Protocol to target a promoter region in human embryonic kidney cells using the CRISPR-dCas9 system for single-locus proteomics.

The unbiased identification of less-abundant transcription factors, which direct the expression of a target gene, is technically challenging. Here, we present a protocol to analyze the locus-specific chromatin-regulating proteome using in situ capture of chromatin interactions by an inactive Cas9 (dCas9). We describe steps for designing guide RNAs and transfection, followed by precipitation of chromatin and associated proteins. In the last step, we describe the elution of DNA and proteins for PCR and mass spectrometric analysis, respectively. For complete details on the use and execution of this protocol, please refer to Alkhayer et al.1.

KLF4-mediated upregulation of the NKG2D ligand MICA in acute myeloid leukemia: a novel therapeutic target identified by enChIP.

The immunoreceptor NKG2D, which is expressed on NK cells and T cell subsets is critically involved in tumor immune surveillance. This applies in particular to acute myeloid leukemia (AML), which evades immune detection by downregulation of NKG2D ligands (NKG2D-L), including MICA. The absence of NKG2D-L on AML cells is moreover associated with leukemia stem cell characteristics. The NKG2D/NKG2D-L system thus qualifies as an interesting and promising therapeutic target.Here we aimed to identify transcription factors susceptible to pharmacological stimulation resulting in the expression of the NKG2D-L MICA in AML cells to restore anti-tumor activity. Using a CRISPR-based engineered ChIP (enChIP) assay for the MICA promoter region and readout by mass spectrometry-based proteomics, we identified the transcription factor krüppel-like factor 4 (KLF4) as associated with the promoter. We demonstrated that the MICA promoter comprises functional binding sites for KLF4 and genetic as well as pharmacological gain- and loss-of-function experiments revealed inducible MICA expression to be mediated by KLF4.Furthermore, induction in AML cells was achieved with the small compound APTO253, a KLF4 activator, which also inhibits MYC expression and causes DNA damage. This induction in turn yielded increased expression and cell surface presentation of MICA, thus rendering AML cells more susceptible to NK cell-mediated killing. These data unravel a novel link between APTO253 and the innate anti-tumor immune response providing a rationale for targeting AML cells via APTO253-dependent KFL4/MICA induction to allow elimination by endogenous or transplanted NK and T cells in vivo. Video Abstract.

Turning Waste into Value: Nanosized Natural Plant Materials of Solanum incanum L. and Pterocarpus erinaceus Poir with Promising Antimicrobial Activities.

Numerous plants are known to exhibit considerable biological activities in the fields of medicine and agriculture, yet access to their active ingredients is often complicated, cumbersome and expensive. As a consequence, many plants harbouring potential drugs or green phyto-protectants go largely unnoticed, especially in poorer countries which, at the same time, are in desperate need of antimicrobial agents. As in the case of plants such as the Jericho tomato, Solanum incanum, and the common African tree Pterocarpus erinaceus, nanosizing of original plant materials may provide an interesting alternative to extensive extraction and isolation procedures. Indeed, it is straightforward to obtain considerable amounts of such common, often weed-like plants, and to mill the dried material to more or less uniform particles of microscopic and nanoscopic size. These particles exhibit activity against Steinernema feltiae or Escherichia coli, which is comparable to the ones seen for processed extracts of the same, respective plants. As S. feltiae is used as a model nematode indicative of possible phyto-protective uses in the agricultural arena, these findings also showcase the potential of nanosizing of crude "waste" plant materials for specific practical applications, especially-but not exclusively-in developing countries lacking a more sophisticated industrial infrastructure.