No abstract

An experimental assay was developed to search for proteins capable of antagonizing histone H1‐mediated general repression of transcription. T7 RNA polymerase templates containing an upstream scaffold‐associated region (SAR) were highly selectively repressed by H1 relative to non‐SAR control templates. This is due to the nucleation of H1 assembly into flanking DNA brought about by the numerous A‐tracts (AT‐rich sequences containing short homopolymeric runs of dA.dT base pairs) of the SAR. Partial, selective titration of these A‐tracts by the high mobility group (HMG) protein HMG‐I/Y led to the complete derepression of transcription from the SAR template by inducing the redistribution of H1 on to non‐SAR templates. SARs are associated with many highly transcribed regulated genes where they may serve to facilitate the HMG‐I/Y‐mediated displacement of histone H1 in chromatin. Indeed, HMG‐I/Y was found to be strongly enriched in the H1‐depleted subfraction which can be isolated from chromatin.

DNA elements termed scaffold‐associated regions (SARs) are AT‐rich stretches of several hundred base pairs which are known to bind specifically to nuclear or metaphase scaffolds and are proposed to specify the base of chromatin loops. SARs contain sequences homologous to the DNA topoisomerase II cleavage consensus and this enzyme is known to be the major structural component of the mitotic chromosome scaffold. We find that purified topoisomerase II preferentially binds and aggregates SAR‐containing DNA. This interaction is highly cooperative and, with increasing concentrations of topoisomerase II, the protein titrates quantitatively first SAR‐containing DNA and then non‐SAR DNA. About one topoisomerase II dimer is bound per 200 bp of DNA. SARs exhibit a Circe effect; they promote in cis topoisomerase II‐mediated double‐strand cleavage in SAR‐containing DNA fragments. The AT‐rich SARs contain several oligo(dA).oligo(dT) tracts which determine their protein‐binding specificity. Distamycin, which is known to interact highly selectively with runs of A.T base pairs, abolishes the specific interaction of SARs with topoisomerase II, and the homopolymer oligo(dA).oligo(dT) is, above a critical length of 240 bp, a highly specific artificial SAR. These results support the notion of an involvement of SARs and topoisomerase II in chromosome structure.

Gamma rays have been used to induce Chinese hamster ovary cell mutants in which the entire locus for dihydrofolate reductase (DHFR) has been eliminated. These mutants were isolated in two steps from a methotrexate-resistant clone (Flintoff, Davidson, and Siminovitch (1976). Somat. Cell Genet. 2, 245-262). The resistant cells contain amplified copies of a mutant dhfr gene that codes for a drug-resistant form of the enzyme. In the first step, methotrexate-sensitive mutants of the amplified line were selected. These mutants exhibit a reduced level of DHFR activity and contain a reduced number of dhfr genes. The remaining DHFR activity is methotrexate-sensitive. These mutants appear to be hemizygotes that have lost all copies of the amplified altered dhfr genes and retain one wild-type allele. In a second mutagenic step, mutants completely deficient in DHFR activity were isolated. Three of four of these mutants were the result of double deletions: they lack all traces of dhfr DNA sequences. The fourth mutant contains a deletion that extends through the 5' half of the dhfr gene. The hemizygotes for dhfr should be useful for the study of mutation at an autosomal mammalian locus without the complications of diploidy.

A series 11 gamma-ray-induced mutants at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary cells has been examined for the types of DNA sequence change brought about by this form of ionizing radiation. All 11 mutants were found to have suffered major structural changes affecting the dhfr gene. In eight of the mutants, all or part of the dhfr gene has been deleted. The extent of these deletions was examined in seven of these mutants and, for comparison, in two deletion mutants that were induced by UV irradiation. For this purpose, probes from an overlapping set of cosmids that span 210 kb of DNA in this region were used. Three of seven gamma-ray-induced mutants and one UV-induced mutant were shown to have deleted the entire 210-kb region. In the remaining mutants, endpoints ranging from within the dhfr gene to 100 kb downstream were observed. No upstream endpoints were detected, so that an upper limit on the size of these large deletions could not be assigned. Three of the 11 gamma-ray-induced mutants contained an interruption in the dhfr gene without any detectable loss of sequence. Restriction analysis of these interrupted mutants showed that at least 8-14 kb of "foreign" DNA sequence became joined to the gene at the point of disruption. Cytogenetic analysis of these mutants showed that in two cases an inversion of the banding pattern on chromosome Z-2 had taken place. The inverted dhfr mutants contain very low amounts of dhfr RNA sequences, and the 5' end of an inversion mutant gene exhibits the same pattern of DNA methylation and DNase I-hypersensitivity as the wild-type gene. Our results suggest that ionizing radiation causes primarily, if not exclusively, large deletions and inversions in mammalian cells.

No abstract

Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer–promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest. Here, we combined chromatin immunoprecipitations and computational approaches to break down the binding signature of the Drosophila boundary element–associated factor (BEAF) subfamily. We identify a dual-core BEAF binding signature at 1,720 sites genome-wide, defined by five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. Dual-cores are tightly linked to hundreds of genes highly enriched in cell-cycle and chromosome organization/segregation annotations. siRNA depletion of BEAF from cells leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses in BEAF-depleted cells show that BEAF controls the expression of dual core–associated genes, including key cell-cycle and chromosome segregation regulators. beaf mutants that impair its insulating function by preventing proper interactions of BEAF complexes with the dual-cores produce similar effects in embryos. Chromatin immunoprecipitations show that BEAF regulates transcriptional activity by restricting the deposition of methylated histone H3K9 marks in dual-cores. Our results reveal a novel role for BEAF chromatin dual-cores in regulating a distinct set of genes involved in chromosome organization/segregation and the cell cycle.

Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occwrs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G+C content and lack of DNA methylation. This region also includes a 214.base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312: [409][410][411][412][413] 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.The mammalian dihydrofolate reductase gene product (DHFR) is one of a group of S-phase-responsive enzymes important for DNA replication. Like other enzymes involved in DNA synthesis, DHFR activity is greater in proliferating cells than in quiescent cells (34). DHFR catalyzes the NADPH-dependent reduction of folate to dihydrofolate and then to tetrahydrofolate. Reduced folates are essential cofactors in the biosynthesis of glycine, purine nucleotides, and thymidylic acid. The de novo synthesis of the DNA precursor thymidylic acid during S phase is the major tetrahydrofolate-consuming reaction, and the cellular requirement for DHFR is highest at this time (34). However, even in rapidly proliferating cells, sufficient DHFR catalytic activity can be maintained by as few as 10 to 20 copies of mRNA per cell (32).Genes coding for low-abundance mRNAs represent the major portion of RNA polymerase II-specific genes that are expressed in any given cell type. Our understanding of the regulation of such genes is only just beginning. Some of the proteins encoded by low-copy mRNAs are tissue-specific enzymes and regulatory proteins involved in metabolic activities unique to a particular cellular phenotype. Others are proteins expressed to some extent in all cells and are commonly known as housekeeping gene products. The dihydrofolate reductase (dhfr) gene is referred to as a house-* Corresponding author. keeping gene because DHFR plays a role in several fundamental biosynthetic reactions, but the ternlinology is not meant to indicate that dhfr gene expression is uniform in all cells or at all times in a given cell. Housekeeping genes do show physiological and tissue-specific variations in expression. The development of cell lines with amplified dhfr genes has greatly facilitated the cloning and characterization of several...