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Histone N-terminal tails play crucial roles in chromatin-related processes. The tails of histones H3 and H4 are highly conserved and well characterized, but much less is known about the functions of the tails of histones H2A and H2B and their sequences are more divergent among eukaryotes. Here we characterized the function of the only highly conserved region in the H2B tail, the H2B repression (HBR) domain. Once thought to play a role only in repression, it also has an uncharacterized function in gene activation and DNA damage responses. We report that deletion of the HBR domain impairs the eviction of nucleosomes at the promoters and open reading frames of genes. A closer examination of the HBR domain mutants revealed that they displayed phenotypes similar to those of histone chaperone complex FACT mutants, including an increase in intragenic transcription and the accumulation of free histones in cells. Biochemical characterization of recombinant nucleosomes indicates that deletion of the HBR domain impairs FACT-dependent removal of H2A-H2B from nucleosomes, suggesting that the HBR domain plays an important role in allowing FACT to disrupt dimer-DNA interactions. We have uncovered a previously unappreciated role for the HBR domain in regulating chromatin structure and have provided insight into how FACT acts on nucleosomes.

Gene expression depends upon the antagonistic actions of chromatin remodeling complexes. While this has been studied extensively for the enzymes that covalently modify the tails of histones, the mechanism of how ATP-dependent remodeling complexes antagonize each other to maintain the proper level of gene activity is not known. The gene encoding a large subunit of ribonucleotide reductase, RNR3, is regulated by ISW2 and SWI/SNF, complexes that repress and activate transcription, respectively. Here, we studied the functional interactions of these two complexes at RNR3. Deletion of ISW2 causes constitutive recruitment of SWI/SNF, and conditional reexpression of ISW2 causes the repositioning of nucleosomes and reduced SWI/SNF occupancy at RNR3. Thus, ISW2 is required for restriction of access of SWI/SNF to the RNR3 promoter under the uninduced condition. Interestingly, the binding of sequence-specific DNA binding factors and the general transcription machinery are unaffected by the status of ISW2, suggesting that disruption of nucleosome positioning does not cause a nonspecific increase in cross-linking of all factors to RNR3. We provide evidence that ISW2 does not act on SWI/SNF directly but excludes its occupancy by positioning nucleosomes over the promoter. Genetic disruption of nucleosome positioning by other means led to a similar phenotype, linking repressed chromatin structure to SWI/SNF exclusion. Thus, incorporation of promoters into a repressive chromatin structure is essential for prevention of the opportunistic actions of nucleosome-disrupting activities in vivo, providing a novel mechanism for maintaining tight control of gene expression.

Saccharomyces cerevisiae mating type switching is a gene conversion event that exhibits donor preference. MATa cells choose HMLalpha for recombination, and MATalpha cells choose HMRa. Donor preference is controlled by the recombination enhancer (RE), located between HMLalpha and MATa on the left arm of chromosome III. A number of a-cell specific noncoding RNAs are transcribed from the RE locus. Mcm1 and Fkh1 regulate RE activity in a cells. Here we show that Mcm1 binding is required for both the transcription of the noncoding RNAs and Fkh1 binding. This requirement can be bypassed by inserting another promoter into the RE. Moreover, the insertion of this promoter increases donor preference and opens the chromatin structure around the conserved domains of RE. Additionally, we determined that the level of Fkh1 binding positively correlates with the level of donor preference. We conclude that the role of Mcm1 in RE is to open chromatin around the conserved domains and activate transcription; this facilitates Fkh1 binding and the level of this binding determines the level of donor preference.

Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies in Saccharomyces cerevisiae yeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.

