Simultaneous mutation of methylated lysine residues in histone H3 causes enhanced gene silencing, cell cycle defects, and cell lethality in Saccharomyces cerevisiae.

The methylation of specific lysine residues in histone H3 is integral to transcription regulation; however, little is known about how combinations of methylated lysine residues act in concert to regulate genome-wide transcription. We have systematically mutated methylated histone lysine residues in yeast and found that the triple mutation of H3K4, H3K36, and H3K79 to arginine (H3 K4,36,79R) is lethal. The histone H3 K4,36,79R mutant causes a mitotic cell cycle delay and a progressive transcription defect that initiates in telomere regions and then spreads into the chromosome. This effect is mediated by the silent information regulator (SIR) silencing complex, as we observe increased binding of the SIR complex to genomic regions adjacent to yeast telomeres in the H3 K4,36,79R mutant and deletion of SIR2, SIR3, or SIR4 rescues the lethal phenotype. Curiously, a yeast strain in which the histone methyltransferase genes are simultaneously deleted is viable. Indeed, deletion of the histone methyltransferase genes can suppress the H3 K4,36,79R lethal phenotype. These and other data suggest that the cause of lethality may in part be due to the association of histone methyltransferase enzymes with a histone substrate that cannot be methylated.

Ctk complex-mediated regulation of histone methylation by COMPASS.

A comparative global proteomic screen identified factors required for COMPASS (complex of proteins associated with Set1)-mediated mono-, di-, and trimethylation of the fourth lysine of histone H3 (H3K4), which included components of a cyclin-dependent protein kinase (Ctk complex) that phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II). Our results indicate that histone H3K4 methylation levels are regulated by the Ctk1, Ctk2, and Ctk3 components of the Ctk complex. We show that loss of Ctk1 kinase activity results in reduced histone H3K4 monomethylation levels, followed by a global increase in histone H3K4 trimethylation levels on chromatin. Ctk1 loss does not appear to have a substantial effect on histone H2B monoubiquitination levels or COMPASS and Paf1 complex phosphorylation. Our chromatin immunoprecipitation studies demonstrate that histone H3 eviction during active transcription is decelerated in a CTK1 deletion strain in response to reduced levels of Pol II recruitment. Our in vitro studies show that the onset of monomethylation on an unmethylated histone H3 by COMPASS is virtually immediate, while the onset of trimethylation occurs upon extended time of association between the histone tail and COMPASS. Our study suggests a role for the Ctk complex in the regulation of the pattern of H3K4 mono-, di-, and trimethylation via COMPASS.

GnRH regulation of Jun and Atf3 requires calcium, calcineurin, and NFAT.

GnRH binds to its receptor on gonadotropes and activates multiple members of the MAPK signaling family that in turn regulates the expression of several immediate early genes (IEGs) including Jun, Fos, Atf3, and Egr1. These IEGs confer hormonal responsiveness to gonadotrope-specific genes including Gnrhr, Cga, Fshb, and Lhb. In this study we tested the hypothesis that GnRH specifically regulates the accumulation of Jun and Atf3 mRNA through a pathway that includes intracellular Ca²âº, calcineurin, and nuclear factor of activated T cells (NFAT). Our results indicate that pretreatment of murine LßT2 cells with 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester, a Ca²âº chelator, reduced the expression of all the IEGs to varying degrees, whereas treatment with thapsigargin, an intracellular Ca²âº protein pump inhibitor, increased the expression of the IEG. Furthermore, cyclosporin A, a calcineurin-specific inhibitor, reduced the ability of GnRH to regulate accumulation of Jun and Atf3 mRNA and to a lesser extent Fos. In contrast, Egr1 mRNA was unaffected. NFATs are transcription factors regulated by calcineurin and were detected in LßT2 cells. GnRH increased luciferase activity of an NFAT-dependent promoter reporter that was dependent on intracellular Ca²âº and calcineurin activity. Additionally, although small interfering RNA specific for Nfat4 only marginally reduced GnRH regulation of Jun, Fos, and Atf3 mRNA accumulation, activity of an activator protein-1-responsive reporter construct was reduced by 48%. Together these data suggest that calcineurin and NFAT are new members of the gonadotrope transcriptional network that confer hormonal responsiveness to several key genes required for gonadotropin synthesis and secretion.

AUG-proximal nucleotides regulate protein synthesis in Leishmania tropica.

Gene expression in the Leishmania is controlled post-transcriptionally, and is likely to be impacted by both 5' and 3' untranslated regions (UTRs). We have investigated the effects of trinucleotides in the AUG-proximal region (APR) (i.e. positions -3 to -1 upstream of an AUG) on two reporter genes in the context of an endogenous intergenic region of Leishmania tropica. The effects of APRs on protein expression were determined in stable transfectants in vivo. Three APRs, namely, C(-3)C(-2)C(-1), ACC and GCC, yielded robust translation, whereas GTA produced low amounts of proteins. A purine at -3 of an APR was not crucial for efficient translation. Steady-state level of reporter mRNA did not correlate directly with the amount of protein detected. Polysome analysis revealed that APRs modulate translation, at least in part, by influencing mRNA association with ribosomes. An analysis of genomic UTRs in L. major showed that (i) the consensus APR is N(-3)N(-2)C(-1) (where N = any nucleotide), and (ii) the most frequently used APRs include ACA, ACC, ATC, GCC, GCG, GTC and CAC, some of which were translation enhancers in our experimental studies.

Cysteine-less glycosylphosphatidylinositol-specific phospholipase C is inhibited competitively by a thiol reagent: evidence for glyco-mimicry by p-chloromercuriphenylsulphonate.

Glycosylphosphatidylinositol (GPI)-specific phospholipases are highly valuable for studying the structure and function of GPIs. GPI-specific phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus are the most widely studied of this class of phospholipases C. Inhibition of protein activity by thiol reagents is indicative of the participation of cysteine residues in biochemical events. The thiol reagent p-chloromercuriphenylsulphonate (pCMPS) inhibits T. brucei GPI-PLC, which has eight cysteine residues. Surprisingly, we found that the activity of B. cereus PI-PLC is also blocked by pCMPS, although the protein does not contain cysteine residues. Inhibition of B. cereus PI-PLC was reversed when pCMPS was size-separated from a preformed pCMPS.PI-PLC complex. In contrast, no activity was recovered when T. brucei GPI-PLC was subjected to a similar protocol. Equimolar beta-mercaptoethanol (beta-ME) reversed the inhibition of PI-PLC activity in a pCMPS.PI-PLC complex. For T. brucei GPI-PLC, however, ultrafiltration of the pCMPS.GI-PLC complex and addition of a large excess of beta-ME was necessary for partial recovery of enzyme activity. Thus T. brucei GPI-PLC is susceptible to inactivation by covalent modification with pCMPS, whereas PI-PLC is not. Kinetic analysis indicated that pCMPS was a competitive inhibitor of PI-PLC when a GPI was a substrate. Curiously, with phosphatidylinositol as substrate, inhibition was no longer competitive. These data suggest that pCMPS is a glyco-mimetic that occupies the glycan binding site of PI-PLC, from where, depending on the substrate, it inhibits catalysis allosterically or competitively.