A new twist on cell growth control

et al. 2020

BMC Cancer

Background Inhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins. Methods We have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen. Results Our primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding. Conclusions The assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.

Section: Introductionmentioning

confidence: 92%

An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1

et al. 2020

BMC Cancer

“…It is also required for normal RNA Polymerase 2 activity. Upon growth stimulation of normal human fibroblasts, the transcript elongation by RNA Polymerase 2 seems to depend on the levels of CGGBP1 [23,34,[37][38][39]. Circumstantial evidence suggests that this H3K9me3 asymmetry in the flanks of CGGBP1dependent CTCF-binding sites may lead to asymmetrical RNA abundance in 10 kb long flankins [23].…”

Section: Introductionmentioning

confidence: 99%

CGGBP1-dependent CTCF-binding sites restrict ectopic transcription

et al. 2021

Cell Cycle

Binding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from the SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones that show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in the synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand-specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription barrier seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. RNA-sequencing reveals that CGGBP1regulated CTCF-binding sites act as barriers to transcription at multiple locations genome-wide. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.

“…It is also required for normal RNA Polymerase 2 activity. Upon growth stimulation of normal human fibroblasts, the transcript elongation by RNA Polymerase 2 seems to depend on the levels of CGGBP1 (Agarwal et al, 2016; Cardiello et al, 2014; Ichiyanagi, 2014; Patel et al, 2019; Singh and Westermark, 2015). Circumstantial evidence suggests that this H3K9me3 asymmetry in the flanks of CGGBP1-dependent CTCF-binding sites may lead to asymmetrical RNA abundance in 10 kb long flanking regions (Patel et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

CGGBP1-dependent CTCF-binding sites restrict ectopic transcription

et al. 2020

Preprint

Binding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones which show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription termination sequence required for the upstream transcription seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.