While constitutive CTCF-binding sites are needed to maintain relatively invariant chromatin structures, such as topologically associating domains, the precise roles of CTCF to control cell type-specific transcriptional regulation remain poorly explored. We examined CTCF occupancy in different types of primary blood cells derived from the same donor to elucidate a new role for CTCF in gene regulation during blood cell development. We identified dynamic, cell type-specific binding sites for CTCF that colocalize with lineage-specific transcription factors. These dynamic sites are enriched for single nucleotide polymorphisms that are associated with blood cell traits in different linages, and they coincide with the key regulatory elements governing hematopoiesis. CRISPR/Cas9-based perturbation experiments demonstrated that these dynamic CTCF-binding sites play a critical role in red blood cell development. Furthermore, precise deletion of CTCF-binding motifs in dynamic sites abolished interactions of erythroid genes, such as RBM38, with their associated enhancers and led to abnormal erythropoiesis. These results suggest a novel, cell type-specific function for CTCF in which it may serve to facilitate interaction of distal regulatory emblements with target promoters. Our study of the dynamic, cell type-specific binding and function of CTCF provides new insights into transcriptional regulation during hematopoiesis.

Background Genomic safe harbors are regions of the genome that can maintain transgene expression without disrupting the function of host cells. Genomic safe harbors play an increasingly important role in improving the efficiency and safety of genome engineering. However, limited safe harbors have been identified. Results Here, we develop a framework to facilitate searches for genomic safe harbors by integrating information from polymorphic mobile element insertions that naturally occur in human populations, epigenomic signatures, and 3D chromatin organization. By applying our framework to polymorphic mobile element insertions identified in the 1000 Genomes project and the Genotype-Tissue Expression (GTEx) project, we identify 19 candidate safe harbors in blood cells and 5 in brain cells. For three candidate sites in blood, we demonstrate the stable expression of transgene without disrupting nearby genes in host erythroid cells. We also develop a computer program, Genomics and Epigenetic Guided Safe Harbor mapper (GEG-SH mapper), for knowledge-based tissue-specific genomic safe harbor selection. Conclusions Our study provides a new knowledge-based framework to identify tissue-specific genomic safe harbors. In combination with the fast-growing genome engineering technologies, our approach has the potential to improve the overall safety and efficiency of gene and cell-based therapy in the near future.

Neuroblastomas (NB) are embryonal childhood tumors that derive from the multipotent neural crest cells (NCCs) of the peripheral nervous system. NB accounts for more than 15% of all childhood cancer-related deaths. Despite the most intensive multimodal therapy, more than 50% of patients with high-risk NB relapse with often fatal, resistant disease. Finding novel treatments, especially for relapse disease, is desperately needed for high-risk NB. In cancers, distinct transcription factors TFs networks forming core regulatory circuitries (CRCs) control gene expression programs that drive cell identity. Recent studies reported the presence of two types of identity states in NB tumors: one establishing a more proliferative adrenergic (ADRN) cell state and a second establishing a more invasive, therapy-resistant mesenchymal (MES) cell state. While most studies are investigating the CRC members for targeting MYCN amplified NB (ADRN) or the MES/NCCs cell states, most primary tumors are heterogeneous, comprised of cells with both identities. Thus, our work is focused on identifying universal factors shared across different lineage states to determine whether targeting both identities could be a valuable strategy for NB treatment. NB was identified from a screen of 673 cancer cell lines as highly sensitive to the BET-inhibitor JQ1. In our work, we identified the class I basic helix-loop-helix transcription factor (bHLH) TCF4 as a critical target of JQ1 mediated cell death in NB. TCF4 functions as a transcriptional hub that heterodimerizes with class II (bHLH) TFs including the proneural gene ASCL1 (ADRN), and TWIST1 a master regulator of the MES state. Heterodimers formed between TCF4 and ASCL1, and TWIST1, have been demonstrated to provide lineage-specific differentiation from embryonic stem cells. Here, we hypothesize that TCF4 is a cell dependency gene in NB that is crucial for determining NB identity. Using RNA seq analysis of TCF4 dox-inducible shRNA stable cell lines, we found that the gene expression changes in TCF4-depleted cells indicate a role for TCF4 in differentiation, cell signaling, and neurodevelopment. Interestingly, gene set enrichment analysis (GSEA) indicates that both identity states are suppressed after silencing of TCF4. Our data also shows that inducible decrease of TCF4 protein resulted in a significant inhibition of NB cell growth, and impaired tumor growth in vivo. Furthermore, we identified TCF4 targets in NB by combined analysis of TCF4 ChIP-seq data and gene expression changes following TCF4 knockdown. ENRICHR analysis suggests that differentially expressed genes in TCF4-knockdown cells that also have a TCF4 ChIP-seq peak are targets of the TFs TWIST1 (MES) and HAND1 (ADRN) that interact directly with TCF4. In conclusion, our preliminary data supports the hypothesis that TCF4 is a master transcriptional regulator of NB oncogenic program that is crucial for maintaining NB identity states. Citation Format: Nour Aljouda, Dewan Shrestha, Megan Walker, Satyanarayana Alleboina, Evan Glazer, Yong Cheng, Kevin Freeman. Defining TCF4’s role as a novel key regulator in mediating neuroblastoma cell identity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5719.