Evidence for histone eviction in trans upon induction of the yeast PHO5 promoter - PubMed (original) (raw)

Evidence for histone eviction in trans upon induction of the yeast PHO5 promoter

Philipp Korber et al. Mol Cell Biol. 2004 Dec.

Abstract

The yeast PHO5 promoter is a model system for the role of chromatin in eukaryotic gene regulation. Four positioned nucleosomes in the repressed state give way to an extended DNase I hypersensitive site upon induction. Recently this hypersensitive site was shown to be devoid of histone DNA contacts. This raises the mechanistic question of how histones are removed from the promoter. A displacement in trans or movement in cis, the latter according to the well established nucleosome sliding mechanism, are the major alternatives. In this study, we embedded the PHO5 promoter into the context of a small plasmid which severely restricts the space for nucleosome sliding along the DNA in cis. Such a construct would either preclude the chromatin transition upon induction altogether, were it to occur in cis, or gross changes in chromatin around the plasmid would be the consequence. We observed neither. Instead, promoter opening on the plasmid was indistinguishable from opening at the native chromosomal locus. This makes a sliding mechanism for the chromatin transition at the PHO5 promoter highly unlikely and points to histone eviction in trans.

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Figures

FIG. 1.

FIG. 1.

(A) Schematic of the chromatin organization at the PHO5 promoter in the repressed (+Pi) and induced state (−Pi). The positioned nucleosomes (circles) are numbered from −5 to +1 with respect to the open reading frame (broad black arrow). The open circles denote the nucleosomes which become remodeled upon induction. Upon induction, the short hypersensitive site (HS; asterisk) becomes extended (HS; dashed line) and the two Pho4 binding sites (black dots) bind Pho4 (striped arches). Remodeling of nucleosomes −1 and −4 is often less complete, as symbolized by stippled circles in the induced state. The position of the TATA box (T) is shown, and the buckled arrow (RNA Pol) symbolizes transcription of the PHO5 gene. The positions of relevant restriction sites are indicated by arrows as well as the relative position of the upstream ApaI-Sau3AI fragment which constitutes probe 1b. (B) Map of plasmid pTAP5C (2,481 bp). The genetic elements (CEN6, TRP1, ARS1, and the PHO5 promoter) are marked by boxes or arrows on the plasmid. The positions of nucleosomes along the plasmid are shown as circles. Nucleosomes are labeled with arabic numbers for the PHO5 promoter region (−5 to +1, as in panel A), with roman numerals for the remainder of the UNF region (36), and alphanumerically for the TRP1 region (T1-T4) and the CEN6 element (C6). The nucleosomes shaded in gray become remodeled upon induction of the PHO5 promoter. Asterisks mark hypersensitive sites. Restriction sites used as markers are shown and the probes used for indirect end labeling (Trp up, Trp down, 63, and 162) are indicated by curved arrows above the plasmid circle. The sites used for secondary cleavage for indirect end labeling are in bold.

FIG. 2.

FIG. 2.

There is no difference between the plasmid and the chromosome locus regarding the chromatin structure of the PHO5 promoter nucleosomes −4 to +1 both under repressed (+Pi) and induced (−Pi) conditions. (A) Nuclei of strain YS1801 carrying pTAP5C were subjected to limited DNase I digestion (wedges on top of the upper panel blot denote increasing DNase I concentration, dashes denote no DNase I addition) and secondary cleavage with HindIII and ApaI for indirect end labeling. The same blot membrane was probed with probe Trp down and reprobed with probe 1b for analysis of the plasmid and the chromosome locus, respectively (see also Fig. 1). The relevant genetic regions as well as schematics of the chromatin structure (labeled and stippled circles, asterisks, and dashed lines for the extended hypersensitive site HS, as in Fig. 1) are indicated at the sides of the blots. The asterisks labeled HS1 and HS3 refer to the hypersensitive sites upstream of the PHO5 promoter and between the PHO5 and the PHO3 gene (3), respectively. (B) Restriction enzyme analysis of nuclei from repressed (+Pi) and induced cells (−Pi) as in panel A with the indicated enzymes at 0.3 and 1.2 U/μl (left and right lane, respectively, for each enzyme) and secondary cleavage with HindIII and ApaI. Probes as in panel A.

