Silencers, the yin to enhancers’ yang, play a pivotal role in fine-tuning gene expression throughout the genome. However, despite their recognized importance, comprehensive identification of these regulatory elements in the genome is still in its early stages. We developed a method called Ss-STARR-seq to directly determine the activity of silencers in the whole genome. In this study, we applied Ss-STARR-seq to human cell lines K562, LNCaP, and 293 T, and identified 134,171, 137,753, and 125,307 silencers on a genome-wide scale, respectively, these silencers function in various cells in a cell-specific manner. Silencers exhibited a substantial enrichment of transcriptional-inhibitory motifs, including REST, and demonstrated overlap with the binding sites of repressor transcription factors within the endogenous environment. Interestingly, H3K27me3 did not reflect silencer activity but facilitated the silencer’s inhibitory role on gene expression. Additionally, the silencer did not have any significant histone markers at the genome-wide level. Our findings unveil that aspect-silencers not only transition into enhancers throughout diverse cell lines but also achieve functional conversion with insulators. Regarding to biological effects, knockout experiments underscored the functional redundancy and specificity of silencers in regulating gene expression and cell proliferation. In summary, this study pioneers the elucidation of the genome-wide silencer landscape in human cells, delineates their global regulatory features, and identifies specific silencers influencing cancer cell proliferation.

Skeletal muscle satellite cells, the resident stem cells in pig skeletal muscle, undergo proliferation and differentiation to enable muscle tissue repair. The proliferative and differentiative abilities of these cells gradually decrease during in vitro cultivation as the cell passage number increases. Despite extensive research, the precise molecular mechanisms that regulate this process are not fully understood. To bridge this knowledge gap, we conducted transcriptomic analysis of skeletal muscle satellite cells during in vitro cultivation to quantify passage number-dependent changes in the expression of genes associated with proliferation. Additionally, we explored the relationships between gene transcriptional activity and chromatin accessibility using transposase-accessible chromatin sequencing. This revealed the closure of numerous open chromatin regions, which were primarily located in intergenic regions, as the cell passage number increased. Integrated analysis of the transcriptomic and epigenomic data demonstrated a weak correlation between gene transcriptional activity and chromatin openness in expressed genic regions; although some genes (e.g., GNB4 and FGD5) showed consistent relationships between gene expression and chromatin openness, a substantial number of differentially expressed genes had no clear association with chromatin openness in expressed genic regions. The p53-p21-RB signaling pathway may play a critical regulatory role in cell proliferation processes. The combined transcriptomic and epigenomic approach taken here provided key insights into changes in gene expression and chromatin openness during in vitro cultivation of skeletal muscle satellite cells. These findings enhance our understanding of the intricate mechanisms underlying the decline in cellular proliferation capacity in cultured cells.

Hi‐C can obtain three‐dimensional chromatin structure information and is widely used for genome assembly. We constructed the GutHi‐C technology. As shown in the graphical abstract, it is a highly efficient and quick‐to‐operate method and can be widely used for human, livestock, and poultry gut microorganisms. It provides a reference for the Hi‐C methodology of the microbial metagenome. DPBS, Dulbecco's phosphate‐buffered saline; Hi‐C, high‐through chromatin conformation capture; LB, Luria‐Bertani; NGS, next‐generation sequencing; PCR, polymerase chain reaction; QC, quality control.

Proper cell fate determination relies on precise spatial and temporal genome-wide cooperation between regulatory elements (REs) and their targeted genes. However, the lengths of REs defined using different methods vary, which indicates that there is sequence redundancy and that the context of the genome may be unintelligible. We developed a method called MAE-seq (Massive Active Enhancers by Sequencing) to experimentally identify functional REs at a 25-bp scale. In this study, MAE-seq was used to identify 626879, 541617 and 554826 25-bp enhancers in mouse embryonic stem cells (mESCs), C2C12 and HEK 293T, respectively. Using ∼1.6 trillion 25 bp DNA fragments and screening 12 billion cells, we identified 626879 as active enhancers in mESCs as an example. Comparative analysis revealed that most of the histone modification datasets were annotated by MAE-Seq loci. Furthermore, 33.85% (212195) of the identified enhancers were identified as de novo ones with no epigenetic modification. Intriguingly, distinct chromatin states dictate the requirement for dissimilar cofactors in governing novel and known enhancers. Validation results show that these 25-bp sequences could act as a functional unit, which shows identical or similar expression patterns as the previously defined larger elements, Enhanced resolution facilitated the identification of numerous cell-specific enhancers and their accurate annotation as super enhancers. Moreover, we characterized novel elements capable of augmenting gene activity. By integrating with high-resolution Hi-C data, over 55.64% of novel elements may have a distal association with different targeted genes. For example, we found that the Cdh1 gene interacts with one novel and two known REs in mESCs. The biological effects of these interactions were investigated using CRISPR-Cas9, revealing their role in coordinating Cdh1 gene expression and mESC proliferation. Our study presents an experimental approach to refine the REs at 25-bp resolution, advancing the precision of genome annotation and unveiling the underlying genome context. This novel approach not only advances our understanding of gene regulation but also opens avenues for comprehensive exploration of the genomic landscape.

Enhancer loci identified by ChIP-Seq or other experimental methods occupy hundreds of base pairs on the genome. It is the paradox comparing with the motif analysis, which usually contains only a few or tens nucleotides, achieved by bioinformatics analysis. To address this issue, we develop an experimental method, termed as massive active enhancer sequencing (MAE-Seq), to designate active enhancer sequences from arbitrary sources of 25bp random DNA libraries. These sequences are constructed in a mini-promoter vector with fluorescent reporter. After transfection, positive cells are sorted out and for sequencing. With the results, we successfully identify hundreds known accurate active enhancer sequences as expected. Besides that, large amounts of unmarked regulatory elements (UREs) without epigenetic features are also been spotted. In conclusion, MAE-Seq would be useful to refine enhancer sequences and precisely annotate the genome in eukaryotes.