Deficiencies or mutations in the human pseudoautosomal SHOX gene are associated with a series of short-stature conditions, including Turner syndrome, Leri-Weill dyschondrosteosis, and Langer mesomelic dysplasia. Although this gene is absent from the mouse genome, the closely related paralogous gene Shox2 displays a similar expression pattern in developing limbs. Here, we report that the conditional inactivation of Shox2 in developing appendages leads to a strong phenotype, similar to the human conditions, although it affects a different proximodistal limb segment. Furthermore, using this mouse model, we establish the cellular etiology of these defects and show that Shox2 acts upstream the Runx2 gene, a key regulator of chondrogenesis.

PU.1 is a hematopoietic-specific transcriptional activator that is absolutely required for the differentiation of B lymphocytes and myeloid-lineage cells. Although PU.1 is also expressed by early erythroid progenitor cells, its role in erythropoiesis, if any, is unknown. To investigate the relevance of PU.1 in erythropoiesis, we produced a line of PU.1-deficient mice carrying a green fluorescent protein reporter at this locus. We report here that PU.1 is tightly regulated during differentiation-it is expressed at low levels in erythroid progenitor cells and down-regulated upon terminal differentiation. Strikingly, PU.1deficient fetal erythroid progenitors lose their self-renewal capacity and undergo proliferation arrest, premature differentiation, and apoptosis. In adult mice lacking one PU.1 allele, similar defects are detected following stress-induced erythropoiesis. These studies identify PU.1 as a novel and critical regulator of erythropoiesis and highlight the versatility of this transcription factor in promoting or preventing differentiation depending on the hematopoietic lineage.

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of β-galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. β-galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial–mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor, which controls adipocyte differentiation. We targeted with homologous recombination the PPARγ2-specific exon B, resulting in a white adipose tissue knockdown of PPARγ. Although homozygous ( PPAR γ hyp/hyp ) mice are born with similar weight as the WT mice, the PPAR γ hyp/hyp animals become growth retarded and develop severe lipodystrophy and hyperlipidemia. Almost half of these PPAR γ hyp/hyp mice die before adulthood, whereas the surviving PPAR γ hyp/hyp animals overcome the growth retardation, yet remain lipodystrophic. In contrast to most lipodystrophic models, the adult PPAR γ hyp/hyp mice only have mild glucose intolerance and do not have a fatty liver. These metabolic consequences of the lipodystrophy are relatively benign because of the induction of a compensatory gene expression program in the muscle that enables efficient oxidation of excess lipids. The PPAR γ hyp/hyp mice unequivocally demonstrate that PPARγ is the master regulator of adipogenesis in vivo and establish that lipid and glucose homeostasis can be relatively well maintained in the absence of white adipose tissue.

The DNA damage‐dependent poly(ADP‐ribose) polymerases, PARP‐1 and PARP‐2, homo‐ and heterodimerize and are both involved in the base excision repair (BER) pathway. Here, we report that mice carrying a targeted disruption of the PARP‐2 gene are sensitive to ionizing radiation. Following alkylating agent treatment, parp‐2−/−‐derived mouse embryonic fibroblasts exhibit increased post‐replicative genomic instability, G2/M accumulation and chromosome mis‐segregation accompanying kinetochore defects. Moreover, parp‐1−/−parp‐2−/− double mutant mice are not viable and die at the onset of gastrulation, demonstrating that the expression of both PARP‐1 and PARP‐2 and/or DNA‐dependent poly(ADP‐ribosyl) ation is essential during early embryogenesis. Interestingly, specific female embryonic lethality is observed in parp‐1+/−parp‐2−/− mutants at E9.5. Meta phase analyses of E8.5 embryonic fibroblasts highlight a specific instability of the X chromosome in those females, but not in males. Together, these results support the notion that PARP‐1 and PARP‐2 possess both overlapping and non‐redundant functions in the maintenance of genomic stability.

