Enzymes catalyzing CpG methylation in DNA, including DNMT1 and DNMT3A/B, are indispensable for mammalian tissue development and homeostasis 1-4. They are also implicated in human developmental disorders and cancers 5-8 , supporting a critical role of DNA methylation during cell fate specification and maintenance. Recent studies suggest that histone posttranslational modifications (PTMs) are involved in specifying patterns of DNMT localization and DNA methylation at promoters and actively transcribed gene bodies 9-11. However, mechanisms governing the establishment and maintenance of intergenic DNA methylation remain poorly understood. Germline mutations in DNMT3A define Tatton-Brown-Rahman syndrome (TBRS), a

The SNF1 protein kinase of Saccharomyces cerevisiae is a member of the SNF1/AMP-activated protein kinase family, which is essential for metabolic control, energy homeostasis, and stress responses in eukaryotes. SNF1 is activated in response to glucose limitation by phosphorylation of Thr210 on the activation loop of the catalytic subunit Snf1. The SNF1 β-subunit contains a glycogen-binding domain that has been implicated in glucose inhibition of Snf1 Thr210 phosphorylation. To assess the role of glycogen, we examined Snf1 phosphorylation in strains with altered glycogen metabolism. A reg1Δ mutant, lacking Reg1-Glc7 protein phosphatase 1, exhibits elevated glycogen accumulation and phosphorylation of Snf1 during growth on high levels of glucose. Unexpectedly, mutations that abolished glycogen synthesis also restored Thr210 dephosphorylation in glucose-grown reg1Δ cells, indicating that elevated glycogen synthesis contributes to activation of SNF1 and that another phosphatase acts on Snf1. We present evidence that Sit4, a type 2A-like protein phosphatase, contributes to dephosphorylation of Snf1 Thr210. Finally, evidence that the effects of glycogen are not mediated by binding to the β-subunit raises the possibility that elevated glycogen synthesis alters glucose metabolism and thereby reduces glucose signaling to the SNF1 pathway.glucose regulation | signal transduction | yeast T he SNF1/AMP-activated protein kinase (AMPK) family is conserved from yeast to mammals and is essential for metabolic control and energy homeostasis (1, 2). The Saccharomyces cerevisiae SNF1 protein kinase is required for adaptation to glucose limitation and other stresses and for growth on carbon sources that are less preferred than glucose; it is named for the sucrose-nonfermenting phenotype of the snf1 mutant (2). SNF1 regulates transcription of a large set of genes (3) and the activity of metabolic enzymes involved in carbohydrate storage and fatty acid metabolism (4-6).

Millions of people worldwide with incurable end-stage lung disease die because of inadequate treatment options and limited availability of donor organs for lung transplantation 1 . Current bioengineering strategies to regenerate the lung have not been able to replicate its extraordinary cellular diversity and complex three-dimensional arrangement, which are indispensable for life-sustaining gas exchange 2 , 3 . Here we report the successful generation of functional lungs in mice through a conditional blastocyst complementation (CBC) approach that vacates a specific niche in chimeric hosts and allows for initiation of organogenesis by donor mouse pluripotent stem cells (PSCs). We show that wild-type donor PSCs rescued lung formation in genetically defective recipient mouse embryos unable to specify (due to Ctnnb1 cnull mutation) or expand (due to Fgfr2 cnull mutation) early respiratory endodermal progenitors. Rescued neonates survived into adulthood and had lungs functionally indistinguishable from those of wild-type littermates. Efficient chimera formation and lung complementation required newly developed culture conditions that maintained the developmental potential of the donor PSCs and were associated with global DNA hypomethylation and increased H4 histone acetylation. These results pave the way for the development of new strategies for generating lungs in large animals to enable modeling of human lung disease as well as cell-based therapeutic interventions 4 – 6 .

