Cecelia D. Trainor

A palindromic regulatory site within vertebrate GATA-1 promoters requires both zinc fingers of the GATA-1 DNA-binding domain for high-affinity interaction.

GATA-1, a transcription factor essential for the development of the erythroid lineage, contains two adjacent highly conserved zinc finger motifs. The carboxy-terminal finger is necessary and sufficient for specific binding to the consensus GATA recognition sequence: mutant proteins containing only the amino-terminal finger do not bind. Here we identify a DNA sequence (GATApal) for which the GATA-1 amino-terminal finger makes a critical contribution to the strength of binding. The site occurs in the GATA-1 gene promoters of chickens, mice, and humans but occurs very infrequently in other vertebrate genes known to be regulated by GATA proteins. GATApal is a palindromic site composed of one complete [(A/T)GATA(A/G)] and one partial (GAT) canonical motif. Deletion of the partial motif changes the site to a normal GATA site and also reduces by as much as eightfold the activity of the GATA-1 promoter in an erythroid precursor cell. We propose that GATApal is important for positive regulation of GATA-1 expression in erythroid cells.

A GATA box in the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors and sites that regulate this gene.

ABSTRACT A region located at kbp −3.9 to −2.6 5′ to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenousGATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL−/− embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.

The N-terminal fingers of chicken GATA-2 and GATA-3 are independent sequence-specific DNA binding domains.

The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N‐terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C‐terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N‐terminal finger of GATA‐1, those of GATA‐2 and GATA‐3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N‐terminal finger. The absence of one of these near the GATA‐1 N‐terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N‐terminal finger may help distinguish GATA–2 and GATA‐3 from GATA‐1 and the other GATA family members in their selective regulatory roles in vivo.

Methylation of human T-cell leukemia virus proviral DNA and viral RNA expression in short- and long-term cultures of infected cells

Leukemic peripheral blood lymphocytes from individuals infected with the human T-cell leukemia/lymphoma virus (HTLV) were found to express little or no viral RNA before being put into tissue culture. Within 24-48 hr, viral RNA expression increased at least four- to eightfold. Established HTLV-infected cell lines constitutively express viral RNA. Southern blots of DNA from HTLV-infected cells digested with the methylation-sensitive restriction enzyme HpaII showed that the proviral DNA was methylated in all of the uncultured peripheral blood cells tested. In contrast, no proviral methylation was detected in any of the cell lines examined, suggesting a functional correlation between methylation and viral RNA expression. However, DNA from HTLV-infected lymphocytes cultured for 48 hr (by which time increases in viral RNA expression are evident) did not differ detectably with respect to proviral DNA methylation from uncultured cells, suggesting that the increase in viral RNA expression after short-term culture is mediated by mechanisms independent of changes in DNA methylation.

Identification of the human T cell lymphoma virus in B cell lines established from patients with adult T cell leukemia.

Cell lines were established from the peripheral blood of two patients with adult T cell leukemia. In contrast to our previous experience, where all such lines expressed T cell markers, these two cell lines expressed B cell antigens and Ig light chains (kappa on CF-2, lambda on HS). Human T cell lymphoma proviral (HTLV) sequences were demonstrated in both cell lines. Since only a portion of the cells in culture expressed Ig light chains, experiments were carried out to exclude the possibility that the cultures were not a mixture of B and T or non-B cells. Cells that expressed kappa- or lambda-light chains were separated by cell sorting from kappa- or lambda-negative cells and replaced in culture. Light chain negative cells reexpressed light chains after time in culture. After 5-azacytidine treatment of the cell lines, all cells expressed Ig light chains. These studies show that the human retrovirus HTLV, which has been demonstrated to be associated with certain T cell malignancies, can infect B cells or B cell precursors.

GATA-1 Self-association Controls Erythroid Development in Vivo

GATA-1 is the key transcription factor for the development of the erythroid, megakaryocytic, eosinophilic, and mast cell lineages. GATA-1 possesses the ability to self-associate, and this characteristic has been suggested to be important for GATA-1 function. To elucidate the roles self-associated GATA-1 plays during hematopoietic cell development in vivo, in this study we prepared GATA-1 mutants in which three lysine residues potentially contributing to the self-association (Lys-245, Lys-246, and Lys-312) are substituted in combination with alanines. Of the mutants, 3KA harboring alanine substitutions in all three lysines showed reduced self-association activity without considerable interference in the modification of GATA-1 by acetylation. We generated transgenic mouse lines that express these GATA-1 mutants utilizing the Gata1 hematopoietic regulatory domain, and crossed the mice to Gata1 knockdown (GATA-1.05) mutant mice. Although NKA (K245A and K246A) and CKA (K312A) mutants almost fully rescued the GATA-1.05 mice from anemia and embryonic lethality, the 3KA mutant only partially rescued the GATA-1.05 mutant mice. Even with the higher than endogenous level expression, GATA-1.05/Y::3KA embryos were prone to die at various stages in mid-to-late gestation. Live birth and an anemic phenotype were restored in some embryos depending on the expression level of the 3KA transgene. The expression of the transferrin receptor and heme biosynthesis enzymes was impaired in the yolk sac and liver of the 3KA-rescued embryos. Immature erythroid cells with insufficient expression of the transferrin receptor accumulated in the livers of 3KA-rescued embryos. These results provide the first convincing line of evidence that the self-association of GATA-1 is important for proper mammalian erythroid development in vivo.

