generation-genetically-modified-organotypic-skin-cultures-using

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

RNA interference (RNAi) is an important means of eliminating mRNAs, but the intracellular location of RNA-induced silencing complex (RISC) remains unknown. We show here that Argonaute 2, a key component of RISC, is not randomly distributed but concentrates in mRNA decay centres that are known as cytoplasmic bodies. The localization of Argonaute 2 in decay centres is not altered by the presence or absence of small interfering RNAs or their targeted mRNAs. However, RNA is required for the integrity of cytoplasmic bodies because RNase eliminates Argonaute 2 localization. In addition, Argonaute 1, another Argonaute family member, is concentrated in cytoplasmic bodies. These results provide new insight into the mechanism of RNAi function.

Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies.

RNA interference constitutes a major means of eliminating mRNAs, yet how the small interfering RNAs (siRNA) within the RNA-induced silencing complex (RISC) finds its homologous target in the cell remains unknown. An attractive hypothesis is that RNA interference is linked to translation which allows RISC ready access to every translated mRNA. To test whether translation could direct siRNAs to mRNAs, chemical and biological inhibitors of translation and their effects on mRNA cleavage were tested. Our results show that mRNA degradation by siRNAs is not dependent on mRNA translation.

mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage.

The use of the RNA interference (RNAi) pathway to eliminate gene products has greatly facilitated the understanding of gene function. Behind this remarkable pathway is an intricate network of proteins that ensures the degradation of the target mRNA. In this review, we explore the history of RNAi as well as highlighting recent discoveries.

A brief history of RNAi: the silence of the genes.

The recent discovery of H3K27me3 demethylases suggests that H3K27me3 may dynamically regulate gene expression, but this potential role in mammalian tissue homeostasis remains uncharacterized. In the epidermis, a tissue that balances stem cell self-renewal with differentiation, H3K27me3, occupies the promoters of many differentiation genes. During calcium-induced differentiation, H3K27me3 was erased at these promoters in concert with loss of PcG protein occupancy and increased binding by the H3K27me3 demethylase, JMJD3. Within epidermal tissue, JMJD3 depletion blocked differentiation, while active JMJD3 dominantly induced it. These results indicate that epigenetic derepression by JMJD3 controls mammalian epidermal differentiation.

Control of differentiation in a self-renewing mammalian tissue by the histone demethylase JMJD3.

Die j&uuml;ngste Entdeckung von H3K27me3 Demethylasen legt nahe, dass H3K27me3 kann dynamisch Genexpression regulieren, aber diese potenzielle Rolle bei der Hom&ouml;ostase S&auml;ugetier-Gewebe uncharakterisiertes bleibt. In der Epidermis belegt eine Gewebe, die Stammzelle Self-Renewal mit Differenzierung, H3K27me3, gleicht die F&ouml;rderer vieler Differenzierung Gene. W&auml;hrend Kalzium-induzierte Differenzierung H3K27me3 auf diese Promotoren im Konzert mit Verlust von PcG-Protein-Belegung gel&ouml;scht wurde und Bindung durch die H3K27me3-Demethylase, JMJD3 gesteigert. Innerhalb epidermale Gewebe blockiert JMJD3 Ersch&ouml;pfung, Differenzierung, aktive JMJD3 induziert es dominant. Diese Ergebnisse zeigen, dass epigenetische Derepression durch JMJD3 Steuerelemente S&auml;ugetier-epidermaler Differenzierung.

Kontrolle der Differenzierung in einem erneuernden S&auml;ugetier-Gewebe durch die Histon-Demethylase JMJD3.

Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

DNMT1 maintains progenitor function in self-renewing somatic tissue.