It has been firmly established that many interphase nuclear functions, including transcriptional regulation, are regulated by chromatin and histones. How mitotic progression and quality control might be influenced by histones is less well characterized. We show that histone H3 plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis. Prior to anaphase, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. Lack of tension due to erroneous attachment activates the spindle assembly checkpoint, which corrects the mistakes and ensures segregation fidelity. A histone H3 mutation impairs the ability of yeast cells to activate the checkpoint in a tensionless crisis, leading to missegregation and aneuploidy. The defects in tension sensing result directly from an attenuated H3-Sgo1p interaction essential for pericentric recruitment of Sgo1p. Reinstating the pericentric enrichment of Sgo1p alleviates the mitotic defects. Histone H3, and hence the chromatin, is thus a key factor transmitting the tension status to the spindle assembly checkpoint.During mitosis, chromatin goes through significant compaction and condensation to form metaphase chromosomes for segregation. While there is a wealth of information on the crucial roles played by chromatin structures and histone modifications in controlling transcription, replication, repair, and recombination (30), much less is known about how individual histones contribute mechanistically to mitotic progression and regulation.Forward and reverse genetic studies have suggested that histones, rather than being merely a part of the cargo during mitotic segregation, may play key roles in cell cycle progression and regulation. A histone H4 allele, hhf1-20, compromises the interaction between H4 and the centromere-specific H3 variant Cse4p, thus impeding centromeric functions and mitosis at the restrictive temperature (50). Two alleles of histone H2A (44) cause cold-sensitive growth defects and a significant increase in ploidy. This hyperploidy phenotype can be suppressed by mutations affecting a histone deacetylase, HDA1 (19). Similarly, the Gcn5p histone acetyltransferase genetically interacts with several inner kinetochore components and is physically mapped to the centromeric regions (57). Deleting the flexible tail domain of H3 and H4 results in mitotic delay (36) via a mechanism that can be suppressed by inhibiting the spindle assembly checkpoint activity (J.L. and M.H.K., unpublished data). Together, these data warrant a more thorough examination of how chromatin may proactively regulate the process of mitotic segregation.The center stage for mitotic segregation and control is the kinetochore, a large proteinaceous complex assembled on centromeres. The ultimate function of the kinetochore is to capture the spindle microtubules during mitosis. The kinetochore-spindle attachment ...

Preeclampsia (PE), a hypertensive disorder of pregnancy, is a leading cause of maternal and fetal morbidity and mortality. Although the etiology is unknown, PE is thought to be caused by defective implantation and decidualization in pregnancy. Pregnant blood pressure high (BPH)/5 mice spontaneously develop placentopathies and maternal features of human PE. We hypothesized that BPH/5 implantation sites have transcriptomic alterations. Next-generation RNA sequencing of implantation sites at peak decidualization, embryonic day (E)7.5, revealed complement gene up-regulation in BPH/5 vs. controls. In BPH/5, expression of complement factor 3 was increased around the decidual vasculature of E7.5 implantation sites and in the trophoblast giant cell layer of E10.5 placentae. Altered expression of VEGF pathway genes in E5.5 BPH/5 implantation sites preceded complement dysregulation, which correlated with abnormal vasculature and increased placental growth factor mRNA and VEGF expression at E7.5. By E10.5, proangiogenic genes were down-regulated, whereas antiangiogenic sFlt-1 was up-regulated in BPH/5 placentae. We found that early local misexpression of VEGF genes and abnormal decidual vasculature preceded sFlt-1 overexpression and increased complement deposition in BPH/5 placentae. Our findings suggest that abnormal decidual angiogenesis precedes complement activation, which in turn contributes to the aberrant trophoblast invasion and poor placentation that underlie PE.-Sones, J. L., Merriam, A. A., Seffens, A., Brown-Grant, D.-A., Butler, S. D., Zhao, A. M., Xu, X., Shawber, C. J., Grenier, J. K., Douglas, N. C. Angiogenic factor imbalance precedes complement deposition in placentae of the BPH/5 model of preeclampsia.

The SNF1/AMP-activated protein kinases are αβγ-heterotrimers that sense and regulate energy status in eukaryotes. They are activated by phosphorylation of the catalytic Snf1/α subunit, and the Snf4/γ regulatory subunit regulates phosphorylation through adenine nucleotide binding. In Saccharomyces cerevisiae, the Snf1 subunit is phosphorylated on the activation-loop Thr-210 in response to glucose limitation. To assess the requirement of the heterotrimer for regulated Thr-210 phosphorylation, we examined Snf1 and a truncated Snf1 kinase domain (residues 1-309) that has partial Snf1 function. Snf1(1-309) does not interact with the β and Snf4/γ regulatory subunits, and its activity was independent of them in vivo. Phosphorylation of both Snf1 and Snf1(1-309) increased in response to glucose limitation in wild-type cells and in cells lacking β-and Snf4/γ-subunits. These results indicate that glucose regulation of activation-loop phosphorylation can occur by mechanism(s) that function independently of the regulatory subunits. We further show that the Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-like phosphatase are largely responsible for dephosphorylation of Thr-210 of Snf1(1-309). Together, these findings suggest that these two phosphatases mediate heterotrimer-independent regulation of Thr-210 phosphorylation.

