HSF1 induces RNA polymerase II synthesis of ribosomal RNA in S. cerevisiae during nitrogen deprivation.

The resource intensive process of accurate ribosome synthesis is essential for cell viability in all organisms. Ribosome synthesis regulation centers on RNA polymerase I (pol I) transcription of a 35S rRNA precursor that is processed into the mature 18S, 5.8S and 25S rRNAs. During nutrient deprivation or stress, pol I synthesis of rRNA is dramatically reduced. Conversely, chronic stress such as mitochondrial dysfunction induces RNA polymerase II (pol II) to transcribe functional rRNA using an evolutionarily conserved cryptic pol II rDNA promoter suggesting a universal phenomenon. However, this polymerase switches and its role in regulation of rRNA synthesis remain unclear. In this paper, we demonstrate that extended nitrogen deprivation induces the polymerase switch via components of the environmental stress response. We further show that the switch is repressed by Sch9 and activated by the stress kinase Rim15. Like stress-induced genes, the switch requires not only pol II transcription machinery, including the mediator, but also requires the HDAC, Rpd3 and stress transcription factor Hsf1. The current work shows that the constitutive allele, Hsf1PO4* displays elevated levels of induction in non-stress conditions while binding to a conserved site in the pol II rDNA promoter upstream of the pol I promoter. Whether the polymerase switch serves to provide rRNA when pol I transcription is inhibited or fine-tunes pol I initiation via RNA interactions is yet to be determined. Identifying the underlying mechanism for this evolutionary conserved phenomenon will help understand the mechanism of pol II rRNA synthesis and its role in stress adaptation.

Genetic Mutations in the S-loop of Human Glutathione Synthetase: Links Between Substrate Binding, Active Site Structure and Allostery.

The second step in the biosynthesis of the cellular antioxidant glutathione (GSH) is catalyzed by human glutathione synthetase (hGS), a negatively cooperative homodimer. Patients with mutations in hGS have been reported to exhibit a range of symptoms from hemolytic anemia and metabolic acidosis to neurological disorders and premature death. Several patient mutations occur in the S-loop of hGS, a series of residues near the negatively cooperative γ-GC substrate binding site. Experimental point mutations and molecular dynamic simulations show the S-loop not only binds γ-GC through a salt bridge and multiple hydrogen bonds, but the residues also modulate allosteric communication in hGS. By elucidating the role of S-loop residues in active site structure, substrate binding, and allostery, the atomic level sequence of events that leads to the detrimental effects of hGS mutations in patients are more fully understood.

Mitotic Activation of a Novel Histone Deacetylase 3-Linker Histone H1.3 Protein Complex by Protein Kinase CK2.

Histone deacetylase 3 (HDAC3) and linker histone H1 are involved in both chromatin compaction and the regulation of mitotic progression. However, the mechanisms by which HDAC3 and H1 regulate mitosis and the factors controlling HDAC3 and H1 activity during mitosis are unclear. Furthermore, as of now, no association between class I, II, or IV (non-sirtuin) HDACs and linker histones has been reported. Here we describe a novel HDAC3-H1.3 complex containing silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor 1 (N-CoR) that accumulated in synchronized HeLa cells in late G2 phase and mitosis. Nonetheless, the deacetylation activity by HDAC3 in the complex was evident only in mitotic complexes. HDAC3 associated with H1.3 was highly phosphorylated on Ser-424 only during mitosis. Isolation of inactive HDAC3-H1.3 complexes from late G2 phase cells, and phosphorylation of HDAC3 in the complexes at serine 424 by protein kinase CK2 (also known as casein kinase 2) activated the HDAC3 in vitro. In vivo, CK2α and CK2α' double knockdown cells demonstrated a significant decrease in HDAC3 Ser-424 phosphorylation during mitosis. HDAC3 and H1.3 co-localized in between the chromosomes, with polar microtubules and spindle poles during metaphase through telophase, and partially co-localized with chromatin during prophase and interphase. H1 has been reported previously to associate with microtubules and, therefore, could potentially function in targeting HDAC3 to the microtubules. We suggest that phosphorylation of HDAC3 in the complex by CK2 during mitosis activates the complex for a dual role: compaction of the mitotic chromatin and regulation of polar microtubules dynamic instability.