ABSTRACT
Copy number variations are genomic structural variants that are frequently associated with human diseases. Among these copy number variations, duplications of DNA segments are often assumed to lead to dosage effects by increasing the copy number of either genes or their regulatory elements. We produced a series of large targeted duplications within a conserved gene desert upstream of the murine HoxD locus. This DNA region, syntenic to human 2q31-32, contains a range of regulatory elements required for Hoxd gene transcription, and it is often disrupted and/or reorganized in human genetic conditions collectively known as the 2q31 syndrome. Unexpectedly, one such duplication led to a transcriptional down-regulation in developing digits by impairing physical interactions between the target genes and their upstream regulatory elements, thus phenocopying the effect obtained when these enhancer sequences are deleted. These results illustrate the detrimental consequences of interrupting highly conserved regulatory landscapes and reveal a mechanism where genomic duplications lead to partial loss of function of nearby located genes.
Subject(s)
DNA Copy Number Variations , Genes, Homeobox , Animals , Base Sequence , Chromosome Inversion , DNA Primers/genetics , Enhancer Elements, Genetic , Evolution, Molecular , Extremities/embryology , Female , Gene Duplication , Gene Expression Regulation, Developmental , Humans , Mice , Mice, Transgenic , Models, Genetic , Multigene Family , PregnancyABSTRACT
The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.
Subject(s)
Enhancer Elements, Genetic , Extremities/embryology , Gene Expression Regulation, Developmental , Genes, Homeobox , Regulatory Sequences, Nucleic Acid , Transcription, Genetic , Animals , Brain/embryology , Brain/metabolism , Extremities/physiology , Homeodomain Proteins , Humans , Mice , Mice, Transgenic , Promoter Regions, Genetic , XenopusABSTRACT
In mammals, male sex determination is controlled by the SRY protein, which drives differentiation of the bipotential embryonic gonads into testes by activating the Sertoli cell differentiation program. The morphological effects of SRY are well documented; however, its molecular mechanism of action remains unknown. Moreover, SRY proteins display high sequence variability among mammalian species, which makes protein motifs difficult to delineate. We previously isolated SIP-1/NHERF2 as a human SRY-interacting protein. SIP-1/NHERF2, a PDZ protein, interacts with the C-terminal extremity of the human SRY protein. Here we showed that the interaction of SIP-1/NHERF2 and SRY via the SIP-1/NHERF2 PDZ1 domain is conserved in mice. However, the interaction occurs via a domain that is internal to the mouse SRY protein and involves a different recognition mechanism than human SRY. Furthermore, we show that mouse and human SRY induce nuclear accumulation of the SIP-1/NHERF2 protein in cultured cells. Finally, a transgenic mouse line expressing green fluorescent protein under the control of the mouse Sry promoter allowed us to show that SRY and SIP-1/NHERF2 are co-expressed in the nucleus of pre-Sertoli cells during testis determination. Taken together, our results suggested that the function of SIP-1/NHERF2 as an SRY cofactor during testis determination is conserved between human and mouse.
Subject(s)
Cytoskeletal Proteins/physiology , Nerve Tissue Proteins/physiology , Sex-Determining Region Y Protein/metabolism , Amino Acid Motifs , Animals , Cell Differentiation , Cell Line , Conserved Sequence , Cytoskeletal Proteins/metabolism , Female , Glutathione Transferase/metabolism , Green Fluorescent Proteins/metabolism , Humans , Immunoprecipitation , Male , Mice , Mice, Transgenic , Microscopy, Fluorescence , NIH 3T3 Cells , Nerve Tissue Proteins/metabolism , Phosphoproteins , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Biosynthesis , Protein Structure, Tertiary , RNA-Binding Proteins , Recombinant Proteins/chemistry , Sertoli Cells/cytology , Sodium-Hydrogen Exchangers , Species Specificity , Subcellular Fractions , Testis/metabolism , TransfectionABSTRACT
During mammalian gonadal development, nuclear import/export of the transcription factor SOX9 is a critical step of the Sry-initiated testis-determining cascade. In this study, we identify a molecular mechanism contributing to the SOX9 nuclear translocation in NT2/D1 cells, which is mediated by the prostaglandin D2 (PGD2) signalling pathway via stimulation of its adenylcyclase-coupled DP1 receptor. We find that activation of cAMP-dependent protein kinase A (PKA) induces phosphorylation of SOX9 on its two S64 and S181 PKA sites, and its nuclear localization by enhancing SOX9 binding to the nucleocytoplasmic transport protein importin beta. Moreover, in embryonic gonads, we detect a male-specific prostaglandin D synthase expression and an active PGD2 signal at the time and place of SOX9 expression. We thus propose a new step in the sex-determining cascade where PGD2 acts as an autocrine factor inducing SOX9 nuclear translocation and subsequent Sertoli cell differentiation.
Subject(s)
Cell Nucleus/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , High Mobility Group Proteins/metabolism , Prostaglandin D2/metabolism , Transcription Factors/metabolism , Animals , Cells, Cultured , Female , Male , Mice , Organ Culture Techniques , Ovary/embryology , Ovary/enzymology , Ovary/metabolism , Phosphorylation , Protein Transport/physiology , SOX9 Transcription Factor , Testis/embryology , Testis/enzymology , Testis/metabolismABSTRACT
SRY, a Y chromosome-encoded DNA-binding protein, is required for testis organogenesis in mammals. Expression of the SRY gene in the genital ridge is followed by diverse early cell events leading to Sertoli cell determination/differentiation and subsequent sex cord formation. Little is known about SRY regulation and its mode of action during testis development, and direct gene targets for SRY are still lacking. In this study, we demonstrate that interaction of the human SRY with histone acetyltransferase p300 induces the acetylation of SRY both in vitro and in vivo at a single conserved lysine residue. We show that acetylation participates in the nuclear localisation of SRY by increasing SRY interaction with importin beta, while specific deacetylation by HDAC3 induces a cytoplasmic delocalisation of SRY. Finally, by analysing p300 and HDAC3 expression profiles during both human or mouse gonadal development, we suggest that acetylation and deacetylation of SRY may be important mechanisms for regulating SRY activity during mammalian sex determination.
