Division of Allergy & Immunology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC 7028, Cincinnati, OH, 45229-3026, USA.
Medical Scientist Training Program (MSTP), University of Cincinnati College of Medicine, Cincinnati, OH, 45267, USA.
BMC Genomics. 2023 May 11;24(1):253. doi: 10.1186/s12864-023-09288-3.
Cis-regulatory elements (CRE) are critical for coordinating gene expression programs that dictate cell-specific differentiation and homeostasis. Recently developed self-transcribing active regulatory region sequencing (STARR-Seq) has allowed for genome-wide annotation of functional CREs. Despite this, STARR-Seq assays are only employed in cell lines, in part, due to difficulties in delivering reporter constructs. Herein, we implemented and validated a STARR-Seq-based screen in human CD4+ T cells using a non-integrating lentiviral transduction system. Lenti-STARR-Seq is the first example of a genome-wide assay of CRE function in human primary cells, identifying thousands of functional enhancers and negative regulatory elements (NREs) in human CD4+ T cells. We find an unexpected difference in nucleosome organization between enhancers and NRE: enhancers are located between nucleosomes, whereas NRE are occupied by nucleosomes in their endogenous locations. We also describe chromatin modification, eRNA production, and transcription factor binding at both enhancers and NREs. Our findings support the idea of silencer repurposing as enhancers in alternate cell types. Collectively, these data suggest that Lenti-STARR-Seq is a successful approach for CRE screening in primary human cell types, and provides an atlas of functional CREs in human CD4+ T cells.
顺式调控元件（CRE）对于协调基因表达程序至关重要，这些程序决定了细胞特异性分化和内稳态。最近开发的自转录活性调控区测序（STARR-Seq）允许对功能 CRE 进行全基因组注释。尽管如此，由于报告基因构建体的传递困难，STARR-Seq 检测仅在细胞系中使用。在此，我们使用非整合慢病毒转导系统在人 CD4+T 细胞中实施和验证了基于 STARR-Seq 的筛选。Lenti-STARR-Seq 是在人原代细胞中进行 CRE 功能全基因组分析的首例实例，鉴定了数千个人 CD4+T 细胞中的功能性增强子和负调控元件（NRE）。我们发现增强子和 NRE 之间核小体组织存在出乎意料的差异：增强子位于核小体之间，而 NRE 则在其内源位置被核小体占据。我们还描述了增强子和 NRE 处的染色质修饰、eRNA 产生和转录因子结合。我们的发现支持了沉默子重新用作替代细胞类型中的增强子的想法。总之，这些数据表明 Lenti-STARR-Seq 是在原代人类细胞类型中进行 CRE 筛选的成功方法，并提供了人类 CD4+T 细胞中功能性 CRE 的图谱。
