Frank Grosveld

Position-independent, high-level expression of the human β-globin gene in transgenic mice

We have constructed a minilocus that contains the 5 and 3 flanking regions of the human beta-globin locus and the beta-globin gene. These regions are characterized by erythroid-specific DNAase I-superhypersensitive sites and are normally located approximately 50 kb 5 and 20 kb 3 of the beta-globin gene. This minilocus is expressed tissue-specifically in transgenic mice at a level directly related to its copy number yet independent of its position of integration in the genome. Moreover, the expression per gene copy is the same in each mouse and as high as that of the endogenous mouse beta-globin gene. These results indicate that the DNA regions flanking the human beta-globin locus contain dominant regulatory sequences that specify position-independent expression and normally activate the complete human multigene beta-globin locus.

Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression.

The human beta‐globin dominant control region (DCR) was previously identified as a region from the 5′ end of the human beta‐globin locus which directs high level, site of integration‐independent, copy number‐dependent expression on a linked human beta‐globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta‐globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta‐globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3′ enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron.

The β-globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner

We have introduced a human beta-globin minilocus, containing the recently described dominant control region (DCR), the beta-globin or Thy-1 gene, and a thymidine kinase (tk)-neoR gene into erythroid and non-erythroid cells. Analysis of the transcription levels of the genes shows that the DCR directs high levels of human beta-globin, Thy-1 and tk-neo expression independent of integration sites in an erythroid-specific manner. The presence of the DNAasel hypersensitive sites at the 5 end of the locus is required for this effect on the homologous and heterologous gene. An analysis of the DCR chromatin in transfected mouse erythroleukemic cells suggests that the formation of the hypersensitive sites in this region precedes beta-globin gene expression.

A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region.

Single‐copy human beta‐globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the locus control region (LCR). Here we show that the 6.5 kbp microlocus LCR cassette reproducibly directs full expression from independent single‐copy beta‐globin transgenes. By testing individual DNase I‐hypersensitive sites (HS) present in the microlocus cassette, we demonstrate that the 1.5 kbp 5′HS2 enhancer fragment does not direct beta‐globin expression from single‐copy transgenes. In contrast, the 1.9 kbp 5′HS3 fragment directs beta‐globin expression in five independent single‐copy transgenic mouse lines. Moreover, the 5′HS3 core element and beta‐globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5′HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5′HS2. We postulate that the different elements of the LCR form a ‘holocomplex’ that interacts with the individual globin genes.

Regulated expression of human Aγ-, β-, and hybrid γβ-globin genes in transgenic mice: manipulation of the developmental expression patterns

We have introduced the human fetal gamma- and adult beta-globin genes into the germ line of mice. Analysis of the resulting transgenic mice shows that the human gamma-globin gene is expressed like an embryonic mouse globin gene; the human beta-globin gene is expressed (as previously shown) like an adult mouse globin gene. These results imply that the regulatory signals for tissue- and developmental stage-specific expression of the globin genes have been conserved between man and mouse but that the timing of the signals has changed. Because the two genes are expressed differently, we introduced a hybrid gamma beta-globin gene construct. The combination of the regulatory sequences resulted in the expression of the hybrid gene at all stages in all the murine erythroid tissues.

Tissue-specific control elements of the Thy-1 gene.

We have exploited the structural homology, but different patterns of expression of the murine and human Thy‐1 genes to map a number of tissue‐specific enhancer elements in the genes. All of these are located downstream from the site of transcriptional initiation. The human gene contains separate elements which direct expression to the kidney or spleen epithelium. The murine gene lacks these elements but instead contains a thymocyte specific enhancer in the third intron. Developmentally‐regulated expression in nerve cells is directed (at least in part) by an atypical element in the first intron. The latter is active on heterologous promoters, but is position and distance dependent.

Isolation of β-globin-related genes from a human cosmid library

A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150 000 recombinant-DNA-containing colonies were screened for the presence of the human beta-globin related genes. Five recombinants were isolated containing the human beta-globin locus and encompassing approx. 70 kb of human DNA.

Cloning and developmental expression of the murine neurofilament gene family

DNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a lambda gt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the entire coding region of 541 amino acids and of an NF-M cDNA that covers 219 amino acids from the internal alpha-helical region and the carboxy-terminal domains of the protein. These cDNA clones were used as probes to screen mouse genomic libraries, and cosmid clones containing both NF-L and NF-M sequences were isolated as well as overlapping cosmids containing the NF-H gene. This strongly suggests that the 3 neurofilament genes are organised in a cluster and derived by gene duplication of a common ancestral gene. RNA blot analyses using specific DNA probes for each of the genes indicate that NF mRNAs are differentially expressed during brain development. The NF-L and NF-M mRNAs are detected early in the embryonal brain, with a progressive increase in their levels during development, while the NF-H mRNA is barely detectable at embryonal stages and accumulates later in the postnatal brain.

Transcriptional regulation of multigene loci: multilevel control.

Recent studies indicate that different levels of control operate within multigene loci. In addition to regulatory sequences immediately flanking the genes, there are also elements that act over long distances on more than one gene. Competition for these elements among genes can influence both the level and timing of gene expression during development.

The two zinc finger-like domains of GATA-1 have different DNA binding specificities.

The GATA‐1 transcription factor has been shown to be important in the regulation of globin and non‐globin genes in erythroid, megakaryocytic and mast cell lineages. It is a member of a family of GATA proteins which both overlap in their expression patterns and bind the motif (A/T)GATA(A/G). The GATA family of proteins are also members of the superfamily of zinc finger‐like domain proteins and have two similar domains of the type Cys‐X2‐Cys‐X17‐Cys‐X2‐Cys which direct the DNA binding of the protein. A random oligonucleotide selection procedure has been employed to further elucidate the mechanism of GATA‐1‐DNA recognition. The resulting oligonucleotides were tested for binding activity to both wild‐type and mutant GATA‐1 proteins. Two classes of GATA‐1‐DNA interaction have been defined, the first requiring only the carboxy finger of GATA‐1 to bind and having the motif GAT(A/T), the second requiring both finger domains to bind and having the core motif (T/C)AAG. By using sequence comparison and depurination analysis it is concluded that the two finger‐like domains of GATA‐1 have different DNA binding recognition motifs. Binding of GATA‐1 to GAT(A/T) motifs is associated with transcriptional activation of linked genes. The only known (T/C)AAG motif is in the distal CAAT‐box promoter region of the human A gamma‐globin gene where the binding of GATA‐1 appears to regulate the correct developmental suppression of gamma‐globin expression.