Human TIF2 (hTIF2) is a member of the p160 family of nuclear receptor coactivators, which includes SRC-1 and p/CIP. Although the functions of hTIF2 and of its mouse homolog (GRIP1 or mTIF2) have been clearly established in vitro, their physiological role remains elusive. Here, we have generated mice lacking mTIF2/GRIP1 and examined their phenotype with a particular emphasis on reproductive functions. TIF2(-/-) mice are viable, but the fertility of both sexes is impaired. Male hypofertility is due to defects in both spermiogenesis (teratozoospermia) and age-dependent testicular degeneration, and TIF2 expression appears to be essential for adhesion of Sertoli cells to germ cells. Female hypofertility is due to a placental hypoplasia that most probably reflects a requirement for maternal TIF2 in decidua stromal cells that face the developing placenta. We conclude that TIF2 plays a critical role in mouse reproductive functions, whereas previous reports have not revealed serious fertility impairment in SRC-1(-/-) or p/CIP(-/-) mutants. Thus, even though the three p160 coactivators exhibit strong sequence homology and similar activity in assays in vitro, they play distinct physiological roles in vivo, as their genetic eliminations result in distinct pathologies.

genesisVolume 32, Issue 2 p. 87-90 ArticleFree Access A conditional floxed (loxP-flanked) allele for the retinoic acid receptor alpha (RARα) gene Benoit Chapellier, Benoit Chapellier Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorManuel Mark, Manuel Mark Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorJean-Marie Garnier, Jean-Marie Garnier Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMarianne LeMeur, Marianne LeMeur Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorPierre Chambon, Corresponding Author Pierre Chambon chambon@igbmc.u-strasbg.fr Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163, 67404 Illkirch Cedex, CU de Strasbourg, FranceSearch for more papers by this authorNorbert B. Ghyselinck, Norbert B. Ghyselinck Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this author Benoit Chapellier, Benoit Chapellier Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorManuel Mark, Manuel Mark Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorJean-Marie Garnier, Jean-Marie Garnier Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMarianne LeMeur, Marianne LeMeur Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorPierre Chambon, Corresponding Author Pierre Chambon chambon@igbmc.u-strasbg.fr Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163, 67404 Illkirch Cedex, CU de Strasbourg, FranceSearch for more papers by this authorNorbert B. Ghyselinck, Norbert B. Ghyselinck Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this author First published: 13 February 2002 https://doi.org/10.1002/gene.10071Citations: 45AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume32, Issue2Special Issue: Conditional Alleles in MiceFebruary 2002Pages 87-90 ReferencesRelatedInformation

TRAF4 belongs to the tumor necrosis factor receptor-associated factor (TRAF) family of proteins but, unlike other family members, has not yet been clearly associated to any specific receptor or signaling pathway. To investigate the biological function of TRAF4, we have generated traf4 -deficient mice by gene disruption. The traf4 gene mutation is embryonic lethal but with great individual variation, as approximately one third of the homozygous mutant embryos died in utero around embryonic day 14, whereas the others reach adulthood. Surviving mutant mice manifest numerous developmental abnormalities; notably, 100% of homozygous mutant mice suffer respiratory disorder and wheezing caused by tracheal ring disruption. Additional malformations concern mainly the axial skeleton, as the ribs, sternum, tail, and vertebral arches are affected, with various degrees of penetrance. Traf4 -deficient mice also exhibit a high incidence of spina bifida, a defect likened to neural tube defects (NTD) that are common congenital malformations in humans. Altogether, our results demonstrate that TRAF4 is required during embryogenesis in key biological processes including the formation of the trachea, the development of the axial skeleton, and the closure of the neural tube. Considering the normal expression pattern of TRAF4 in neural tissues, we can conclude that TRAF4 participates in neurulation in vivo .

