CRISPR Reveals a Distal Super-Enhancer Required for Sox2 Expression in Mouse Embryonic Stem Cells

December CRISPR Reveals a Distal Super-Enhancer Required for Sox2 Expression in Mouse Embryonic Stem Cells Yan Li 0 4 5 Chloe M. Rivera 0 1 4 5 Haruhiko Ishii 0 4 5 Fulai Jin 0 4 5 Siddarth Selvaraj 0 4 5 Ah 4 5 Young Lee 0 4 5 Jesse R. Dixon 0 2 4 5 Bing Ren * 0 3 4 5 0 Ludwig Institute for Cancer Research, San Diego, California, United States of America, 1 The Biomedical Sciences Graduate Program, University of California San Diego, School of Medicine , San Diego, California , United States of America, 2 Medical Scientist Training Program, University of California San Diego, School of Medicine , San Diego, California , United States of America, 3 Department of Cellular and Molecular Medicine, Institute of Genome Medicine, Moores Cancer Center, University of California San Diego, School of Medicine , San Diego, California , United States of America 4 Funding: This work is supported by funds from the Ludwig Institute for Cancer Research, and US National Institutes of Health (U54 HG006997) to BR. CMR was supported in part by the UCSD Genetics Training Program through an institutional training grant from the National Institute of General Medical Sciences, T32 GM008666. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 5 Editor: Qiang Wu, National University of Singapore , Singapore The pluripotency of embryonic stem cells (ESCs) is maintained by a small group of master transcription factors including Oct4, Sox2 and Nanog. These core factors form a regulatory circuit controlling the transcription of a number of pluripotency factors including themselves. Although previous studies have identified transcriptional regulators of this core network, the cis-regulatory DNA sequences required for the transcription of these key pluripotency factors remain to be defined. We analyzed epigenomic data within the 1.5 Mb gene-desert regions around the Sox2 gene and identified a 13kb-long super-enhancer (SE) located 100kb downstream of Sox2 in mouse ESCs. This SE is occupied by Oct4, Sox2, Nanog, and the mediator complex, and physically interacts with the Sox2 locus via DNA looping. Using a simple and highly efficient double-CRISPR genome editing strategy we deleted the entire 13-kb SE and characterized transcriptional defects in the resulting monoallelic and biallelic deletion clones with RNA-seq. We showed that the SE is responsible for over 90% of Sox2 expression, and Sox2 is the only target gene along the chromosome. Our results support the functional significance of a SE in maintaining the pluripotency transcription program in mouse ESCs. - Sox2 is one of the three core transcription factors (Oct4, Sox2, Nanog) responsible for maintaining ESC pluripotency. These core pluripotency factors form autoregulatory loops and transcriptionally induce a cohort of other key pluripotency genes [1-3]. Besides ESCs, Sox2 is also expressed in various types of adult stem cells [4, 5]. Recently, it has been reported that Sox2-expressing cancer stem cells may drive tumor initiation and relapse in medulloblastoma and skin tumor [6, 7]. Therefore, elucidating the transcriptional regulation mechanisms of Sox2 gene is important for the understanding of both pluripotency and tumorigenesis. Enhancers play a critical role in regulating metazoan gene transcription [8-11]. Recent genome-wide analyses have revealed that enhancers are very abundant in the genome. Additionally, highly specific enhancer landscapes are responsible for cellular identity as they regulate distinct transcriptional programs in different cell types [12-16]. Clusters of enhancer constituents that together generate a domain of MED1-, and transcription factor-binding, and H3K27ac enrichment have been identified as super-enhancers (SE) or stretch enhancers [17-19]. Compelling genomic evidence has predicted that SEs play a particularly important role in the control of cell identity and diseases [17-19], but direct functional evidence is lacking. Until recently, validating the in vivo function of a putative enhancer has proven technically difficult. Reporter assays confirm that the sequence can function as an enhancer exogenously, but it does not demonstrate whether a distal target gene can be activated by the enhancer in vivo. Knocking out the enhancer sequence is the gold standard approach, but conventional methods using homologous recombination are inefficient and labor-intensive. The recently developed CRISPR/Cas9 system [20, 21] has proven to be a highly efficient genome editing technique and offers a promising method to validate enhancer functions in vivo. A recent study used CRISPR to validate the importance of a SE near GATA2 in chronic myeloid leukemia by demonstrating it is responsible for 80% of GATA2 expression [22]. With this recent advance, we have attempted to examine the role of SEs in pluripotency and normal development. In the mouse genome, Sox2 is located within a,1.5Mb gene desert region. It has been postulated that regulatory sequences may lie in such gene deserts and modulate gene expression over long distances [23]. In this study, we examined the gene desert near Sox2 and identified a putative distal super enhancer that is marked by active histone marks only in ESCs. This 13kb-long enhancer is occupied by multiple pluripotency factors and forms a long-distance DNA loop with the Sox2 promoter from 100kb away. Using a double-CRISPR excision strategy, we studied the in vivo function of this SE and demonstrated that it is responsible for over 90% of Sox2 gene expression in mouse ESCs. Our results therefore provide direct evidences on the key roles of a SE in governing ESC pluripotency. Methods and Materials Cell culture and transfection experiments The F1 Mus musculus castaneus 6 S129/SvJae mouse ESC line (F123 line) was a gift from the laboratory of Dr. Edith Heard and was previously described [24]. The cells were cultured as described previously [25]. Importantly, cells were passaged twice on 0.1% gelatin-coated feeder-free plates before harvesting. F123 cells were plated at a density of 0.5 million/mL on 0.1% gelatin-coated feeder-free plates 24 hours before transfection. Cells were triple transfected using the Mouse ES Cell Nucleofector Kit (Lonza) and Amaxa Nucleofector with 7.5ug of each CRISPR plasmid and 5ug of pBABE-Puro. Post-transfection, cells were immediately plated on puromycin-resistant MEF feeders (GlobalStem) for recovery. 48 hours after transfection 2 mg/mL of puromycin (Sigma) was supplemented to the cell culture media for 3 days to select for transfected cells. Alkaline phosphatase staining was performed using mESC culture in the presence of MEF feeder cells with the Alkaline Phosphatase Staining kit (Stemgent). For growth curve analysis, control and mutant ES cells were plated at 16105 cells in triplicates on 12-well plates and counted for six consecutive days. The cells were passaged on day 4 by making 1:3 dilutions. (...truncated)