GLI genes: Cis-Acting Regulatory Elements
Towards the elucidation of genetic mechanisms, by which the transcription of GLI genes is regulated during early embryonic development, in this study one of the members of this family, i.e. human GLI3, was selected. By employing multispecies sequence alignment, an anciently conserved (tetrapodteleo...
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|Zusammenfassung:||Towards the elucidation of genetic mechanisms, by which the transcription of GLI genes is regulated during early embryonic development, in this study one of the members of this family, i.e. human GLI3, was selected. By employing multispecies sequence alignment, an anciently conserved (tetrapodteleost) non-coding architecture within the introns of GLI3 was identified. To search for possible enhancers of expression, 11 of these tetrapod-teleost conserved non-coding elements (CNEs) were selected for functional analysis. A GLI3 specific regulatory function of these deeply conserved intronic sequences was detected in human cell lines and model organisms: zebrafish, chicken (limb-bud), and mouse. In particular, this study has defined two distinct enhancers apparently recapitulating the entire known aspects of endogenous GLI3 expression within cartilaginous and non cartilaginous mesenchyme of embryonic limbs in a non-redundant manner. In vivo data from zebrafish, chicken and mice suggests that one limb specific anciently conserved enhancer might have been co-opted during the course of evolution to regulate GLI3 expression within more modern aspects of vertebrate appendicular structure, i.e. within hands and feet (autopodia). In contrast with respect to fin/limb specificity a second limb specific enhancer region preserved its ancient functions, dictating GLI3 expression within ancient fin/limb domains, i.e. stylopod and zeugopod. To sort out the evolutionary patterns of GLI coding sequences, a molecular evolutionary analysis in silico was carried out employing GLI sequences from representative members of tetrapod and teleost lineages. This analysis confirmed that changes experienced by the GLI sequences during the course of vertebrate evolution are largely in agreement with the reported similarities and differences in their functions within and between the species. In the present study, in an attempt to elucidate those ancient evolutionary events which brought the human GLI paralogs and members of many other gene families in the physical proximity of HOX clusters in three or four collinear regions of different chromosomes (Hsa2,7, 12 and 17; HOX cluster paralogon), the phylogenetic history of 11 multigene families with three or more of their representatives linked to human HOX clusters was analyzed. The results from this analysis suggest that that extensive triplicate or quadruplicate synteny that is seen on the present day human HOX-bearing chromosomes is the result of ancient small-scale duplications (segmental or gene-clusters) and subsequent genomic rearrangement events which occurred at different time points during chordate evolution. Significance: Congruent with the complex role of GLI3 in a multitude of patterning steps during vertebrate embryonic development, in this study, an ancient regulatory network comprising multiple distinctly acting cis-acting elements was revealed. In particular, the elucidation of a GLI3 specific cis-regulatory catalog offers a new perspective on understanding the genetic mechanisms by which the downstream effectors of SHH signaling cascade might themselves be regulated at correct place and precise time to direct pattern formation along the body axis during embryogenesis. For instance, this data could help to understand the mechanisms by which a proper balance between SHH and GLI3 transcripts is established in complementary domains within the developing limb and neural tube, and also during organogenesis. In addition, these cis-regulatory elements might contribute to understanding the genetic basis of those potentially GLI3-associated human birth defects which cannot be attributed to a mutation in the coding sequence of this gene. In such cases, these enhancers can be searched for mutations that can potentially affect the availability of GLI3 transcripts during embryogenesis.|