Overexpression of Hoxc13 in differentiating keratinocytes results in downregulation of a novel hair keratin gene cluster and alopecia

Studying the roles of Hox genes in normal and pathological development of skin and hair requires identification of downstream target genes in genetically defined animal models. We show that transgenic mice overexpressing Hoxc13 in differentiating keratinocytes of hair follicles develop alopecia, accompanied by a progressive pathological skin condition that resembles ichthyosis. Large-scale analysis of differential gene expression in postnatal skin of these mice identified 16 previously unknown and 13 known genes as presumptive Hoxc13 targets. The majority of these targets are downregulated and belong to a subgroup of genes that encode hair-specific keratin-associated proteins (KAPs). Genomic mapping using a mouse hamster radiation hybrid panel showed these genes to reside in a novel KAP gene cluster on mouse chromosome 16 in a region of conserved linkage with human chromosome 21q22.11. Furthermore, data obtained by Hoxc13/lacZ reporter gene analysis in mice that overexpress Hoxc13 suggest negative autoregulatory feedback control of Hoxc13 expression levels, thus providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hox gene expression. Combined, these results provide a framework for understanding molecular mechanisms of Hoxc13 function in hair growth and development.     

Expression analysis of KAP9.2 and Hoxc13 genes during different cashmere growth stages by qRT-PCR method

Keratin-associated protein 9.2 (KAP9.2) and Homeobox C13 (Hoxc13) genes were chosen to study because of their biological functions involving hair formation. KAP9.2 gene belongs to the ultra high sulfur KAPs, which is important for hair formation and may have association with cashmere. Hoxc13 takes part in the formation of cashmere keratin and maintaining the normal structure of follicle. It has been reported that Hoxc13 gene exists binding site of KP and KAP genes at its promoter regions in mouse. So the expression of KAP9.2 and Hoxc13 genes was detected at anagen stage vs telogen stage by qRT-PCR. The data showed that KAP9.2 and Hoxc13 gene had similar expression trend at different stages, which indicated that there was interaction between them. KAP9.2 and Hoxc13 gene had lower expression level in anagen than that of in telogen of cashmere growth. In anagen, KAP9.2 and Hoxc13 expressed lower in high cashmere yield individuals than that of in low cashmere yield ones. In telogen, the result was reverse. The study would provide the evidence of involvement of KAP9.2 and Hoxc13 in hair periodic growth.     

Hoxc13在毛囊发育中的作用

Hoxc13属于Hox(Homobox)基因家族Abd-B类成员之一, 与毛囊形成和毛发生长密切相关。毛发结构蛋白KP(角蛋白)和KAP(角蛋白关联蛋白)的表达都受Hoxc13的严格调控, Hoxc13表达水平会直接影响毛发的特性, 对维持毛囊的正常形态也至关重要。文章就Hoxc13的表达水平对毛囊发育和毛发生长的影响及Hoxc13与相关基因的调控进行了综述。     

A Cluster of Keratin-Associated Proteins on Mouse Chromosome 10 in the Region of Conserved Linkage with Human Chromosome 21

A gene cluster of three to five high-cysteine keratin-associated proteins (KAPs) has been identified on mouse Chromosome 10 (MMU10) in the region of conserved linkage with human chromosome 21 (HSA21). One of these genes,Krtap12-1,has been sequenced in its entirety and shown to be an intronless gene encoding a predicted 130-amino-acid protein.Krtap12-1is most closely related to two previously identified KAP4 genes, but variation in sequence and cysteine content suggests that it represents a new KAP family.Krtap12-1is expressed in the skin of a 3-day-old mouse. The corresponding region of HSA21, betweenITGB2(integrin β2) andPFKL(the liver isoform of phosphofructokinase), has proven refractory to cloning, and thus mapping of this region at high resolution has been problematic. Based on the KAP gene cluster position in mouse, evidence has been found for an orthologous human KAP cluster on HSA21q22.3, reinforcing the observation that comparative genomics can play an essential and practical role in determining mammalian genome organization.     

Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein (KAP) genes on chromosome 21q22.1

Analysis of the EBI/GeneBank(TM) data base using non-human hair keratin-associated protein (KAP) cDNA sequences as a query resulted in the identification of a first domain of high glycine-tyrosine and high sulfur KAP genes located on human chromosome 21q22.1. This domain, present on the DNA accession numbers and, was approximately 535 kb in size and contained 17 high glycine-tyrosine and 7 high sulfur KAP genes, as well as 9 KAP pseudogenes. Based on amino acid sequence comparisons of the encoded proteins, the KAP genes could be divided into seven high glycine-tyrosine gene families (KAP6-KAP8, and KAP19-KAP22) and four high sulfur gene families (KAP11, KAP13, KAP15, and KAP23). The high glycine-tyrosine genes described here appear to represent the complete set of this type of KAP genes present in the human genome. Both systematic cDNA isolation studies from an arrayed scalp cDNA library and in situ hybridization expression studies of all of the KAP genes identified in the 21q22.1 region revealed varying degrees and regions of expression of 11 members of the high tyrosine-glycine genes and 6 members of the high sulfur KAP genes in the hair forming compartment.     

Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the type I keratin gene domain on chromosome 17q12-21

Low stringency screening of a human P1 artificial chromosome library using a human hair keratin-associated protein (hKAP1.1A) gene probe resulted in the isolation of six P1 artificial chromosome clones. End sequencing and EMBO/GenBank(TM) data base analysis showed these clones to be contained in four previously sequenced human bacterial artificial chromosome clones present on chromosome 17q12-21 and arrayed into two large contigs of 290 and 225 kilobase pairs (kb) in size. A fifth, partially sequenced human bacterial artificial chromosome clone data base sequence overlapped and closed both of these contigs. One end of this 600-kb cluster harbored six gene loci for previously described human type I hair keratin genes. The other end of this cluster contained the human type I cytokeratin K20 and K12 gene loci. The center of the cluster, starting 35 kb downstream of the hHa3-I hair keratin gene, contained 37 genes for high/ultrahigh sulfur hair keratin-associated proteins (KAPs), which could be divided into a total of 7 KAP multigene families based on amino acid homology comparisons with previously identified sheep, mouse, and rabbit KAPs. To date, 26 human KAP cDNA clones have been isolated through screening of an arrayed human scalp cDNA library by means of specific 3'-noncoding region polymerase chain reaction probes derived from the identified KAP gene sequences. This screening also yielded four additional cDNA sequences whose genes were not present on this gene cluster but belonged to specific KAP gene families present on this contig. Hair follicle in situ hybridization data for single members of five different KAP multigene families all showed localization of the respective mRNAs to the upper cortex of the hair shaft.     

Identification of two novel clusters of ultrahigh-sulfur keratin-associated protein genes on human chromosome 11

We analyzed two novel clusters of keratin-associated protein (KAP) genes on human chromosome 11 (11p15.5 and 11q13.5) in which we identified two known human KRTAP5 genes, KerA (=KRN1) and KerB, and nine novel KRTAP5 family genes. RT-PCR analysis of these KAP genes showed preferential expression in human hair root, suggesting these gene products are required for hair formation. Based on the deduced amino acid sequences, all these KAP proteins were classified into an ultrahigh-sulfur (UHS) type KAP with high cysteine content (> 30 mol%). These KAPs also showed high glycine and serine contents (average 24.30 and 21.13 mol%, respectively), distinguishing from other UHS/HS KAP families located on human chromosomes 17 and 21. Dot-matrix analysis revealed a significant similarity between these two KAP gene clusters. We postulated a mechanism by which these two KAP gene clusters are generated via genomic duplication of a primordial gene cluster followed by genetic modification during evolution.     

