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SOCS7 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS1-SOCS7 and CIS). SOCS proteins are composed of an N-terminal domain of variable length, a central Src homology 2 domain, and a C-terminal SOCS box. Biochemical and genetic studies have revealed that SOCS1, SOCS2, SOCS3, and CIS play an important role in the termination of cytokine and growth factor signaling. However, the biological actions of other SOCS proteins are less well defined. To investigate the physiological role of SOCS7, we have used gene targeting to generate mice that lack expression of the Socs7 gene. [Socs7.sup.-/-] mice were born in expected numbers, were fertile, and did not exhibit defects in hematopoiesis or circulating glucose or insulin concentrations. However, [Socs7.sup.-/-] mice were 7-10% smaller than their wild-type littermates, and within 15 weeks of age [approximately equal to] 50% of the [Socs7.sup.-/-] mice died as a result of hydrocephalus that was characterized by cranial distortion, dilation of the ventricular system, reduced thickness of the cerebral cortex, and disorganization of the subcommissural organ. In situ hybridization studies revealed prominent expression of Socs7 in the brain, suggestive of an important functional role of SOCS7 in this organ.

Author(s): Donald Metcalf; Christopher J. Greenhalgh; Elizabeth Viney; Tracy A. Willson; Robyn Starr; Nicos A. Nicola; Douglas J. Hilton; Warren S. Alexander (corresponding author) Suppressor of cytokine signalling-2 (SOCS-2) is [...]

Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is an epigenetic regulator that mediates gene expression silencing at targeted sites across the genome. Our current understanding of SMCHD1's molecular mechanism, and how substitutions within SMCHD1 lead to the diseases, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS), are only emerging. Recent structural studies of its two component domains — the N-terminal ATPase and C-terminal SMC hinge — suggest that dimerization of each domain plays a central role in SMCHD1 function. Here, using biophysical techniques, we demonstrate that the SMCHD1 ATPase undergoes dimerization in a process that is dependent on both the N-terminal UBL (Ubiquitin-like) domain and ATP binding. We show that neither the dimerization event, nor the presence of a C-terminal extension past the transducer domain, affect SMCHD1's in vitro catalytic activity as the rate of ATP turnover remains comparable to the monomeric protein. We further examined the functional importance of the N-terminal UBL domain in cells, revealing that its targeted deletion disrupts the localization of full-length SMCHD1 to chromatin. These findings implicate UBL-mediated SMCHD1 dimerization as a crucial step for chromatin interaction, and thereby for promoting SMCHD1-mediated gene silencing. [ABSTRACT FROM AUTHOR]

Development of a branching tree in the embryonic lung is crucial for the formation of a fully mature functional lung at birth. Sox9+ cells present at the tip of the primary embryonic lung endoderm are multipotent cells responsible for branch formation and elongation. We performed a genetic screen in murine primary cells and identified aurora kinase b (Aurkb) as an essential regulator of Sox9+ cells ex vivo. In vivo conditional knockout studies confirmed that Aurkb was required for lung development but was not necessary for postnatal growth and the repair of the adult lung after injury. Deletion of Aurkb in embryonic Sox9+ cells led to the formation of a stunted lung that retained the expression of Sox2 in the proximal airways, as well as Sox9 in the distal tips. Although we found no change in cell polarity, we showed that loss of Aurkb or chemical inhibition of Aurkb caused Sox9+ cells to arrest at G2/M, likely responsible for the lack of branch bifurcation. This work demonstrates the power of genetic screens in identifying novel regulators of Sox9+ progenitor cells and lung branching morphogenesis. [ABSTRACT FROM AUTHOR]

Illuminating dimerization: Proteins of the SMC family are chromosomal organizers involved in sister chromatid cohesion, chromosome condensation, and DNA repair. Unlike other eukaryotic family members, SMCHD1 forms homodimers, rather than heterodimers, and has a distinct domain architecture. Dysregulation of SMCHD1 function results in a form of muscular dystrophy and a developmental disorder. Chen et al. solved the x-ray crystal structure of the Smchd1 hinge domain, which is important for homodimerization and nucleic acid binding. Site-directed mutagenesis studies identified critical residues involved in SMCHD1 function in cells. Together, these data suggest how mutations in the SMCHD1 hinge domain contribute to human disease. Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is an epigenetic regulator in which polymorphisms cause the human developmental disorder, Bosma arhinia micropthalmia syndrome, and the degenerative disease, facioscapulohumeral muscular dystrophy. SMCHD1 is considered a noncanonical SMC family member because its hinge domain is C-terminal, because it homodimerizes rather than heterodimerizes, and because SMCHD1 contains a GHKL-type, rather than an ABC-type ATPase domain at its N terminus. The hinge domain has been previously implicated in chromatin association; however, the underlying mechanism involved and the basis for SMCHD1 homodimerization are unclear. Here, we used x-ray crystallography to solve the three-dimensional structure of the Smchd1 hinge domain. Together with structure-guided mutagenesis, we defined structural features of the hinge domain that participated in homodimerization and nucleic acid binding, and we identified a functional hotspot required for chromatin localization in cells. This structure provides a template for interpreting the mechanism by which patient polymorphisms within the SMCHD1 hinge domain could compromise function and lead to facioscapulohumeral muscular dystrophy. [ABSTRACT FROM AUTHOR]

