A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance.

In fungal species, lysine 56 of newly synthesized histone H3 molecules is modified by the acetyltransferase Rtt109, which promotes resistance to genotoxic agents. To further explore how H3 K56ac contributes to genome stability, we conducted screens for suppressors of the DNA damage sensitivity of budding yeast rtt109 Delta mutants. We recovered a single extragenic suppressor mutation that efficiently restored damage resistance. The suppressor is a point mutation in the histone H3 gene HHT2, and converts lysine 56 to glutamic acid. In some ways, K56E mimics K56ac, because it suppresses other mutations that interfere with the production of H3 K56ac and restores histone binding to chromatin assembly proteins CAF-1 and Rtt106. Therefore, we demonstrate that enhanced association with chromatin assembly factors can be accomplished not only by acetylation-mediated charge neutralization of H3K56 but also by the replacement of the positively charged lysine with an acidic residue. These data suggest that removal of the positive charge on lysine 56 is the functionally important consequence of H3K56 acetylation. Additionally, the suppressive function of K56E requires the presence of a second H3 allele, because K56E impairs growth when it is the sole source of histones, even more so than does constitutive H3K56 acetylation. Our studies therefore emphasize how H3 K56ac not only promotes chromatin assembly but also leads to chromosomal malfunction if not removed following histone deposition.

Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

Histone H3-K56 acetylation is catalyzed by histone chaperone-dependent complexes.

Acetylation of histone H3 on lysine 56 occurs during mitotic and meiotic S phase in fungal species. This acetylation blocks a direct electrostatic interaction between histone H3 and nucleosomal DNA, and the absence of this modification is associated with extreme sensitivity to genotoxic agents. We show here that H3-K56 acetylation is catalyzed when Rtt109, a protein that lacks significant homology to known acetyltransferases, forms an active complex with either of two histone binding proteins, Asf1 or Vps75. Rtt109 binds to both these cofactors, but not to histones alone, forming enzyme complexes with kinetic parameters similar to those of known histone acetyltransferase (HAT) enzymes. Therefore, H3-K56 acetylation is catalyzed by a previously unknown mechanism that requires a complex of two proteins: Rtt109 and a histone chaperone. Additionally, these complexes are functionally distinct, with the Rtt109/Asf1 complex, but not the Rtt109/Vps75 complex, being critical for resistance to genotoxic agents.

Nuclear import of the stem-loop binding protein and localization during the cell cycle.

A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem-loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Impalpha/Impbeta and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Impalpha/Impbeta binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Impalpha/Impbeta pathway contributes to SLBP nuclear import in HeLa cells.

Nuclear export of metazoan replication-dependent histone mRNAs is dependent on RNA length and is mediated by TAP.

Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated, ending instead in a conserved stem-loop sequence. Histone pre-mRNAs lack introns and are processed in the nucleus by a single cleavage step, which produces the mature 3' end of the mRNA. We have systematically examined the requirements for the nuclear export of a mouse histone mRNA using the Xenopus oocyte system. Histone mRNAs were efficiently exported when injected as mature mRNAs, demonstrating that the process of 3' end cleavage is not required for export factor binding. Export also does not depend on the stem-loop binding protein (SLBP) since mutations of the stem-loop that prevent SLBP binding and competition with a stem-loop RNA did not affect export. Only the length of the region upstream of the stem-loop, but not its sequence, was important for efficient export. Histone mRNA export was blocked by competition with constitutive transport element (CTE) RNA, indicating that the mRNA export receptor TAP is involved in histone mRNA export. Consistent with this observation, depletion of TAP from Drosophila cells by RNAi resulted in the restriction of mature histone mRNAs to the nucleus.

SLBP is associated with histone mRNA on polyribosomes as a component of the histone mRNP.

The stem-loop binding protein (SLBP) binds the 3' end of histone mRNA and is present both in nucleus, and in the cytoplasm on the polyribosomes. SLBP participates in the processing of the histone pre-mRNA and in translation of the mature message. Histone mRNAs are rapidly degraded when cells are treated with inhibitors of DNA replication and are stabilized by inhibitors of translation, resulting in an increase in histone mRNA levels. Here, we show that SLBP is a component of the histone messenger ribonucleoprotein particle (mRNP). Histone mRNA from polyribosomes is immunoprecipitated with anti-SLBP. Most of the SLBP in cycloheximide-treated cells is present on polyribosomes as a result of continued synthesis and transport of the histone mRNP to the cytoplasm. When cells are treated with inhibitors of DNA replication, histone mRNAs are rapidly degraded but SLBP levels remain constant and SLBP is relocalized to the nucleus. SLBP remains active both in RNA binding and histone pre-mRNA processing when DNA replication is inhibited.

