Biomedical subjects
Publications and source records attributed to N Heintz.
Specific DNA-binding and in vitro transcription activities of H4TF-1 and H4TF-2 are inactivated by chelating agents. Binding activity is restored by addition of Zn2+, and H4TF-2 is also reactivated by Fe2+. In contrast, preformed factor-DNA complexes are resistant to chelators. Therefore, metal ions are a required component of the H4TF-1 and H4TF-2 DNA-binding domains.
We have previously shown that the human 70-kilodalton heat shock protein gene (hsp70) is induced by the adenovirus E1A gene product and during the S-G2 phase of the cell cycle. In this study, we investigated the effect of E1A on the expression of other human hsp genes. A gene encoding one form of the hsp89 protein (hsp89 alpha) was activated during an adenovirus infection with kinetics similar to those of activation of hsp70. The induction required a functional E1A gene. However, the hsp89 transcript was not cell cycle regulated. Genes encoding another form of hsp89 and the hsp27 protein were not induced by E1A or during the cell cycle. Further examination of hsp70 expression revealed a greater complexity than previously seen. S1 nuclease analysis using an hsp70 cDNA as well as a distinct hsp70 genomic clone demonstrated three related hsp70 transcripts; two were induced by E1A, and one was not. Both of the E1A-inducible genes were regulated during the cell cycle. All three were induced by heat shock. These results suggest common aspects of control among certain members of this family of cellular genes distinct from heat shock control. Finally, using viruses that express the individual E1A proteins, we found that the hsp70 gene is induced by the 12S and the 13S E1A products. The efficiency of induction by the 12S product was somewhat less than that by the 13S product but only by a factor of less than 2. This is in contrast to the induction of early viral genes, for which the 13S product is considerably more efficient than the 12S product.
Two proteins specifically binding to separate regions of the human histone H4 promoter were identified in nuclear extracts prepared from synchronized S-phase HeLa cells. Competition experiments with H4 promoter mutants and DNase protection assays ("footprinting") demonstrate that these factors bind to regions of the H4 promoter that are essential for maximal expression in vitro. One of these factors (H4TF-1) binds to sequences between -80 and -110 base pairs upstream of the H4 cap site, whereas the other (H4TF-2) binds to the H4 subtype-specific sequence element immediately upstream from the "TATA" homology. Neither of these activities can efficiently bind to any of the other histone gene subtypes or simian virus 40 DNA. Binding of H4TF-1 to the distal region of the pHu4A histone H4 promoter is inhibited competitively with varying efficiency by four of six human histone H4 genes cloned in this laboratory, whereas efficient competition for binding of H4TF-2 is exhibited by five of the six H4 genes. Since both of these factors bind to significant regions of the pHu4A histone H4 promoter and can be bound by several different human H4 genes, we believe that they are important for maximal transcription of the gene and that they may be involved in its regulated expression during the cell cycle.
As part of our studies on the cell cycle regulation of human histone gene expression, we examined the elements governing transcription of a human histone H2B gene in nuclear extracts derived from human HeLa cells. Circular templates were transcribed at 5- to 10-fold higher levels than were linear templates. A series of deletion, linker-substitution, and point mutants defined cis-acting promoter sequences that were recognized in nuclear extracts. These sequences extended from 118 to 21 base pairs 5' to the transcription initiation site. Elements recognized included (from 5' to 3') a series of direct repeats, a CCAAT homology, a human histone-specific hexamer, an H2B consensus element, and a TATA box. Sequence elements 5' to the hexamer were required for its function. In contrast, the H2B consensus element could function independently of more-5' promoter elements and in turn was essential for the function of upstream elements. An interesting feature of this consensus is that its core octanucleotide (ATTTGCAT) is found in several nonhistone genes. By comparison with functional elements in an H4 promoter, we infer that a combinatorial interaction of general and gene-specific factors may contribute to the S-phase elevation of H2B transcription.
Mouse L cells containing integrated copies of a human histone H4 gene have been obtained by cotransfection with the herpesvirus thymidine kinase gene. Nuclease S1 assays of RNA from several independent cell lines show that the expression of the introduced H4 gene is regulated during the cell cycle. One of these cell lines (line 6-8) contains more than 60 human H4 gene copies per haploid genome and does not express the endogenous mouse histone H4 mRNA. In contrast, the expression of the mouse H2a and H3 mRNAs in this cell line is not perturbed. In cell revertants that have lost the majority of the human H4 gene copies, the expression of the mouse H4 mRNA is restored, demonstrating that the mouse genes remain functional although not expressed. The rate of transcription of the histone H4 genes in clone 6-8 is at least 10-fold greater than that of the parental cell line and it is regulated during traversal of the cell cycle. These results show that the expression of mammalian histone H4 genes involves both a trans-acting transcriptional regulatory factor and an H4-specific activity. We propose that cell cycle regulation of histone gene expression may be effected through subtype-specific transcriptional regulatory proteins.
