Selective Functions of Individual Zinc Fingers Within the DNA-Binding Domain of Ikaros (RNA-seq: Thymocytes)

The C2H2 zinc finger is the most prevalent DNA-binding motif in the mammalian proteome, with DNA-binding domains usually containing more tandem fingers than are needed for stable sequence-specific DNA recognition. To examine the reason for the frequent presence of multiple zinc fingers, we generated mice lacking finger 1 or finger 4 of the 4-finger DNA-binding domain of Ikaros, a critical regulator of lymphopoiesis and leukemogenesis. Each mutant strain exhibited a specific subset of the phenotypes observed with Ikaros null mice. Of particular relevance, fingers 1 and 4 contributed to distinct stages of B- and T-cell development and finger 4 was selectively required for tumor suppression in thymocytes and in a new model of BCR-ABL+ acute lymphoblastic leukemia. These results, combined with transcriptome profiling (this GEO submission: RNA-Seg of whole thymus from wt and the two ZnF mutants), reveal that different subsets of fingers within multi-finger transcription factors can regulate distinct target genes and biological functions, and they demonstrate that selective mutagenesis can facilitate efforts to elucidate the functions and mechanisms of action of this prevalent class of factors. RNA-Seq from Whole Thymus comparing wt (3 replicates), Ikaros-ZnF1-/- mutant (2 replicates) and Ikaros-ZnF4-/- mutant (2 replicates) RPKM_Thymocytes.txt (linked below as a supplementary file) reports the relative mRNA expression levels (RPKM)values for all annotated Refseq genes that had at least one read in at least one of the samples, with duplicates for the same gene (different transcripts for same gene) filtered out. RPKM (Mortazavi et al., 2008) were calculated based on exonic reads obtained by using the software SeqMonk (Babraham Bioinformatics) and reference genome annotations from NCBI (mm9).C2H2锌指（C2H2 zinc finger）是哺乳动物蛋白质组中最为普遍的DNA结合基序，其DNA结合结构域所含的串联锌指数量，通常多于实现稳定序列特异性DNA识别所需的数目。为探究多锌指频繁存在的内在原因，我们构建了缺失Ikaros（调控淋巴细胞生成与白血病发生的关键因子）的4指DNA结合结构域第1指或第4指的基因工程小鼠。每个突变品系均表现出Ikaros敲除小鼠表型的特定子集。尤为值得关注的是，第1指与第4指分别参与B细胞与T细胞发育的不同阶段，而第4指是胸腺细胞及一种新型BCR-ABL+急性淋巴细胞白血病模型中肿瘤抑制所选择性必需的功能元件。上述结果结合转录组谱分析（transcriptome profiling）数据（本项提交至基因表达综合数据库（Gene Expression Omnibus，GEO）的内容：野生型（wild type，wt）与两种锌指（ZnF）突变小鼠的全胸腺RNA测序（RNA-Seq）数据）表明，多锌指转录因子内不同的锌指子集可调控不同的靶基因与生物学功能，同时证实选择性诱变可助力阐明这类广泛存在的转录因子家族的功能与作用机制。本项比对野生型（wt，3个生物学重复）、Ikaros-ZnF1-/-突变体（2个生物学重复）与Ikaros-ZnF4-/-突变体（2个生物学重复）的全胸腺RNA测序数据，以及作为补充文件链接于下方的RPKM_Thymocytes.txt，均报告了所有至少在任一样本中存在至少一条测序读数的注释参考序列（RefSeq）基因的相对mRNA表达水平（RPKM）值，同一基因的重复项（同一基因的不同转录本）已被过滤去除。RPKM（每百万比对reads中每千碱基转录本的读数条数，Reads Per Kilobase of transcript per Million mapped reads，RPKM；Mortazavi等，2008）通过使用SeqMonk（巴布拉姆生物信息学，Babraham Bioinformatics）软件获取的外显子测序读数，并结合美国国家生物技术信息中心（National Center for Biotechnology Information，NCBI）的mm9版本参考基因组注释计算得到。