) following the manufacturer 0 s protocol and was used to clone the 5 0 -upstream regions of KRTAP3-3 and KRTAP11-1. The 6,404 bp and 5,954 bp sequences upstream of the translation initiation sites (ATG) of KRTAP3-3 and KRTAP11-1, respectively, were obtained. The KRTAP3-3p F primer contained an Xho I site, and the KRTAP3-3p R primer included a Sac II site. The KRTAP11-1p F/R primers contained a Sac II site and a BamH I site, respectively (in lowercase, see S3 Table). After amplification, digestion and purification, the two promoter sequences containing 5 0 flanking/5 0 untranslated regions were cloned into the pZsGreen1-1 vector (Clontech, Mountain View, CA, USA), which is a promoterless vector encoding the green fluorescent protein ZsGreen1. The constructed vectors used for transgenic mouse generation were named KRTAP3-3-ZsGreen1 and KRTAP11-1-ZsGreen1. The transgenic mice were created through a commercial service (BGI Art Biotechnology Co. LTD., Shenzhen, China). In brief, the constructed KRTAP3-3-ZsGreen1 and KRTAP11-1-ZsGreen1 vectors were linearized using the restriction enzymes Xho I and Afl II, respectively. After extraction and purification using the phenol chloroform, the concentrations were all 18 ng/μl. The transgenes were microinjected into the fertilized Kunming White mouse eggs and were subsequently re-implanted into the pseudo-pregnant females. The mice were housed in the Laboratory Animal Research Center at Huazhong Agricultural University in Wuhan.

Validation of transgenic mice
First, genomic DNA was isolated from tail biopsies from founder mice using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) according to the manufacturer 0 s protocol. The genomic DNA was used to screen the transgenic mice. PCR primers (S3 Table) were designed based on the promoter sequences of the KRTAP3-3-ZsGreen1 and KRTAP11.1-ZsGreen1 constructs. Second, the sheared dorsal hair from PCR-positive founder mice was visualized using a fluorescence inversion microscope system (NIKON, Japan) to detect the green fluorescent signal. Founder mice with green fluorescent signal-positive hair were used to establish the transgenic line. Third, the offspring that showed a strong green fluorescent signal in the hair were anesthetized and imaged using a whole-body fluorescence imaging system (Maestro In-vivo Imaging Systems, CRI, Inc., Woburn, MA, USA) to detect the expression of the ZsGreen1 protein. Then, the hair and eleven internal organs were removed from the mice and imaged using the same system. A negative mouse was as a negative control (NC).

In situ hybridization and immunohistochemical analysis
Sheep skin containing wool follicles was collected from three 1-year-old Tibetan sheep during the anagen phase and fixed in 4% paraformaldehyde for 24 h. Then, 5 μm paraffin sections were hybridized with a custom-designed QuantiGene ViewRNA probe against sheep KRTAP11-1 (Affymetrix). Nonradioactive ISH (In situ hybridization) was performed using the QuantiGene ViewRNA FFPE Assay (Affymetrix/Panomics), according to the manufacturer's instructions and a previously described method [20]. The hybridization signals were recorded using an Olympus BX53F light microscope (OLYMPUS, Tokyo, Japan).
Immunohistochemical analysis (IHC) was performed to determine the presence of ZsGreen1 driven by the sheep KRTAP11-1 promoter in mouse hair follicles. Skin containing hair follicles was collected from green fluorescent signal-positive mice during the anagen phase (postnatal day 14) and embedded in a mixture of beeswax and paraffin. IHC of fresh sections was performed according to a previously described method [20]. The primary antibody was the rabbit recombinant Living Colors1 full-length ZsGreen1 Polyclonal Antibody (1:100, 632474; Clontech, Mountain View, CA, USA), and the secondary antibody was a biotinylated secondary antibody (1:100, SA1022; Boster Corporation). All of the sections were stained immunohistochemically under the same conditions. Images were collected using an Olympus BX53F light microscope (OLYMPUS, Tokyo, Japan).

Investigation of the transcriptional regulatory region of the KRTAP11-1 promoter
First, 5 0 -RACE (rapid amplification of cDNA ends) was performed to determine the transcription start site of the sheep KRTAP11-1 gene. Total RNA isolated from the skin was used for the first-strand cDNA synthesis, according to the manufacturer 0 s instructions (SMART TM RACE cDNA Amplification Kit, Clontech, Mountain View, CA, USA). A reverse gene-specific primer designed based on the coding sequence was used to amplify the 5 0 end of sheep KRTAP11-1 (S2 Table). The PCR products were cloned into the pMD19-T vector (Takara Bio, Inc., Dalian, China) and sequenced in two directions.