We describe an assay employing the competitive binding of estrogen receptor (ER) with basal transcription factors on a constitutive promoter (cytomegalovirus-hormone response element[s]-chloramphenicol acetyltransferase [CMV-(HRE)n-CAT, containing a hormone response element(s) between the TATA box and the start site of transcription]) to examine the DNA-binding ability of the human ER in whole cells. We used this promoter interference assay to examine the DNA binding of ER in cell lines containing high and low levels of endogenous ER, as well as in CHO cells expressing wild-type and mutant ERs from cotransfected expression vectors. The ER is capable of binding to the promoter interference constructs in the absence of added ligand, and estrogen (estradiol) or antiestrogen (trans-hydroxytamoxifen or ICI 164,384) enhances or stabilizes this interaction. The binding of unoccupied ER to reporter gene activation plasmids results in ligand-independent transactivation, presumably due to the TAF-1 function of the receptor. DNA binding of ER in the absence of ligand is observed in cells containing endogenous ER, or expressed ER, and occurs in cells with high or low receptor contents. Although estrogen- and antiestrogen-occupied ER complexes bind to DNA and reduce the template promoter activity, the extent of suppression achieved by ICI-bound ERs is consistently less than that achieved with the other ligands, presumably caused by the fact that ICI rapidly reduces the level of ER in most of the cells examined. However, the ICI-ER complexes that remain are in sufficient quantity to bind to gene activation reporter constructs, and in these cells, ICI still behaves as a pure antagonist of gene transcription and does not activate reporter genes. Hence, obstruction of ER DNA binding or reduction of ER in target cells may contribute to, but cannot fully explain, the pure antagonist character of the antiestrogen ICI 164,384. In addition, DNA binding by the ER alone is clearly not sufficient for ensuring full activation of transcription and argues for an intermediate in the receptor activation of promoters.

In Saccharomyces cerevisiae, the repressor Crt1 and the global corepressor Ssn6-Tup1 repress the DNA damage-inducible ribonucleotide reductase (RNR) genes. Initiation of DNA damage signals causes the release of Crt1 and Ssn6-Tup1 from the promoter, coactivator recruitment, and derepression of transcription, indicating that Crt1 plays a crucial role in the switch between gene repression and activation. Here we have mapped the functional domains of Crt1 and identified two independent repression domains and a region required for gene activation. The N terminus of Crt1 is the major repression domain, it directly binds to the Ssn6-Tup1 complex, and its repression activities are dependent upon Ssn6-Tup1 and histone deacetylases (HDACs). In addition, we identified a C-terminal repression domain, which is independent of Ssn6-Tup1 and HDACs and functions at native genes in vivo. Furthermore, we show that TFIID and SWI/SNF bind to a region within the N terminus of Crt1, overlapping with but distinct from the Ssn6-Tup1 binding and repression domain, suggesting that Crt1 may have activator functions. Crt1 mutants were constructed to dissect its activator and repressor functions. All of the mutants were competent for repression of the DNA damage-inducible genes, but a majority were “derepression-defective” mutants. Further characterization of these mutants indicated that they are capable of receiving DNA damage signals and releasing the Ssn6-Tup1 complex from the promoter but are selectively impaired for TFIID and SWI/SNF recruitment. These results imply a two-step activation model of the DNA damage-inducible genes and that Crt1 functions as a signal-dependent dual-transcription activator and repressor that acts in a transient manner.

DNA microarray and genetic studies of Saccharomyces cerevisiae have demonstrated that histone deacetylases (HDACs) are required for transcriptional activation and repression, but the mechanism by which they activate transcription remains poorly understood. We show that two HDACs, RPD3 and HOS2, are required for the activation of DNA damage-inducible genes RNR3 and HUG1. Using mutants specific for the Rpd3L complex, we show that the complex is responsible for regulating RNR3. Furthermore, unlike what was described for the GAL genes, Rpd3L regulates the activation of RNR3 by deacetylating nucleosomes at the promoter, not at the open reading frame. Rpd3 is recruited to the upstream repression sequence of RNR3, which surprisingly does not require Tup1 or Crt1. Chromatin remodeling and TFIID recruitment are largely unaffected in the Deltarpd3/Deltahos2 mutant, but the recruitment of RNA polymerase II is strongly reduced, arguing that Rpd3 and Hos2 regulate later stages in the assembly of the preinitiation complex or facilitate multiple rounds of polymerase recruitment. Furthermore, the histone H4 acetyltransferase Esa1 is required for the activation of RNR3 and HUG1. Thus, reduced or unregulated constitutive histone H4 acetylation is detrimental to promoter activity, suggesting that HDAC-dependent mechanisms are in place to reset promoters to allow high levels of transcription.