Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription.

We have investigated regulatory sequences in noncoding human DNA that are associated with repression of an integrated human immunodeficiency virus type 1 (HIV-1) promoter. HIV-1 integration results in the formation of precise and homogeneous junctions between viral and host DNA, but integration takes place at many locations. Thus, the variation in HIV-1 gene expression at different integration sites reports the activity of regulatory sequences at nearby chromosomal positions. Negative regulation of HIV transcription is of particular interest because of its association with maintaining HIV in a latent state in cells from infected patients. To identify chromosomal regulators of HIV transcription, we infected Jurkat T cells with an HIV-based vector transducing green fluorescent protein (GFP) and separated cells into populations containing well-expressed (GFP-positive) or poorly expressed (GFP-negative) proviruses. We then determined the chromosomal locations of the two classes by sequencing 971 junctions between viral and cellular DNA. Possible effects of endogenous cellular transcription were characterized by transcriptional profiling. Low-level GFP expression correlated with integration in (i) gene deserts, (ii) centromeric heterochromatin, and (iii) very highly expressed cellular genes. These data provide a genome-wide picture of chromosomal features that repress transcription and suggest models for transcriptional latency in cells from HIV-infected patients.

Regulation of brain fatty acid-binding protein expression by differential phosphorylation of nuclear factor I in malignant glioma cell lines.

Brain fatty acid-binding protein (B-FABP) is expressed in the radial glial cells of the developing central nervous system as well as in a subset of human malignant glioma cell lines. Most of the malignant glioma lines that express B-FABP also express GFAP, an intermediate filament protein found in mature astrocytes. We are studying the regulation of the B-FABP gene to determine the basis for its differential expression in malignant glioma lines. By DNase I footprinting, we have identified five DNA-binding sites located within 400 base pairs (bp) of the B-FABP transcription start site, including two nuclear factor I (NFI)-binding sites at -35 to -58 bp (footprint 1, fp1) and -237 to -260 bp (fp3), respectively. Competition experiments, supershift experiments with anti-NFI antibody, and methylation interference experiments all indicate that the factor binding to fp1 and fp3 is NFI. By site-directed mutagenesis of both NFI-binding sites, we show that the most proximal NFI site is essential for B-FABP promoter activity in transiently transfected malignant glioma cells. Different band shift patterns are observed with nuclear extracts from B-FABP(+) and B-FABP(-) malignant glioma lines, with the latter generating complexes that migrate more slowly than those obtained with B-FABP(+) extracts. All bands are converted to a faster migrating form with potato acid phosphatase treatment, indicating that NFI is differentially phosphorylated in B-FABP(+) and B-FABP(-) lines. Our results suggest that B-FABP expression in malignant glioma lines is determined by the extent of NFI phosphorylation which, in turn, is controlled by a phosphatase activity specific to B-FABP(+) lines.

Differential expression of AP-2alpha and AP-2beta in the developing chick retina: repression of R-FABP promoter activity by AP-2.

Retinal fatty acid binding protein (R-FABP) is the avian counterpart of murine brain FABP implicated in glial cell differentiation and neuronal cell migration. R-FABP is highly expressed in the undifferentiated retina and brain of chick embryos. We have previously shown by in vitro studies that the AP-2 transcription factor binds to a consensus AP-2 binding site in the R-FABP promoter region. Based on the expression pattern of AP-2 in the developing retina and on mutational analysis of the AP-2 binding site in DNA transfection experiments, we proposed that AP-2 could be involved in the down-regulation of R-FABP transcription. Here, we describe the cDNA isolation of two members of the AP-2 family expressed in the chick retina, AP-2alpha and AP-2beta. We show that R-FABP mRNA and the AP-2 factors are expressed in mutually exclusive patterns in the differentiating retina: whereas AP-2alpha and AP-2beta are selectively expressed either in amacrine, or in amacrine and horizontal cells, respectively, R-FABP mRNAis found in Muller glial cells and/or bipolar cells. Furthermore, a decrease in R-FABP-dependent expression is obtained upon cotransfection of primary retinal cultures with AP-2 expression vectors and a CAT reporter construct. The early and cell-specific expression of AP-2alpha and AP-2beta in the developing retina suggest a role for this transcription factor family in the early steps of amacrine and horizontal cell differentiation. Repression of the R-FABP gene in these cells may be an important component of their developmental program.

Correlation of B-FABP and GFAP expression in malignant glioma.

