Histones serve as protein spools for winding the DNA in the nucleosome. High variability of their post-translational modifications result in a unique code system often responsible for the pathomechanisms of epigenetics-based diseases. Decoding is performed by reader proteins via complex formation with the N-terminal peptide tails of histones. Determination of structures of histone-reader complexes would be a key to unravel the histone code and the design of new drugs. However, the large number of possible histone complex variations imposes a true challenge for experimental structure determination techniques. Calculation of such complexes is difficult due to considerable size and flexibility of peptides and the shallow binding surfaces of the readers. Moreover, location of the binding sites is often unknown, which requires a blind docking search over the entire surface of the target protein. To accelerate the work in this field, a new approach is presented for prediction of the structure of histone H3 peptide tails docked to their targets. Using a fragmenting protocol and a systematic blind docking method, a collection of well-positioned fragments of the H3 peptide is produced. After linking the fragments, reconstitution of anchoring regions of the target-bound H3 peptide conformations was possible. As a first attempt of combination of blind and fragment docking approaches, our new method is named fragment blind docking (FBD).
Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and an indicator of poor disease outcome. Understanding the effect of pharmacotherapy on ABCG2 expression is critical in guiding therapy recommendations and may help rational drug development. Engineered reporter cell lines are useful in monitoring gene regulation and protein activity in live cells but rely on precise targeting to preserve native regulatory functions. Here, we generated a number of precision‐engineered, fluorescent human cell lines that interrogate ABCG2 regulation in live lung cancer cells. Using homology‐based CRISPR engineering, we targeted a promoterless eGFP to the translational start site of the ABCG2 gene, generating ABCG2 knockout and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that anti‐cancer medications, HDAC inhibitors and hypoxia‐mimicking agents induce ABCG2 expression. In addition, we provide evidence that the glucocorticoid receptor is a specific, positive regulator of the ABCG2 gene. To our knowledge, this is the first fluorescent reporter assay designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may direct therapy recommendations and assist rational drug design.
Support or Funding Information
This research has been supported mainly by OTKA‐NKFIH grant NK 115375, but also by NKFIH K104903 and the NVKP_16‐1‐2016‐0005 program from the National Research, Development and Innovation Office. This work has also been supported by research grant OTKA‐NKFIH K128011, awarded to GV. A Borsy was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (HA: BO/00579/17/5), through the New National Excellence Program of the Ministry of Human Resources (HA: ÚNKP‐18‐4‐SE‐11).
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