Mutations in PHF8 are associated with X linked mental retardation and cleft lip/cleft palate.

Truncating mutations were found in the PHF8 gene (encoding the PHD finger protein 8) in two unrelated families with X linked mental retardation (XLMR) associated with cleft lip/palate (MIM 300263). Expression studies showed that this gene is ubiquitously transcribed, with strong expression of the mouse orthologue Phf8 in embryonic and adult brain structures. The coded PHF8 protein harbours two functional domains, a PHD finger and a JmjC (Jumonji-like C terminus) domain, implicating it in transcriptional regulation and chromatin remodelling. The association of XLMR and cleft lip/palate in these patients with mutations in PHF8 suggests an important function of PHF8 in midline formation and in the development of cognitive abilities, and links this gene to XLMR associated with cleft lip/palate. Further studies will explore the specific mechanisms whereby PHF8 alterations lead to mental retardation and midline defects.

Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice.

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A + C + 0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A + C + 0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.

Bidirectional activity and orientation-dependent specificity of the rat aldolase C promoter in transgenic mice.

We previously reported that the rat aldolase C 115 bp promoter is sufficient to ensure the brain specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice. We identify in a further reduced 84 bp promoter several putative binding sites for the transcriptional factors Sp1, USF, AP1, and AP2. Deletion or mutation of these partially overlapping binding sites results in inactivation of the cognate transgenes. Moreover, we show that the 115 bp sequence is able to direct bidirectional transcription in vivo but, surprisingly, transcriptional activity in the opposite direction is no more brain specific.

Upstream elements involved in vivo in activation of the brain-specific rat aldolase C gene. Role of binding sites for POU and winged helix proteins.

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a 115-base pair (bp) promoter fragment was able to ensure the brain-specific expression of the chloramphenicol acetyltransferase (CAT) reporter gene in transgenic mice, but only at a low level (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). Here we show that in vivo activation of this promoter at a high level requires cooperation between an upstream 0.6-kilobase pair (kb) fragment and far upstream sequences. In the 0.6-kb region, a 28-bp DNA element is shown to include overlapping in vitro binding sites for POU domain regulatory proteins and for the Winged Helix hepatocyte nuclear factor-3beta factor. An hepatocyte nuclear factor-3beta-binding site previously described in the short proximal promoter fragment is also shown to interact in vitro with POU proteins, although with a lower affinity than the 28-bp motif. Additional binding sites for POU factors were detected in the upstream 0.6-kb sequences. Progressive deletion in this region resulted in decreased expression levels of the transgenes in mice, suggesting synergistic interactions between these multiple POU-binding sites. We propose that DNA elements characterized by a dual binding specificity for both POU domain and Winged Helix transcription factors could play an essential role in the brain-specific expression of the aldolase C gene and other neuronal genes.

Muscle-specific transcriptional activation by CArG box requires either homophilic or heterophilic interactions of the CArG box binding factors.

CArG boxes (CC(A/T)6GG sequences) are present in various promoters and are able to confer two different types of transcriptional responsiveness: serum inducibility and muscle-specific activation. Inserted upstream from the ubiquitous HSV thymidine kinase promoter, multimerized HCA1 boxes (human cardiac alpha-actin proximal CArG box) behave as strong muscle-specific activating elements. Transient expression assay was used to determine whether the muscle-specific transcriptional activation by the CArG boxes depends on the presence in the vicinity of other specific cis-acting DNA elements. Our results show that no specific association between different regulatory binding sites is required for the myogenic activity of a CArG box and that CArG elements are able to stimulate transcription in myogenic cells through either homophilic or heterophilic interactions of the CArG box binding factors.

Activation of gene expression via CArG boxes during myogenic differentiation.