Hoxc12 expression pattern in developing and cycling murine hair follicles

We examine the Hoxc12 RNA expression pattern during both hair follicle morphogenesis and cycling in direct comparison to its only upstream neighbor, Hoxc13. Expression of both genes is restricted to the epidermal part of the follicle excluding the outer root sheath and interfollicular epidermis in a distinct stage-dependent and cyclical manner. During the progressive growth phase (anagen) of developing and cycling follicles, the distinct proximo-distal expression domain of Hoxc12 overlaps only proximally, at the upper-most region of the bulb, with the more proximally restricted Hoxc13 domain. This arrangement of the expression domains of the two genes along the proximal-toward-distal axis of increasing follicular differentiation correlates with the sequential expression of first Hoxc13 and then Hoxc12. This indicates a reversal of the typical temporal colinearity of Hox gene activation otherwise observed along the anterior-posterior morphogenetic axis of the embryo (review: Cell 78 (1994) 191).     

Genetic and biochemical aspects of keratin synthesis by hair follicles

In this review article the data about synthesis and gene regulation of keratin by hair follicles have been summarized. It has been shown that both differentiation of hair follicle matrix cells and normal growth of hair require the coordinated activities of the genes encoding structural proteins. The keratin genes are clustered in families and are usually 5-10 kb in the genome. The separate clusters of two keratin IF gene families and five KAP gene families have been discovered and some of them have been mapped. The close relation between these clusters suggests that the "global" regulatory domains might govern their expression.     

Structural dynamics and epigenetic modifications of Hoxc loci along the anteroposterior body axis in developing mouse embryos

Hox genes are organized as clusters and specify regional identity along the anteroposterior body axis by sequential expression at a specific time and region during embryogenesis. However, the precise mechanisms underlying the sequential spatio-temporal, collinear expression pattern of Hox genes are not fully understood. Since epigenetic modifications such as chromatin architecture and histone modifications have become crucial mechanisms for highly coordinated gene expressions, we examined such modifications. E14.5 mouse embryos were dissected into three parts along the anteroposterior axis: brain, trunk-anterior, and trunk-posterior. Then, structural changes and epigenetic modifications were analyzed along the Hoxc cluster using chromosome conformation capture and chromatin immunoprecipitation-PCR methods. Hox non-expressing brain tissues had more compact, heterochromatin-like structures together with the strong repressive mark H3K27me3 than trunk tissues. In the trunk, however, a more loose euchromatin-like topology with a reduced amount of H3K27me3 modifications were observed along the whole cluster, regardless of their potency in gene activation. The active mark H3K4me3 was rather closely associated with the collinear expression of Hoxc genes; at trunk-anterior tissues, only 3' anterior Hoxc genes were marked by H3K4me3 upon gene activation, whereas whole Hoxc genes were marked by H3K4me3 and showed expression in trunk-posterior tissues. Altogether, these results indicated that loosening of the chromatin architecture and removing H3K27me3 were not sufficient for, but rather the concomitant acquisition of H3K4me3 drove the collinear expression of Hoxc genes.     

Characterization and expression analysis of KAP7.1, KAP8.2 gene in Liaoning new-breeding cashmere goat hair follicle

Keratin-associated protein is one of the major structural proteins of the hair, whose content in hair has important effect on the quality of cashmere. In order to study the relationship between HGTKAP gene expression and cashmere fineness, the quantitative real-time RT–PCR (qRT–PCR) was firstly used to detect the levels of KAP7.1, KAP8.2 gene expression in the primary and secondary hair follicles; semi-quantitative RT–PCR was used to detect whether KAP7.1, KAP8.2 gene are expressed in heart, liver, spleen, lung, kidney tissues; and in situ hybridization(ISH) to detect KAP7.1 gene expression location. qRT–PCR result showed that the expression of both KAP7.1 and KAP8.2 gene in the secondary hair follicles are significantly higher than that in the primary follicles, relative quantitative analysis obtained that KAP7.1 was 2.28 times, while KAP8.2 was 2.71 times. Semi-quantitative RT–PCR results revealed that KAP 7.1 and KAP8.2 mRNA were not detected in the heart, liver, spleen, lung and kidney tissues, demonstrating that KAP7.1 and KAP8.2 were specially expressed in hair follicles, participating in hair formation. Moreover, KAP7.1 gene has a strong expression in the cortical layer, inner root sheath of the primary follicles and the cortical layer, inner root sheath and hair matrix of the secondary hair follicles by ISH analysis. Taken together, the evidence presented here indicated that in the formation of cashmere and wool, differential expression of these two genes in the primary and secondary hair follicles may have an important role in regulating the fiber diameter.     

