Cell-type-specific transcriptome and histone modification dynamics during cellular reprogramming in the Arabidopsis stomatal lineage.

Plant cells maintain remarkable developmental plasticity, allowing them to clonally reproduce and to repair tissues following wounding; yet plant cells normally stably maintain consistent identities. Although this capacity was recognized long ago, our mechanistic understanding of the establishment, maintenance, and erasure of cellular identities in plants remains limited. Here, we develop a cell-type-specific reprogramming system that can be probed at the genome-wide scale for alterations in gene expression and histone modifications. We show that relationships among H3K27me3, H3K4me3, and gene expression in single cell types mirror trends from complex tissue, and that H3K27me3 dynamics regulate guard cell identity. Further, upon initiation of reprogramming, guard cells induce H3K27me3-mediated repression of a regulator of wound-induced callus formation, suggesting that cells in intact tissues may have mechanisms to sense and resist inappropriate dedifferentiation. The matched ChIP-sequencing (seq) and RNA-seq datasets created for this analysis also serve as a resource enabling inquiries into the dynamic and global-scale distribution of histone modifications in single cell types in plants.

The plant stomatal lineage at a glance.

Stomata are structures on the surfaces of most land plants that are required for gas exchange between plants and their environment. In Arabidopsis thaliana, stomata comprise two kidney bean-shaped epidermal guard cells that flank a central pore overlying a cavity in the mesophyll. These guard cells can adjust their shape to occlude or facilitate access to this pore, and in so doing regulate the release of water vapor and oxygen from the plant, in exchange for the intake of carbon dioxide from the atmosphere. Stomatal guard cells are the end product of a specialized lineage whose cell divisions and fate transitions ensure both the production and pattern of cells in aerial epidermal tissues. The stomatal lineage is dynamic and flexible, altering stomatal production in response to environmental change. As such, the stomatal lineage is an excellent system to study how flexible developmental transitions are regulated in plants. In this Cell Science at a Glance article and accompanying poster, we will summarize current knowledge of the divisions and fate decisions during stomatal development, discussing the role of transcriptional regulators, cell-cell signaling and polarity proteins. We will highlight recent work that links the core regulators to systemic or environmental information and provide an evolutionary perspective on stomata lineage regulators in plants.

The INO80 chromatin remodeler sustains metabolic stability by promoting TOR signaling and regulating histone acetylation.

Chromatin remodeling complexes are essential for gene expression programs that coordinate cell function with metabolic status. However, how these remodelers are integrated in metabolic stability pathways is not well known. Here, we report an expansive genetic screen with chromatin remodelers and metabolic regulators in Saccharomyces cerevisiae. We found that, unlike the SWR1 remodeler, the INO80 chromatin remodeling complex is composed of multiple distinct functional subunit modules. We identified a strikingly divergent genetic signature for the Ies6 subunit module that links the INO80 complex to metabolic homeostasis. In particular, mitochondrial maintenance is disrupted in ies6 mutants. INO80 is also needed to communicate TORC1-mediated signaling to chromatin, as ino80 mutants exhibit defective transcriptional profiles and altered histone acetylation of TORC1-responsive genes. Furthermore, comparative analysis reveals subunits of INO80 and mTORC1 have high co-occurrence of alterations in human cancers. Collectively, these results demonstrate that the INO80 complex is a central component of metabolic homeostasis that influences histone acetylation and may contribute to disease when disrupted.

The Chinese herbs Scutellaria baicalensis and Fritillaria cirrhosa target NFκB to inhibit proliferation of ovarian and endometrial cancer cells.