We have studied gene activation via CArG boxes in the context of myogenesis. The proximal CArG box of the human cardiac actin gene (HCA1) stimulates transcription from the herpes simplex virus thymidine kinase (TK) promoter in a tissue-specific fashion. Thus in transient transfection assays, when the expression of chloramphenicol acetyltransferase (CAT) from p(HCA1)4 TKCAT is compared to that derived from p(M1)4 TKCAT which contains an inactive mutated version (M1) of the HCA1 element, high levels of expression are seen in C2 mouse myoblasts and myotubes, and in the T4 myoblast cell line derived from the C3H10T1/2 cell line by 5-azacytidine treatment, whereas only low levels of expression are seen in the mouse L fibroblast cell line. The parental C3H10T1/2 cell line shows intermediate levels of expression. A similar situation is seen in stably transfected cell lines. Gene activation via CArG boxes was also analyzed in the course of myogenic conversion of C3H10T1/2 cells treated with 5-azacytidine. Our results indicate that activation of the CAT gene from the HCA1 element is slightly posterior to the appearance of the first MyoD1 and myogenin transcripts, concomitant with the appearance of cardiac alpha-actin transcripts, but clearly precedes the accumulation of myosin light-chain 1a transcripts and the appearance of troponin T-positive cells. These results further establish that CArG boxes can be seen as muscle-specific cis-acting regulatory element prior to terminal differentiation.

Insulin/insulin-like growth factor I induce actin transcription in mouse fibroblasts expressing constitutively myc gene.

Quiescent benzo[alpha]pyrene-transformed BALB/c 3T3 fibroblasts (line BP-A31), continue to express 'competence' genes (such as c-myc) and do not require platelet-derived growth factor ('competence' factor) in order to resume the cell division cycle. Insulin-like growth factor I (IGF-I), as well as insulin (at high concentrations, where it interacts with IGF-I-receptors) are potent mitogens in these cells. In contrast with the original non-transformed A31 cell line, we show that insulin/IGF-I (even in the absence of de novo protein synthesis) induce actin transcription in BP-A31 cells. We have verified that 'CArG' boxes, major actin promoter elements, can act as insulin-inducible elements in BP-A31 cells. Insulin-induced actin transcription is also observed in quiescent A31 cells stably transfected with a myc expression vector, suggesting a correlation between constitutive myc expression and insulin-induced actin transcription.

CC Ar GG boxes, cis-acting elements with a dual specificity. Muscle-specific transcriptional activation and serum responsiveness.

The influence of different CC Ar GG boxes derived from either muscle-specific or serum-responsive genes, on the specificity of different promoters has been investigated. Inserted upstream from an 85 base-pair long minimal promoter of the human cardiac alpha-actin gene, a single copy of both the cognate CC Ar GG element (HCA1) and the c-fos gene serum response element (SRE) stimulate transcription four- to fivefold more efficiently in C2 myogenic cells than in L fibroblastic cells, SRE being two- to threefold more active than HCA1. Inserted upstream from the ubiquitous Herpes simplex thymidine kinase (HSV-tk) promoter, multimerized CC Ar GG boxes behave as strong muscle-specific activating elements, about 20-fold more active in myogenic C2 cells than in L fibroblasts and hepatoma HepG2 cells. They also confer serum responsiveness on the HSV-tk promoter. Efficiency of HCA1 and SRE tetramers in conferring both muscle specificity and serum responsiveness is roughly similar. It appears, therefore, that regardless of their origin (either muscle-specific or serum-responsive genes) CC Ar GG boxes behave by themselves as both muscle-specific activating and serum-responsive elements.

The 'CC.Ar.GG' box. A protein-binding site common to transcription-regulatory regions of the cardiac actin, c-fos and interleukin-2 receptor genes.

We have previously suggested that a repeated sequence motif in the upstream region of the human cardiac actin gene 'CC.Ar.GG', where Ar is an (A + T)-rich six-base-pair-sequence, may be important in the muscle-specific expression of this gene [Minty, A. & Kedes, L. (1986) Mol. Cell Biol. 6, 2125-2136]. Here we show that this sequence binds a nuclear protein, and that binding is abolished by mutating either the CC and GG dinucleotides or the (A + T)-rich centre. Mutation of the CC and GG nucleotides also abolishes the transcription-stimulating activity of this sequence on the cardiac actin promoter. A similar sequence has been implicated in the serum-response of the c-fos gene [Treisman, R. (1986) Cell 46, 567-574]. We show that this c-fos 'CC.Ar.GG' sequence competes with the cardiac actin sequence for factor binding. Our results suggest that the minimum sequence requirements for binding of the serum response factor may correspond to the 'CC.Ar.GG' box sequence. Using this criterion, we predict and confirm the existence of such a binding site in a regulatory region of the interleukin-2 receptor gene. It appears therefore that interactions between 'CC.Ar.GG' boxes and similar proteic factors could be involved in the control of different genes responding to different stimuli, e.g. muscle differentiation (cardiac actin gene) or growth stimulation (c-fos, cytoskeletal actin or interleukin-2 receptor genes).

