ABSTRACT
Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.
Subject(s)
Chromatin/genetics , Human Embryonic Stem Cells/enzymology , Poly(ADP-ribose) Polymerases/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Adaptor Proteins, Signal Transducing , CCCTC-Binding Factor , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatin Immunoprecipitation , Circadian Rhythm , Embryoid Bodies/enzymology , Epistasis, Genetic , Gene Expression Regulation , Gene Regulatory Networks , HCT116 Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nuclear Lamina/metabolism , Poly (ADP-Ribose) Polymerase-1 , Protein Binding , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolismABSTRACT
Stem cells display a fundamentally different mechanism of proliferation control when compared to somatic cells. Uncovering these mechanisms would maximize the impact in drug discovery with a higher translational applicability. The unbiased approach used in phenotype-based drug discovery (PDD) programs can offer a unique opportunity to identify such novel biological phenomenon. Here, we describe an integrated phenotypic screening approach, employing a combination of in vitro and in vivo PDD models to identify a small molecule increasing stem cell proliferation. We demonstrate that a combination of both in vitro and in vivo screening models improves hit identification and reproducibility of effects across various PDD models. Using cell viability and colony size phenotype measurement we characterize the structure activity relationship of the lead molecule, and identify that the small molecule inhibits phosphorylation of ERK2 and promotes stem cell proliferation. This study demonstrates a PDD approach that employs combinatorial models to identify compounds promoting stem cell proliferation.