Third, genomic DNA from the skin of Tibetan sheep was used to amplify the ovine KRTAP11-1 5 0 -deletion fragments. The PCR fragments were generated using a common reverse primer that included a BamH I site and ten different forward primers that contained a Sac II site (S4 Table). Each PCR product was digested separately with the Sac II and BamH I restriction enzymes and cloned into the pZsGreen1-1 vector (Clontech, Mountain View, CA, USA). The positive clones were sequenced by TSINGKE Biological Technology Co., Ltd. (Beijing, China).

Identification of highly expressed genes in wool follicles
We previously analyzed the transcriptome profiles of the Tibetan sheep wool follicle bulb [21] and other four tissues (muscle, liver, spleen and lung; unpublished data). Comparative analyses of these data revealed candidate genes that were expressed at higher levels in the wool follicle bulb (data not shown). Subsequently, we investigated the expression patterns of these genes in fifteen tissues (wool follicle, skin, heart, live, spleen, lung, kidney, rumen, abomasums, duodenum, rectum, muscle, lymph, testis and ovary) using RT-PCR. The results showed that KRTAP3-3 and KRTAP11-1 mRNA expression was detected at high levels in the wool follicle and skin but was not detected in the remaining tissues ( Fig 1A). Third, given that it is difficult to completely separate the wool follicles from the skin, the skin samples we used contained wool follicles. Therefore, we performed quantitative real time RT-PCR (qRT-PCR) to verify whether the mRNA expression levels of KRTAP3-3 and KRTAP11-1 genes were higher in wool follicles than in the skin. The KRT83 and KRT5, which were demonstrated to be expressed specifically in wool follicles and skin, respectively, were considered to be the reference genes in this study [10,14] (Fig 1B). We found that the mRNA expression levels of the KRTAP3-3 and KRTAP11-1 genes were significantly higher in wool follicles than in the skin, indicating that the observed expression patterns were consistent with that of KRT83 ( Fig 1C). Thus, our data revealed strong expression of KRTAP3-3 and KRTAP11-1 in sheep wool follicles.
cDNA cloning and sequence analysis of sheep KRTAP 3-3 and KRTAP 11-1 We isolated the cDNA sequences of KRTAP3-3 and KRTAP11-1 from Tibetan sheep skin (S1 Appendix). A BLAST search of the Ovine Genome assembly v3.1 revealed that KRTAP3-3 and KRTAP11-1 were localized to sheep chromosome 11 and chromosome 1, respectively. The The results showed that sheep KAP3-3 and KAP11-1 were grouped with bovine KAP3-3 and KAP11-1, respectively, at a relatively high bootstrap confidence value, indicating that ovine KAP3-3 and KAP11-1 have the highest homology with Bos taurus and Capra hircus and the lowest homology with Homo sapiens (Fig 2).

Generation and characterization of transgenic mice
To identify the upstream regions that regulate the transcriptional activity of the two genes, the 6,404 bp sequence upstream of the ATG of the KRTAP3-3 gene and the 5,954 bp sequence upstream of the ATG of the KRTAP11-1 gene (with the A of the ATG start codon numbered as +1) were isolated from sheep skin genomic DNA (S2 Appendix). Then, each of the two isolated 5 0 flanking/5 0 untranslated sequences was ligated into the pZsGreen1-1 vector (Fig 3). Then the KRTAP3-3-ZsGreen1 and KRTAP11-1-ZsGreen1 transgenic mice were generated, respectively. The transgenic founders were characterized via a PCR assay using genomic DNA from tail biopsies. The sheared dorsal hair from the transgenic positive offspring mice was visualized under a fluorescent microscope. Strong ZsGreen1 fluorescent signals were detected in the sheared dorsal hair from KRTAP11-1-ZsGreen1 transgenic mice. However, strong but scattered green fluorescent signals were observed in the sheared dorsal hair from KRTAP3-3-ZsGreen1 transgenic mice (Fig 4). Therefore, only the KRTAP11-1-ZsGreen1 transgenic mice were subsequently anesthetized and imaged using the Maestro in vivo imaging system to detect the expression of the ZsGreen1 protein. We found that the expression of ZsGreen1 was strongly visible along the hair of the transgenic positive mouse but undetectable in the negative mouse ( Fig 5). In addition, fluorescence imaging also revealed that the green fluorescent signals were strong in the sheared dorsal hair but undetectable in the other 11 tissues investigated from the KRTAP11-1-ZsGreen1 transgenic mouse (Fig 6). Furthermore, by using a nonradioactive in situ hybridization technique, we demonstrated that the KRTAP11-1 mRNA was uniquely expressed in the cortex of the sheep hair shaft. No signals were detected in the inner and outer root sheath, the cuticle region or other adjacent skin tissues (Fig 7). We further defined the expression location of the ZsGreen1 protein in the hair follicles of the positive transgenic mouse. Immunohistochemical assays showed that the ZsGreen1 protein exhibited a strong signal in the cortex of the hair follicle (Fig 8). Thus, the localization of ZsGreen1 is consistent with that of the endogenous KRTAP11-1 gene in sheep, indicating that the promoter sequence of sheep KRTAP11-1 could drive ZsGreen1 to be expressed exclusively in the hair cortex in the transgenic mouse. and an optimal Kozak consensus sequence (ACCATGG) was identified around the translation start codon. Subsequently, the minimal promoter region was predicted using the Promoter 2.0 and Promoter Scan software programs. Within the minimal promoter region, the TATA box and CCAAT box were located 31 bp and 253 bp upstream of the transcription start site, respectively.

