George L. Sen

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

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.

DNMT1 maintains progenitor function in self-renewing somatic tissue

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.

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

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.

Restriction enzyme-generated siRNA (REGS) vectors and libraries.

Small interfering RNA (siRNA) technology facilitates the study of loss of gene function in mammalian cells and animal models, but generating multiple siRNA vectors using oligonucleotides is slow, inefficient and costly. Here we describe a new, enzyme-mediated method for generating numerous functional siRNA constructs from any gene of interest or pool of genes. To test our restriction enzyme–generated siRNA (REGS) system, we silenced a transgene and two endogenous genes and obtained the predicted phenotypes. REGS generated on average 34 unique siRNAs per kilobase of sequence. REGS enabled us to create enzymatically a complex siRNA library (>4 × 105 clones) from double-stranded cDNA encompassing known and unknown genes with 96% of the clones containing inserts of the appropriate size.

A brief history of RNAi: the silence of the genes

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.—Sen, G. L., Blau, H. M. A brief history of RNAi: the silence of the genes. FASEB J. 20, 1293–1299 (2006)

DNA methylation and epigenetic control of cellular differentiation.

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.1-3 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.4 DNA methylation confers distinct epigenetic states via several mechanisms.5, 6 Here we discuss fundamental mechanisms of DNA methylation and their interplay with several regulatory pathways that define cellular physiology and differentiation.

Active tissue-specific DNA demethylation conferred by somatic cell nuclei in stable heterokaryons

DNA methylation is among the most stable epigenetic marks, ensuring tissue-specific gene expression in a heritable manner throughout development. Here we report that differentiated mesodermal somatic cells can confer tissue-specific changes in DNA methylation on epidermal progenitor cells after fusion in stable multinucleate heterokaryons. Myogenic factors alter regulatory regions of genes in keratinocyte cell nuclei, demethylating and activating a muscle-specific gene and methylating and silencing a keratinocyte-specific gene. Because these changes occur in the absence of DNA replication or cell division, they are mediated by an active mechanism. Thus, the capacity to transfer epigenetic changes to other nuclei is not limited to embryonic stem cells and oocytes but is also a property of highly specialized mammalian somatic cells. These results suggest the possibility of directing the reprogramming of readily available postnatal human progenitor cells toward specific tissue cell types.

PTH/PTHrP and Vitamin D Control Antimicrobial Peptide Expression and Susceptibility to Bacterial Skin Infection

Vitamin D and parathyroid hormone work together to maximize innate immune resistance to skin infections. A Sunny Solution to Infectious Disease A role for vitamin D in immune defense has been hypothesized on the basis of observations made over several decades that increased vitamin D intake, often through increased sunlight exposure, provides a therapeutic effect in fighting infectious diseases. However, a critical role for vitamin D status in immune function has not been established by controlled clinical trials. The current study by Muehleisen et al. sought to determine whether parathyroid hormone (PTH), or PTH-related peptide (PTHrP), might also act together with vitamin D in immune defense against bacterial skin infections and thus confound interpretations based solely on vitamin D status. The authors found that human skin keratinocytes showed increased expression of PTHrP in response to bacterial products. PTH or PTHrP was then observed to induce expression of cathelicidin antimicrobial peptide by keratinocytes in culture. When PTH was administered to mice, it enhanced their resistance to skin infection by group A Streptococcus. If vitamin D was absent from the diet of normal mice, they responded with an increase in PTH production and an increase in antimicrobial peptide expression. However, mice lacking the capacity to convert vitamin D to 1,25-vitamin D failed to induce cathelicidin in response to restriction of dietary vitamin D and became much more susceptible to invasive bacterial infection. Because 1,25-dihydroxyvitamin D was found necessary for keratinocytes to express the PTH/PTHrP receptor, this increase in susceptibility to infection may reflect the inability of PTH or PTHrP to induce cathelicidin in a setting of low vitamin D. These findings show that PTH/PTHrP is immunologically active, can boost innate immunity, and may compensate for low vitamin D status. Understanding this system provides a new target for studying the immunological functions of vitamin D. 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 D3 (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 D3 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.

Genomic Profiling of a Human Organotypic Model of AEC Syndrome Reveals ZNF750 as an Essential Downstream Target of Mutant TP63

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.

SNAI2 Controls the Undifferentiated State of Human Epidermal Progenitor 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. Stem Cells 2014;32:3209–3218