Tyrosine kinases in normal human blood cells. Platelet but not erythrocyte band 3 tyrosine kinase is p60c-src.

The major tyrosine kinase from platelets was purified as a 51 kDa active enzyme which was shown to be a degradation product of the protooncogene product p60c-src. Immuno-depletion experiments using a monoclonal antibody recognizing p60c-src failed to remove band 3 phosphorylating activity from red blood cell membranes. The erythrocyte tyrosine kinase was not at all immunoprecipitated by this antibody under conditions where the platelet enzyme was immunoprecipitated.

Protein kinases in human leukemic cells.

Protein kinase activities and cyclic AMP binding capacity were investigated in human peripheral blood cells from leukemic patients and normal controls. Using [gamma 32P] ATP as phosphoryldonor, the phosphorylating activities were not found to be significantly different in either normal or leukemic cells when measured on both artificial basic and acidic substrates. In contrast, the GTP-dependent casein kinase activity, CK2, which is almost undetectable in normal granulocytes, was markedly increased in highly proliferating myeloblastic cells from patients with acute myelogenous leukemia (AML) or with chronic myelogenous leukemia in blastic crisis (BC-CML). Levels of endogenous phosphotyrosine were not higher in leukemic cells than in normal peripheral lymphocytes or granulocytes. Finally, cAMP binding capacity was found to be increased in several types of proliferating leukemic cells, due to a higher amount of the R1-type regulatory subunit of the cAMP-dependent protein kinases. Specific patterns of cAMP binding proteins observed in the different types of normal blood cells were rather blurred in leukemic cells. In conclusion, modifications observed in human leukemic cells seem to be more related to proliferation or blockage in normal differentiation than to their cellular origin.

Characterization of human red blood cell tyrosine kinase.

A new tyrosine kinase in human red blood cells has been characterized and partially purified. The major substrate was a protein of molecular weight 93 K which could be phosphorylated both in whole red blood cells incubated with inorganic [32P] orthophosphate and in ghost preparations incubated with [gamma 32P] ATP. This tyrosine kinase displayed an alkaline isoelectric pH (around 8.5), a molecular weight of 32-33 K and does not seem to be autophosphorylable. Some kinetics of the enzyme are reported. This red blood cell tyrosine kinase is unrelated to EGF and insulin or insulin-like receptor subunits. This enzyme may represent a novel class of tyrosine kinases.

Protein kinases in normal human blood cells.

Protein kinases active on basic and acidic artificial substrates were investigated in normal human erythrocytes, platelets, polymorphonuclear and mononuclear cells. These two types of protein kinases were partially purified by affinity chromatography, then assayed for their enzymatic activity using [gamma-32P]ATP or GTP as phosphoryl donor. Partially purified kinases active on acidic substrates were subjected to high-performance liquid chromatography (HPLC). Protein kinases active on basic substrates were analyzed by cellulose acetate electrophoresis of crude cellular extracts and the influence of 3'5' cyclic AMP was studied. Three forms of casein-phosvitin kinases could be distinguished according to their molecular weight (165 K, 38 K, and 31 K). The 165 K species, in contrast to the light species, can use GTP instead of ATP as phosphoryl donor and corresponds to the "so-called" casein kinase 2. This form is very sensitive to proteolysis and, when partial purification is performed without the addition of various antiproteolytic agents, it is degraded into 120-135 K and 105-115 K active species; this artefactual degradative process is especially active in platelet extracts. As many as eight different active bands of histone and protamine kinases can be separated by cellulose acetate electrophoresis, several of them being stimulated by cyclic AMP. Isozymic patterns of protein kinases, levels of activity on the different substrates, and utilization of ATP and GTP were found to be specific for each cell type. These results suggest the possibility of using protein kinases as markers for cell differentiation.

