Life with nucleosomes: chromatin remodelling in gene regulation.

In the past year, the role of chromatin has emerged at the forefront of transcription research. Discovery and characterisation of the chromatin modifying machinery have significantly advanced our understanding of the molecular activities that establish a transcriptionally competent substrate in vivo, and have underscored the importance of the part played by chromatin in the regulation of transcription.

Transcription factors vs nucleosomes: regulation of the PHO5 promoter in yeast.

Activation of the Saccharomyces cerevisiae PHO5 gene is accompanied by the disruption of four positioned nucleosomes at the promoter. The chromatin transition requires a DNA-binding protein, Pho4, and its transactivation domain. The mechanism of nucleosome disruption and the contribution of the nucleosomes to PHO5 regulation are reviewed.

Regulation of gene expression by nucleosomes.

During the past year, the characterization of mechanisms and factors capable of disrupting nucleosomes during transcriptional activation has been a recurrent theme in studies which address the contribution of nucleosome structure to gene regulation. In vivo studies using yeast and Drosophila together with biochemical purification schemes using nucleosome perturbation assays have provided evidence for the existence of multiprotein complexes that are able to alleviate nucleosome repression. At the same time, new insights into the mechanism of heterochromatin formation have been gained, which have direct links to nucleosome structure.

Histones, nucleosomes and transcription.

It is becoming increasingly clear that nucleosome structure is integrally involved in gene regulation. In particular, the study of inducible genes has shown that nucleosomes not only contribute to a repressed basal state, but can also be rearranged in response to induction. The mechanism of this process is just beginning to be elucidated, and genetic studies have implicated several proteins in the modulation of nucleosome structure.

The chromatin structure at the promoter of a glyceraldehyde phosphate dehydrogenase gene from Saccharomyces cerevisiae reflects its functional state.

The chromatin structure of TDH3, one of three genes encoding glyceraldehyde phosphate dehydrogenases in Saccharomyces cerevisiae, was analyzed by nuclease digestion. A large hypersensitive region was found at the TDH3 promoter extending from the RNA initiation site at position -40 to position -560. This hypersensitive domain is nucleosome free and includes all putative cis-acting regulatory DNA elements. It is equally present in cells grown on fermentable as well as nonfermentable carbon sources. In a mutant which lacks the trans-activating protein GCR1 and which as a consequence expresses TDH3 at less than 5% of the wild-type level, the chromatin structure is different. Hypersensitivity between -40 and -370 is lost, due to the deposition of nucleosomes on a stretch that is nucleosome free in wild-type cells. Hypersensitivity is retained, however, further upstream (from -370 to -560). A similarly altered chromatin structure, as in a ger1 mutant, is found in wild-type cells when they approach stationary phase. This is the first evidence for a growth-dependent regulation of the TDH3 promoter.

The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.

The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PHO4 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASp1) and the other located at -239 to -262 (UASp2). Exonuclease III footprint experiments revealed stops at -349 and -368 (UASp1) as well as at -245 and -260 (UASp2). Gel retardation assays with the PHO2 protein revealed a binding region that lay between the two PHO4-binding sites. DNase I footprint analysis suggested a PHO2-binding site covering the region between -277 and -296.

Cooperative Pho2-Pho4 interactions at the PHO5 promoter are critical for binding of Pho4 to UASp1 and for efficient transactivation by Pho4 at UASp2.

The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes to PHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

Requirements for chromatin modulation and transcription activation by the Pho4 acidic activation domain.

Perhaps the best characterized example of an activator-induced chromatin transition is found in the activation of the Saccharomyces cerevisiae acid phosphatase gene PHO5 by the basic helix-loop-helix (bHLH) transcription factor Pho4. Transcription activation of the PHO5 promoter by Pho4 is accompanied by the remodeling of four positioned nucleosomes which is dependent on the Pho4 activation domain but independent of transcription initiation. Whether the requirements for transcription activation through the TATA sequence are different from those necessary for the chromatin transition remains a major outstanding question. In an attempt to understand better the ability of Pho4 to activate transcription and to remodel chromatin, we have initiated a detailed characterization of the Pho4 activation domain. Using both deletion and point mutational analysis, we have defined residues between positions 75 and 99 as being both essential and sufficient to mediate transcription activation. Significantly, there is a marked concordance between the ability of mutations in the Pho4 activation domain to induce chromatin opening and transcription activation. Interestingly, the requirements for transcription activation within the Pho4 activation domain differ significantly if fused to a heterologous bHLH-leucine zipper DNA-binding domain. The implications for transcription activation by Pho4 are discussed.

Analysis of the promoter of the human prostatic acid phosphatase gene.

From the analysis of two overlapping cosmid clones prepared from human genomic DNA libraries, a contig of 44 kb containing a 5' portion of the PAP gene and 17 kb of the upstream region was established. It was characterized by restriction mapping and sequence analysis of 2.5 kb upstream of the initiation codon. Two major transcription initiation sites were found to be located around 56 and 91 bp upstream of the initiation codon, as determined by nuclease S1 and primer extension mapping. Expression of the PAP gene was measured by Northern blots in the androgen responsive LNCaP cell line. It was found to be induced 2-3-fold by the addition of the synthetic androgen mibolerone to the cells. The induced mRNA levels were approx. 10-times lower than those for the prostate-specific antigen (PSA) in LNCaP cells.

DNA engineering shows that nucleosome phasing on the African green monkey alpha-satellite is the result of multiple additive histone-DNA interactions.

