Prospects for Knockout of MYB60, a Transcriptional Repressor of Anthocyanin Biosynthesis, in Brassicaceae Plants by Genome Editing

The Brassicaceae plant family contains many economically important crops such as Brassica napus L., Brassica rapa L., Brassica oleracea L., Brassica juncea L., Eruca sativa Mill., Camelina sativa L. and Raphanus sativus L. Insufficient data on the genetic regulation of agronomic traits in these species complicates the editing of their genomes. In recent years, the attention of the academic community has been drawn to anthocyanin hyperaccumulation. This trait is not only beneficial for human health, but can also increase plant resistance to stress. MYB transcription factors are the main regulators of flavonoid biosynthesis in plants. Some of them are well studied in Arabidopsis thaliana. The AtMYB60 gene is a transcriptional repressor of anthocyanin biosynthesis, and it also negatively impacts plant responses to drought stress. Myb60 is one of the least studied transcription factors with similar functions in Brassicaceae. There is a high degree of homology between predicted MYB60 genes of A. thaliana and related plant species. However, functions of these homologous genes have never been studied. Gene knockout by CRISPR/Cas technology remains the easiest way to perform genome editing in order to discover the role of individual plant genes. Disruption of genes acting as negative regulators of anthocyanin biosynthesis could result in color staining of plant tissues and an increase in stress tolerance. In the present study, we investigated the AtMYB60 gene and its homologs in Brassicaceae plants and suggested universal gRNAs to knockout these genes. Keywords: CRISPR, Brassicaceae, MYB60, knockout, anthocyanin

Inhibition of β-catenin dependent WNT signalling upregulates the transcriptional repressor NR0B1 and downregulates markers of an A9 phenotype in human embryonic stem cell-derived dopaminergic neurons: Implications for Parkinson’s disease

In this study we investigate how β-catenin-dependent WNT signalling impacts midbrain dopaminergic neuron (mDA) specification. mDA cultures at day 65 of differentiation responded to 25 days of the tankyrase inhibitor XAV969 (XAV, 100nM) with reduced expression of markers of an A9 mDA phenotype (KCNJ6, ALDH1A1 and TH) but increased expression of the transcriptional repressors NR0B1 and NR0B2. Overexpression of NR0B1 and or NR0B2 promoted a loss of A9 dopaminergic neuron phenotype markers (KCNJ6, ALDH1A1 and TH). Overexpression of NR0B1, but not NR0B2 promoted a reduction in expression of the β-catenin-dependent WNT signalling pathway activator RSPO2. Analysis of Parkinson’s disease (PD) transcriptomic databases shows a profound PD-associated elevation of NR0B1 as well as reduced transcript for RSPO2. We conclude that reduced β-catenin-dependent WNT signalling impacts dopaminergic neuron identity, in vitro, through increased expression of the transcriptional repressor, NR0B1. We also speculate that dopaminergic neuron regulatory mechanisms may be perturbed in PD and that this may have an impact upon both existing nigral neurons and also neural progenitors transplanted as PD therapy.

Neutrophil DREAM come true: The not-so-impossible quest for mechanisms of neutrophil function and heterogeneity

Neutrophil functions and responses are heterogeneous, and the nature and categorization of this heterogeneity is achieving considerable interest. Work by Li et al. in this issue of JEM (2021. J. Exp. Med.https://doi.org/10.1084/jem.20211083) identifies how a transcriptional repressor, DREAM, regulates adhesion of neutrophils to endothelial cells and their transmigration into tissue. This study offers a mechanism for heterogeneity in this critical response of neutrophils to inflammatory stimuli.

Growth-dependent signals drive an increase in early G1 cyclin concentration to link cell cycle entry with cell growth

Entry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 is thought to initiate cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that control cell growth and size. Together, the data are consistent with models in which Cln3 is a crucial link between cell growth and the cell cycle.

Knockdown of long noncoding RNA AC245100.4 inhibits the tumorigenesis of prostate cancer cells via the STAT3/NR4A3 axis

Aim: To explore the role and mechanism of long noncoding RNA AC245100.4 and NR4A3 in prostate cancer (PCa). Methods: RNA-sequencing analysis was used to detect the downstream genes of AC245100.4. A series of gain- and loss-of-function approaches were used to investigate the roles of AC245100.4 and NR4A3. RNA immunoprecipitation was performed to examine the interaction between AC245100.4 and STAT3. Results: AC245100.4 was significantly upregulated in PCa cells and tissues. Knockdown of AC21500.4 significantly inhibited the tumorigenesis of PCa cells. Mechanistically, AC245100.4 deregulated the transcription of NR4A3 via increasing p-STAT3, which acted as a transcriptional repressor of NR4A3. Conclusion: Knockdown of lncRNA AC245100.4 inhibits the tumorigenesis of PCa cells via the STAT3/ NR4A3 axis.

A histidine kinase and a response regulator provide phage resistance to Marinomonas mediterranea via CRISPR-Cas regulation

CRISPR-Cas systems are used by many prokaryotes to defend against invading genetic elements. In many cases, more than one CRISPR-Cas system co-exist in the same cell. Marinomonas mediterranea MMB-1 possesses two CRISPR-Cas systems, of type I–F and III-B respectively, which collaborate in phage resistance raising questions on how their expression is regulated. This study shows that the expression of both systems is controlled by the histidine kinase PpoS and a response regulator, PpoR, identified and cloned in this study. These proteins show similarity to the global regulators BarA/UvrY. In addition, homologues to the sRNAs CsrB and CsrC and the gene coding for the post-transcriptional repressor CsrA have been also identified indicating the conservation of the elements of the BarA/UvrY regulatory cascade in M. mediterranea. RNA-Seq analyses have revealed that all these genetics elements are regulated by PpoS/R supporting their participation in the regulatory cascade. The regulation by PpoS and PpoR of the CRISPR-Cas systems plays a role in phage defense since mutants in these proteins show an increase in phage sensitivity.