Michael Sheffery

PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc gamma RIIIA promoter.

Expression of the low‐affinity Fc receptor for IgG (murine Fc gamma RIIIA) is restricted to cells of myelomonocytic origin. We report here the promoter structure, the proximal DNA sequences responsible for transcription of Fc gamma RIIIA in macrophages and the protein factors which interact with these sequences. A 51 bp sequence, termed the myeloid restricted region (MRR), was both necessary and sufficient for conferring cell type‐specific expression in macrophages. Reporter constructs containing mutations in this sequence result in the loss of MRR activity upon transfection into the macrophage cell line, RAW264.7. Two cis‐acting elements have been identified and are required for full promoter function. These same elements analyzed by EMSA define two binding sites recognized by nuclear factors derived from macrophages. A 3′ purine tract (‐50 to ‐39) within the MRR binds the macrophage and B cell‐specific factor, PU.1, and a second E box‐like element, termed MyE, upstream of the PU.1 box (‐88 to ‐78) binds the HLH factors TFE3 and USF. EMSA studies using RAW cell extracts suggest that both PU.1 and MyE factors may bind simultaneously to the MRR resulting in a ternary complex that is responsible, in part, for the myeloid‐specific activity of the Fc gamma RIIIA promoter.

Molecular cloning of the alpha-globin transcription factor CP2.

CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1.

Nucleosome disruption precedes transcription and is largely limited to the transcribed domain of globin genes in murine erythroleukemia cells

We used micrococcal nuclease to separate murine erythroleukemia cell (MELC) chromatin into soluble and insoluble fractions which differ in gene content and chromatin structure. Genes that are not expressed in the erythroid lineage, such as the Ig alpha and albumin genes, distribute preferentially into the soluble rather than the insoluble fraction, and are organized into nucleosomes in both fractions. Both alpha 1- and beta maj-globin genes are enriched in the insoluble fraction and are organized into structures that are partially devoid of nucleosomes in uninduced MELC, when the genes are transcriptionally inactive. Following chemical induction of MELC and the onset of globin gene transcription, globin gene enrichment and nucleosome disruption in the insoluble chromatin fraction increase. Using seven DNA subclones that span the beta maj-globin gene we show that insolubility and nucleosome disruption are largely limited to DNA sequences lying within the transcribed domain. Non-transcribed, flanking sequences are soluble and organized into nucleosomes. In addition, the globin genes found in insoluble, non-nucleosomal chromatin contain previously engaged RNA polymerases which can elongate globin RNA chains in vitro in a pattern qualitatively and quantitatively similar to intact nuclei. These results are discussed in terms of a model for globin gene activation during erythropoeisis.

Identification and Characterization of a Critical CP2-binding Element in the Human Interleukin-4 Promoter

Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells.

Purification of multiple erythroid cell proteins that bind the promoter of the alpha-globin gene.

Three erythroid cell factors that bind the murine alpha-globin promoter were enriched more than 1,000-fold by conventional and DNA sequence affinity chromatography. Visualization of enriched polypeptides revealed simple patterns suggesting that each binding activity was purified. Two of the purified proteins, alpha-CP1 and alpha-CP2, have been shown previously to interact with distinct binding sites that overlap in the alpha-globin CCAAT box. Affinity purification of alpha-CP1 revealed seven polypeptides with Mrs raging from 27,000 to 38,000. In contrast, purified alpha-CP2 was made up of a polypeptide doublet with Mrs of 64,000 and 66,000. The third purified binding activity, alpha-IRP, interacted with sequences that formed an inverted repeat (IR) between the alpha-globin CCAAT and TATAA boxes. Affinity-purified alpha-IRP was made up of a single polypeptide with an Mr of 85,000. We confirmed that the purified polypeptides corresponded to alpha-CP1-, alpha-CP2-, and alpha-IRP-binding activities by UV cross-linking experiments (alpha-CP2 and alpha-IRP) or by renaturation of binding activity after elution of polypeptides from sodium dodecyl sulfate-polyacrylamide gels (alpha-CP1 and alpha-CP2). The apparent complexity of the polypeptides accounting for alpha-CP1 binding activity prompted a further physical characterization of this factor. Sedimentation of affinity-purified alpha-CP1 in glycerol gradients containing 100 mM KCl showed that all seven polypeptides migrated as a complex that cosedimented with alpha-CP1-binding activity. In contrast, when sedimented in glycerol gradients containing 500 mM KCl, alpha-CP1 dissociated into at least two components. Under these conditions, alpha-CP1-binding activity was reduced or lost. Activity was reconstituted, however, by combining fractions that were enriched in the two components. These results were confirmed by experiments in which we showed that alpha-CP1-binding activity can be recovered only by combining distinct sets of polypeptides that were isolated and renatured from sodium dodecyl sulfate-polyacrylamide gels. Our results suggest that the seven polypeptides visualized after affinity purification of alpha-CP1 interact to form a heterotypic complex (or set of complexes) required for alpha-CP1-binding activity.

Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene.

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.

Partial purification of a nuclear protein that binds to the CCAAT box of the mouse alpha 1-globin gene.

We enriched a fraction from nuclear extracts of murine erythroleukemia cells which contains a protein able to form stable complexes with the promoter region of the alpha 1-globin gene. Binding activity, which is present in mouse brain and a variety of cultured mouse and human cell lines, is not erythroid cell specific. Binding studies with alpha-globin gene promoter deletion mutants as well as DNase I footprinting and dimethyl sulfate protection studies showed that the factor bound specifically to the CCAAT box of the alpha 1 promoter. Enriched factor preparations exhibited weak binding to the promoter region of the beta maj-globin gene. This suggests that this protein could bind differentially to these two promoters in vivo. The enriched factor may be a ubiquitous nuclear protein involved in the differential regulation of the alpha 1- and beta maj-globin genes.

Induced transcription of the mouse β-globintranscription unit in erythroleukemia cells: Time-course of induction and of changes in chromatin structure

The transcription of the β-globin genes in mouse erythroleukemia cells has beenexamined by hybridizing labeled RNA obtained from isolated nuclei after chain elongation in the presence of [α- 32 P]UTP. There is induction of at least 30-fold of β maj globin transcription after cells are treated with either dimethylsulfoxide or hexamethylene bisacetamide. The induction requires 36 to 48 hours to be maximal, during which time the cells double about three to four times. During this time, a site in the β maj DNA region becomes hypersensitive to DNase. The development of this hypersensitive site is co-ordinate with the transcriptional increase. The induced transcripts in the β-globin region are α-amanitin-sensitive (and therefore are RNA polymerase II products). An examination of weak transcriptional signals to DNA fragments upstream of the β maj globin gene in uninduced mouse erythroleukemia cells and in cells that do not make globin is also reported. The low level of hybridization to the upstream regions in uninduced erythroleukemia cells, in L cells (a fibroblast) and in a strain of erythroleukemia cells that no longer make globin are not equally sensitive to α-amanitin as in the induced signal. These experiments help define the inducible transcription unit for β maj globin mRNA production.

The expression of integrated plasmid DNA depends on copy number.

The effect of copy number, integration site, and enhancers on the expression of stably integrated exogenous DNA was examined in Chinese hamster cells. Three similar plasmids were constructed with the mouse beta maj-globin promoter fused to the galK gene either with no enhancer or with the SV40 or Harvey sarcoma virus (HaSV) enhancer. Eighteen stable cell lines were obtained and characterized with respect to plasmid copy number and galactokinase activity. At copy numbers of four or less, the enhancers showed detectable activity and a DNase I hypersensitive site was present. Above four copies, gene activity decreased as the copy number increased, the enhancer sequences were apparently inactive, and the DNase I hypersensitive site disappeared. These data suggest that, at least in this model system, when exogenous DNA is integrated as multiple head-to-tail copies, the entire multigene unit expresses poorly and inappropriately. When the same exogenous DNA integrates as a single (or low number) copy, expression appears to be relatively normal as judged by enhancer stimulation and DNase I hypersensitivity.