We have examined the nucleotide sequences necessary for transcription of a human histone H4 gene in vitro. Maximal transcription of the H4 promoter requires, in addition to the TATA box and cap site, promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site. These distal promoter elements are recognized preferentially in extracts from synchronized S-phase HeLa cells. The inability of non-S-phase nuclear extracts to recognize the H4 upstream sequences reflects a specific lack of a transcription factor which interacts with those sequences. These results indicate that the cell cycle regulation of human histone gene expression involves both a specific transcription factor and distal transcription signals in the H4 promoter.
The gene encoding the human 70-kilodalton heat shock protein (HSP70) is subject to activation by the adenovirus E1A gene product and appears to be regulated in the absence of heat shock by a cellular activity similar to E1A. Given the relation of E1A to alteration of growth control, we have investigated the expression of the HSP70 gene during the cell cycle. Assay of mRNA levels after release from a thymidine-aphidicolin block revealed a 20-fold increase in mRNA abundance, reaching a peak level in the post-S-phase period. Upon reaching this peak level, the abundance of the mRNA then declined as the cells entered the next cycle. Control of the abundance of the mRNA during the cell cycle appeared to be primarily at the level of transcription as measured in nuclear runoff assays. Very similar results were obtained by analyzing the expression of the HSP70 gene in the adenovirus-transformed 293 cell line. Furthermore, the E1A gene was also found to be cell cycle regulated; the activation and peak level of the E1A mRNA occurred at an earlier time than those of the heat shock mRNA, consistent with, but not proof of, the hypothesis that E1A is responsible for the cell cycle control of the HSP70 expression. We therefore suggest that the E1A-like cellular activity may govern certain aspects of cell cycle transcription.
Nuclear extracts were prepared from synchronized HeLa cells at various times during the cell cycle and assayed for the ability to transcribe several cellular and viral genes. The efficiency of transcription of a human histone H4 gene is 3- to 10-fold greater in nuclear extracts from S phase nuclei than in extracts from non-S phase cells. In contrast, the adenovirus virus type 2 (Ad2) major late promoter is utilized 3- to 20-fold more efficiently in nuclear extracts from non-S phase cells. Transcription of other genes, including a human histone H3 and the simian virus 40 late transcription unit, is equally efficient in S and non-S phase extracts. Mixing experiments demonstrate that the rate-limiting activities for histone H4 and Ad2 major late transcription function independently and that the effects of these activities are additive. Competition studies suggest that the H4-specific transcription activity can be sequestered by preincubation with the H4 template DNA. These data support the concept that cell cycle regulation of human histone gene transcription may depend in part on soluble transcription activities that are modulated during the cell cycle. Further, in addition to the H4-specific transcription activity, there may exist other transcription factors whose activity can fluctuate according to the cell cycle or according to the growth state of the cells.
We have examined the effects of protein synthesis inhibition on histone gene expression during the HeLa cell cycle. Histone mRNAs, which normally are rapidly degraded in the absence of DNA synthesis, persist and increase in concentration when translation is inhibited before DNA replication is halted. This is not a function of polysomal shielding of these mRNAs from active degradation mechanisms since inhibitors of translation initiation alone effect stabilization and induction. The superinduction of histone mRNAs by protein synthesis inhibition is effective at the G1/S border, and in the S-phase and non-S-phase periods of the cell cycle. However, the relative increase in histone mRNA is greater when cells not synthesizing DNA are treated with a protein synthesis inhibitor than when S-phase cells are so treated. Non-histone mRNAs examined are not superinduced by translation inhibition. Transcription rates from both histone and non-histone genes increase after protein synthesis inhibition. Although the decrease in histone gene transcription associated with DNA synthesis inhibition is prevented and reversed by protein synthesis inhibition, we have no evidence that histone gene-specific transcriptional regulation is dependent on protein synthesis. Transcriptional increases may contribute to the superinduction effect but cannot explain its differential extent during the cell cycle, since these increases are similar when replicating or nonreplicating cells are treated with a protein synthesis inhibitor. We believe that changes in histone mRNA stability can account for much of the differential superinduction effect. Our results indicate a requirement for continuing protein synthesis in the cell cycle regulation of histone mRNAs.
The complete nucleotide sequence of four human histone genes has been determined. Each gene codes for a core histone protein which is very homologous with the corresponding calf thymus of rat histones. The 5' and 3' flanking regions of the human histone genes contain previously identified concensus sequences: the TATA box, the GACTTC element; the CCAAT sequence; the 3' terminal dyad symmetry element thought to be involved in transcription termination; and a recently identified H2b specific upstream sequence. A putative H2a specific upstream sequence 5'-TTCTTGGACTCCTCTTTTC-3' is present approximately 40 base pairs upstream from the TATA box in the human H2a gene promoter. Nuclease S1 analysis of the human histone mRNAs encoded within each of these clones demonstrates that the mRNA terminii map to the expected positions relative to the known concensus sequences, and that the abundance of each mRNA is regulated during the HeLa cell cycle. Finally, in contrast to the H2b, H3 and H4 mRNAs encoded within clones pHh 4A/pHh4C, pHh5B and pHu4A, respectively, the H2a mRNA encoded by Hh5G is not present in human placental RNA.