In mammals the genome is shaped by epigenetic regulation to manifest numerous cellular identities. The term epigenetics has been used to refer to changes in gene expression, which are heritable through multiple cell division cycles that are not due to variations in primary DNA sequence. Stable suppression of differentiation genes is required to sustain the undifferentiated state in cells ranging from embryonic stem cells to somatic stem cell progenitors that constantly replenish self-renewing tissues. However, the epigenetic mechanisms behind the maintenance of cellular dedifferentiation are not yet fully understood. Major effectors of epigenetic control include regulators of DNA methylation and histone modification as well as ATP-dependent chromatin remodeling enzymes. These interact with other regulators, such as DNA sequence-specific transcription factors and noncoding RNAs to landscape the genome during development, differentiation and cancer. DNA methylation is a classic and powerful example of the epigenetic inheritance of cellular identity that is widely used in eukaryotes. DNA methylation confers distinct epigenetic states via several mechanisms. Here we discuss fundamental mechanisms of DNA methylation and their interplay with several regulatory pathways that define cellular physiology and differentiation.

DNA methylation and epigenetic control of cellular differentiation.

Stem cells serve a vital role in multicellular organisms by constantly replenishing tissue with their differentiated progeny during normal homeostasis or damage. How these cells maintain their identities throughout the life of an organism is an area of intense research. In this review, we explore recent emerging evidence that stem cell fate decisions are based on their epigenome and specific epigenetic factors.

Remembering one's identity: the epigenetic basis of stem cell fate decisions.

Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting that it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process.

ZNF750 is a p63 target gene that induces KLF4 to drive terminal epidermal differentiation.

The production of antimicrobial peptides is essential for protection against a wide variety of microbial pathogens and plays an important role in the pathogenesis of several diseases. The mechanisms responsible for expression of antimicrobial peptides are incompletely understood, but a role for vitamin D as a transcriptional inducer of the antimicrobial peptide cathelicidin has been proposed. We show that 1,25-dihydroxyvitamin D(3) (1,25-D3) acts together with parathyroid hormone (PTH), or the shared amino-terminal domain of PTH-related peptide (PTHrP), to synergistically increase cathelicidin and immune defense. Administration of PTH to mouse skin decreased susceptibility to skin infection by group A Streptococcus. Mice on dietary vitamin D(3) restriction that responded with an elevation in PTH have an increased risk of infection if they lack 1,25-D3. These results identify PTH/PTHrP as a variable that serves to compensate for inadequate vitamin D during activation of antimicrobial peptide production.

PTH/PTHrP and vitamin D control antimicrobial peptide expression and susceptibility to bacterial skin infection.

Stem and progenitor cells maintain the tissue they reside in for life by regulating the balance between proliferation and differentiation. How this is done is not well understood. Here, we report that the human exosome maintains progenitor cell function. The expression of several subunits of the exosome were found to be enriched in epidermal progenitor cells, which were required to retain proliferative capacity and to prevent premature differentiation. Loss of PM/Scl-75 also known as EXOSC9, a key subunit of the exosome complex, resulted in loss of cells from the progenitor cell compartment, premature differentiation, and loss of epidermal tissue. EXOSC9 promotes self-renewal and prevents premature differentiation by maintaining transcript levels of a transcription factor necessary for epidermal differentiation, GRHL3, at low levels through mRNA degradation. These data demonstrate that control of differentiation specific transcription factors through mRNA degradation is required for progenitor cell maintenance in mammalian tissue.

Progenitor function in self-renewing human epidermis is maintained by the exosome.

The basis for impaired differentiation in TP63 mutant ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome is unknown. Human epidermis harboring AEC TP63 mutants recapitulated this impairment, along with downregulation of differentiation activators, including HOPX, GRHL3, KLF4, PRDM1, and ZNF750. Gene-set enrichment analysis indicated that disrupted expression of epidermal differentiation programs under the control of ZNF750 and KLF4 accounted for the majority of disrupted epidermal differentiation resulting from AEC mutant TP63. Chromatin immunoprecipitation (ChIP) analysis and ChIP-sequencing of TP63 binding in differentiated keratinocytes revealed ZNF750 as a direct target of wild-type and AEC mutant TP63. Restoring ZNF750 to AEC model tissue rescued activator expression and differentiation, indicating that AEC TP63-mediated ZNF750 inhibition contributes to differentiation defects in AEC. Incorporating disease-causing mutants into regenerated human tissue can thus dissect pathomechanisms and identify targets that reverse disease features.