Nuclear export of mRNA: from the site of transcription to the cytoplasm.

Cellular mRNAs are produced in the nucleus and must be exported to the cytoplasm to allow for their translation into proteins. Recruitment of export factors to nascent mRNA starts cotranscriptionally and involves elaborate systems of quality control. Correctly processed mRNAs are committed for export in the form of large ribonucleoprotein complexes (mRNPs). Translocation of mRNPs through the nuclear pore complex (NPC) is mediated by a conserved heterodimeric transport receptor (NXF1/p15 in metazoa and Mex67p/Mtr2p in yeast) that bridges the interaction between the mRNP and the NPC. In this review, we describe the cis- and trans-requirements for mRNA export as well as the different mechanisms of recruiting export factors to mRNPs. We also discuss the significance of linking mRNA export with both downstream and upstream events in gene expression.

Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo.

The stem-loop binding protein (SLBP) binds to the 3' end of histone mRNA and participates in 3'-processing of the newly synthesized transcripts, which protects them from degradation, and probably also promotes their translation. In proliferating cells, translation of SLBP mRNA begins at G1/S and the protein is degraded following DNA replication. These post-transcriptional mechanisms closely couple SLBP expression to S-phase of the cell cycle, and play a key role in restricting synthesis of replication-dependent histones to S-phase. In contrast to somatic cells, replication-dependent histone mRNAs accumulate and are translated independently of DNA replication in oocytes and early embryos. We report here that SLBP expression and activity also differ in mouse oocytes and early embryos compared with somatic cells. SLBP is present in oocytes that are arrested at prophase of G2/M, where it is concentrated in the nucleus. Upon entry into M-phase of meiotic maturation, SLBP begins to accumulate rapidly, reaching a very high level in mature oocytes arrested at metaphase II. Following fertilization, SLBP remains abundant in the nucleus and the cytoplasm throughout the first cell cycle, including both G1 and G2 phases. It declines during the second and third cell cycles, reaching a relatively low level by the late 4-cell stage. SLBP can bind the histone mRNA-stem-loop at all stages of the cell cycle in oocytes and early embryos, and it is the only stem-loop binding activity detectable in these cells. We also report that SLBP becomes phosphorylated rapidly following entry into M-phase of meiotic maturation through a mechanism that is sensitive to roscovitine, an inhibitor of cyclin-dependent kinases. SLBP is rapidly dephosphorylated following fertilization or parthenogenetic activation, and becomes newly phosphorylated at M-phase of mitosis. Phosphorylation does not affect its stem-loop binding activity. These results establish that, in contrast to Xenopus, mouse oocytes and embryos contain a single SLBP. Expression of SLBP is uncoupled from S-phase in oocytes and early embryos, which indicates that the mechanisms that impose cell-cycle-regulated expression of SLBP in somatic cells do not operate in oocytes or during the first embryonic cell cycle. This distinctive pattern of SLBP expression may be required for accumulation of histone proteins required for sperm chromatin remodelling and assembly of newly synthesized embryonic DNA into chromatin.

A novel zinc finger protein is associated with U7 snRNP and interacts with the stem-loop binding protein in the histone pre-mRNP to stimulate 3'-end processing.

The stem-loop binding protein (SLBP) is the posttranscriptional regulator of histone mRNA in metazoan cells. SLBP binds histone pre-mRNAs and facilitates 3'-end processing by promoting stable association of U7 snRNP with the pre-mRNA. To identify other factors involved in histone pre-mRNA processing, we used a modified yeast two-hybrid assay in which SLBP and its RNA target were coexpressed as bait. A novel zinc finger protein, hZFP100, which interacts with the SLBP/RNA complex but not with free SLBP, was cloned. The interaction requires regions of SLBP that are important for histone pre-mRNA processing. Antibodies to hZFP100 precipitate U7 snRNA, and expression of hZFP100 in Xenopus oocytes stimulates processing of histone pre-mRNA, showing that hZFP100 is a component of the processing machinery.