FIG. 3.

FIG. 3.

Chromatin immunoprecipitation analysis of the PHO5 promoter region on the plasmid shows reduced histone occupancy upon induction. (A) Immunoprecipitated DNA from strain YS18018 carrying pTAP5C using antibodies against the C terminus of H2B, H3, or H4 was quantified by real-time PCR with amplicons in the PHO5 promoter (UASp2) and in the open reading frame (ORF) of the TRP1 gene (TRP1). The PCR signals were controlled for amplicon efficiency with input DNA (T. Luckenbach, data not shown) and normalized versus the signal of an amplicon at the telomere. These normalized values represent the relative histone occupancy in the respective region and were determined for repressed (+Pi) and induced (−Pi) conditions. (B) Data as in panel A, but the values of amplicon UASp2 are divided by the values of amplicon TRP1 for each antibody and condition.

FIG. 4.

FIG. 4.

A change in superhelicity confirms the loss of nucleosomes from the plasmid upon induction. (A) Genomic DNA from strain YS1801 (wt) and YS3101 (pho80) both carrying the plasmid pTAP5CΔTATA was analyzed in an agarose gel containing 33 μM chloroquine. Three independent but parallel cultures were grown logarithmically in high-phosphate media for each strain. The arrowheads mark each maximum of the Gaussian distribution as averaged over the three lanes for each strain (closed arrowhead, YS1801; open arrowhead, YS3101). The difference in linking number between the repressed and induced states was 2.5 ± 0.4. Lanes 1 and 8 contain linearized plasmid as a marker and the position of the nicked circular and the linear form of the plasmid is indicated on the right side of the gel. (B) Trace profiles of lane 3 (top) and lane 5 (bottom) are shown and the respective maxima of the Gaussian fit indicated by arrowheads. The position of the nicked circular form was used as the origin for the trace profile.

FIG. 5.

FIG. 5.

The chromatin structure of regions adjacent to the PHO5 promoter on plasmid pTAP5C remain largely unchanged upon induction. Nuclei of strain YS18018 carrying pTAP5C and grown under repressive (+Pi) or inducing (−Pi) conditions were analyzed by DNase I digestion and indirect end labeling (wedges and dashes, as in Fig. 2A). HindIII was used for secondary cleavage in panel A and ClaI in panels B and C, and probes were as indicated on the left of the blots (see also Fig. 1B). The relevant genetic regions as well as schematics of the chromatin structure (labeled and stippled circles, asterisks, and dashed lines for the extended hypersensitive site HS, as in Fig. 1A and B) are indicated at the sides of the blot. The blot of panel A was also probed with probe Trp down with the same result as in the “Plasmid” panel in Fig. 2A (P. Korber and D. Blaschke, data not shown). The two middle lanes of all blots contain markers for different probes. Only one of the two lanes is relevant for a particular probe and is labeled accordingly. The marker lane for probe Trp up (A) was underloaded and the position of the restriction fragments as indicated by dashes was deduced from a longer exposure. Panels B and C show the same blot which was probed with two different probes. The marker lane for probe 63 (B) was overloaded; therefore, a shorter exposure of this lane is shown in panel B and it was cut out in panel C. The pound sign in panel B denotes an artifact band.

FIG. 6.

FIG. 6.

The chromatin structures of regions adjacent to the PHO5 promoter on plasmid pTAP5CΔTATA remain unchanged upon induction. Chromatin analysis was done by using DNase I digestion with indirect end labeling for the plasmid pTAP5CΔTATA in strain YS18018 under repressive and inducing conditions. The labeling is analogous to Fig. 2A and 5. Compare panel A with the “Plasmid” panel in Fig. 2A and panels B, C, and D with panel A, B, and C of Fig. 5, respectively. The marker bands in panel B are rather weak and are therefore indicated by dashes. The marker for probe 63 in panel C was overloaded and a shorter exposure of this lane is shown in panel C, whereas the lane was cut out in panel D.

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