Human DNA was denatured, annealed to low C0t, and fractionated by centrifugation to equilibrium on gradients of NaI. Three well-defined zones resulted. The topmost, least dense zone contained satellite DNA. The bottom zone contained molecules bearing infrequent sequences and short repeated DNA. The middle zone contained molecules bearing infrequent sequences, short repeated DNA, and long repeated DNA. The NaI pattern was independent of single-strand DNA chain length from 5 to 30 kilobases. Structural genes were found in the organization bearing long repeats.
We have analyzed the kinetics of accumulation of each of the individual core histone mRNAs throughout the HeLa cell cycle in cells synchronized by sequential thymidine and aphidicolin treatments. These analyses showed that during the S phase there was a 15-fold increase in the levels of histone mRNAs and that this resulted from both an increased rate of synthesis and a lengthening of the half-life of histone mRNAs. A comparison of the kinetics of accumulation of histone mRNA in the total cellular and nuclear RNA populations suggested an increased transcription rate through the S phase. Within 30 min after the inhibition of DNA synthesis in mid-S phase, the steady-state concentration and the rate of synthesis of histone mRNA each declined to their non-S-phase levels. Reactivation of histone mRNA accumulation could occur even after an extended mid-S-phase block in DNA synthesis. These results suggest that the mechanisms responsible for histone mRNA synthesis are not restricted to the G1/S boundary of the HeLa cell cycle, but can operate whenever DNA synthesis is occurring.
There are six classes of SPO1 transcripts which are, at least partially, regulated independently of each other. Analysis of proteins made in infections by phage mutants defective in DNA synthesis, or in genes which positively control transcription, permitted each protein to be assigned to one transcription class. Most of the late proteins belong to transcription class m2l. There seem to be few, if any, phage proteins in the l class whose mRNA synthesis depends absolutely on phage DNA synthesis, UV irradiation of host cells allowed the detection of many additional early proteins. The early proteins detected in vivo were compared with proteins synthesized in vitro, using bacterial or gp28 phage-modified RNA polymerase in an Escherichia coli cell-free system. Proteins characterized in vivo as belonging to the e transcription class could be made efficiently in vitro only when transcription was performed by bacterial RNA polymerase. em proteins could be elicited through the use of either bacterial or gp28 polymerase, indicating that their genes can be transcribed in either the early or the middle mode.
We have isolated five clones containing human histone genes from a human genomic DNA library, using a cloned X. laevis histone H4 cDNA probe (pX1ch4). Each genomic clone has been mapped and the locations of the histone genes have been determined by blot hybridization and hybridization-selection of human histone mRNA. In contrast to the organization of previously characterized histone genes, the human histone genes are clustered in the genome but are not arranged into recognizable repeating units. The extreme lack of organization of the human histone genes may reflect the diminished requirement for rapid synthesis of large quantities of histone proteins during mammalian early development.
The Xenopus laevis histone genes have been isolated from a bacteriophage lambda library using a cloned cDNA probe to X. laevis H4 mRNA (pX1ch4). Their structural organization has been determined by restriction mapping, blot hybridization and hybridization selection and translation of Xenopus histone mRNAs. They are clustered and probably tandemly arranged but, in contrast to invertebrate histone genes (Kedes, 1979), there has been extensive sequence divergence in the spacer regions of the clusters. Also, the order of the genes within individual clusters is not conserved. We report the isolation of two variant histone H1 gene and find that the order of the nucleosomal core histone genes within a cluster containing an H1B gene is different from that found in two clusters containing the H1A gene.
Rat hepatocyte cultures (RL-PR-C) were tested with the (+)- or (-)-trans-7,8-dihydrodiol isomers of benzo(a)pyrene [B(a)P] and were assessed for growth inhibition, chromosomal damage, growth in soft agar, and tumor formation. Because early-passage cells have a noninducible low specific activity, aryl hydrocarbon hydroxylase parallel inhibition studies were performed on aryl hydrocarbon hydroxylase-inducible late-passage cultures. Early-passage cells exhibited little inhibition in the presence of B(a)P or either isomer. Comparable later-passage cells demonstrated inhibitory effects with B(a)P and the (+)- and (-)-trans-7,8-dihydrodiol metabolites. No treated cultures grew in soft agar, and the modal number of chromosomes was unaffected by carcinogen treatment. However, both (+) and (-) isomer-treated early-passage cells formed tumors in isogeneic animals, the (+) isomer being more efficient in this regard. These results indicate that noninhibitory doses of either the (+)- or (-)-trans-7,8-dihydrodiol isomer of B(a)P are nonetheless capable of malignantly transforming hepatocyte cells in vitro.
We have tested the sensitivity of a cloned rat hepatocyte line, RL-PR-C, to aflatoxin B1 and benzo(a)pyrene as a function of population-doubling level. The cells were much more sensitive to the cytotoxic action of these agents subsequent to 230 population doublings. This sensitivity corresponded to the enhanced inducibility of aryl hydrocarbon hydroxylase activity by 3-methylcholanthrene.
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