Constrained transcriptional polarity in the organization of mammalian Hox gene clusters (4C-seq). Constrained transcriptional polarity in the organization of mammalian Hox gene clusters (4C-seq)

n many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, for the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters towards a tighter structure during the evolution of the vertebrate lineage. Here we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster such as to invert one or several transcription unit(s), with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this genetic system, and the distinct problems elicited by these inversion alleles illustrate various aspects of the intricate regulations implemented at these loci. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in-cis. Overall design: 4C-seq analysis of Hoxd9 and lacZ interactions in E13.5 metanephros in two HoxD mutant alleles.

在诸多具有两侧对称体型的动物物种中，Hox基因（Hox genes）通常成簇分布于单个或多个基因组座位（genomic loci）上。此类成簇组织具有功能相关性：对各基因的转录调控，取决于其在基因簇内的相对位置。既往研究表明，脊椎动物的Hox基因簇的基因组组织程度远高于无脊椎动物的同源基因簇。脊椎动物Hox簇始终比无脊椎动物的更为紧凑，通常不含重复序列与嵌入基因，且所有基因均由同一条DNA链转录，这提示在脊椎动物谱系的演化过程中，特定因素促使这些基因簇形成更为紧密的结构。
本研究通过对HoxD基因簇内的多个等位基因进行工程改造，翻转单个或多个转录单元（transcription unit），可伴随或不携带相邻的CTCF（CCCTC结合因子）位点，以此探究统一转录方向的重要性。我们发现，哺乳动物Hox基因簇的紧密结构与其调控机制之间的关联，使得此类翻转操作大概率会对该遗传系统的正常运行产生不利影响；而这些翻转等位基因引发的不同问题，也揭示了这些基因组座位上复杂调控机制的多个维度。
我们提出，脊椎动物Hox基因簇的整合（包括转录极性的形成），是伴随着侧翼调控区域介导的全局基因调控机制的出现而共同演化的，以此优化选定的靶基因亚组在顺式（cis）作用下的协同应答。
实验设计：对两个HoxD突变等位基因的E13.5天胚胎后肾（metanephros）中，Hoxd9与lacZ的相互作用进行4C-seq（环形染色体构象捕获测序，Circular Chromosome Conformation Capture sequencing）分析。