Discussion
Hair fibers originate from the follicle bulb. The differentiation of follicle bulb cells into either cortical or cuticle hair keratinocytes is determined primarily by the activation of keratin and KRTAP genes in a sequential and spatial manner. In this study, the sheep KRTAP3-3 and KRTAP11-1 genes were demonstrated to be highly expressed specifically in the wool follicle bulb via qRT-PCR and cDNA cloning. Subsequently, to define the regulatory functional region located upstream of the translation initiation sites of the two sheep genes, we established two transgenic mice (KRTAP3-3-ZsGreen1 and KRTAP11-1-ZsGreen1) and found that the two isolated promoter fragments encompassing the immediate 5 0 flanking/5 0 UTR sequences could direct the exclusive expression of the ZsGreen1 protein in the hair. Compared to KRTAP3-3-ZsGreen1 transgenic mice, a much stronger and more uniformly expressed green fluorescent signal was observed in the KRTAP11-1-ZsGreen1 transgenic mice. Furthermore, the KRAP11-1 gene was demonstrated to be expressed symmetrically in the entire cortex of wool by using in situ hybridization. Consistently, immunohistochemical analysis revealed that the 5,954 bp     Two Sheep Keratin-Associated Protein Genes for Hair Cortex-Specific Expression upstream sequence of the sheep KRTAP11-1 gene also drove the expression of the green fluorescent protein in the entire hair cortex in transgenic mice. Moreover, in vitro analysis suggested that some common regulatory elements, including NF-kappaB (p65), AP-1, GATA3, Cart-1 and Oct-1, were present in the region between -1,570 bp and -545 bp.
The KRTAP3-3 and KRTAP11-1 genes both belong to the high sulfur group and are located on sheep chromosomes 11 and 1, respectively [22,23]. KRTAP3-3 is a member of the KRTAP3 family, which consists of three protein-coding genes and one pseudogene in humans [24,25]. We obtained a 956 bp cDNA sequence for KRTAP3-3 from sheep skin tissue and found that the included 297 bp coding region exhibited 99% identity with the previously reported sheep BIIIB4 gene [25], indicating that the sheep KRTAP3-3 gene is a protein-coding gene. In addition, the expression of the KRTAP3-3 mRNA and protein has been demonstrated to occur in the wool fiber cortex or hair [2,26]. As the only known member of the KRTAP11 family [27], the genomic sequence of the sheep KRTAP11-1 gene, which consists of only one exon, was reported previously. Our results concerning the isolated KRTAP11-1 cDNA sequence further confirmed that the KRTAP11-1 gene was expressed in the sheep skin tissue. Furthermore, both the human and mouse KRTAP11-1 genes were reported to be expressed in the entire cortex [6,28]. We also found that the sheep KRTAP11-1 gene showed uniform cortical expression and was clearly absent from the central medulla of the wool fiber. Taken together, our study revealed that, similar to their orthologs, the sheep KRTAP11-1 and KRTAP3-3 genes are strongly expressed exclusively in the cortical region of wool fibers.