genesisVolume 32, Issue 2 p. 105-108 ArticleFree Access A Hoxa2 mutant conditional allele generated by Flp- and Cre-mediated recombination Shu-Yue Ren, Shu-Yue Ren Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMassimo Pasqualetti, Massimo Pasqualetti Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorAndrée Dierich, Andrée Dierich Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMarianne Le Meur, Marianne Le Meur Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorFilippo M. Rijli, Corresponding Author Filippo M. Rijli rijli@igbmc.u-strasbg.fr Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceIGMBC, Parc d'Innovation, 1, rue Laurent Fries, 67404 Illkirch Cedex, FranceSearch for more papers by this author Shu-Yue Ren, Shu-Yue Ren Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMassimo Pasqualetti, Massimo Pasqualetti Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorAndrée Dierich, Andrée Dierich Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMarianne Le Meur, Marianne Le Meur Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorFilippo M. Rijli, Corresponding Author Filippo M. Rijli rijli@igbmc.u-strasbg.fr Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceIGMBC, Parc d'Innovation, 1, rue Laurent Fries, 67404 Illkirch Cedex, FranceSearch for more papers by this author First published: 13 February 2002 https://doi.org/10.1002/gene.10052Citations: 13AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume32, Issue2Special Issue: Conditional Alleles in MiceFebruary 2002Pages 105-108 ReferencesRelatedInformation

genesisVolume 32, Issue 2 p. 109-111 ArticleFree Access A Hoxa2 knockin allele that expresses EGFP upon conditional Cre-mediated recombination Massimo Pasqualetti, Massimo Pasqualetti Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorShu-Yue Ren, Shu-Yue Ren Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMartine Poulet, Martine Poulet Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMarianne LeMeur, Marianne LeMeur Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorAndrée Dierich, Andrée Dierich Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorFilippo M. Rijli, Corresponding Author Filippo M. Rijli rijli@igbmc.u-strasbg.fr Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceIGBMC, Parc d'Innovation, 1, rue Laurent Fries, 67404 Illkirch Cedex, FranceSearch for more papers by this author Massimo Pasqualetti, Massimo Pasqualetti Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorShu-Yue Ren, Shu-Yue Ren Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMartine Poulet, Martine Poulet Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorMarianne LeMeur, Marianne LeMeur Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorAndrée Dierich, Andrée Dierich Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceSearch for more papers by this authorFilippo M. Rijli, Corresponding Author Filippo M. Rijli rijli@igbmc.u-strasbg.fr Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Strasbourg, FranceIGBMC, Parc d'Innovation, 1, rue Laurent Fries, 67404 Illkirch Cedex, FranceSearch for more papers by this author First published: 13 February 2002 https://doi.org/10.1002/gene.10053Citations: 21AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. LITERATURE CITED Abu-Abed S, Dolle P, Metzger D, Beckett B, Chambon P, Petkovich M. 2001. The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures. Genes Dev 15: 226– 240. Barrow JR, Stadler HS, Capecchi MR. 2000. Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse. Development 127: 933– 944. Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP. 1998. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol 8: 1323– 1326. Davenne M, Maconochie MK, Neun R, Pattyn A, Chambon P, Krumlauf R, Rijli FM. 1999. Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal development in the rostral hindbrain. Neuron 22: 677– 691. Dupe V, Davenne M, Brocard J, Dolle P, Mark M, Dierich A, Chambon P, Rijli FM. 1997. In vivo functional analysis of the Hoxa-1 3′ retinoic acid response element (3′RARE). Development 124: 399– 410. Gendron-Maguire M, Mallo M, Zhang M, Gridley T. 1993. Hoxa-2 mutant mice exhibit homeotic transformation of skeletal elements derived from cranial neural crest. Cell 75: 1317– 1331. Godwin AR, Stadler HS, Nakamura K, Capecchi MR. 1998. Detection of targeted GFP-Hox gene fusions during mouse embryogenesis. Proc Natl Acad Sci U S A 95: 13042– 13047. Gu H, Zou YR, Rajewsky K. 1993. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73: 1155– 1164. Hunt P, Gulisano M, Cook M, Sham MH, Faiella A, Wilkinson D, Boncinelli E, Krumlauf R. 1991. A distinct Hox code for the branchial region of the vertebrate head. Nature 353: 861– 864. Rijli FM, Mark M, Lakkaraju S, Dierich A, Dolle P, Chambon P. 1993. A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene. Cell 75: 1333– 1349. Tan DP, Ferrante J, Nazarali A, Shao X, Kozak CA, Guo V, Nirenberg M. 1992. Murine Hox-1.11 homeobox gene structure and expression. Proc Natl Acad Sci U S A 89: 6280– 6284. Citing Literature Volume32, Issue2Special Issue: Conditional Alleles in MiceFebruary 2002Pages 109-111 ReferencesRelatedInformation