Phylogenetic tests of the hypothesis of block duplication of homologous genes on human chromosomes 6, 9, and 1

There are 10 gene families that have members on both human chromosome 6 (6p21.3, the location of the human major histocompatibility complex [MHC]) and human chromosome 9 (mostly 9q33-34). Six of these families also have members on mouse chromosome 17 (the mouse MHC chromosome) and mouse chromosome 2. In addition, four of these families have members on human chromosome 1 (1q21-25 and 1p13), and two of these have members on mouse chromosome 1. One hypothesis to explain these patterns is that members of the 10 gene families of human chromosomes 6 and 9 were duplicated simultaneously as a result of polyploidization or duplication of a chromosome segment ("block duplication"). A subsequent block duplication has been proposed to account for the presence of representatives of four of these families on human chromosome 1. Phylogenetic analyses of the 9 gene families for which data were available decisively rejected the hypothesis of block duplication as an overall explanation of these patterns. Three to five of the genes on human chromosomes 6 and 9 probably duplicated simultaneously early in vertebrate history, prior to the divergence of jawed and jawless vertebrates, and shortly after that, all four of the genes on chromosomes 1 and 9 probably duplicated as a block. However, the other genes duplicated at different times scattered over at least 1.6 billion years. Since the occurrence of these clusters of related genes cannot be explained by block duplication, one alternative explanation is that they cluster together because of shared functional characteristics relating to expression patterns.      

qPCR array检测分析 C57BL/6小鼠胚胎后肢发育基因表达谱

目的:胚胎生育过程中因肢体发育异常造成的出生缺陷比率不低,其相关基因表达模式尚不明确。本实验通过建立实时定量PCR芯片(Real-time quantitative polymerasechain reaction array,qPCR array)检测方案,研究C57BL/6品系小鼠后肢发育相关基因的表达谱。方法:以同源异形盒基因家族(Hox)、Wnt5a、配对同源结构域基因(Pitx1)、成纤维生长因子(Fgf8)、音猬因子(Shh)等小鼠肢体发育相关的重要基因制作基因检测表达谱,以C57BL/6品系怀孕雌鼠为材料,取胚胎肢芽发育的四个关键时期(E10.5,E11.5,E12.5,E13.5)的胎鼠后肢,利用qPCR array方案检测表达谱中基因的相对表达水平差异。结果:通过已建立的qPCR array检测了C57BL/6品系小鼠胚胎后肢发育时期Hox家族、Wnt5a、Pitx1、Fgf8、Shh等基因的表达差异。以E10.5为对照,检测出在后肢发育时期基因呈三种表达模式,即Hoxb6、Hoxb8、Hoxc8、Hoxc9、Hoxc10、Hoxd9和Shh基因的表达水平呈上调;Hoxa11、Hoxa13、Hoxc12、Hoxc13、Hoxd13等基因表达出现下调;Hoxc9、Hoxc10、Hoxc11、Hoxd9、Hoxd12、Fgf8和Pitx1等基因的相对表达量呈先上调后下调的曲线表达模式,且有少部分基因在小鼠后肢发育时期表达水平无明显变化。结论:Hox家族、Wnt5a、Pitx1、Fgf8、Shh等基因在小鼠后肢发育时期表达,并且表达模式存在明显差异。     

Biparental expression of IGFBP1 and IGFBP3 renders their involvement in the etiology of Silver-Russell syndrome unlikely.