The murine brain fatty acid binding protein (B-FABP) is encoded by a developmentally regulated gene that is expressed in radial glial cells and immature astrocytes. We have cloned the human B-FABP gene and have mapped it to chromosome 6q22-23. We show that B-FABP mRNA is expressed in human malignant glioma tumor biopsies and in a subset of malignant glioma cell lines, as well as in human fetal retina and brain. Malignant glioma tumors are characterized by cytoplasmic bundles of glial fibrillary acidic protein (GFAP), a protein normally expressed in mature astrocytes. Establishment of malignant glioma cell lines often results in loss of GFAP. The subset of malignant glioma cell lines that express GFAP mRNA also express B-FABP mRNA. Co-localization experiments in cell lines indicate that the same cells produce both GFAP and B-FABP. We suggest that some malignant gliomas may be derived from astrocytic precursor cells which can express proteins that are normally produced at different developmental stages in the astrocytic differentiation pathway.

Involvement of AP-2 in regulation of the R-FABP gene in the developing chick retina.

Little is known regarding the molecular pathways that underlie the retinal maturation process. We are studying the regulation of the retinal fatty-acid-binding protein (R-FABP) gene, highly expressed in retinal precursor cells, to identify DNA regulatory elements and transcriptional factors involved in retinal development. Although the upstream sequence of the R-FABP gene is extremely GC rich, CpG methylation in this region is not implicated in the regulation of this gene because the 5' flanking DNA remains unmethylated with tissue differentiation when there is a dramatic decrease in R-FABP transcript levels. Using a combination of DNase I hypersensitivity experiments, gel shift assays, and DNase I footprinting, we have found three sites of DNA-protein interaction within 205 bp of 5' flanking DNA in the undifferentiated retina and four sites in the differentiated retina. DNA transfection analysis indicates that the first two footprints located within 150 bp of 5' flanking DNA are required for high levels of transcription in primary undifferentiated retinal cultures. The first footprint includes a putative TATA box and Spl binding sites while the second footprint contains a consensus AP-2 DNA binding site. Supershift experiments using antibodies to AP-2 and methylation interference experiments indicate that an AP-2-like transcription factor present in both late-proliferative-stage retina and differentiated retina binds to the upstream region of the R-FABP gene. A combination of data including the expression profile of AP-2 during retinal development and DNA transfection analysis using constructs mutated at critical residues within the AP-2 binding site suggests that AP-2 is a repressor of R-FABP transcription.

Localization of a fatty acid binding protein and its transcript in the developing chick retina.

The undifferentiated chick retina has elevated levels of fatty acid binding protein (R-FABP) mRNA. Tissue maturation is accompanied by a 50-100-fold decrease in transcript levels. To determine the location of the R-FABP transcript and its encoded protein in the developing retina, in situ hybridization and immunohistochemical analyses were carried out using chick embryos at different stages of development. The R-FABP mRNA and protein were found throughout the retina from day 3 to day 7 of incubation. Accumulation of R-FABP in the neurites of ganglion cells could readily be detected at early developmental stages. By day 11, R-FABP transcript levels were considerably reduced in the retina, while the protein was primarily found in the inner nuclear layer, inner plexiform layer and optic nerve fiber layer of the retina. As well, R-FABP mRNA and protein were abundant in the non-pigmented ciliary epithelium, which represents the forward prolongation of the retina in the anterior eye. Immunoelectron microscopy revealed the presence of R-FABP in both the nucleus and cytoplasm of day 4 retinal cells. In the day 13 retina, R-FABP was abundant in the processes of neuronal cells. These results suggest that, early in retinal development, there is a requirement for FABP in the nucleus as well as the cytoplasm of all retinal cells. At later stages, the concentration of R-FABP in the processes of neuronal cells would suggest a biochemical or structural role related to neurite extension and synapse formation.

A human DEAD box protein with partial homology to heterogeneous nuclear ribonucleoprotein U.

In a previous report, we showed that a novel DEAD box protein-encoding gene, DDX1, was amplified in two retinoblastoma (RB) cell lines. In addition to the eight conserved motifs that characterize all DEAD box proteins, the deduced amino acid sequence of DDX1 contains a subregion with considerable homology to heterogeneous nuclear ribonucleoprotein U.

Amplification of the gene encoding the alpha-subunit of the mitochondrial ATP synthase complex in a human retinoblastoma cell line.

A cDNA clone encoding the precursor of the alpha-subunit of the human mitochondrial ATP synthase (F1-ATPS) complex was isolated from a library prepared from the poly(A)+ RNA present in a retinoblastoma (RB) cell line. Northern blot analysis of RNAs derived from a variety of transformed cell lines as well as from normal human fetal tissues indicated that RNA expression was significantly higher in two of the four RB cell lines analysed, Y79 (10- to 30-fold) and RB522A (3- to 8-fold), than in other cell lines or tissues. The increased mRNA level was apparently the result of gene amplification in Y79, but not in RB522A.