Mouse embryonic stem (ES) cells remain “pluripotent” in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from “differentiation-committed” ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

A group of specialized genes has been defined to govern the molecular mechanisms controlling the circadian clock in mammals. Their expression and the interactions among their products dictate circadian rhythmicity. Three genes homologous to Drosophila period exist in the mouse and are thought to be major players in the biological clock. Here we present the generation of mice in which the founding member of the family, Per1, has been inactivated by homologous recombination. These mice present rhythmicity in locomotor activity, but with a period almost 1 h shorter than wild‐type littermates. Moreover, the expression of clock genes in peripheral tissues appears to be delayed in Per1 mutant animals. Importantly, light‐induced phase shifting appears conserved. The oscillatory expression of clock genes and the induction of immediate‐early genes in response to light in the master clock structure, the suprachiasmatic nucleus, are unaffected. Altogether, these data demonstrate that Per1 plays a distinct role within the Per family, as it may be involved predominantly in peripheral clocks and/or in the output pathways of the circadian clock.

Metazoan genomes encode two related proteins, TBP and the TBP-like factor (TLF/TRF2), sharing a highly conserved saddle-like domain. TLF is highly expressed in a finely regulated pattern in the mouse testis during spermatogenesis. The murine TLF gene has been inactivated using homologous recombination. TLF-/- mice are viable, but mutant male mice are sterile due to a late, complete arrest of spermiogenesis. In mutant animals, spermatogonia and spermatocytes develop normally, but round spermatids undergo apoptosis at step 7. Although the expression of the transcriptional activator CREM and many other postmeiotic genes was unaltered in TLF null mice, several spermiogenesis genes transcribed in late round spermatids appeared to be under TLF control. Hence, TLF is not required for embryonic development in the mouse but is essential for spermiogenesis.

Deletion of the murine survival of motor neuron gene (SMN) exon 7, the most frequent mutation found in spinal muscular atrophy (SMA) patients, directed to neurons but not to skeletal muscle, enabled generation of a mouse model of SMA providing evidence that motor neurons are the primary target of the gene defect. Moreover, the mutated SMN protein (SMNDeltaC15) is dramatically reduced in the motor neuron nuclei and causes a lack of gems associated with large aggregates of coilin, a coiled-body-specific protein. These results identify the lack of the nuclear targeting of SMN as the biochemical defect in SMA.

In the mammalian pancreas, the endocrine cell types of the islets of Langerhans, including the alpha-, beta-, delta-, and pancreatic polypeptide cells as well as the exocrine cells, derive from foregut endodermal progenitors. Recent genetic studies have identified a network of transcription factors, including Pdx1, Isl1, Pax4, Pax6, NeuroD, Nkx2.2, and Hlxb9, regulating the development of islet cells at different stages, but the molecular mechanisms controlling the specification of pancreatic endocrine precursors remain unknown. neurogenin3 (ngn3) is a member of a family of basic helix-loop-helix transcription factors that is involved in the determination of neural precursor cells in the neuroectoderm. ngn3 is expressed in discrete regions of the nervous system and in scattered cells in the embryonic pancreas. We show herein that ngn3-positive cells coexpress neither insulin nor glucagon, suggesting that ngn3 marks early precursors of pancreatic endocrine cells. Mice lacking ngn3 function fail to generate any pancreatic endocrine cells and die postnatally from diabetes. Expression of Isl1, Pax4, Pax6, and NeuroD is lost, and endocrine precursors are lacking in the mutant pancreatic epithelium. Thus, ngn3 is required for the specification of a common precursor for the four pancreatic endocrine cell types.

MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu66-Leu67) and a tyrosine residue (Tyr89) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s).