Nonchev, S. | Search bioRxiv

bioRxiv2018

The abnormal, mis-localizated HR bmh protein associates with members of the protein processing machinery in the cytoplasm

We have recently mapped a 296 bp deletion at the mouse hairless locus that causes the mutation hairless rhino bald Mill Hill, (Hrrhbmh), (ID:MGI:3039558; J:89321), removing the stop codon and generating a larger mutant protein HR bmh with an additional sequence of 117 amino acids. The mutant hairless gene mRNA is expressed during the embryonic and post-natal development of the hair follicle. A mutant HR protein was identified in bmh mouse skin at different stages of development by a specific antibody. We demonstrated that the HR bmh protein is able to interact with the vitamin D receptor, but is not able to repress VDR-mediated transactivation. Immunofluorescence analysis revealed that HR bmh protein displays an abnormal cellular localization in transfected cell lines, as well as in the epidermis and hair follicle of bmh mutant mice. Here we analyse the patterns of HR bmh extra-nuclear localization in cell transfection experiments. Using a candidate approach, double transfection experiments, immunofluorescent staining and IP protocols we established that HR bmh co-localizes specifically with the proteins HDAC6 in the cytoplasm is able to physically interact with it. Blast analysis allowed to show that HDAC6 and share high sequence homologies with specific motifs of HR bmh. We studied the association of these potential interactors with various cytoplasmic compartments. We discuss the relevance of the mutant hairless protein mis-localization in endosomal processing and in proteasome related pathways with respect to the specific skin phenotype of mouse hairless mutants.

cell biology↗

bioRxiv2018

Functional mapping of the mouse hairless gene promoter region

The mouse hairless gene (Hr) encodes a protein of 127 kDa, acting as corepressor of nuclear hormone receptors. The Hairless protein (HR) is involved in the control of the cellular transition to the first hair cycle in adult Mammals. In its absence hair follicles disintegrate leading to a complete and irreversible hair loss with formation of cutaneous cysts. The hairless phenotype is therefore linked to defective proliferation and migration of the hair follicle stem cells apparently unable to respond to various signalling molecules. The Hr gene is expressed at high levels in skin and brain, and hairless transcripts were detected in gonads, thymus and colon. Although the patterns of Hr expression appear to be spatially and temporally regulated, very little is known about the molecular basis of the transcriptional control underlying Hr gene function. In this work we determine the precise transcriptional initiation start site of the mouse Hr gene and identify a new 1,1 kb cis-control element (RE1) that encompasses the promoter region and is able to drive luciferase reporter expression in skin and brain derived cell lines. We performed a deletion analysis and explored functionally regulatory motifs within this fragment to show that the role of this upstream regulatory region is linked to the presence of TRE and VDRE binding sites. We find that a TRE situated at -300 bp from the cap site is essential for gene expression in both skin NIH 3T3 and GHFT1 cells, while a VDRE positioned 94 bp upstream of the TRE modulates reporter expression specifically in skin derived cell lines. In addition, we define a novel cis-regulatory motif UE60, situated at the 5-end of RE1 and likely to interact with both TRE and VDRE. Our data complete previous results on the possible existence of an autoregulatory pathway, implicated in Hr gene regulation. Taken together these findings reveal a complex molecular network that potentially links several signalling pathways in hair follicle formation. We discuss the organisation of the regulatory modules in the mouse Hr gene upstream DNA sequences in the light of the high homology of this region in mouse, rat and human.

molecular biology↗