Haro Endika, Watson Billy A, Feenstra Jennifer M, Tegeler Luke, Pira Charmaine U, Mohan Subburaman, Oberg Kerby C
Department of Pathology and Human Anatomy, Loma Linda University, Loma Linda, CA 92354, USA.
Department of Basic Sciences, Loma Linda University, Loma Linda, CA 92354, USA.
Development. 2017 Jun 1;144(11):2009-2020. doi: 10.1242/dev.146332. Epub 2017 Apr 28.
Lmx1b is a homeodomain transcription factor responsible for limb dorsalization. Despite striking double-ventral (loss-of-function) and double-dorsal (gain-of-function) limb phenotypes, no direct gene targets in the limb have been confirmed. To determine direct targets, we performed a chromatin immunoprecipitation against Lmx1b in mouse limbs at embryonic day 12.5 followed by next-generation sequencing (ChIP-seq). Nearly 84% (=617) of the Lmx1b-bound genomic intervals (LBIs) identified overlap with chromatin regulatory marks indicative of potential -regulatory modules (PCRMs). In addition, 73 LBIs mapped to CRMs that are known to be active during limb development. We compared Lmx1b-bound PCRMs with genes regulated by Lmx1b and found 292 PCRMs within 1 Mb of 254 Lmx1b-regulated genes. Gene ontological analysis suggests that Lmx1b targets extracellular matrix production, bone/joint formation, axonal guidance, vascular development, cell proliferation and cell movement. We validated the functional activity of a PCRM associated with joint-related that provides a mechanism for Lmx1b-mediated joint modification and a PCRM associated with that suggests a role in autoregulation. This is the first report to describe genome-wide Lmx1b binding during limb development, directly linking Lmx1b to targets that accomplish limb dorsalization.
Lmx1b是一种负责肢体背侧化的同源域转录因子。尽管出现了显著的双腹侧（功能丧失）和双背侧（功能获得）肢体表型，但肢体中尚未确认直接的基因靶点。为了确定直接靶点，我们在胚胎第12.5天对小鼠肢体进行了针对Lmx1b的染色质免疫沉淀，随后进行了下一代测序（ChIP-seq）。所鉴定的与Lmx1b结合的基因组区间（LBI）中，近84%（=617个）与指示潜在调控模块（PCRM）的染色质调控标记重叠。此外，73个LBI映射到已知在肢体发育过程中活跃的CRM。我们将与Lmx1b结合的PCRM与受Lmx1b调控的基因进行比较，发现在254个受Lmx1b调控的基因的1 Mb范围内有292个PCRM。基因本体分析表明，Lmx1b的靶点包括细胞外基质产生、骨/关节形成、轴突导向、血管发育、细胞增殖和细胞运动。我们验证了与关节相关的一个PCRM的功能活性，该PCRM为Lmx1b介导的关节修饰提供了一种机制，还验证了与一个相关的PCRM的功能活性，该PCRM提示其在自我调节中的作用。这是第一份描述肢体发育过程中全基因组Lmx1b结合情况的报告，直接将Lmx1b与实现肢体背侧化的靶点联系起来。
