On the importance of taking a firm position. Chromatin structure and gene expression sponsored by Fundación Juan March, Madrid, Spain September 24-26, 1990.
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Animals↗
Biomedical subjects
S C Elgin
Publications and source records attributed to S C Elgin.
Optimal heat-induced expression of the Drosophila hsp26 gene requires a promoter sequence containing (CT)n.(GA)n repeats.
We report here the analysis of the sequence requirements for the heat-induced expression of the Drosophila melanogaster hsp26 gene using germline transformation. Heat-induced expression is augmented fivefold by a homopurine/homopyrimidine region from -85 to -134 that is devoid of heat-shock elements but contains numerous (dC-dT).(dG-dA) repeats. Sequences within this interval have been shown to assume a nuclease S1-hypersensitive structure in vitro. In this paper, we extend those in vitro observations, demonstrating that the S1-hypersensitive structure is triple-helical H-DNA formed by a symmetric (dC-dT).(dG-dA) sequence. Thus, the sequences that form H-DNA in vitro are also required in vivo for optimal hsp26 transcription. However, mutational analysis and diethylpyrocarbonate modification experiments in isolated nuclei suggest that the (dC-dT).(dG-dA) sequence does not form H-DNA in vivo and argue against a role for H-DNA in the heat-induced expression of hsp26.
Animals↗
Conservation of intron position indicates separation of major and variant H2As is an early event in the evolution of eukaryotes.
Genomic clones of Drosophila and Tetrahymena histone H2A variants were isolated using the corresponding cDNA clones (van Daal et al. 1988; White et al. 1988). The site corresponding to the initiation of transcription was defined by primer extension for both Drosophila and Tetrahymena genomic sequences. The sequences of the genomic clones revealed the presence of introns in each of the genes. The Drosophila gene has three introns: one immediately following the initiation codon, one between amino acids 26 and 27 (gln and phe), and one between amino acids 64 and 65 (glu and val). The Tetrahymena gene has two introns, the positions of which are identical to the first two introns of the Drosophila gene. The chicken H2A.F variant gene has been recently sequenced and it contains four introns (Dalton et al. 1989). The first three of these are in the same positions as the introns in the Drosophila gene. The fourth intron interrupts amino acid 108 (gly). In all cases the sizes and the sequences of the introns are divergent. However, the fact that they are in conserved positions suggests that at least two of the introns were present in the ancestral gene. A phylogenetic tree constructed from the sequences of the variant and major cell cycle-regulated histone H2A proteins from several species indicates that the H2A variant proteins are evolutionarily separate and distinct from the major cell cycle-regulated histone H2A proteins. The ancestral H2A gene must have duplicated and diverged before fungi and ciliates diverged from the rest of the eukaryote lineage. In addition, it appears that the variant histone H2A proteins analyzed here are more conserved than the major histone H2A proteins.
Amino Acid Sequence↗
Chromatin structure and gene activity.
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Animals↗
Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster.
We report here that a point mutation in the gene which encodes the heterochromatin-specific nonhistone chromosomal protein HP-1 in Drosophila melanogaster is associated with dominant suppression of position-effect variegation. The mutation, a G-to-A transition at the first nucleotide of the last intron, causes missplicing of the HP-1 mRNA. This suggests that heterochromatin-specific proteins play a central role in the gene suppression associated with heterochromatic position effects.
Alleles↗
UV cross-linking identifies four polypeptides that require the TATA box to bind to the Drosophila hsp70 promoter.
A protein fraction that requires the TATA sequence to bind to the hsp70 promoter has been partially purified from nuclear extracts of Drosophila embryos. This TATA factor produces a large DNase I footprint that extends from -44 to +35 on the promoter. A mutation that changes TATA to TATG interferes both with the binding of this complex and with the transcription of the hsp70 promoter in vitro, indicating that this interaction is important for transcriptional activity. Using a highly specific protein-DNA cross-linking assay, we have identified four polypeptides that require the TATA sequence to bind to the hsp70 promoter. Polypeptides of 26 and 42 kilodaltons are in intimate contact with the TATA sequence. Polypeptides of 150 and 60 kilodaltons interact within the region from +24 to +47 in a TATA-dependent manner. Both the extended footprint and the polypeptides identified by UV cross-linking indicate that the Drosophila TATA factor is a multicomponent complex.
Animals↗
Nonenzymatic cleavage of chromatin.
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Animals↗
Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila.
