HOX13 activity reprograms cis-regulatory modules during digit development (ChIP-Seq)

The combinatorial expression of the Hox genes along the body axes, referred to as the HOX code, is a major determinant of cell fate and plays a prevailing role in generating the animal body plan. In developing limb buds, the paralogous group 13 genes of the HoxA and HoxD clusters are essential for patterning the distal-most limb structures, the digits. Inactivation of HOXA13 and HOXD13 transcription factors (HOX13) leads to complete digit agenesis in mice, but how HOX13 regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here we performed genome-wide profiling of HOX13 by chromatin immunoprecipitation and analyzed the transcriptome and chromatin state of wild type early and late-distal limb buds, as well as Hoxa13-/-;Hoxd13-/- compound mutant limb buds. Our results show that inactivation of HOX13 impairs the activation and repression of putative cis-regulatory modules specific to the late-distal limb cells. Loss of HOX13 also disrupts the specific, spatial patterning of gene expression along the proximal-distal axis of the developing limb buds. These results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules. Overall design: dissected distal parts of e11.5 forelimb, of wild-type as well as Hoxa13-/-;Hoxd13-/- mice同源框基因（Hox genes）沿躯体轴的组合表达模式，即所谓的HOX密码（HOX code），是调控细胞命运的核心决定因素之一，在动物体构型构建过程中发挥主导作用。在发育中的肢芽内，HoxA与HoxD基因簇的旁系同源组13基因，对于塑造肢体最远端结构——指（趾）的模式至关重要。失活HOXA13与HOXD13转录因子（统称为HOX13）会完全导致小鼠指（趾）发育缺失，但HOX13如何调控转录结局并赋予肢体远端细胞身份，迄今仍未明晰。本研究通过染色质免疫沉淀（chromatin immunoprecipitation）开展了HOX13的全基因组谱分析，并对野生型早期、晚期远端肢芽，以及Hoxa13-/-;Hoxd13-/-复合突变体肢芽的转录组与染色质状态进行了分析。结果显示，HOX13的失活会破坏晚期远端肢芽细胞特异性顺式调控模块（cis-regulatory modules）的激活与抑制过程。HOX13的缺失还会扰乱发育肢芽沿近-远轴的基因表达特异性空间模式。上述结果表明，早期肢体转录程序的正常终止与晚期远端肢体转录程序的激活，是通过HOX13对顺式调控模块的双重调控作用协同完成的。实验设计概述：解剖获取胚胎第11.5天（e11.5）野生型及Hoxa13-/-;Hoxd13-/-小鼠的前肢远端组织。