RESUMO
Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel "sister" assays are examined either for gene expression by RNA sequencing (RNA-seq) or for fate in culture. We identified, and then validated using CRISPR, genes that controlled fate bias for different dendritic cell (DC) subtypes. This included Bcor as a suppressor of plasmacytoid DC (pDC) and conventional DC type 2 (cDC2) numbers during Flt3 ligand-mediated emergency DC development. We then developed SIS-skew to examine development of wild-type and Bcor-deficient siblings of the same clone in parallel. We found Bcor restricted clonal expansion, especially for cDC2s, and suppressed clonal fate potential, especially for pDCs. Therefore, SIS-seq and SIS-skew can reveal the molecular and cellular mechanisms governing clonal fate.
Assuntos
Células Dendríticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Feminino , Expressão Gênica/genética , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Células-Tronco/metabolismoRESUMO
Estrogen receptors (ERs) regulate gene transcription by interacting with regulatory elements. Most information regarding how ER activates genes has come from studies using a small set of target genes or simple consensus sequences such as estrogen response element, activator protein 1, and Sp1 elements. However, these elements cannot explain the differences in gene regulation patterns and clinical effects observed with estradiol (E(2)) and selective estrogen receptor modulators. To obtain a greater understanding of how E(2) and selective estrogen receptor modulators differentially regulate genes, it is necessary to investigate their action on a more comprehensive set of native regulatory elements derived from ER target genes. Here we used chromatin immunoprecipitation-cloning and sequencing to isolate 173 regulatory elements associated with ERalpha. Most elements were found in the introns (38%) and regions greater than 10 kb upstream of the transcription initiation site (38%); 24% of the elements were found in the proximal promoter region (<10 kb). Only 11% of the elements contained a classical estrogen response element; 23% of the elements did not have any known response elements, including one derived from the naked cuticle homolog gene, which was associated with the recruitment of p160 coactivators. Transfection studies found that 80% of the 173 elements were regulated by E(2), raloxifene, or tamoxifen with ERalpha or ERbeta. Tamoxifen was more effective than raloxifene at activating the elements with ERalpha, whereas raloxifene was superior with ERbeta. Our findings demonstrate that E(2), tamoxifen, and raloxifene differentially regulate native ER-regulatory elements isolated by chromatin immunoprecipitation with ERalpha and ERbeta.