Membrane dynamics in human leukemia and lymphoma cells. pH dependency of diphenylhexatriene fluorescence polarization.

Membrane dynamics of human leukemia and lymphoma cell lines were analyzed by investigating the effect of pH on fluorescence polarization (P) of the lipophilic probe diphenylhexatriene (DPH). The degree of P varied as a function of pH, depending on the cell lines. These variations were not detected in phospholipid vesicles. In addition, they were prevented by treatments with glutaraldehyde, sodium azide or phenylmethylsulfanyl fluoride, a specific protease inhibitor. Therefore, these P value changes might be influenced by protein modification.

T cell subpopulations defined by monoclonal antibodies in autoimmune hemolytic anemia.

T cell subsets were studied using monoclonal anti-T cell antibodies in 10 patients with IgM cold agglutinins and 30 patients with IgG warm autoantibodies. Two of the 10 patients with cold agglutinin disease had abnormally low helper/suppressor T cell ratio. In the 30 patients with IgG warm autoantibodies this ratio was abnormally low in 7 and abnormally high in 7 other patients. Treatment by steroids or immunosuppressive agents tended to decrease OKT4/OKT8 ratio since high ratios were essentially found in untreated patients. This report documents the high incidence of T cell imbalance in autoimmune hemolytic anemia due to IgG warm autoantibodies and comments on its significance in the light of the great heterogeneity of this syndrome.

Molecular identification of human T-lymphocyte antigens defined by the OKT5 and OKT8 monoclonal antibodies.

Three human lymphocyte differentiation antigens, specific of the entire T-cell population, of the helper/inducer T-cell subset, and of the cytotoxic/suppressor T-cell subset have been identified, using mouse monoclonal antibodies obtained from Dr. P. Kung. Various T-cell populations were radio-labelled, the antigens were isolated by immunoprecipitation with the monoclonal antibodies and the resulting immune complexes subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The OKT3 antigen, present on peripheral T-lymphocytes and on functionally mature thymocytes has been identified as an oligomeric protein, composed of 23,000 mol. wt subunits. The OKT4 antigen, specific for the helper/inducer subset, is a single protein of 53,000 mol. wt. The OKT8/OKT5 antigen, defining the cytotoxic/suppressor subpopulation is composed of two subunits of 31,000 and 33,000. From co-capping experiments and biochemical data, the hypothesis is established that OKT5 recognizes a dimer of 140,000 mol. wt and OKT8 recognizes a determinant present on both the monomer 70,000 and the dimer. This hypothesis could explain the OKT5- OKT8+ phenotype of some T-cells.

Unusual phenotypes of human inducer T cells as measured by OKT4 and related monoclonal antibodies.

OKT4, a murine monoclonal antibody, was previously shown to react with inducer/helper T cells in man. We now report the absence of this reactivity in 2 subjects of African ancestry and the production of 4 new monoclonal antibodies (OKT4A-D) that detect distinct antigens on human inducer/helper T cells.

T-cell subset analysis by monoclonal antibodies in primary immunodeficiencies.

T-cell subsets have been analysed in 23 cases of primary immunodeficiency with monoclonal antibodies. Functional assays investigating T-cell function--proliferative response to mitogens, antigens, and allogeneic cells, cytotoxicity generated against allogeneic target cells and helper function to pokeweek mitogen-induced immunoglobulin synthesis--were performed in parallel in the same patients. The results enabled us to delineate four groups of patients. The first group consisted of patients in whom marker and function studies show concordant data, with either normal or strongly decreased T-cell number and function. The second group consisted of patients in whom various degrees of functional abnormality coexist with subnormal T-cell number and increased suppressor T-cell proportion. In the third group, we collected all patients who showed functional deficiencies without marker abnormalities. Finally, there was a small group composed of patients whose T-cell pattern was strongly suggestive of abnormal differentiation.