A 4.2 kb Upstream Region of the Human Corneodesmosin Gene Directs Site-Specific Expression in Hair Follicles and Hyperkeratotic Epidermis of Transgenic Mice

Keratins K5 and K14 form the extensive intermediate filament network of mitotically active basal cells in all stratified epithelia. We have explored the regulatory mechanisms governing cell-type-specific and differentiation stage-specific expression of the human K5 gene in transiently transfected keratinocytes in vitro and in transgenic mice in vivo. Six thousand base pairs of 5' upstream K5 sequence directed proper basal cell-specific expression in all stratified epithelia. Surprisingly, as few as 90 bp of the K5 promoter still directed expression to stratified epithelia, with expression predominantly in epidermis, hair follicles, and tongue. Despite keratinocyte-preferred expression, the truncated K5 promoter displayed departures from basal to suprabasal expression in epidermis and from outer root sheath to inner root sheath expression in the follicle, with some regional variations in expression as well. To begin to elucidate the molecular controls underlying the keratinocyte specificity of the truncated promoter, we examined protein-DNA interactions within this region. A number of keratinocyte nuclear proteins bind to a K5 gene segment extending from -90 to +32 bp and are functionally involved in transcriptional regulation in vitro. Interestingly, several of these factors are common to both the K5 and K14 promoters, although they appear to be distinct from those previously implicated in keratinocyte specificity. Mutagenesis studies indicate that factors binding in the vicinity of the TATA box and transcription initiation are responsible for the cell type specificity of the truncated K5 promoter.

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Skin-specific expression of a truncated E1a oncoprotein binding to p105-Rb leads to abnormal hair follicle maturation without increased epidermal proliferation.

The Journal of Cell Biology ◽

10.1083/jcb.121.5.1109 ◽

1993 ◽

Vol 121 (5) ◽

pp. 1109-1120 ◽

Cited By ~ 19

Author(s):

C Missero ◽

C Serra ◽

K Stenn ◽

G P Dotto

Keyword(s):

Transgenic Mice ◽

Hair Follicle ◽

Cultured Cells ◽

Hair Follicles ◽

Tgf Beta ◽

Specific Expression ◽

Adenovirus E1a ◽

Keratin 5 ◽

Interfollicular Epidermis

In cultured cells, mutants of the Adenovirus E1a oncoprotein which bind to a reduced set of cellular proteins, including p105-Rb, p107, and p60-cyclin A, are transformation defective but can still interfere with exogenous growth inhibitory and differentiating signals, such as those triggered by TGF-beta. We have tested the ability of one such mutant, NTdl646, to interfere with keratinocyte growth and differentiation in vivo, in the skin of transgenic mice. Keratinocyte-specific expression of the transgene was achieved by using a keratin 5 promoter. Two independent lines of transgenic mice were obtained which expressed E1a specifically in their skin and exhibited an aberrant hair coat phenotype with striking regional variations. Affected hair shafts were short and crooked and hair follicles exhibited a dystrophic or absent inner root sheath. Interfollicular epidermis was normal, but its hyperplastic response to acute treatment with TPA (12-O-tetradecanoylphorbol-13-acetate) was significantly reduced. Primary keratinocytes derived from these animals were partially resistant to the effects of TPA and TGF-beta. The rate of spontaneous or chemically induced skin tumors in the transgenic mice was not increased. Thus, expression of a transgene which interferes with known negative growth regulatory proteins causes profound disturbances of keratinocyte maturation into a highly organized structure such as the hair follicle but does not lead to increased and/or neoplastic proliferation.

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Exploiting the Keratin 17 Gene Promoter To Visualize Live Cells in Epithelial Appendages of Mice

Molecular and Cellular Biology ◽

10.1128/mcb.25.16.7249-7259.2005 ◽

2005 ◽

Vol 25 (16) ◽

pp. 7249-7259 ◽

Cited By ~ 41

Author(s):

Nicholas Bianchi ◽

Daryle DePianto ◽

Kevin McGowan ◽

Changhong Gu ◽

Pierre A. Coulombe

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Fluorescent Protein ◽

Gene Promoter ◽

Hair Follicles ◽

Live Cells ◽

Upstream Region ◽

Regulatory Sequence ◽

Gfp Fluorescence ◽

Keratin 17

ABSTRACT Keratin genes afford, given their large number (>50) and differential regulation, a unique opportunity to study the mechanisms underlying specification and differentiation in epithelia of higher metazoans. Moreover, the small size and regulation in cis of many keratin genes enable the use of their regulatory sequence to achieve targeted gene expression in mice. Here we show that 2 kilobases of 5′ upstream region from the mouse keratin 17 gene (mK17) confers expression of green fluorescent protein (GFP) in major epithelial appendages of transgenic mice. Like that of mK17, onset of [mK17 5′]-GFP reporter expression coincides with the appearance of ectoderm-derived epithelial appendages during embryonic development. In adult mice, [mK17 5′]-GFP is appropriately regulated within hair, nail, glands, and oral papilla. Tracking of GFP fluorescence allows for the visualization of growth cycle-related changes in hair follicles, and the defects engendered by the hairless mutation, in live skin tissue. Deletion of an internal 48-bp interval, which encompasses a Gli-responsive element, from this promoter results in loss of GFP fluorescence in most appendages in vivo, suggesting that sonic hedgehog participates in K17 regulation. The compact mK17 gene promoter provides a novel tool for appendage-preferred gene expression and manipulation in transgenic mice.

