JMJD3 activated hyaluronan synthesis drives muscle regeneration in an inflammatory environment.

Muscle stem cells (MuSCs) reside in a specialized niche that ensures their regenerative capacity. Although we know that innate immune cells infiltrate the niche in response to injury, it remains unclear how MuSCs adapt to this altered environment for initiating repair. Here, we demonstrate that inflammatory cytokine signaling from the regenerative niche impairs the ability of quiescent MuSCs to reenter the cell cycle. The histone H3 lysine 27 (H3K27) demethylase JMJD3, but not UTX, allowed MuSCs to overcome inhibitory inflammation signaling by removing trimethylated H3K27 (H3K27me3) marks at the Has2 locus to initiate production of hyaluronic acid, which in turn established an extracellular matrix competent for integrating signals that direct MuSCs to exit quiescence. Thus, JMJD3-driven hyaluronic acid synthesis plays a proregenerative role that allows MuSC adaptation to inflammation and the initiation of muscle repair.

Pygo2 regulates ß-catenin-induced activation of hair follicle stem/progenitor cells and skin hyperplasia.

Understanding the epigenetic mechanisms that control the activation of adult stem cells holds the promise of tissue and organ regeneration. Hair follicle stem cells have emerged as a prime model to study stem cell activation. Wnt/ß-catenin signaling controls multiple aspects of skin epithelial regeneration, with its excessive activity promoting the hyperactivation of hair follicle stem/progenitor cells and tumorigenesis. The contribution of chromatin factors in regulating Wnt/ß-catenin pathway function in these processes is unknown. Here, we show that chromatin effector Pygopus homolog 2 (Pygo2) produced by the epithelial cells facilitates depilation-induced hair regeneration, as well as ß-catenin-induced activation of hair follicle stem/early progenitor cells and trichofolliculoma-like skin hyperplasia. Pygo2 maximizes the expression of Wnt/ß-catenin targets, but is dispensable for ß-catenin-mediated expansion of LIM/homeobox protein Lhx2(+) cells, in the stem/early progenitor cell compartment of the hair follicle. Moreover, ß-catenin and Pygo2 converge to induce the accumulation and acetylation of tumor suppressor protein p53 upon the cell cycle entry of hair follicle early progenitor cells and in cultured keratinocytes. These findings identify Pygo2 as an important regulator of Wnt/ß-catenin function in skin epithelia and p53 activation as a prominent downstream event of ß-catenin/Pygo2 action in stem cell activation.

Transcriptional mechanisms link epithelial plasticity to adhesion and differentiation of epidermal progenitor cells.

During epithelial tissue morphogenesis, developmental progenitor cells undergo dynamic adhesive and cytoskeletal remodeling to trigger proliferation and migration. Transcriptional mechanisms that restrict such a mild form of epithelial plasticity to maintain lineage-restricted differentiation in committed epithelial tissues are poorly understood. Here, we report that simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 results in expansion and blocked terminal differentiation of embryonic epidermal progenitor cells. Conversely, mice overexpressing Ovol2 in their skin epithelia exhibit precocious differentiation accompanied by smaller progenitor cell compartments. We show that Ovol1/Ovol2-deficient epidermal cells fail to undertake α-catenin-driven actin cytoskeletal reorganization and adhesive maturation and exhibit changes that resemble epithelial-to-mesenchymal transition (EMT). Remarkably, these alterations and defective terminal differentiation are reversed upon depletion of EMT-promoting transcriptional factor Zeb1. Collectively, our findings reveal Ovol-Zeb1-α-catenin sequential repression and highlight Ovol1 and Ovol2 as gatekeepers of epithelial adhesion and differentiation by inhibiting progenitor-like traits and epithelial plasticity.

Mammary morphogenesis and regeneration require the inhibition of EMT at terminal end buds by Ovol2 transcriptional repressor.

Epithelial cells possess remarkable plasticity, having the ability to become mesenchymal cells through alterations in adhesion and motility (epithelial-to-mesenchymal transition [EMT]). However, how epithelial plasticity is kept in check in epithelial cells during tissue development and regeneration remains to be fully understood. Here we show that restricting the EMT of mammary epithelial cells by transcription factor Ovol2 is required for proper morphogenesis and regeneration. Deletion of Ovol2 blocks mammary ductal morphogenesis, depletes stem and progenitor cell reservoirs, and leads epithelial cells to undergo EMT in vivo to become nonepithelial cell types. Ovol2 directly represses myriad EMT inducers, and its absence switches response to TGF-ß from growth arrest to EMT. Furthermore, forced expression of the repressor isoform of Ovol2 is able to reprogram metastatic breast cancer cells from a mesenchymal to an epithelial state. Our findings underscore the critical importance of exquisitely regulating epithelial plasticity in development and cancer.

Chromatin effector Pygo2 mediates Wnt-notch crosstalk to suppress luminal/alveolar potential of mammary stem and basal cells.

