Histone gene transcription factor binding in extracts of normal human cells.

Transcriptional regulation of mammalian histone genes during S phase is achieved through activation of specific factors which interact with subtype-specific histone gene promoter sequences. It has previously been shown that in HeLa cells this induction is not mediated by obligatory changes in the DNA binding activity of histone gene transcription factors as cells progress through the cell cycle. Recently, it has been reported that the DNA binding properties of a putative histone gene transcription factor may be quite different in normal and transformed cells (J. Holthuis, T. A. Owen, A. J. van Wijnen, K. L. Wright, A. Ramsey-Ewing, M. B. Kennedy, R. Carter, S. C. Cosenza, K. J. Soprano, J. B. Lian, J. L. Stein, and G. S. Stein, Science 247:1454-1457, 1990). To determine whether the properties of well-characterized histone gene transcription factors are altered in transformed versus normal cells, we have examined the DNA binding activity of human histone transcription factors during the WI38 (a primary line of normal human fetal lung fibroblasts) cell cycle. The results demonstrate that the properties of Oct1, H4TF1, and H4TF2 are similar in WI38 and HeLa cells and that their DNA binding activities are constitutive during interphase of both normal and transformed cell lines. Although it remains possible that these factors are directly or indirectly perturbed as a result of cellular transformation, it appears unlikely that transformation results in gross changes in DNA binding activity as cells progress toward division.

Histone H1 subtype-specific consensus elements mediate cell cycle-regulated transcription in vitro.

In this study we used nuclear extracts from centrifugally elutriated cell populations to study histone H1 transcriptional regulation during the cell cycle. Analysis of mutations within the H1 promoter establish that both of the H1 subtype-specific consensus elements participate in induction of transcription upon entry into S phase. The DNA binding activity of H1TF2, which specifically interacts with the H1 proximal subtype-specific element, is increased in S-phase nuclear extracts, whereas no increase in DNA binding is observed for the H1 distal subtype-specific DNA transcription factor H1TF1 or the H2b subtype-specific factor OTF1. These data strongly support the idea that histone gene subtype-specific transcription factors are important for S-phase-dependent expression of histone genes. Further studies of these factors will be important for increased understanding of the transition from G1 to S phase of the mammalian cell cycle.

Characterization and purification of H1TF2, a novel CCAAT-binding protein that interacts with a histone H1 subtype-specific consensus element.

Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.

Cell type-specific expression of a human histone H1 gene.

The expression of a human histone H1 gene (Hh8c H1) was studied in several different human cell lines. Nuclease S1 analysis revealed that this gene is expressed at high levels in KB cells and that it is regulated during the cell cycle, but that it is not expressed in several other human cell lines (HeLa, HEK, 293, and Hep G2). The differential expression of the Hh8c H1 gene in vivo was not due to gross chromosomal rearrangements or to activation of the entire histone gene cluster at this genomic locus. Thus, the cell type-specific expression of this human H1 gene must result from either critical differences in the promoter sequences of this gene in KB and HeLa cells, or from cell-specific utilization of trans-acting transcription factors.

Differential replication of SV40 and polyoma DNAs in Chinese hamster ovary cells.

We have investigated the ability of CHO cells to allow growth of papovaviruses by analyzing viral DNA replication after transfection using the calcium-phosphate co-precipitation technique. These analyses showed that when SV40-containing plasmids were introduced into CHO cells, viral DNA replicated to a level of approximately 1000 copies per T antigen-expressing cell, and neither late proteins nor virus progeny were produced. When polyoma (Py)-containing plasmids were transfected into CHO cells, a ten-fold higher level of Py DNA was present per T antigen-positive cell, and viral capsid proteins and progeny virus were detected, indicating that CHO cells are not equally restricted for all papovaviruses. Infection with intact virions was restricted in both cases. These results indicate that either SV40 or Py DNA introduced into CHO cells are able to express their early viral functions, and that different interactions of cellular proteins involved in the replication machinery with viral nucleic acids and proteins result in different levels of viral DNA synthesis and virus progeny production. We propose that, because of their favorable genetic characteristics, CHO cells should, therefore, provide a valuable experimental system for definition of the cellular contributions to papovavirus replication.

Changes in the levels of viral and cellular gene-transcripts in the cell cycle of SV40 transformed mouse cells.

We have analyzed the regulation of transcription of integrated SV40 DNA and of five cellular genes during the cell cycle of two lines of SV40 transformed mouse 3T3 cells. These cells (ts SV3T3) are temperature sensitive for the expression of the transformed phenotype and at the nonpermissive temperature (39 degrees C) become arrested in G1 at low serum concentrations. SV40 specific RNAs are not detected either in the nuclear or in the cytoplasmic poly(A+)RNA of quiescent cells, suggesting control at the level of transcription. After serum stimulation, however, viral transcription increases and reaches its maximum during S-phase. The expression of a group of selected housekeeping genes has received parallel analysis to determine whether other cellular genes, beside the integrated SV40, are shut off in G1 arrested cells or are expressed in restricted periods of the cell cycle. We have found that, while the mRNAs for collagen, adenosinphosphoribosiltransferase (APRT) and the mouse major histocompatibility complex (H2) are present throughout the cell cycle, the genes coding for the multifunctional protein CAD and dehydrofolate reductase are cell-cycle regulated.

High mobility group proteins 1 and 2 are present in simian virus 40 provirions, but not in virions.

Viral particles at the late stages of SV40 morphogenesis were examined for the presence of HMG proteins 1 and 2, by an immunochemical method involving the transfer of proteins from polyacrylamide gels to nitrocellulose membranes. It was found that these proteins are present in SV40 provirions, in which histone H1 is still associated with viral chromatin, but absent in mature SV40 virions.

Late modifications of simian virus 40 chromatin during the lytic cycle occur in an immature form of virion.

Two main modifications of the simian virus 40 chromatin were found to occur during the lytic cycle. One was the progressive increase in the acetylation level in the four non-H1 histones as the 75S deoxynucleoprotein complexes (minichromosomes) became assembled into heavier structures. The other was the final elimination from viral chromatin of histone H1. An important stage in the course of these changes was represented by an intracellular simian virus 40 particle, in which the virus-coded proteins were already assembled, but properties distinct from those of mature virions were still present. This particle resembled the mature virions in morphology, sedimentation rate, and buoyant density. It was distinguished by the instability, the presence of histone H1, the uptake of radioactive acetate, and the lower infectivity. Its significance appears to be that of an immature virion on the basis of these characters and of the consistent kinetic behavior during the lytic cycle.