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Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3176 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3176-3190 ◽

Cited By ~ 102

Author(s):

C Byrne ◽

E Fuchs

Keyword(s):

Transgenic Mice ◽

Transcription Initiation ◽

Gene Segment ◽

Hair Follicles ◽

Cell Type ◽

Outer Root Sheath ◽

Specific Expression ◽

Root Sheath ◽

Stratified Epithelia

Keratins K5 and K14 form the extensive intermediate filament network of mitotically active basal cells in all stratified epithelia. We have explored the regulatory mechanisms governing cell-type-specific and differentiation stage-specific expression of the human K5 gene in transiently transfected keratinocytes in vitro and in transgenic mice in vivo. Six thousand base pairs of 5' upstream K5 sequence directed proper basal cell-specific expression in all stratified epithelia. Surprisingly, as few as 90 bp of the K5 promoter still directed expression to stratified epithelia, with expression predominantly in epidermis, hair follicles, and tongue. Despite keratinocyte-preferred expression, the truncated K5 promoter displayed departures from basal to suprabasal expression in epidermis and from outer root sheath to inner root sheath expression in the follicle, with some regional variations in expression as well. To begin to elucidate the molecular controls underlying the keratinocyte specificity of the truncated promoter, we examined protein-DNA interactions within this region. A number of keratinocyte nuclear proteins bind to a K5 gene segment extending from -90 to +32 bp and are functionally involved in transcriptional regulation in vitro. Interestingly, several of these factors are common to both the K5 and K14 promoters, although they appear to be distinct from those previously implicated in keratinocyte specificity. Mutagenesis studies indicate that factors binding in the vicinity of the TATA box and transcription initiation are responsible for the cell type specificity of the truncated K5 promoter.

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Neuronal subtype-specific expression directed by the GABAA receptor δ subunit gene promoter/upstream region in transgenic mice and in cultured cells

Molecular Brain Research ◽

10.1016/s0169-328x(97)00242-8 ◽

1997 ◽

Vol 51 (1-2) ◽

pp. 197-211 ◽

Cited By ~ 16

Author(s):

Bernhard Lüscher ◽

Remo Häuselmann ◽

Sabine Leitgeb ◽

Thomas Rülicke ◽

Jean-Marc Fritschy

Keyword(s):

Transgenic Mice ◽

Gabaa Receptor ◽

Cultured Cells ◽

Gene Promoter ◽

Upstream Region ◽

Subunit Gene ◽

Specific Expression ◽

Neuronal Subtype

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Molecular Dissection of DNA Sequences and Factors Involved in Slow Muscle-Specific Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8490-8503.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8490-8503 ◽

Cited By ~ 45

Author(s):

Soledad Calvo ◽

Detlef Vullhorst ◽

Pratap Venepally ◽

Jun Cheng ◽

Irina Karavanova ◽

...

Keyword(s):

Transcription Factor ◽

Transgenic Mice ◽

Dna Sequences ◽

Muscle Development ◽

Fiber Type ◽

Regulatory Element ◽

Slow Muscle ◽

Upstream Region ◽

Fiber Types ◽

Specific Expression

ABSTRACT Transcription is a major regulatory mechanism for the generation of slow- and fast-twitch myofibers. We previously identified an upstream region of the slow TnI gene (slow upstream regulatory element [SURE]) and an intronic region of the fast TnI gene (fast intronic regulatory element [FIRE]) that are sufficient to direct fiber type-specific transcription in transgenic mice. Here we demonstrate that the downstream half of TnI SURE, containing E box, NFAT, MEF-2, and CACC motifs, is sufficient to confer pan-skeletal muscle-specific expression in transgenic mice. However, upstream regions of SURE and FIRE are required for slow and fast fiber type specificity, respectively. By adding back upstream SURE sequences to the pan-muscle-specific enhancer, we delineated a 15-bp region necessary for slow muscle specificity. Using this sequence in a yeast one-hybrid screen, we isolated cDNAs for general transcription factor 3 (GTF3)/muscle TFII-I repeat domain-containing protein 1 (MusTRD1). GTF3 is a multidomain nuclear protein related to initiator element-binding transcription factor TF II-I; the genes for both proteins are deleted in persons with Williams-Beuren syndrome, who often manifest muscle weakness. Gel retardation assays revealed that full-length GTF3, as well as its carboxy-terminal half, specifically bind the bicoid-like motif of SURE (GTTAATCCG). GTF3 expression is neither muscle nor fiber type specific. Its levels are highest during a period of fetal development that coincides with the emergence of specific fiber types and transiently increases in regenerating muscles damaged by bupivacaine. We further show that transcription from TnI SURE is repressed by GTF3 when overexpressed in electroporated adult soleus muscles. These results suggest a role for GTF3 as a regulator of slow TnI expression during early stages of muscle development and suggest how it could contribute to Williams-Beuren syndrome.

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