The herbs Scutellaria baicalensis (SB) and Fritillaria cirrhosa (FC) are widely used in Chinese medicine to treat several aliments and as an adjuvant to chemotherapy of lung cancer. No information is available regarding the two herbs' influence on ovarian and endometrial cancer. To fill this data gap we compared cell growth responses to SB and FC in ovarian and endometrial cancer cell lines. Dose-dependent cell growth inhibition was observed following higher doses in all cell lines while lower doses stimulated growth in only endometrial cell lines. Higher doses of SB and FC significantly decreased cell growth on soft agar and decreased the invasive potential of cancer cells. Treatment of cells with both herbs resulted in activation of caspase-3, G0 /G1 phase cell cycle arrest, downregulation of cyclins D1 and D3 and induction of p27. Both herbs decreased NFκB DNA binding, reduced expression of phosphorylated IκBα, abrogated NFκB activation, and downregulated NFκB-regulated metastasis-promoting proteins in cancer cells. Furthermore, knockdown of NFκB attenuated SB- and FC-induced cell growth inhibition. These results suggest that inhibition of NFκB activation may be an important mechanism for growth suppression by SB and FC. Data indicate that these herbs may represent a new source of agents for NFκB inhibition in cancer therapy.

Glutathione accelerates the cell cycle and cellular reprogramming in plant regeneration.

The plasticity of plant cells underlies their wide capacity to regenerate, with increasing evidence in plants and animals implicating cell cycle dynamics in cellular reprogramming. To investigate the cell cycle during cellular reprogramming, we developed a comprehensive set of cell cycle phase markers in the Arabidopsis root. Using single-cell RNA-seq profiles and live imaging during regeneration, we found that a subset of cells near an ablation injury dramatically increases division rate by truncating G1. Cells in G1 undergo a transient nuclear peak of glutathione (GSH) prior to coordinated entry into S phase followed by rapid divisions and cellular reprogramming. A symplastic block of the ground tissue impairs regeneration, which is rescued by exogenous GSH. We propose a model in which GSH from the outer tissues is released upon injury licensing an exit from G1 near the wound to induce rapid cell division and reprogramming.

Morphology and life history divergence in cave and surface populations of Gammarus lacustris (L.).

Cave animals provide a unique opportunity to study contrasts in phenotype and life history in strikingly different environments when compared to surface populations, potentially related to natural selection. As such, we compared a permanent cave-living Gammarus lacustris (L.) population with two lake-resident surface populations analyzing morphology (eye- and antennal characters) and life-history (size at maturity, fecundity and egg-size). A part of the cytochrome c oxidase subunit I gene in the mitochondrion (COI) was analyzed to contrast genetic relationship of populations and was compared to sequences in GenBank to assess phylogeography and colonization scenarios. In the cave, a longer life cycle was implied, while surface populations seemed to have a shorter life cycle. Egg size, and size at maturity for both sexes, were larger in the cave than in surface populations, while fecundity was lower in the cave than in surface populations. The cave population had longer first- and second antennae with more articles, longer first- and second peduncles, and fewer ommatidia than surface populations. The cold low-productive cave environment may facilitate different phenotypic and life-history traits than in the warmer and more productive surface lake environments. The trait divergences among cave and surface populations resembles other cave-surface organism comparisons and may support a hypothesis of selection on sensory traits. The cave and Lake Ulvenvann populations grouped together with a sequence from Slovenia (comprising one genetic cluster), while Lake Lille Lauarvann grouped with a sequence from Ukraine (comprising another cluster), which are already recognized phylogenetic clusters. One evolutionary scenario is that the cave and surface populations were colonized postglacially around 9 000-10 000 years ago. We evaluate that an alternative scenario is that the cave was colonized during an interstadial during the last glaciation or earlier during the warm period before onset of the last glaciation.

Progesterone potentiates the growth inhibitory effects of calcitriol in endometrial cancer via suppression of CYP24A1.

Here, we evaluated the expression of CYP24A1, a protein that inactivates vitamin D in tissues. CYP24A1 expression was increased in advanced-stage endometrial tumors compared to normal tissues. Similarly, endometrial cancer cells expressed higher levels of CYP24A1 than immortalized endometrial epithelial cells. RT-PCR and Western blotting were used to examine CYP24A1 mRNA and protein levels in endometrial cancer cells after 8, 24, 72, and 120 h of exposure to progesterone, progestin derivatives and calcitriol, either alone or in combination. Progestins inhibited calcitriol-induced expression of CYP24A1 and splice variant CYP24SV mRNA and protein in cancer cells. Furthermore, actinomycin D, but not cycloheximide, blocked calcitriol-induced CYP24A1 splicing. siRNA-induced knockdown of CYP24A1 expression sensitized endometrial cancer cells to calcitriol-induced growth inhibition. These data suggest that CYP24A1 overexpression reduces the antitumor effects of calcitriol in cancer cells and that progestins may be beneficial for maintaining calcitriol's anti-endometrial cancer activity.

