In response to harsh environmental conditions, ascomycetes produce stress-resistant spores to promote survival. As sporulation requires a diploid DNA content, species with a haploid lifestyle, such as Kluyveromyces lactis, first induce mating in response to stress. In K. lactis, mating and mating-type switching are induced by the DNA-binding protein Mts1. Mts1 expression is known to be upregulated by nutrient limitation, but the mechanism is unknown. We show that a ras2 mutation results in a hyperswitching phenotype. In contrast, strains lacking the phosphodiesterase Pde2 had lower switching rates compared to that of the wild type (WT). As Ras2 promotes cyclic AMP (cAMP) production and Pde2 degrades cAMP, these data suggest that low cAMP levels induce switching. Because the MTS1 regulatory region contains several Msn2 binding sites and Msn2 is a transcription factor that is activated by low cAMP levels, we investigated if Msn2 regulates MTS1 transcription. Consistently with this idea, an msn2 mutant strain displayed lower switching rates than the WT strain. The transcription of MTS1 is highly induced in the ras2 mutant strain. In contrast, an msn2 ras2 double mutant strain displays WT levels of the MTS1 transcript, showing that Msn2 is a critical inducer of MTS1 transcription. Strains lacking Msn2 and Pde2 also exhibit mating defects that can be complemented by the ectopic expression of Mts1. Finally, we show that MTS1 is subjected to negative autoregulation, presumably adding robustness to the mating and switching responses. We suggest a model in which Ras2/cAMP/Msn2 mediates the stress-induced mating and mating-type switching responses in K. lactis.

Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of the unscheduled meiotic gene expression 6 (UME6) gene in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HMLα and HMRa. Chromatin immunoprecipitation (ChIP) suggested that Ume6 acted directly by binding the cis-regulatory silencers of these loci. Unexpectedly, a MATa ume6 strain was mating proficient, whereas a MATα ume6 strain was sterile. This observation was explained by the fact that ume6 derepressed HMLα2 only weakly, but derepressed HMRa1 strongly. Consistently, two a/α-repressed genes (MTS1 and STE4) were repressed in the MATα ume6 strain, but were expressed in the MATa ume6 strain. Surprisingly, ume6 partially suppressed the mating defect of a MATa sir2 strain. MTS1 and STE4 were repressed in the MATa sir2 ume6 double-mutant strain, indicating that the suppression acted downstream of the a1/α2-repressor. We show that both STE12 and the MATa2/HMRa2 genes were overexpressed in the MATa sir2 ume6 strain. Consistent with the idea that this deregulation suppressed the mating defect, ectopic overexpression of Ste12 and a2 in a MATa sir2 strain resulted in efficient mating. In addition, Ume6 served as a block to polyploidy, since ume6/ume6 diploids mated as pseudo a-strains. Finally, Ume6 was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors.

We explored the regulation of mating type switching in Kluyveromyces lactis. Using an assay dependent on loss of a URA3 gene inserted into the MATa locus, we determined that the switching rate of a wild type strain grown in rich media was ~6X10^-4 events/generation. In a genetic selection for identifying strains with increased switching rates, we found a strain with an insertion in the K. lactis RAS1 gene, encoding a small GTPase with a central role in growth regulation. Compromised Ras1 function leads to a lower cAMP level suggesting a role for cAMP in promoting switching. Consistent with this idea, a strain lacking the PDE2 gene, which encodes an enzyme that degrades cAMP, resulted in decreased switching rates. To explore how cAMP regulated switching, we investigated the transcription of the MTS1 gene, encoding an inducer of switching. The ras1 mutant strain contained 20-fold higher levels of the MTS1 mRNA compared to wild type, but in the pde2 mutant strain MTS1 transcription was repressed 5-fold. In addition, strains lacking the MSN2 gene, which encodes a transcription factor that binds the stress response element (STRE), expressed less MTS1 mRNA and had decreased switching rates. We suggest a model in which nutrient limitation induces switching through cAMP and Msn2-dependent transcriptional induction of the MTS1 gene.

Yen1 is nuclease that can cleave the Holliday junction (HJ), an important DNA intermediate formed during homologous recombination. Here, we show that Yen1 interacts molecularly with Uls1, a SUMO targeted ubiquitin ligase that also belongs to the SWI/SNF-family of DNA-dependent ATPases. We demonstrate that Yen1 is SUMO modified in its carboxyl terminus and that this modification strengthens the interaction between Yen1 and Uls1. Absence of Uls1 increased the steady-state levels of Yen1, but only after extensive DNA damage, suggesting that Uls1 has a role in damage-induced degradation of Yen1. Consistent with a shared role for Uls1 and Yen1, mutations in the two enzymes display similar phenotypes. Both uls1 and yen1 have a negative genetic interaction with the alternative HJ-cleaving nuclease Mus81. This negative genetic interaction is manifested in supersensitivity to DNA damaging agents, but also in a meiotic defect. Neither mus81 uls1 nor mus81 yen1 double mutant diploids can complete meiosis. Moreover, both uls1 and yen1 exacerbates the chromosome mis-segregation phenotype of mus81. However, the mus81 uls1 yen1 triple mutant strain was slightly more sensitive to DNA damage compared to any double mutant combination, indicating that Uls1 and Yen1 also have independent roles in DNA repair. Point mutant alleles of Uls1 (uls1K975R and uls1C1330S/C1333S) that inactivates the ATPase and potential ubiquitin ligase activities are also supersensitive to DNA damage when combined with mus81, indicating that both activities of Uls1 are essential for function. We suggest that Yen1 and Uls1 are involved in an alternative pathway that is responsible for resolving complex DNA repair intermediates in the absence of Mus81.

