Genome-wide transcription factor binding and chromatin methylation marks in the G1ME megakaryocytic progenitor model cell line. Mus musculus

There are many examples of transcription factor families whose members control gene expression profiles of diverse cell types. However, the mechanism by which closely related factors occupy distinct regulatory elements and impart lineage specificity is largely undefined. Here we demonstrate on a genome wide scale that the hematopoietic GATA factors GATA-1 and GATA-2 bind overlapping sets of genes, often at distinct sites, as a means to differentially regulate target gene expression and to regulate the balance between proliferation and differentiation. We also reveal that the GATA switch, which entails a chromatin occupancy exchange between GATA2 and GATA1 in the course of differentiation, operates on more than a third of GATA1 bound genes. The switch is equally likely to lead to transcriptional activation or repression and, in general, GATA1 and GATA2 act oppositely on switch target genes. In addition, we reveal that genomic regions co-occupied by GATA2 and the ETS factor ETS1 are strongly enriched for regions marked by H3K4me3 and occupied by Pol II. Finally, by comparing GATA1 occupancy in erythroid cells and megakaryocytes, we find that the presence of ETS factor motifs is a major discriminator of megakaryocyte versus red cell specification. Overall design: We used Illumina ChIP-Seq to examine binding of GATA1, GATA2, and ETS1 transcription factors as well as the genomic locations of two histone methylation marks, H3K4me3 and H3K27me3. Except H3K4me3 (1 sample), all data were generated from at least 2 biological replicates of immunoprecipitations from megakaryocyte progenitor cells, G1ME. Input DNA was prepared and sequenced along with each immunoprecipitation and used as a control dataset for binding site identification.

已有诸多研究表明，转录因子（transcription factor）家族成员可调控多种细胞类型的基因表达谱。但结构紧密相关的转录因子如何结合不同调控元件、并赋予细胞谱系特异性的分子机制，目前仍未得到充分阐明。本研究在全基因组范围内证实，造血系统GATA家族因子GATA-1与GATA-2可结合存在重叠的基因集，且二者通常结合于不同位点，以此实现靶基因表达的差异化调控，并维持细胞增殖与分化的平衡。本研究同时发现，在分化过程中伴随GATA2与GATA1染色质结合位点置换的GATA开关（GATA switch），可调控超过三分之一的GATA-1结合靶基因。该开关既可介导靶基因的转录激活，也可引发转录抑制，且总体而言GATA-1与GATA-2对开关靶基因的调控作用完全相反。此外，本研究还发现，同时被GATA2与ETS因子（ETS factor）结合的基因组区域，显著富集于带有H3K4me3（组蛋白H3第4位赖氨酸三甲基化）标记且结合RNA聚合酶II（Pol II）的区域。最后，通过比较红系细胞与巨核细胞中GATA-1的结合位点，本研究发现ETS因子基序的存在是区分巨核细胞与红细胞谱系特化的关键标志。实验整体设计：本研究采用Illumina平台的染色质免疫共沉淀测序（ChIP-Seq）技术，检测GATA-1、GATA2与ETS1转录因子的结合位点，同时定位两种组蛋白甲基化修饰标记H3K4me3与H3K27me3的基因组区域。除H3K4me3仅设置1个样本外，其余所有数据均来自巨核细胞祖细胞G1ME的免疫沉淀实验至少2次生物学重复。我们将每份免疫沉淀样本配套制备输入DNA并进行测序，以此作为结合位点识别的对照数据集。