Maternal uniparental disomy (UPD) of chromosome 7 has recently been reported in about 10% of Silver-Russell (SRS) patients. It can therefore be concluded that at least one gene on chromosome 7 is imprinted and mutations in this gene/these genes might contribute to the phenotype of the disease. Two genes which are involved in growth and localised in 7p12-13 are the insulin-like growth factor binding proteins 1 and 3 (IGFBP1; IGFBP3). Comparison to the mouse genome shows that the syntenic region on mouse chromosome 11 is imprinted, UPD of this region leads to deviations in growth in mice. In the present study we investigated whether the genes for IGFBP1 and IGFBP3 might be involved in the etiology of SRS: after exclusion of SRS specific mutations we could demonstrate biparental expression of both genes in lymphocytes of an SRS patient without UPD7 as well as expression in a patient with maternal UPD7. Our results as well as those from other groups show biparental expression of IGFBP1 in fetal tissues and expression of IGFBP3 in nearly every tissue during puberty and adult life. Thus, no evidence is given for an involvement of the two genes in SRS.     

Axial skeletal patterning in mice lacking all paralogous group 8 Hox genes

We present a detailed study of the genetic basis of mesodermal axial patterning by paralogous group 8 Hox genes in the mouse. The phenotype of Hoxd8 loss-of-function mutants is presented, and compared with that of Hoxb8- and Hoxc8-null mice. Our analysis of single mutants reveals common features for the Hoxc8 and Hoxd8 genes in patterning lower thoracic and lumbar vertebrae. In the Hoxb8 mutant, more anterior axial regions are affected. The three paralogous Hox genes are expressed up to similar rostral boundaries in the mesoderm, but at levels that strongly vary with the axial position. We find that the axial region affected in each of the single mutants mostly corresponds to the area with the highest level of gene expression. However, analysis of double and triple mutants reveals that lower expression of the other two paralogous genes also plays a patterning role when the mainly expressed gene is defective. We therefore conclude that paralogous group 8 Hox genes are involved in patterning quite an extensive anteroposterior (AP) axial region. Phenotypes of double and triple mutants reveal that Hoxb8, Hoxc8 and Hoxd8 have redundant functions at upper thoracic and sacral levels, including positioning of the hindlimbs. Interestingly, loss of functional Hoxb8 alleles partially rescues the phenotype of Hoxc8- and Hoxc8/Hoxd8-null mutants at lower thoracic and lumbar levels. This suggests that Hoxb8 affects patterning at these axial positions differently from the other paralogous gene products. We conclude that paralogous Hox genes can have a unique role in patterning specific axial regions in addition to their redundant function at other AP levels.     

Expression patterns of Hox10 paralogous genes during lumbar spinal cord development

We have examined the expression of three paralogous Hox genes from E11.5 through E15.5 in the mouse spinal cord. These ages coincide with major phases of spinal cord neurogenesis, neuronal differentiation, cell migration, gliogenesis, and motor neuron cell death. The three genes, Hoxa10, Hoxc10, and Hoxd10, are all expressed in the lumbar spinal cord and have distinct expression patterns. Mutations in these three genes are known to affect motor neuron patterning. All three genes show lower levels of expression at the rostral limits of their domains, with selective regions of higher expression more caudally. Hoxa10 and Hoxd10 expression appears confined to postmitotic cell populations in the intermediate and ventral gray, while Hoxc10 is also expressed in proliferating cells in the dorsal ventricular zone. Hoxc10 and Hoxd10 expression is clearly excluded from the lateral motor columns at rostral lumbar levels but is present in this region more caudally. Double labeling demonstrates that Hoxc10 expression is correlated with ventrolateral LIM gene expression in the caudal part of the lumbar spinal cord.