The SIR2 homologues HST3 and HST4 have been implicated in maintenance of genome integrity in the yeast Saccharomyces cerevisiae. We find that Hst3 has NAD-dependent histone deacetylase activity in vitro and that it functions during S phase to deacetylate the core domain of histone H3 at lysine 56 (H3K56). In response to genotoxic stress, Hst3 undergoes rapid Mec1-dependent phosphorylation and is targeted for ubiquitinmediated proteolysis, thus providing a mechanism for the previously observed checkpoint-dependent accumulation of Ac-H3K56 at sites of DNA damage. Loss of Hst3-mediated regulation of H3K56 acetylation results in a defect in the S phase DNA damage checkpoint. The pathway that regulates H3K56 acetylation acts in parallel with the Rad9 pathway to transmit a DNA damage signal from Mec1 to Rad53. We also observe that loss of Hst3 function impairs sister chromatid cohesion (SCC). Both S phase checkpoint and SCC defects are phenocopied by H3K56 point mutants. Our findings demonstrate that Hst3-regulated H3K56 acetylation safeguards genome stability by controlling the S phase DNA damage response and promoting SCC.Post-translational modification of histone proteins has been implicated in controlling many aspects of chromosome biology. Among the recently described histone modifications is acetylation of histone H3 on lysine 56 (Ac-H3K56) located at the lateral surface of the nucleosome core (1-4). Ac-H3K56 appears in S phase on newly synthesized histone H3 prior to its deposition onto chromatin, and Lys 56 is deacetylated later in S phase following its deposition (5). Acetylation of H3K56 requires a histone chaperone, Asf1 (6, 7), and is carried out by a recently characterized acetyltransferase Rtt109 (8 -11). Regulation of acetylation is critical, since cells with a mutant form of histone H3 that cannot be acetylated are DNA damage-and hydroxyurea (HU) 3 -sensitive. In the presence of DNA damage, persistent checkpoint-dependent H3K56 acetylation suggested an interplay between classical DNA damage checkpoint proteins (e.g. Mec1) and regulators of H3K56 acetylation.In response to genotoxic stress, Mec1, acting as a DNA damage sensor, activates the DNA damage response network by phosphorylating Rad53. Rad9 serves as an adaptor (12) that facilitates Mec1-mediated phosphorylation of Rad53 throughout the cell cycle. In addition, and specifically during S phase, Mec1 is capable of efficiently phosphorylating Rad53 in a RAD9-independent fashion (13, 14). The ability of Mec1 to transmit a DNA damage signal during S phase in the absence of RAD9 depends on several genes (e.g. TOF1, CSM3, and MRC1) that constitute an S phase-specific branch of the DNA damage response network (15). In addition to DNA damage signal transduction from Mec1 to Rad53, the members of the S phasespecific branch of the DNA damage response network play a separate role in ensuring the stability of the stalled replication forks (16,17) and enabling the establishment of faithful sister chromatid cohesion SCC (18,19). Their role in SCC appears to be ...

Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein β-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations in the X chromosomal gene Methyl-CpG-binding Protein 2 (MECP2) (1). RTT treatment so far is symptomatic. Mecp2 disruption in mice phenocopies major features of the syndrome (2) that can be reversed upon re-expression of Mecp2 (3. It has recently been reported that transplantation of wild type (WT) bone marrow (BMT) into lethally irradiated Mecp2tm1.1Jae/y mice prevented neurologic decline and early death by restoring microglial phagocytic activity against apoptotic targets (4). Based on this report, clinical trials of BMT for patients with RTT have been initiated (5). We aimed to replicate and extend the BMT experiments in three different RTT mouse models but found that despite robust microglial engraftment, BMT from WT donors did not rescue early death or ameliorate neurologic deficits. Furthermore, early and specific genetic expression of Mecp2 in microglia did not rescue Mecp2-deficient mice. In conclusion our experiments do not support BMT as therapy for RTT.