Epigenetic mechanisms regulating lineage differentiation of mammary stem cells (MaSCs) remain poorly understood. Pygopus 2 (Pygo2) is a histone methylation reader and a context-dependent Wnt/ß-catenin coactivator. Here we provide evidence for Pygo2's function in suppressing luminal/alveolar differentiation of MaSC-enriched basal cells. We show that Pygo2-deficient MaSC/basal cells exhibit partial molecular resemblance to luminal cells, such as elevated Notch signaling and reduced mammary repopulating capability upon transplantation. Inhibition of Notch signaling suppresses basal-level and Pygo2-deficiency-induced luminal/alveolar differentiation of MaSC/basal cells, whereas activation of Wnt/ß-catenin signaling suppresses luminal/alveolar differentiation and Notch3 expression in a Pygo2-dependent manner. We show that Notch3 is a direct target of Pygo2 and that Pygo2 is required for ß-catenin binding and maintenance of a poised/repressed chromatin state at the Notch3 locus in MaSC/basal cells. Together, our data support a model where Pygo2-mediated chromatin regulation connects Wnt signaling and Notch signaling to restrict the luminal/alveolar differentiation competence of MaSC/basal cells.

An updated steroid benchmark set and its application in the discovery of novel nanomolar ligands of sex hormone-binding globulin.

A benchmark data set of steroids with known affinity for sex hormone-binding globulin (SHBG) has been widely used to validate popular molecular field-based QSAR techniques. We have expanded the data set by adding a number of nonsteroidal SHBG ligands identified both from the literature and in our previous experimental studies. This updated molecular set has been used herein to develop 4D QSAR models based on "inductive" descriptors and to gain insight into the molecular basis of protein-ligand interactions. Molecular alignment was generated by means of docking active compounds into the active site of the SHBG. Surprisingly, the alignment of the benchmark steroids contradicted the classical ligand-based alignment utilized in previous CoMFA and CoMSIA models yet afforded models with higher statistical significance and predictive power. The resulting QSAR models combined with CoMFA and CoMSiA models as well as structure-based virtual screening allowed discovering several low-micromolar to nanomolar nonsteroidal inhibitors for human SHBG.

Steroid conjugates of dichloro(6-aminomethylnicotinate)platinum(II): effects on DNA, sex hormone binding globulin, the estrogen receptor, and various breast cancer cell lines.

Esters of 6-aminomethylnicotinic acid with various steroidal alcohols were treated with K(2)PtCl(4) to give the N,N-chelated dichloroplatinum(II) complex conjugates 4. Their interaction with plasmid DNA was monitored by electrophoretic mobility measurements. Their affinities towards sex hormone binding globulin (SHBG) and towards the nuclear estrogen receptor ER(alpha) were assessed by competitive displacement radioassays. The inhibitory effect of 4 on breast tumour cells MCF-7 ER(+)/ER(-) and MDA-MB-231 was investigated in vitro. Conjugates with 3-O-linked estrogens 4 a,b or 17-O-linked androgens 4 g bound strongly to SHBG. The conjugate complex 4 b, featuring a 3-O-linked estradiol, also bound strongly and agonistically to the estrogen receptor. It also elicited distinct growth retardation of MCF-7 (ER(+)) cells, presumably by a mechanism different from that of cisplatin.

Progressive docking: a hybrid QSAR/docking approach for accelerating in silico high throughput screening.

A combination of protein-ligand docking and ligand-based QSAR approaches has been elaborated, aiming to speed-up the process of virtual screening. In particular, this approach utilizes docking scores generated for already processed compounds to build predictive QSAR models that, in turn, assess hypothetical target binding affinities for yet undocked entries. The "progressive docking" has been tested on drug-like substances from the NCI database that have been docked into several unrelated targets, including human sex hormone binding globulin (SHBG), carbonic anhydrase, corticosteroid-binding globulin, SARS 3C-like protease, and HIV1 reverse transcriptase. We demonstrate that progressive docking can reduce the amount of computations 1.2- to 2.6-fold (when compared to traditional docking), while maintaining 80-99% hit recovery rates. This progressive-docking procedure, therefore, substantially accelerates high throughput screening, especially when using high accuracy (slower) docking approaches and large-sized datasets, and has allowed us to identify several novel potent nonsteroidal SHBG ligands.

'Inductive' charges on atoms in proteins: comparative docking with the extended steroid benchmark set and discovery of a novel SHBG ligand.

We have developed a novel iterative approach for calculation of partial charges in proteins within the framework of the 'molecular capacitance' model. The method operates by an effective 'inductive' electronegativity scale derived from a number of the conventional charge systems including CHARMM, AMBER, MMFF, OPLS, and PEOE among others. Our novel 'inductive' electronegativity equalization procedure allows rapid and conformation sensitive computation of adequate partial charges in proteins. Accuracy of the 'inductive' values was confirmed by their correlation with DFT-computed partial charges in common amino acids. A comparative docking study with an extended steroid data set not only illustrated the adequacy of 'inductive' protein charges but also demonstrated their superior performance compared to several conventional protein charging systems. Subsequent docking with 'inductive' charges resulted in identification of five potential leads as human Sex Hormone Binding Globulin (SHBG) ligands from a commercial library of natural compounds. When the selected substances were evaluated for their ability to bind SHBG in vitro, three of them displaced testosterone from the SHBG steroid-binding site, and with one compound this was achieved at micromolar concentrations.

