Chromosomal Dynamics at the Shh Locus: Limb Bud-Specific Differential Regulation of Competence and Active Transcription

The expression of Sonic hedgehog (Shh) in mouse limb buds is regulated by a long-range enhancer 1 Mb upstream of the Shh promoter. We used 3D-FISH and chromosome conformation capture assays to track changes at the Shh locus and found that long-range promoter-enhancer interactions are specific to limb bud tissues competent to express Shh. However, the Shh locus loops out from its chromosome territory only in the posterior limb bud (zone of polarizing activity or ZPA), where Shh expression is active. Notably, while Shh mRNA is detected throughout the ZPA, enhancer-promoter interactions and looping out were only observed in small fractions of ZPA cells. In situ detection of nascent Shh transcripts and unstable EGFP reporters revealed that active Shh transcription is likewise only seen in a small fraction of ZPA cells. These results suggest that chromosome conformation dynamics at the Shh locus allow transient pulses of Shh transcription

Chromosomal Dynamics at the Shh Locus: Limb Bud-Specific Differential Regulation of Competence and Active Transcription

SummaryThe expression of Sonic hedgehog (Shh) in mouse limb buds is regulated by a long-range enhancer 1 Mb upstream of the Shh promoter. We used 3D-FISH and chromosome conformation capture assays to track changes at the Shh locus and found that long-range promoter-enhancer interactions are specific to limb bud tissues competent to express Shh. However, the Shh locus loops out from its chromosome territory only in the posterior limb bud (zone of polarizing activity or ZPA), where Shh expression is active. Notably, while Shh mRNA is detected throughout the ZPA, enhancer-promoter interactions and looping out were only observed in small fractions of ZPA cells. In situ detection of nascent Shh transcripts and unstable EGFP reporters revealed that active Shh transcription is likewise only seen in a small fraction of ZPA cells. These results suggest that chromosome conformation dynamics at the Shh locus allow transient pulses of Shh transcription

Members of a novel gene family, Gsdm, are expressed exclusively in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner

AbstractGasdermin (Gsdm) was originally identified as a candidate causative gene for several mouse skin mutants. Several Gsdm-related genes sharing a protein domain with DFNA5, the causative gene of human nonsyndromic hearing loss, have been found in the mouse and human genomes, and this group is referred to as the DFNA5–Gasdermin domain family. However, our current comparative genomic analysis identified several novel motifs distinct from the previously reported domain in the Gsdm-related genes. We also identified three new Gsdm genes clustered on mouse chromosome 15. We named these genes collectively the Gsdm family. Extensive expression analysis revealed exclusive expression of Gsdm family genes in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner. Further database searching revealed the presence of other related genes with a similar N-terminal motif. These results suggest that the Gsdm family and related genes have evolved divergent epithelial expression profiles

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

AbstractMammal–fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements

青森県民の健康寿命アップ対策としての「心疾患10年リスク」の活用について(青森県の健康寿命アップと保健大学の取り組み, 第2回青森県立保健大学学術研究集会)

publisher青森市国立情報学研究所の「学術雑誌公開支援事業」により電子化されました

Two Types of Etiological Mutation in the Limb-Specific Enhancer of Shh

An enhancer named MFCS1 regulates Sonic hedgehog (Shh) expression in the posterior mesenchyme of limb buds. Several mutations in MFCS1 induce ectopic Shh expression in the anterior limb bud, and these result in preaxial polydactyly (PPD). However, the molecular basis of ectopic Shh expression remains elusive, although some mutations are known to disrupt the negative regulation of Shh expression in the anterior limb bud. Here, we analyzed the molecular mechanism of ectopic Shh expression in PPD including in a mouse mutation—hemimelic extra toes (Hx)—and in other MFCS1 mutations in different species. First, we generated transgenic mouse lines with a LacZ reporter cassette flanked with tandem repeats of 40 bp MFCS1 fragments harboring a mutation. The transgenic mouse line with the Hx-type fragment showed reporter expression exclusively in the anterior, but not in the posterior margins of limb buds. In contrast, no specific LacZ expression was observed in lines carrying the MFCS1 fragment with other mutations. Yeast one-hybrid assays revealed that the msh-like homeodomain protein, MSX1, bound specifically to the Hx sequence of MFCS1. Thus, PPD caused by mutations in MFCS1 has two major types of molecular etiology: loss of a cis-motif for negative regulation of Shh, and acquisition of a new cis-motif binding to a preexisting transcription factor, as represented by the Hx mutation

Two Types of Etiological Mutation in the Limb-Specific Enhancer of Shh

An enhancer named MFCS1 regulates Sonic hedgehog (Shh) expression in the posterior mesenchyme of limb buds. Several mutations in MFCS1 induce ectopic Shh expression in the anterior limb bud, and these result in preaxial polydactyly (PPD). However, the molecular basis of ectopic Shh expression remains elusive, although some mutations are known to disrupt the negative regulation of Shh expression in the anterior limb bud. Here, we analyzed the molecular mechanism of ectopic Shh expression in PPD including in a mouse mutation—hemimelic extra toes (Hx)—and in other MFCS1 mutations in different species. First, we generated transgenic mouse lines with a LacZ reporter cassette flanked with tandem repeats of 40 bp MFCS1 fragments harboring a mutation. The transgenic mouse line with the Hx-type fragment showed reporter expression exclusively in the anterior, but not in the posterior margins of limb buds. In contrast, no specific LacZ expression was observed in lines carrying the MFCS1 fragment with other mutations. Yeast one-hybrid assays revealed that the msh-like homeodomain protein, MSX1, bound specifically to the Hx sequence of MFCS1. Thus, PPD caused by mutations in MFCS1 has two major types of molecular etiology: loss of a cis-motif for negative regulation of Shh, and acquisition of a new cis-motif binding to a preexisting transcription factor, as represented by the Hx mutation