DNA methylation and expression of HLA-DR alpha.

B-cell lines established from two individuals with T-cell acute lymphocytic leukemia (T-ALL) express HLA-DR antigens, whereas the isogenic T-cells do not. The lack of expression correlates with a lack of detectable HLA-DR mRNA. All of the DR alpha DNA sequences detected by a cloned DR alpha cDNA probe are contained in a BglII fragment which varies slightly in size (4.0 to 4.8 kilobases) from one individual to another. In DNA from the T-cells not expressing DR alpha mRNA, all of the potential HpaII sites within the BglII fragment appeared to be methylated. In contrast, at least some of these sites were not methylated in DNA from the B-cells expressing high levels of DR alpha mRNA. Treatment of these T-cells with 5-azacytidine resulted in the induction of DR surface antigen expression, the appearance of DR alpha mRNA, and the partial demethylation of the DR alpha DNA sequences. T-cell lines established from human T-cell leukemia-lymphoma virus associated T-cell neoplasias, in contrast to the T-cell acute lymphocytic leukemia cell lines, expressed both DR antigens and DR alpha mRNA; the HpaII sites within the BglII fragment of DR alpha DNA of these human T-cell leukemia-lymphoma virus-positive T-cell lines were in all cases at least partially unmethylated. Uncultured peripheral blood T-cells from human T-cell leukemia-lymphoma virus-infected individuals expressed DR antigens at a low level, and the DR alpha locus was partially unmethylated. After 48 h in culture, DR antigen expression was substantially increased, but no significant changes were observed in methylation of the DR alpha locus or in the amount of DR mRNA which was present. This suggests that expression of DR antigens also can be modulated post-transcriptionally.

Precipitation of nucleic acids with cetyltrimethylammonium bromide: A method for preparing viral and cellular DNA polymerase products for cesium sulfate density gradient analysis

Abstract Precipitation with cetyltrimethylammonium bromide followed by precipitation with ethanol:salt provides a quick, simple, and effective method for preparing labelled nucleic acid products of DNA polymerase reactions for analysis by Cs 2 SO 4 equilibrium density gradient centrifugation. Results obtained using this treatment show that this approach is superior to other methods of preparing products of endogenous DNA polymerase reactions from relatively crude particulate fractions such as disrupted virions.

Association analysis indicates that a variant GATA-binding site in the PIK3CB promoter is a Cis-acting expression quantitative trait locus for this gene and attenuates insulin resistance in obese children.

OBJECTIVE—In search of functional polymorphisms associated with the genetics of insulin resistance, we studied a variant in the promoter of PIK3CB, the gene coding for the catalytic p110β subunit of phosphatidylinositol (PI) 3-kinase, a major effector of insulin action. RESEARCH DESIGN AND METHODS—The rs361072 C/T variant was selected among single nucleotide polymorphisms of the PIK3CB region because we suspected that its common C allele (allelic frequency ∼50% in Europeans) could create a GATA-binding motif and was genotyped in five cohorts of obese (n = 1,876) and two cohorts of nonobese (n = 1,490) European children. To estimate insulin resistance in these children, the homeostasis model assessment for insulin resistance (HOMA-IR) index was measured in strict nutritional conditions. GATA-binding and functional effects of rs361072 were explored in transfected cell lines and in lymphocytes from obese children. RESULTS—The rs361072 C/T variant was associated with HOMA-IR in the obese children cohorts (1.7 × 10−12 < P < 2 × 10−4 for C/C vs. T/T using regression analysis). HOMA-IR averaged 3.3 ± 0.1 in C/C and 4.5 ± 0.2 in T/T obese children (P = 4.5 × 10−6 by ANOVA). C/T patients had intermediate values. As shown by the interaction between BMI and genotype (P = 2.1 × 10−9), the association of rs361072 with HOMA-IR depended on BMI and was only marginal in nonobese children (P = 0.04). At the molecular level, the C allele of rs361072 was found to create a GATA-binding site able to increase transcription of PIK3CB. CONCLUSIONS—We postulate that the C allele of rs361072 is a causal variant capable of attenuating insulin resistance in obese children through increased expression of p110β.

Developmental regulation of globin gene expression.

Summary We have used the globin family of genes in chicken to study developmental regulation of gene expression, both at the level of individual interaction of trans-acting factors with local promoters and enhancers, and at the level of chromatin structure. Regulation of all members of the α- and β-globin clusters is affected by the erythroid regulatory factor GATA-1. Separate mechanisms exist for regulation of individual members of the family. As an example, we describe the control mechanisms that play a role in the expression of the ρ-globin gene, which is expressed only in primitive lineage erythroid cells. In addressing the involvement of chromatin structure in gene activation, we have examined the role of locus control elements, and also considered the way in which RNA polymerase molecules might accommodate to the presence of nucleosomes on transcribed genes.