Activation of the cell surface receptor, c-Mpl, by the cytokine, thrombopoietin (TPO), underpins megakaryocyte and platelet production in mammals. In humans, mutations in c-Mpl have been identified as the molecular basis of Congenital Amegakaryocytic Thrombocytopenia (CAMT). Here, we show that CAMT-associated mutations in c-Mpl principally lead to defective receptor presentation on the cell surface. In contrast, one CAMT mutant c-Mpl, F104S, was expressed on the cell surface, but showed defective TPO binding and receptor activation. Using mutational analyses, we examined which residues adjacent to F104 within the membrane-distal cytokine receptor homology module (CRM) of c-Mpl comprise the TPO-binding epitope, revealing residues within the predicted Domain 1 E-F and A-B loops and Domain 2 F′-G′ loop as key TPO-binding determinants. These studies underscore the importance of the c-Mpl membrane-distal CRM to TPO-binding and suggest that mutations within this CRM that perturb TPO binding could give rise to CAMT. [ABSTRACT FROM AUTHOR]

Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling. [ABSTRACT FROM AUTHOR]

Ets-related gene (ERG), which encodes a member of the Ets family of transcription factors, is a potent oncogene. Chromosomal rearrangements involving ERG are found in acute myeloid leukemia, acute lymphoblastic leukemia, Ewing's sarcoma and more than half of all prostate cancers; however, the normal physiological function of Erg is unknown. We did a sensitized genetic screen of the mouse for regulators of hematopoietic stem cell function and report here a germline mutation of Erg. We show that Erg is required for definitive hematopoiesis, adult hematopoietic stem cell function and the maintenance of normal peripheral blood platelet numbers. [ABSTRACT FROM AUTHOR]

The four mammalian SPRY domain–containing SOCS box proteins (SSB-1 to SSB-4) are characterized by a C-terminal SOCS box and a central SPRY domain. We have determined the first SPRY-domain structure, as part of SSB-2, by NMR. This domain adopts a novel fold consisting of a β-sandwich structure formed by two four-stranded antiparallel β-sheets with a unique topology. We demonstrate that SSB-1, SSB-2 and SSB-4, but not SSB-3, bind prostate apoptosis response protein-4 (Par-4). Mutational analysis of SSB-2 loop regions identified conserved structural determinants for its interaction with Par-4 and the hepatocyte growth factor receptor, c-Met. Mutations in analogous loop regions of pyrin and midline-1 SPRY domains have been shown to cause Mediterranean fever and Opitz syndrome, respectively. Our findings provide a template for SPRY-domain structure and an insight into the mechanism of SPRY-protein interaction. [ABSTRACT FROM AUTHOR]

Abstract: Mixl1 is a paired-type homeodomain protein that plays a crucial role in morphogenesis and endoderm differentiation in the murine embryo. To understand how Mixl1 directs embryogenesis, we studied the regulation of Mixl1 expression at a transcriptional level. In HepG2 cells, a genomic fragment encompassing the Mixl1 promoter conferred strong TGF-β-induced transcription that was dependent on the presence of the DNA-binding protein FoxH1. Further analysis of the Mixl1 promoter identified a proximal response element (PRE) containing SMAD- and FoxH1-binding sites required for TGF-β responsiveness. The PRE was also responsive to signalling by Nodal, a TGF-β ligand required for normal embryonic patterning. These results demonstrate for the first time a functional role for TGF-β ligands in regulation of mammalian Mixl1, identify FoxH1 as an essential transcriptional co-activator, and implicate Nodal as the embryonic regulator of Mixl1 in mesendoderm morphogenesis. [Copyright &y& Elsevier]

SOCS7 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCSI-SOCS1 and CS). SOCS proteins are composed of an N-terminal domain of variable length, a central Src homology 2 domain, and a C-terminal SOCS box. Biochemical and genetic studies have revealed that SOCS1, SOCS2, SOCS3, and CIS play an important role in the termination of cytokine and growth factor signaling. However, the biological actions of other SOCS proteins are less well defined. To investigate the physiological role of SOCS7, we have used gene targeting to generate mice that lack expression of the Socs7 gene. Socs7-/- mice were born in expected numbers, were fertile, and did not exhibit defects in hematopoiesis or circulating glucose or insulin concentrations. However, Socs7-/- mice were 7-10% smaller than their wild-type littermates, and within 15 weeks of age ≈50% of the Socs7-/- mice died as a result of hydrocephalus that was characterized by cranial distortion, dilation of the ventricular system, reduced thickness of the cerebral cortex, and disorganization of the subcommissural organ. In situ hybridization studies revealed prominent expression of Socs7 in the brain, suggestive of an important functional role of SOCS7 in this organ. [ABSTRACT FROM AUTHOR]

