ABSTRACT
Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells. We have performed high-content screening with small interfering RNAs targeting 300 chromatin-associated factors and extracted colony-level quantitative features. This revealed five morphological phenotypes in early reprogramming, including one displaying large round colonies exhibiting an early block of reprogramming. Using RNA sequencing, we identified transcriptional changes associated with these phenotypes. Furthermore, double knockdown epistasis experiments revealed that BRCA1, BARD1, and WDR5 functionally interact and are required for the DNA damage response. In addition, the mesenchymal-to-epithelial transition is affected in Brca1, Bard1, and Wdr5 knockdowns. Our data provide a resource of chromatin-associated factors in early reprogramming and underline colony morphology as an important high-dimensional readout for reprogramming quality.
Subject(s)
BRCA1 Protein/genetics , Cellular Reprogramming/genetics , DNA Damage , Epithelial-Mesenchymal Transition/genetics , Intracellular Signaling Peptides and Proteins/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Animals , BRCA1 Protein/metabolism , Chromatin/genetics , Chromatin/metabolism , DNA Repair , Gene Expression Profiling , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Phenotype , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
INTRODUCTION: We conducted a phase Ib proof of mechanism trial to determine whether bexarotene (Targretin) increases central nervous system (CNS) apolipoprotein E (apoE) levels and alters Aß metabolism in normal healthy individuals with the APOE ε3/ε3 genotype. METHODS: We used stable isotope labeling kinetics (SILK-ApoE and SILK-Aß) to measure the effect of bexarotene on the turnover rate of apoE and Aß peptides and stable isotope spike absolute quantitation (SISAQ) to quantitate their concentrations in the cerebrospinal fluid (CSF). Normal subjects were treated for 3 days with bexarotene (n = 3 women, 3 men, average 32 years old) or placebo (n = 6 women, average 30.2 years old) before administration of C13-leucine and collection of plasma and CSF over the next 48 hours. Bexarotene concentrations in plasma and CSF were also measured. RESULTS: Oral administration of bexarotene resulted in plasma levels of 1 to 2 µM; however, only low nM levels were found in CSF. Bexarotene increased CSF apoE by 25% but had no effect on metabolism of Aß peptides. DISCUSSION: Bexarotene has poor CNS penetration in normal human subjects. Drug treatment resulted in a modest increase in CSF apoE levels. The absence of an effect on Aß metabolism is likely reflective of the low CNS levels of bexarotene achieved. This study documents the utility of SILK-ApoE technology in measuring apoE kinetics in humans. TRIAL REGISTRATION: This trial is registered at clinicaltrials.gov (NCT02061878).