To investigate the controlling sequences involved in the regulation of sheep KRTAP11-1 and KRTAP3-3 expression and their effects on wool quality, we investigated the activity of the isolated 5 0 upstream sequences of the two genes in controlling the expression of the ZsGreen1 gene in transgenic mice. The results showed that both sequences could govern proper ZsGreen1 gene expression in the hair of the transgenic mice, but the degree of activity differed between the two constructs. The transgenic mice were generated via the pronuclear microinjection of constructs in this study. The two main disadvantages of the system are the variability of transgene expression, which is caused by random integration and the unpredictable number of copies that eventually incorporate into the host genome [29][30][31]. Thus, the scatter distribution of fluorescent signals along the hair of the KRTAP3-3-ZsGreen1 transgenic mice may be primarily a technical artifact. Additionally, the observed pattern may also be caused by the absence of the necessary controlling elements in the KRTAP3-3-ZsGreen1 construct or by other regulatory factors, especially those that are functionally conserved between mammals. On the other hand, a strong and uniform distribution of fluorescent signals was evident in the hair of the KRTAP11-1-ZsGreen1 transgenic mice. Further characterization demonstrated that the ZsGreen1 protein was expressed in the mouse hair fiber cortex, which is consistent with the expression pattern of endogenous KRTAP11-1 mRNA in sheep wool fibers.
Due to the lack of a hair cortical cell line in which KRTAP11-1 is expressed, SEF and HHORS cells, in which the endogenous KRTAP11-1 gene is not expressed, were used instead to investigate the regulatory elements in the 5,954 bp upstream sequence of KRTAP11-1. Transient transfection assays showed that the region located between -5,954 bp and -1,571 bp was not active. However, detectable transcriptional activity was observed within the region between -1,570 bp and -545 bp (1,026 bp in length) in both cell types, which implied that this region plays a role in the ubiquitous expression of KRTAP11-1. The proximal 1,026 bp of the sheep KRTAP11-1 gene 5 0 promoter region contains putative regulatory motifs, including NF-kappaB (p65), AP-1, GATA3, Cart-1 and Oct-1 motifs (S3 Appendix). Sequence comparisons revealed that the putative regulatory motifs were highly conserved across sheep, humans, and mice. The activation of the five transcription factors is not cell type-specific, and of the identified factors, NF-kappaB (p65) and AP-1 have been suggested to influence the expression of human KRT6 expression in HeLa cells or epidermal keratinocytes [32]. In addition, sheep KRTAP6-1 is another gene with hair cortical keratinocyte-specific expression [33]. Similar to our results, Yang et al reported that the proximal 1.5 kb of the sheep KRTAP6-1 gene 5 0 promoter region contained an NF-kappaB binding site and was capable of enhancing the transcription of the reporter gene in sheep fetal fibroblast cells. In contrast, the sequences outside the 1.5 kb proximal promoter showed significantly decreased transcriptional activity [18], suggesting that the region plays a dispensable role [34]. Thus, our findings indicated the possibility that the regulatory elements required for the hair cortical keratinocyte-specific activity of the sheep KRTAP11-1 promoter may exist outside of the 1,026 bp proximal promoter region. A recent study used an organ culture system to reveal that the mouse KAP11-1 protein may participate in the assembly of micro/macro fibrils and consequently contribute to the formation of the appropriate hair structure. That study also found an altered expression pattern of the KAP11-1 protein without impaired expression in the hair cortex in forkhead box N1 (Foxn1, also named Whn) nude mice [28], indicating that the expression of the KAP11-1 protein is at least partially dependent on Foxn1 activity. Foxn1 is a transcription activator that is transcribed primarily in the hair cortex and cuticle and has been established to play a role in the regulation of hair keratin gene expression [35][36][37][38]. It is possible that Foxn1 is a cofactor responsible for the activation of KAP11-1. Thus, further characterization of the KRTAP11-1 5 0 region in a transgenic system is needed.
In conclusion, we characterized an~5 kb sequence of the 5 0 upstream region of sheep KRTAP11-1 that can strongly direct the expression of ZsGreen1 in the mouse hair fiber cortex, corresponding to the location of endogenous KRTAP11-1 expression in sheep wool fibers. In addition, the regulatory region of the sheep KRTAP11-1 gene that governs hair keratinocyte specificity was investigated in vitro. Our findings represent a first step toward understanding the role of sheep KAP11-1 in keratin intermediate filament assembly in the wool cortex. In addition, the characterized promoter of KRTAP11-1 could be a candidate for targeting the expression of foreign genes to manipulate specific hair/wool traits.