Genetic screens in lower organisms, particularly those that identify modifiers of preexisting genetic defects, have been used successfully to order components of complex signaling pathways. To date, similar suppressor screens have not been used in vertebrates. To define the molecular pathways regulating platelet production, we have executed a large-scale modifier screen with genetically thrombocytopenic Mpt-/- mice by using N-ethyl-N-nitrosourea mutagenesis. Here we show that mutations in the c-Myb gene cause a myeloproliferative syndrome and supraphysiological expansion of megakaryocyte and platelet production in the absence of thrombopoietin signaling. This screen demonstrates the utility of large-scale N-ethyl-N-nitrosourea mutagenesis suppressor screens in mice for the simultaneous discovery and in vivo validation of targets for therapeutic discovery in diseases for which mouse models are available. [ABSTRACT FROM AUTHOR]

Members of the suppressor of cytokine signaling (SOCS) family are potentially key physiological negative regulators of interleukin-6 (IL-6) signaling. To examine whether SOCS3 is involved in regulating this signaling, we have used conditional gene targeting to generate mice lacking Socs3 in the liver or in macrophages. We show that Socs3 deficiency results in prolonged activation of signal transducer and activator of transcription 1 (STAT1) and STAT3 after IL-6 stimulation but normal activation of STAT1 after stimulation with interferon-γ (IFN-γ). Conversely, IL-6-induced STAT activation is normal in Socs1-deficient cells, whereas STAT1 activation induced by IFN-γ is prolonged. Microarray analysis shows that the pattern of gene expression induced by IL-6 in Socs3deficient livers mimics that induced by IFN-γ. Our data indicate that SOCS3 and SOCS1 have reciprocal functions in IL-6 and IFN-γ regulation and imply that SOCS3 has a role in preventing IFN-γ-like responses in cells stimulated by IL-6. [ABSTRACT FROM AUTHOR]

Examines the impact of Suppressor of Cytokine Signaling-1 (SOCS-1) on inhibition of cytokine action in vivo. Correlation between SOCS-1 and Janus kinase; Impact of lymphoid and macrophage infiltration on pancreatic acinar tissue; Determination of the ratio of granulocytes to mononuclear.

Leukaemia inhibitory factor (LIF) is a pleiotropic growth factor active in diverse cell systems in both the adult and the embryo. The LIF gene from a number of mammalian species is highly conserved. The ovine and porcine LIF genes were cloned, sequenced and compared to the previously published murine and human LIF gene sequences. While the coding regions of the LIF gene are highly conserved, the non-coding regions are largely non-conserved. In a region of ≈ 340 bp, the 5' end of the translational initiation codon is highly conserved (84%). This region includes four conserved TATA boxes, two transcriptional start-sites identified in the murine gene and the minimal region required to function as the LIF promoter. A sequence in the murine gene adjacent to this highly conserved region which appears to contain a negative control element is, however, poorly conserved between the four species compared, except for a sequence of 16 conserved nucleotides. Within the largely non-conserved first intron, there is a block of ≈ 150 nucleotides which is highly conserved between all four species (≈ 72%). However, a sequence in intron I of the murine LIF gene which corresponds to an alternative exon of a putative variant LIF transcript is very poorly conserved between species, with only relics of this exon evident in the other three species. A comparison of the five LIF protein sequences available (murine, rat, human, ovine and porcine) revealed that the protein displays a high degree of similarity, ranging from 74% between mouse and sheep to 92% between rat and mouse. Several large blocks of absolutely conserved amino acid sequence were identified. The ovine LIF gene was modified to allow production of recombinant ovine LIF in yeast cells, which was shown to be biologically active on murine cells. [ABSTRACT FROM AUTHOR]

This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial olil gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular olil mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39 → Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfennentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the olil gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35 → Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23 → Ser or Leu53 → Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N-terminal membrane-embedded stem lying at residue 34. The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear-encoded subunit of the ATPase complex. [ABSTRACT FROM AUTHOR]

Summary We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist -HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation. Graphical Abstract Highlights • Smchd1 does not bind endogenous RNA with detectable sequence specificity • Smchd1 depends on the Xist-HnrpnK-PRC1 pathway for recruitment to the inactive X • Smchd1 depends on histone H2A lysine 119 ubiquitination for inactive X localization • Smchd1 protein stability depends on histone H2A lysine 119 ubiquitination Jansz et al. report that the chromatin protein Smchd1 depends on polycomb repressive complex 1-mediated ubiquitylation of histone H2A for its recruitment to the inactive X chromosome and for its protein stability. These data have implications for Smchd1 targeting genome-wide. [ABSTRACT FROM AUTHOR]