We have previously reported the identification of a nonhistone chromosomal protein (nhcp-19; now called HP1) preferentially associated with the heterochromatin of Drosophila melanogaster. A detailed study of the HP1 distribution pattern on polytene chromosomes by immunofluorescent staining, using monoclonal antibody C1A9, has been carried out. The results indicate that this protein is found within the centric beta-heterochromatin, in cytological regions 31, 41 and 80, and throughout polytene chromosome 4. Staining of telomeres is frequently observed, those of chromosome arms 2R and 3R and the X chromosome being the most conspicuous. Analysis of a fourth chromosome insertional translocation T(3;4)f/In(3L)P confirms an autonomous interaction with chromosome 4 material. Similarly, the beta-heterochromatin distal to light on chromosome arm 2L, moved to position 97D2 on chromosome arm 3R in the rearrangement ltx13, is prominently stained using the C1A9 antibody. Staining of intact salivary glands indicates that this rearranged segment of beta-heterochromatin is not associated with the polytene chromocenter, but provides an independent structural reference point. HP1 is not observed in the nuclei of the early syncytial embryo, but becomes concentrated in the nuclei at the syncytial blastoderm stage (ca. nuclear division cycle 10). This suggests that heterochromatin formation occurs at approximately the same stage at which nuclei first become transcriptionally competent. Thus, the C1A9 antibody may serve as a useful marker for both structural and functional studies of the Drosophila nucleus.
Animals↗
The formation and function of DNase I hypersensitive sites in the process of gene activation.
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Chromatin↗
Drosophila has a single copy of the gene encoding a highly conserved histone H2A variant of the H2A.F/Z type.
The Tetrahymena histone H2A variant designated hv1 is localized exclusively in the transcriptionally active macronucleus and is absent from the quiescent micronucleus (1). A cDNA clone of the hv1 gene (2) was used to screen a Drosophila cDNA library. A cross-hybridizing clone was recovered and shown by sequence analysis to code for a protein homologous to hv1 as well as to the chicken H2A variant, H2A.F (3), the sea urchin H2A variant, H2A.F/Z (4) and the mammalian H2A variant H2A.Z (5). Southern analysis of Drosophila genomic DNA indicates that the H2AvD (H2A variant Drosophila) gene is present in one copy. In situ hybridization places the locus at 97CD on chromosome 3, while the S-phase regulated histone genes are on chromosome 2 (6). Thus the Drosophila H2A variant should be accessible to genetic analysis, which will enable its function to be determined.
Amino Acid Sequence↗
Protein/DNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter.
Genomic footprinting on the Drosophila hsp26 promoter in isolated nuclei has shown that a TATA box binding factor is present before and after induction by heat shock, while three of the seven heat shock consensus sequences 5' of the gene are occupied (presumably by heat shock factor, HSF) specifically on heat shock. The sites of HSF interaction are separated by greater than 200 bp of which approximately 150 bp are bound to the surface of a nucleosome. The juxtaposition of these various macromolecules on the DNA suggests a basis for the major DNase I hypersensitive site 5' of hsp26 and a novel tertiary structure for the promoter complex.
Animals↗
TATA box-dependent protein-DNA interactions are detected on heat shock and histone gene promoters in nuclear extracts derived from Drosophila melanogaster embryos.
We monitored protein-DNA interactions that occur on the hsp26, hsp70, histone H3, and histone H4 promoters in nuclear extracts derived from frozen Drosophila melanogaster embryos. All four of these promoters were found to be transcribed in vitro at comparable levels by extracts from both heat-shocked and non-heat-shocked embryos. Factors were detected in both types of extracts that block exonuclease digestion from a downstream site at ca. +35 and -20 base pairs from the start of transcription of all four of these promoters. In addition, factors in extracts from heat-shocked embryos blocked exonuclease digestion at sites flanking the heat shock consensus sequences of hsp26 and hsp70. Competition experiments indicated that common factors cause the +35 and -20 barriers on all four promoters in both extracts. The formation of the barriers at +35 and -20 required a TATA box but did not appear to require specific sequences downstream of +7. We suggest that the factors responsible for the +35 and -20 barriers are components whose association with the promoter precedes transcriptional activation.
Animals↗
Isolation and distribution of a Drosophila protein preferentially associated with inactive regions of the genome.
The distribution patterns of chromosomal proteins from Drosophila can be observed by immunofluorescent staining of the polytene chromosomes from larval salivary glands. We have purified a non-histone chromosomal protein of Mr = 69,000 molecular weight which has a high affinity for DNA with little sequence specificity. Immunofluorescent staining indicates that this protein is preferentially associated with the inactive portions of the genome, including the centric heterochromatin and the condensed bands within the euchromatic arms of the chromosomes. Observation of both the heat shock loci 87A and 87C and the developmentally regulated loci 74EF and 75B shows an inverse correlation between immunofluorescent staining for the Mr = 69,000 protein and for RNA polymerase. The presence of this protein appears to be correlated with the packaging of the chromatin in an inactive form.
Animals↗
Hsp28stl: a P-element insertion mutation that alters the expression of a heat shock gene in Drosophila melanogaster.
We have identified and cloned a mutant allele of the small heat shock gene Hsp28 of Drosophila melanogaster. This allele, which we have called Hsp28stl, produces small amounts of a single aberrantly large, heat-inducible transcript in heat-shocked flies, while a normal-sized Hsp28 transcript is present only in fertile females. No Hsp28 transcript at all is detected in mutant prepupae, a stage when wildtype flies show high levels of Hsp28 RNA. We have cloned the Hsp28stl allele, and have found that a 1.3-kb defective P-element is present 5' to Hsp28 in the mutant line. The site of P-element insertion lies between the Hsp28 "TATA box" sequence and the Hsp28 RNA cap site; in contrast to previously described P-element insertions, the element at Hsp28stl is flanked by a two base pair duplication of the insertional target sequence. The results suggest that this insert may separate elements regulating heat-inducible and developmental expression of Hsp28, leading to the different patterns of transcription observed.