Progesterone inhibits endometrial cancer invasiveness by inhibiting the TGFß pathway.

Increased expression of TGFß isoforms in human endometrial cancer correlates with decreased survival and poor prognosis. Progesterone has been shown to exert a chemoprotective effect against endometrial cancer, and previous animal models have suggested that these effects are accompanied by changes in TGFß. The goal of this study was to characterize the effect of progesterone on TGFß signaling pathway components and on TGFß-induced protumorigenic activities in endometrial cancer cell lines. Progesterone significantly decreased expression of three TGFß isoforms at 72 hours after treatment except for TGFß2 in HEC-1B and TGFß3 in Ishikawa cells. Progesterone treatment for 120 hours attenuated expression of the three isoforms in all cell lines. Progesterone exposure for 72 hours reduced expression of TGFß receptors in HEC-1B cells and all but TGFßR1 in Ishikawa cells. Progesterone reduced TGFßR3 expression in RL-95 cells at 72 hours, but TGFßR1 and ßR2 expression levels were not affected by progesterone at any time point. SMAD2/3 and pSMAD2/3 were substantially reduced at 72 hours in all cell lines. SMAD4 expression was reduced in RL-95 cells at 24 hours and in HEC-1B and Ishikawa cells at 72 hours following progesterone treatment. Furthermore, progesterone effectively inhibited basal and TGFß1-induced cancer cell viability and invasion, which was accompanied by increased E-cadherin and decreased vimentin expression. An inhibitor of TGFßRI blocked TGFß1-induced effects on cell viability and invasion and attenuated antitumor effects of progesterone. These results suggest that downregulation of TGFß signaling is a key mechanism underlying progesterone inhibition of endometrial cancer growth.

Progesterone enhances calcitriol antitumor activity by upregulating vitamin D receptor expression and promoting apoptosis in endometrial cancer cells.

Human studies suggest that progesterone and calcitriol may prove beneficial in preventing or inhibiting oncogenesis, but the underlying mechanism is not fully understood. The current study investigates the effects of progesterone, calcitriol, and their combination on immortalized human endometrial epithelial cells and endometrial cancer cells and identifies their targets of action. Combination treatment with both agents enhanced vitamin D receptor expression and inhibited cell proliferation through caspase-3 activation and induction of G0-G1 cell-cycle arrest with associated downregulation of cyclins D1 and D3 and p27 induction. We used mass spectrometry-based proteomics to measure protein abundance differences between calcitriol-, progesterone-, or combination-exposed endometrial cells. A total of 117 proteins showed differential expression among these three treatments. Four proteins were then selected for validation studies: histone H1.4 (HIST1H1E), histidine triad nucleotide-binding protein 2 (HINT2), IFN-induced, double-stranded RNA-activated protein kinase (EIF2AK2), and Bcl-2-associated X protein (BAX). Abundance levels of selected candidates were low in endometrial cancer cell lines versus the immortalized endometrial epithelial cell line. All four proteins displayed elevated expression in cancer cells upon exposure to calcitriol, progesterone, or the combination. Further BAX analysis through gain- or loss-of-function experiments revealed that upregulation of BAX decreased cell proliferation by changing the BAX:BCL-2 ratio. Knockdown of BAX attenuated progesterone- and calcitriol-induced cell growth inhibition. Our results showed that progesterone and calcitriol upregulate the expression of BAX along with other apoptosis-related proteins, which induce inhibition of endometrial cancer cell growth by apoptosis and cell-cycle arrest.