RESUMEN
Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories.
Asunto(s)
Cromatina , Linfocitos T Reguladores , Humanos , Cromatina/genética , Leucocitos Mononucleares , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , TranscriptomaRESUMEN
BACKGROUND: Environmental enrichment (EE) has been shown to modulate behavior and hippocampal gene expression; however, the currently available literature does not explain the differential effects that may relate to the duration of EE. AIM: To investigate the differential effects of short- and long-term EE on locomotion, anxiety-, depressive- and cognition-like behaviors, and hippocampal gene expression under physiological conditions. METHODS: We assigned either short-term or long-term intervention with respective controls to healthy C57BL/6 mice (n = 12-16/group). The short-term EE group received EE for four weeks starting at eight months of age, while the long-term EE group received EE for six months starting at three months of age. Differential effects of the duration of EE on various behaviors and hippocampal gene expression at nine months of age were measured using an established behavioral battery and high-throughput RT-qPCR, respectively. RESULTS: Both short-term and long-term EE significantly enhanced locomotion in the home cage and reduced depressive-like behavior in the forced-swim test. Long-term EE, however, reduced locomotion in the open-field test. Additionally, short-term EE reduced the mean body weight and showed anxiolytic effects in the elevated-zero maze (EZM), while these effects were lost after long-term EE. There were no effects of either short-term or long-term EE on the expression of 43 hippocampal genes of interest tested at adjusted p < 0.05. CONCLUSION: Both short and long-term EE are equally beneficial for baseline locomotor activity and depressive-like behavior. However, long-term EE affects locomotion adversely in a threatening environment and is anxiogenic.