Genomic profiling of a human organotypic model of AEC syndrome reveals ZNF750 as an essential downstream target of mutant TP63.

Cell fate commitment during development is achieved through the expression of lineage-specific transcription factors. Recent studies have suggested that the expression of combinations of these lineage-specific transcription factors can convert adult somatic cells from one type to another. Here we report that the combination of p63, a master regulator of epidermal development and differentiation, and KLF4, a regulator of epidermal differentiation, is sufficient to convert dermal fibroblasts to a keratinocyte phenotype. Induced keratinocytes (KCs) expressed KC-specific proteins and had a transcriptome similar to KCs. Reprogramming to a KC phenotype was rapid and efficient with a vast majority of cells morphologically resembling and expressing KC-specific genes within a week of p63 and KLF4 transduction. Furthermore, p63 and KLF4 are capable of inducing a KC phenotype even in a cancerous cell line, highlighting their importance for epidermal specification. The robustness of the conversion process also allows the use of this as a model system to study the mechanisms of reprogramming.

Highly rapid and efficient conversion of human fibroblasts to keratinocyte-like cells.

The transcription factor, SNAI2, is an inducer of the epithelial to mesenchymal transition (EMT) which mediates cell migration during development and tumor invasion. SNAI2 can also promote the generation of mammary epithelial stem cells from differentiated luminal cells when overexpressed. How SNAI2 regulates these critical and diverse functions is unclear. Here, we show that the levels of SNAI2 expression are important for epidermal cell fate decisions. The expression of SNAI2 was found to be enriched in the basal layer of the interfollicular epidermis where progenitor cells reside and extinguished upon differentiation. Loss of SNAI2 resulted in premature differentiation whereas gain of SNAI2 expression inhibited differentiation. SNAI2 controls the differentiation status of epidermal progenitor cells by binding to and repressing the expression of differentiation genes with increased binding leading to further transcriptional silencing. Thus, the levels of SNAI2 binding to genomic targets determine the differentiation status of epithelial cells with increased levels triggering EMT and dedifferentiation, moderate (physiological) levels promoting epidermal progenitor function, and low levels leading to epidermal differentiation.

SNAI2 controls the undifferentiated state of human epidermal progenitor cells.

Epithelial to mesenchymal transition transcription factors (EMT-TFs) such as SNAI2 have been found to be expressed endogenously in epidermal stem and progenitor cells and downregulated upon differentiation. The presence of SNAI2 in progenitor cells is necessary to repress the expression of differentiation genes by binding directly to their promoters. SNAI2 is downregulated upon differentiation which allows expression of differentiation genes. Furthermore overexpression of SNAI2 can block the differentiation process suggesting that the levels of SNAI2 are crucial to epidermal cell fate decisions. To address on a genome wide level the genes that are impacted by changing the levels of SNAI2, we performed microarray analysis on SNAI2 knockdown and overexpressing epidermal progenitor cells. Here we provide a detailed methods and analysis on these microarray data which has been deposited in Gene Expression Omnibus (GEO): GSE55269.

Transcriptional profiling of SNAI2 regulated genes in primary human keratinocytes.

PAX6 is a master regulatory gene involved in neuronal cell fate specification. It also plays a critical role in early eye field and subsequent limbal stem cell (LSC) determination during eye development. Defects in Pax6 cause aniridia and LSC deficiency in humans and the Sey (Small eye) phenotype in mice (Massé, K., Bhamra, S., Eason, R., Dale, N., and Jones, E. A. (2007) Nature 449, 1058-1062). However, how PAX6 specifies LSC and corneal fates during eye development is not well understood. Here, we show that PAX6 is expressed in the primitive eye cup and later in corneal tissue progenitors in early embryonic development. In contrast, p63 expression commences after that of PAX6 in ocular adnexal and skin tissue progenitors and later in LSCs. Using an in vitro feeder-free culture system, we show that PAX6 knockdown in LSCs led to up-regulation of skin epidermis-specific keratins concomitant with differentiation to a skin fate. Using gene expression analysis, we identified the involvement of Notch, Wnt, and TGF-β signaling pathways in LSC fate determination. Thus, loss of PAX6 converts LSCs to epidermal stem cells, as demonstrated by a switch in the keratin gene expression profile and by the appearance of congenital dermoid tissue.

