DNase I-defined chromatin configuration of the human CD3 gene cluster.

The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.     

Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction

The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.     

Q vectors, bicistronic retroviral vectors for gene transfer

We have developed a retroviral vector that incorporates unique features of some previously described vectors. This vector includes: 3' long terminal repeats (LTRs) of the self-inactivating class; a 5' LTR that is a hybrid of the cytomegalovirus (CMV) enhancer and the mouse sarcoma virus promoter; an internal CMV immediate early region promoter to drive expression of the transduced gene and the neomycin phosphotransferase selectable marker; an expanded multiple cloning site and an internal ribosome entry site. An SV40 ori was introduced into the vector backbone to promote high copy number replication in packaging cell lines that express the SV40 large T antigen. We demonstrate that these retroviral constructs, designated Q vectors, can be used in applications where high viral titers and high level stable or transient gene expression are desirable.     

Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.     

Remodelling of the S3 PA700 proteasome activator gene chromatin structure during thymocyte differentiation

The proteasome is the major cytosolic protease, composed of a 20S catalytic core that associates with either the 19S (PA700) activator or the 11S (PA28) regulator complex. The 19S complex is thought to promote protein substrate unfolding and subsequent degradation, but precise functions for the individual subunits remain undefined. The chromatin structure and regulation of the S3 (P91A) subunit of the 19S activator was examined as a novel approach towards understanding its role in the complex. DNase I hypersensitivity (HS) analysis of S3 chromatin revealed a ubiquitous DNase I HS site mapping to the promoter region. Examination of the S3 chromatin structure in thymocytes, a dynamic population that undergo substantial proliferation, apoptosis, and differentiation, revealed an additional DNase I HS site mapping to the sixth intron of the genomic sequence. This second site was demonstrated to be associated with CD4(+)CD8(+) double-positive (DP) but not CD4(+) single-positive (SP) thymoma cell lines, and may correlate with a downregulation of S3 message. When a DP thymic cell line was induced to differentiate through retroviral transduction with Notch-1, the second DNase I HS site was dramatically diminished, illustrating that S3 chromatin is developmentally regulated during thymocyte positive selection.     

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5'' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.     

Identification of a candidate regulatory region in the human CD8 gene complex by colocalization of DNase I hypersensitive sites and matrix attachment regions which bind SATB1 and GATA-3

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.     

Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution.

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.     

Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.     

At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI- and EFII-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.     

Control of organ-specific demethylation by an element of the T-cell receptor-alpha locus control region

DNA methylation is important for mammalian development and the control of gene expression. Recent data suggest that DNA methylation causes chromatin closure and gene silencing. During development, tissue specifically expressed gene loci become selectively demethylated in the appropriate cell types by poorly understood processes. Locus control regions (LCRs), which are cis-acting elements providing stable, tissue-specific expression to linked transgenes in chromatin, may play a role in tissue-specific DNA demethylation. We studied the methylation status of the LCR for the mouse T-cell receptor alpha/delta locus using a novel assay for scanning large distances of DNA for methylation sites. Tissue-specific functions of this LCR depend largely on two DNase I-hypersensitive site clusters (HS), HS1 (T-cell receptor alpha enhancer) and HS1'. We report that these HS induce lymphoid organ-specific DNA demethylation in a region located 3.8 kilobases away with little effect on intervening, methylated DNA. This demethylation is impaired in mice with a germline deletion of the HS1/HS1' clusters. Using 5'-deletion mutants of a transgenic LCR reporter gene construct, we show that HS1' can act in the absence of HS1 to direct this tissue-specific DNA demethylation event. Thus, elements of an LCR can control tissue-specific DNA methylation patterns both in transgenes and inside its native locus.     

DNA and chromatin structure of the human alpha 1 (I) collagen gene

The human alpha 1 (I) collagen gene and 48 kilobase pairs of flanking DNA have been isolated on two overlapping cosmids. The alpha 1 (I) gene is 18 kilobase pairs long and contains a single repetitive element of the Alu family; at least 15 repetitive elements are present in the flanking DNA. Analysis of chromatin structure in nuclei isolated from cultured fibroblasts demonstrated a single chromatin domain greater than 65 kilobase pairs in length that contained 9 DNase I-hypersensitive sites. The pattern of hypersensitive sites was also determined in nuclei derived from placental tissue. Five of the DNase I-hypersensitive sites were observed in both placental and fibroblast chromatin including one site near the 5' end and another near the 3' end of alpha 1 (I). An additional two sites located near the 3' end of the alpha 1 (I) gene in fibroblast chromatin are associated with the tissue-specific use of different polyadenylation sites. Two DNase I-hypersensitive sites found only in fibroblast chromatin and one site found only in placental chromatin were located more than 10 kilobase pairs away from the alpha 1 (I) gene and may be related to tissue-specific expression of other genes in the domain. However, the only abundant placental mRNAs from the 65-kilobase pair domain were those transcribed from the alpha 1 (I) gene. These findings suggest that physical linkage does not play a predominant role in controlling coordinate expression of collagen genes.