The mechanism underlying sequence-specific positioning of nucleosomes on DNA was investigated. African green monkey alpha-satellite DNA was reconstituted in vitro with histones. Histone octamers were found to adopt one major and several minor positions on the satellite repeat unit, very similar to those positions found previously in vitro, demonstrating that sequence-specific histone-DNA interactions are responsible for nucleosome positioning on this DNA. In order to understand the nature of these interactions in more detail, we have constructed a variant satellite fragment containing an insertion of half a helical DNA turn. The parent fragment directs histones to one major and two overlapping minor positions that are all affected by the insertion. All three frames respond in a unique fashion to the additional five base-pairs. From a quantitative analysis of the nucleosome positions on the engineered fragment, consensus "phasing boxes" as the basis for nucleosome positioning can be ruled out. Instead, our results argue very strongly that nucleosome positioning is due to the independent contribution of many different DNA-histone contacts along the entire core particle, in an apparently additive fashion.

Nucleosomes are positioned on mouse satellite DNA in multiple highly specific frames that are correlated with a diverged subrepeat of nine base-pairs.

Nucleosome phasing on highly repetitive DNA was investigated using a novel strategy. Nucleosome cores were prepared from mouse liver nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population that contains satellite sequences was then purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value and hydroxyapatite chromatography to separate the renatured satellite fraction. After end-labeling, the termini of the satellite core DNA fragments were mapped with an accuracy of +/- 1 base-pair relative to known restriction sites on the satellite DNA. Sixteen dominant nucleosome positions were detected. There is a striking correlation between these nucleosome frames and an internal highly diverged 9 base-pair subrepeat of the satellite DNA. The results are consistent with a sequence-dependent association of histone octamers with the satellite DNA. Our finding that histone octamers can interact with a given DNA in a number of different defined frames has important implications for the possible biological significance of nucleosome phasing.

Structural and functional requirements for the chromatin transition at the PHO5 promoter in Saccharomyces cerevisiae upon PHO5 activation.

The PHO5 promoter from Saccharomyces cerevisiae can exist in two chromatin configurations depending on its state of activity. In the repressed promoter a short hypersensitive site containing a binding site for the transcription factor PHO4 is flanked by specifically positioned nucleosomes. After induction two nucleosomes upstream and two downstream of the hypersensitive site are disrupted, and the entire promoter becomes accessible. We have investigated mechanisms responsible for setting up the structure of the repressed state and for the transition. Episomal centromeric plasmids bearing the PHO5 promoter show the same chromatin structure as the endogenous chromosomal copy arguing that the chromosomal context is not essential and that the nucleosomal organization is not set up from a distance. Deleting most of the hypersensitive region including the PHO4 binding site also leaves the positioning of the adjacent nucleosomes in the repressed promoter unchanged indicating that histone-DNA interactions play an important role in setting up nucleosome positions. However, when half of the DNA of a nucleosome is deleted a new nucleosome forms at the same location with respect to the neighboring nucleosome indicating that boundary effects also contribute to nucleosome positioning in the native promoter. Disruption of the nucleosomes under activating conditions is shown to require interaction of PHO4 with its binding site located within the hypersensitive region. This disruption takes place also in two independent constructs in which the TATA box had been deleted and as a result the gene was not transcribed. This result shows for the first time that the generation of active chromatin at a regulated promoter is not the result of gene expression but occurs prior to transcription.

Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work, the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nM of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease S1 analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcription 96 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to cells results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in vivo, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression.

Specificity of cleavage by restriction nuclease from Bacillus subtilis.

The restriction nuclease from B. subtilis (Bsu) which cleaves in the middle of the tetra-nucleotide sequence 5'-GGCC-3' 3'-CCGG-5' has been found to decrease its substrate specificity at high nuclease concentrations. There are special conditions, high pH, low ionic strength, and high glycerol content, which strongly enhance splitting with decreased specificity and also lead to splitting of single-stranded DNA. By sequence analyses it is shown that the reduction in specificity of Bsu corresponds to cleavage predominantly at 5'-GC-3' 3'-CG-5' sequences. No comparable change in specificity has been observed in a restriction nuclease from Haemophilus aegyptius (HaeIII), and isoschizomer of Bsu.

Experimental study on the biocompatibility of lining cements based on glass ionomer as compared with calcium hydroxide.

An in vitro study with cultured fibroblast-like cells of the pulp was used to assess the biological tolerance of three lining cements based on glass ionomers and two hard liners containing calcium hydroxide. The results indicate that strong cytotoxic components of all g.-i. lining cements tested can be eluted even after hardening for 48 hours. In contrast, the two liners based on calcium hydroxide proved to be of greater biocompatibility after the same hardening period. Therefore, use of calcium hydroxide liners is recommended for placement in deep cavities near the pulp.

Reaction of cultured pulp cells to eight different cements based on glass ionomers.

A comparative study was carried out to determine the cytotoxicity of eight different glass-ionomer cements by means of cell culture. Only fibroblast-like cells from the pulp of the same subcultures were used. Factors for the evaluation of the biological tolerance were, first, adhesion of cells to the slides as well as to the material, second, relative growth rates of cultures, and, in addition, cytomorphology. The cultured cells reacted quite differently to the glass-ionomer cements tested. The results imply that cytotoxic substances are eluted from some products even after a hardening period of 48 hours. In contrast, three of the glass-ionomer cements tested had only slight cytotoxic effects on the cultured pulp cells.