Slow diffusion on the monolayer culture enhances auto/paracrine effects of Noggin in differentiation of human iPS cells induced by BMP

Auto/paracrine factors secreted from cells affect differentiation of human pluripotent stem cells (hPSCs). However, the molecular mechanisms underlying the role of secreted factors are not well known. We previously showed that pattern formation in hPSCs induced by BMP4 could be reproduced by a simple reaction-diffusion of BMP and Noggin, a cell-secreted BMP4 inhibitor. However, the amount of Noggin secreted is unknown.In this study, we measured the concentration of Noggin secreted during the differentiation of hPSCs induced by BMP4. The Noggin concentration in the supernatant before and after differentiation was constant at approximately 0.69 ng/mL, which is approximately 50–200 times less than expected in the model. To explain the difference between the experiment and model, we assumed that macromolecules such as heparan sulfate proteoglycan on the cell surface act as a diffusion barrier structure, where the diffusion slows down to 1/400. The model with the diffusion barrier structure reduced the Noggin concentration required to suppress differentiation in the static culture model. The model also qualitatively reproduced the pattern formation, in which only the upstream but not the downstream hPSCs were differentiated in a one-directional perfusion culture chamber, with a small change in the amount of secreted Noggin resulting in a large change in the differentiation position. These results suggest that the diffusion barrier on the cell surface might enhance the auto/paracrine effects on monolayer hPSC culture.     

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

Quantitative Phosphoproteomics Analysis Uncovers PAK2- and CDK1-Mediated Malignant Signaling Pathways in Clear Cell Renal Cell Carcinoma

Clear cell Renal Cell Carcinoma (ccRCC) is among the 10 most common cancers in both men and women and causes more than 140,000 deaths worldwide every year. In order to elucidate the underlying molecular mechanisms orchestrated by phosphorylation modifications, we performed a comprehensive quantitative phosphoproteomics characterization of ccRCC tumor and normal adjacent tissues. Here, we identified 16,253 phosphopeptides, of which more than 9000 were singly quantified. Our in-depth analysis revealed 600 phosphopeptides to be significantly differentially regulated between tumor and normal tissues. Moreover, our data revealed that significantly up-regulated phosphoproteins are associated with protein synthesis and cytoskeletal re-organization which suggests proliferative and migratory behavior of renal tumors. This is supported by a mesenchymal profile of ccRCC phosphorylation events. Our rigorous characterization of the renal phosphoproteome also suggests that both epidermal growth factor receptor and vascular endothelial growth factor receptor are important mediators of phospho signaling in RCC pathogenesis. Furthermore, we determined the kinases p21-activated kinase 2, cyclin-dependent kinase 1 and c-Jun N-terminal kinase 1 to be master kinases that are responsible for phosphorylation of many substrates associated with cell proliferation, inflammation and migration. Moreover, high expression of p21-activated kinase 2 is associated with worse survival outcome of ccRCC patients. These master kinases are targetable by inhibitory drugs such as fostamatinib, minocycline, tamoxifen and bosutinib which can serve as novel therapeutic agents for ccRCC treatment.     

Elevated granulocyte-colony stimulating factor and hematopoietic stem cell mobilization in Niemann-Pick type C1 disease

Niemann-Pick type C1 (NPC1) disease is a progressive lysosomal storage disorder caused by mutations of the NPC1 gene. While neurodegeneration is the most severe symptom, a large proportion of NPC1 patients also present with splenomegaly, which has been attributed to cholesterol and glycosphingolipid accumulation in late endosomes and lysosomes. However, recent data also reveal an increase in the inflammatory monocyte subset in the Npc1^nih mouse model expressing an Npc1 null allele. We evaluated the contribution of hematopoietic cells to splenomegaly in NPC1 disease under conditions of hypercholesterolemia. We transplanted Npc1^nih (Npc1 null mutation) or Npc1^wt bone marrow (BM) into Ldlr^?/? mice and fed these mice a cholesterol-rich Western-type diet. At 9 weeks after BM transplant, on a chow diet, the Npc1 null mutation increased plasma granulocyte-colony stimulating factor (G-CSF) by 2-fold and caused mild neutrophilia. At 18 weeks after BM transplant, including 9 weeks of Western-type diet feeding, the Npc1 mutation increased G-csf mRNA levels by ～5-fold in splenic monocytes/macrophages accompanied by a ～4-fold increase in splenic neutrophils compared with controls. We also observed ～5-fold increased long-term and short-term hematopoietic stem cells (HSCs) in the spleen, and a ～30–75% decrease of these populations in BM, reflecting HSC mobilization, presumably downstream of elevated G-CSF. In line with these data, four patients with NPC1 disease showed higher plasma G-CSF compared with age-matched and gender-matched healthy controls. In conclusion, we show elevated G-CSF levels and HSC mobilization in the setting of an Npc1 null mutation and propose that this contributes to splenomegaly in patients with NPC1 disease.