Alleles↗
Localization of specific topoisomerase I interactions within the transcribed region of active heat shock genes by using the inhibitor camptothecin.
Camptothecin stabilizes the topoisomerase I-DNA covalent intermediate that forms during the relaxation of torsionally strained DNA. By mapping the position of the resultant DNA nicks, we analyzed the distribution of the covalent intermediates formed on heat shock genes in cultured Drosophila melanogaster cells. Topoisomerase I was found to interact with the transcriptionally active genes hsp22, hsp23, hsp26, and hsp28 after heat shock but not with the inactive genes before heat shock. The interaction occurred predominantly within the transcribed region, with specific sites occurring on both the transcribed and nontranscribed strands of the DNA. Little interaction was seen with nontranscribed flanking sequences. Camptothecin only partially inhibited transcription of the hsp28 gene during heat shock, causing a reduced level of transcripts which were nonetheless full length. Topoisomerase I also interacted with the DNA throughout the transcriptionally active hsp83 gene, including an intron, in both heat-shocked and non-heat-shocked cells. The results point to a dynamic set of interactions at the active locus.
Animals↗
Association of topoisomerase I with transcriptionally active loci in Drosophila.
Immunofluorescence staining of the polytene chromosomes of Drosophila shows high levels of topoisomerase I associated with transcriptionally active regions. A photocrosslinking technique demonstrates the presence of topoisomerase I in the region of transcription of the active heat-shock genes. Camptothecin stabilizes the topoisomerase I-DNA covalent intermediate that forms during the relaxation of torsionally strained DNA. By mapping the position of the resultant DNA nicks, topoisomerase I is found to interact with the transcriptionally active genes hsp23, hsp26, and hsp28 after heat shock but not with the inactive genes prior to heat shock. The interaction occurs predominantly within the transcribed region, with specific sites observed on both the transcribed and nontranscribed strands of the DNA. Little interaction is seen with nontranscribed flanking sequences. Camptothecin only partially inhibits transcription of the hsp28 gene during heat shock, causing a reduced level of transcripts which are nonetheless full length. The results point to a dynamic set of interactions at the active locus.
Animals↗
Characterization of a supercoil-dependent S1 sensitive site 5' to the Drosophila melanogaster hsp 26 gene.
We have analyzed the prominent supercoil-dependent S1 nuclease cleavage site 5' to hsp 26 in the plasmid 88B13, which contains 11.7 kilobases from the Drosophila locus 67B1. The double stranded cleavage product is generated by initial nicking on the purine strand, six preferred sites occurring between positions -96 and -90 (relative to the start of transcription) with weaker ones extending to position -84, followed by cleavage on the pyrimidine strand at positions -86 and -84. A derivative of 88B13, 88B13-X, was generated by insertion of an Xho I linker at position -84; this does not affect the positions or strand specificity of the S1 cleavage in that region. A small deletion, delta 41.1, removes the homopurine/homopyrimidine stretch from positions -86 to -132 and is no longer sensitive to cleavage by S1 nuclease 5' to hsp 26. Mung bean and P1 nucleases recognize the same site 5' to hsp 26 and give the same general pattern of cleavage. All three nucleases show an initial cleavage of 88B13 DNA at this site at pH 5.5 but not at pH 6.5, indicating that the DNA structure there may be pH dependent in vitro.
Animals↗
Micrococcal nuclease as a DNA structural probe: its recognition sequences, their genomic distribution and correlation with DNA structure determinants.
We have analyzed micrococcal nuclease (MNase) DNA cleavage patterns at the sequence level by examining 2.3 X 10(3) base-pairs of data derived from the Drosophila melanogaster 44D larval cuticle locus. Within this region, MNase preferentially cleaved 140 sites. Clusters of these sites appear to generate the preferential MNase eukaryotic DNA cleavage sites seen on agarose gels at roughly 100 to 300 base-pair intervals. These clusters of preferential cleavage sites rarely occur within gene coding regions. The analysis revealed that duplex DNA sequences preferentially cleaved by MNase are generally determined by a single strand sequence: d(A-T)n, where n greater than or equal to 1, flanked by a 5' dC or dG. Cleavage of the other strand is generally staggered 5' by several nucleotides and occurs even if such sequences are absent on that strand. An empirical predictive DNA cleavage model derived from a statistical analysis of the sequence level data was applied to seven eukaryotic gene loci of known sequence. The predicted patterns were in good general agreement with the previously observed eukaryotic gene/spacer cleavage pattern. Statistical analysis also revealed that sites of predicted preferential DNA cleavage occur less frequently in protein coding regions than for randomized sequences of the same length and nucleotide content. Comparison of the MNase cleavage patterns to the sequence-dependent pattern of binding energies between duplex DNA strands indicates that MNase preferentially cleaves sequences with low helix stability.
Animals↗