Successful in silico discovery of novel nonsteroidal ligands for human sex hormone binding globulin.

Using "in silico" drug design methodologies, we have discovered several nonsteroidal compounds of natural origin that bind to human sex hormone binding globulin (SHBG) with affinity constants of 0.1 x 10(6) to 1.2 x 10(6) M(-1). The computational solutions we developed involved pharmacophore-aided database search, virtual protein-ligand docking, and structure-activity modeling with "inductive" QSAR descriptors. By screening 23 836 natural substance structures, we identified 29 potential SHBG ligands, and eight of these bound the protein in vitro. These nonsteroidal ligands belong to four classes of molecular scaffolds with several available substitution positions that could allow chemical modification to enhance SHBG-binding activity. Interestingly, one of these compounds is structurally similar to a dicyclohexane derivative that binds to rat SHBG and causes azospermia when administered to male rats. Taken together, the in silico strategy we have developed will aid in the discovery of nonsteroidal ligands of SHBG with novel pharmacological properties.

Fate of mitochondrially located S19 ribosomal protein genes after transfer of a functional copy to the nucleus in cereals.

Mitochondrial genes for ribosomal proteins undergo relatively frequent transfer to the nucleus during plant evolution, and when migration is successful the mitochondrial copy becomes redundant and can be lost. We have examined the status of the mitochondrial rps19 gene for ribosomal protein S19 in closely related cereals. In oat, the mitochondrial rps19 reading frame is blocked by a premature termination codon and lacks abundant transcripts, whereas in the mitochondria of wheat and rye rps19 is a 5'-truncated pseudogene which is co-transcribed with the downstream nad4L gene. In barley and maize, rps19 sequences are completely absent from the mitochondrion. All five of these cereals differ from rice, in which an intact, transcriptionally active mitochondrial rps19 gene is found, and this is preceded by rpl2 in an organization reminiscent of that seen in bacteria. Based on EST sequence data for maize, barley and wheat, it can be inferred that a functional rps19 gene was transferred to the nucleus prior to the divergence of the maize and rice lineages (approximately 50 million years ago), and the present-day nuclear copies encode an N-terminal sequence related to the mitochondrial targeting signal of Hsp70 (heat shock protein) in cereals. Subsequent evolutionary events have included independent losses of the mitochondrial copies in the barley and maize lineages. In the rice lineage, on the other hand, the nuclear copy was lost. This is reflected in the persistence of the mitochondrial rps19 after a period during which rps19 genes coexisted in both compartments. These observations illustrate the dynamic nature of the location and structure of genes for mitochondrial ribosomal proteins in flowering plants.

Ovol1 regulates meiotic pachytene progression during spermatogenesis by repressing Id2 expression.

Previous studies have shown that a targeted deletion of Ovol1 (previously known as movo1), encoding a member of the Ovo family of zinc-finger transcription factors, leads to germ cell degeneration and defective sperm production in adult mice. To explore the cellular and molecular mechanism of Ovol1 function, we have examined the mutant testis phenotype during the first wave of spermatogenesis in juvenile mice. Consistent with the detection of Ovol1 transcripts in pachytene spermatocytes of the meiotic prophase, Ovol1-deficient germ cells were defective in progressing through the pachytene stage. The pachytene arrest was accompanied by an inefficient exit from proliferation, increased apoptosis and an abnormal nuclear localization of the G2-M cell cycle regulator cyclin B1, but was not associated with apparent chromosomal or recombination defects. Transcriptional profiling and northern blot analysis revealed reduced expression of pachytene markers in the mutant, providing molecular evidence that pachytene differentiation was defective. In addition, the expression of Id2 (inhibitor of differentiation 2), a known regulator of spermatogenesis, was upregulated in Ovol1-deficient pachytene spermatocytes and repressed by Ovol1 in reporter assays. Taken together, our studies demonstrate a role for Ovol1 in regulating pachytene progression of male germ cells, and identify Id2 as a Ovol1 target.

The LEF1/beta -catenin complex activates movo1, a mouse homolog of Drosophila ovo required for epidermal appendage differentiation.

Drosophila ovo/svb (dovo) is required for epidermal cuticle/denticle differentiation and is genetically downstream of the wg signaling pathway. Similarly, a mouse homolog of dovo, movo1, is required for the proper formation of hair, a mammalian epidermal appendage. Here, we provide biochemical evidence that movo1 encodes a nuclear DNA binding protein (mOvo1a) that binds to DNA sequences similar to those that dOvo binds to, further supporting the notion that mOvo1a and dOvo are genetically and biochemically homologous proteins. Additionally, we show that the movo1 promoter is activated by the lymphoid enhancer factor 1 (LEF1)/beta-catenin complex, a transducer of wnt signaling. Collectively, our findings suggest that movo1 is a developmental target of wnt signaling during hair morphogenesis in mice, and that the wg/wnt-ovo link in epidermal appendage regulatory pathways has been conserved between mice and flies.