Cis-regulatory atlas of primary human CD4+?T cells

Naïve CD4+ T Cells are capable of differentiating into numerous T helper effector lineages depending on the provided local cytokines during activation. Cis-regulatory elements (CRE) are critical for cell differentiation, homeostasis, and function; however, CRE functional annotation (e.g. silencers, enhancers, and insulators) from existing genomic libraries remains an active need. Genome wide screens, including Transcribing Active Regulatory Region Sequencing (STARR-Seq) provides quantifies enhancer activity. However, these screens are mainly conducted in immortalized cell lines. Therefore, we have modified STARR-Seq using a non-integrating lentiviral transduction system (Lenti-STARR-seq) to investigate CRE in human CD4+ T cells. We identify and validate functional enhancers and negative regulatory elements (NRE). These elements differences stark differences in chromatin modification, TF binding, and nucleosome positioning. Additionally, STARR-Seq enhancers, but not NRE, exhibit transcription of enhancer RNA. Collectively these data suggest that Lenti-STARR-Seq may be a useful tool in the screening of primary human cell types for CRE function, and provides an atlas of functional CRE in human CD4+ T Cells. Overall design: Freshly isolated hCD4 T Cells are suspended to 6.6E6 cells per mL in CTS Optimizer, then combined 0.33mL concentrated lentivirus per mL cells. Cells are spinfected in low-bind 6-well plates (Corning #3471) ant 7ug/mL polybrene (MilliporeSigma #TR1003G) at 950g, 32°C, for 2 hours. Immediately following spinfection, cells are gently resuspended and cultured for 2 hours at 37°C, 5% CO2. Cells collected and centrifuged and suspended cultured for 24 hours. Cells are treated with 100U DNaseI (NEB M0303S) for 30 minutes at 37°C. Total RNA is purified from transduced cells (Qiagen #75144) and concentrated 7.5x using RNA Cleanup (NEB #T2040L). RNA was Reverse Transcribed according to manufacturer guidelines for STARR-Seq transcript specific primers (Supplemental File 1) with SuperScript IV (Invitrogen #18090010), using twice the recommended RNA input. cDNA was purified using a Reaction Cleanup Kit (Qiagen #28206). cDNA was amplified using custom library primers (Supplemental File 1) as previously described.1 Diluted Plasmid control was amplified using an input concentration which yielded the same amplification cycle number as the STARR library for hCD4+ Donor I. Library was purified using PCR cleanup columns (Qiagen #28206), and quantified for PE-150bp sequencing (Novogene).