To ensure genome stability during cell division, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies before segregation. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. In budding yeast, the residue Gly44 of histone H3 is critical for retaining the conserved Shugoshin protein Sgo1p at the pericentromeres for monitoring the tension status during mitosis. Studies carried out in this work showed that Lys42, Gly44, and Thr45 of H3 form the core of a tension sensing motif (TSM). Similar to the previously reported G44S mutant, K42A, G44A, and T45A alleles all rendered cells unable to respond to erroneous spindle attachment, a phenotype suppressed by Sgo1p overexpression. TSM functions by physically recruiting or retaining Sgo1p at pericentromeres as evidenced by chromatin immunoprecipitation and by in vitro pulldown experiments. Intriguingly, the function of TSM is likely regulated by multiple histone modifying enzymes, including the histone acetyltransferase Gcn5p, and deacetylases Rpd3p and Hos2p. Defects caused by TSM mutations can be suppressed by the expression of a catalytically inactive mutant of Gcn5p. Conversely, G44S mutant cells exhibit prominent chromatin instability phenotype in the absence of RPD3. Importantly, the gcn5 2 suppressor restores the tension sensing function in tsm 2 background in a fashion that bypasses the need of stably associating Sgo1p with chromatin. These results demonstrate that the TSM of histone H3 is a key component of a mechanism that ensures faithful segregation, and that interaction with chromatin modifying enzymes may be an important part of the mitotic quality control process.KEYWORDS histone H3; chromatin; mitosis; Shugoshin; Saccharomyces cerevisiae F AITHFUL partitioning of the genome duplicates in mitosis requires that the sister chromatids be engaged in bipolar attachment to mitotic spindle. Once all chromosomes are appropriately captured by the spindles, anaphase starts with the action of separase that cleaves the cohesin complex, thus separating the two sister chromosomes (Nasmyth 2002). Premature anaphase onset causes aneuploidy, a common trait associated with spontaneous abortion, birth defects, and cancer. Chromosome biorientation is a result of sister chromatid cohesion by the cohesin complex, stable attachment of spindles to kinetochores, and that the two sister kinetochores each attach to spindles emanating from different spindle pole bodies (Kschonsak and Haering 2015). Erroneous attachment of spindles to kinetochore activates the spindle assembly checkpoint (SAC), which prevents metaphase-to-anaphase transition so that errors can be corrected (Akera and Watanabe 2016). The two essential elements of biorientation are the spindle-kinetochore interaction and the tension between sister chromatids (Goshima and Yanagida 2000;Pinsky and Biggins 2005;Wang et al. 2014). The latter results from the physical cohesion of the sister chromatids that resi...

Gcn5 protein is a prototypical histone acetyltransferase that controls transcription of multiple yeast genes. To identify molecular functions that act downstream of or in parallel with Gcn5 protein, we screened for suppressors that rescue the transcriptional defects of HIS3 caused by a catalytically inactive mutant Gcn5, the E173H mutant. One bypass of Gcn5 requirement gene (BGR) suppressor was mapped to the REG1 locus that encodes a semidominant mutant truncated after amino acid 740. Reg1(1-740) protein does not rescue the complete knockout of GCN5, nor does it suppress other gcn5 ؊ defects, including the inability to utilize nonglucose carbon sources. Reg1(1-740) enhances HIS3 transcription while HIS3 promoter remains hypoacetylated, indicating that a noncatalytic function of Gcn5 is targeted by this suppressor protein. Reg1 protein is a major regulator of Snf1 kinase that phosphorylates Ser10 of histone H3. However, whereas Snf1 protein is important for HIS3 expression, replacing Ser10 of H3 with alanine or glutamate neither attenuates nor augments the BGR phenotypes. Overproduction of Snf1 protein also preferentially rescues the E173H allele. Biochemically, both Snf1 and Reg1(1-740) proteins copurify with Gcn5 protein. Snf1 can phosphorylate recombinant Gcn5 in vitro. Together, these data suggest that Reg1 and Snf1 proteins function in an H3 phosphorylation-independent pathway that also involves a noncatalytic role played by Gcn5 protein.Histone acetylation is a well-studied modification of chromatin (67) and has been linked to transcriptional regulation, recombination, DNA replication, and damage repair (13). GNAT (Gcn5 protein-related N-acetyltransferases) and MYST (MOZ-Ybf2/Sas3-Sas2-Tip60) families of histone acetyltransferases (HATs) generate both targeted and global acetylation of the chromatin (78). Other HATs, such as TAF1 (formerly TAF II 250) and nuclear hormone receptor coactivators, though not belonging to either family, have also been shown to play critical chromatin-related functions via their HAT activities (78).The Saccharomyces cerevisiae Gcn5 protein is the catalytic subunit of several chromatographically distinct HAT complexes, including SAGA, ADA (32), SALSA, and SLIK (70,71,85). SAGA is recruited to the promoter by certain transcriptional activators and causes promoter-specific nucleosomal hyperacetylation leading to transcriptional activation (4,5,48,51,72). The SAGA complex also performs HAT-independent functions, such as TATA binding protein (TBP) recruitment and histone deubiquitinylation (8,9,19,24,38,44,55,75,86). SAGA and SALSA/SLIK complexes share TBP-associated factors with TFIID (33). Low-resolution electron microscopic studies showed that the architectures of SAGA and TFIID complexes are highly similar (3,11,91,103). TFIID is critical for mostly housekeeping gene expression, and the SAGA-dominated genes (ϳ10% of the nuclear genes) are largely stressinduced and are under the coordinated control of multiple chromatin and transcriptional regulators (43).Although the promoter-spec...