Transcription Factor PAX6 (Paired Box 6) Controls Limbal Stem Cell Lineage in Development and Disease.

SOX2 expression inhibits terminal epidermal differentiation.

In adult tissues, stem and progenitor cells must balance proliferation and differentiation to maintain homeostasis. How this is done is unclear. Here, we show that the DEAD box RNA helicase, DDX6 is necessary for maintaining adult progenitor cell function. DDX6 loss results in premature differentiation and decreased proliferation of epidermal progenitor cells. To maintain self-renewal, DDX6 associates with YBX1 to bind the stem loops found in the 3' UTRs of regulators of proliferation/self-renewal (CDK1, EZH2) and recruit them to EIF4E to facilitate their translation. To prevent premature differentiation of progenitor cells, DDX6 regulates the 5' UTR of differentiation inducing transcription factor, KLF4 and degrades its transcripts through association with mRNA degradation proteins. Our results demonstrate that progenitor function is maintained by DDX6 complexes through two distinct pathways that include the degradation of differentiation-inducing transcripts and by promoting the translation of self-renewal and proliferation mRNAs.

Article	Year
Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nature cell biology\| PubMed ID: 15908945	2005
mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage. Differentiation; research in biological diversity\| PubMed ID: 16138829	2005
A brief history of RNAi: the silence of the genes. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 16816104	2006
Control of differentiation in a self-renewing mammalian tissue by the histone demethylase JMJD3. Genes & development\| PubMed ID: 18628393	2008
DNMT1 maintains progenitor function in self-renewing somatic tissue. Nature\| PubMed ID: 20081831	2010
DNA methylation and epigenetic control of cellular differentiation. Cell cycle (Georgetown, Tex.)\| PubMed ID: 20890116	2010
Remembering one's identity: the epigenetic basis of stem cell fate decisions. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 21441350	2011
ZNF750 is a p63 target gene that induces KLF4 to drive terminal epidermal differentiation. Developmental cell\| PubMed ID: 22364861	2012
PTH/PTHrP and vitamin D control antimicrobial peptide expression and susceptibility to bacterial skin infection. Science translational medicine\| PubMed ID: 22623742	2012
Progenitor function in self-renewing human epidermis is maintained by the exosome. Cell stem cell\| PubMed ID: 22770246	2012
Genomic profiling of a human organotypic model of AEC syndrome reveals ZNF750 as an essential downstream target of mutant TP63. American journal of human genetics\| PubMed ID: 22922031	2012
Highly rapid and efficient conversion of human fibroblasts to keratinocyte-like cells. The Journal of investigative dermatology\| PubMed ID: 23921950	2014
SNAI2 controls the undifferentiated state of human epidermal progenitor cells. Stem cells (Dayton, Ohio)\| PubMed ID: 25100569	2014
Transcriptional profiling of SNAI2 regulated genes in primary human keratinocytes. Genomics data\| PubMed ID: 25793155	2015
Transcription Factor PAX6 (Paired Box 6) Controls Limbal Stem Cell Lineage in Development and Disease. The Journal of biological chemistry\| PubMed ID: 26045558	2015
SOX2 expression inhibits terminal epidermal differentiation. Experimental dermatology\| PubMed ID: 26344931	2015
DDX6 Orchestrates Mammalian Progenitor Function through the mRNA Degradation and Translation Pathways. Molecular cell\| PubMed ID: 26412305	2015

George L. Sen

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