Yen1 is a nuclease identified in Saccharomyces cerevisiae that cleaves the Holliday junction (HJ) intermediate formed during homologous recombination. Alternative routes to disjoin HJs are performed by the Mus81/Mms4- and Sgs1/Top3/Rmi1-complexes. Here, we investigate the role of the Yen1 protein in the yeast Kluyveromyces lactis. We demonstrate that both yen1 mus81 and yen1 sgs1 double mutants displayed negative genetic interactions in the presence of DNA-damaging chemicals. To test if these phenotypes required the catalytic activity of Yen1, we introduced point mutations targeting the catalytic site of Yen1, which abolished the nuclease activity in vitro. Remarkably, catalytically inactive Yen1 did not exacerbate the hydroxyurea sensitivity of the sgs1Δ strain, which the yen1Δ allele did. In addition, overexpression of catalytically inactive Yen1 partially rescued the DNA damage sensitivity of both mus81 and sgs1 mutant strains albeit less efficiently than WT Yen1. These results suggest that Yen1 serves both a catalytic and non-catalytic role in its redundant function with Mus81 and Sgs1. Diploids lacking Mus81 had a severe defect in sporulation efficiency and crossover frequency, but diploids lacking both Mus81 and Yen1 showed no further reduction in spore formation. Hence, Yen1 had no evident role in meiosis. However, overexpression of WT Yen1, but not catalytically inactive Yen1 partially rescued the crossover defect in mus81/mus81 mutant diploids. Yen1 is a member of the RAD2/XPG-family of nucleases, but genetic analyses revealed no genetic interaction between yen1 and other family members (rad2, exo1 and rad27). In addition, yen1 mutants had normal nonhomologous end-joining efficiency. We discuss the similarities and differences between K. lactis Yen1 and Yen1/GEN1 from other organisms.

Condensed chromatin hinders proteins from accessing the DNA, hence posing a block to processes like DNA repair. In this study, we investigate how histone modifications influence DNA double-strand break (DSB) repair. We show that blocking phosphorylation of serine 129 of histone H2A impairs DSB-repair, probably by reducing the efficiency of homologous recombination (HR). The lysine residues of histone H3 and H4 are subjected to reversible acetylation and methylation and we exchanged the lysines for either arginine (mimicking non-acetylated lysine) or glutamine (mimicking acetylated lysine). A histone H3 mutant with five N-terminal lysines exchanged for arginine showed reduced gene conversion and perturbed cell cycle progression. Leaving a single lysine residue intact was sufficient for protecting cells from DNA damage. In addition, exchanging the five lysines for glutamine did not result in these defects, indicating that one lysine residue in the histone H3 N-terminus must be acetylated for efficient DSB-repair. We find no evidence for that histone modification reduces the efficiency of nonhomologous end joining. Furthermore, the histone H3 K9, 14, 18, 23, 27R mutation is not defective in transcription of DSB repair genes indicating that the defects we observe in DSB-repair is unlikely to be due to indirect regulatory effects. These findings indicate that both histone H2A phosphorylation and histone H3 acetylation is important for the efficiency of the HR-pathway.

Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associatedtranscriptome and many orphan snoRNAs are associated with protein coding genes in the genome ofDrosophila melanogaster. We have studied a specific chromatin-associated snoRNA, snoRNA:U3:9B,that binds to immune response genes. Using a Sindbis virus replicon model, we have shownthat snoRNA:U3:9B depletion in S2 cells leads to reduced immune response gene expression andreduced chromatin accessibility at target immune response genes. We have used CRISPR/Cas9 tocreate a snoRNA:U3:9B knock-out fly strain and revealed that snoRNA:U3:9B-deficient larvae areviable in control conditions, but fail to develop into pupae when challenged by expression of the Sindbisvirus replicon, which suggests that this snoRNA is essential for the activation of an effective antiviralresponse. In agreement with this proposal, the chromatin decompaction and gene activation normallyobserved at immune response gene loci in response to Sindbis replicon expression are abolished inthe snoRNA:U3:9B-deficient larvae, as shown by ATAC-qPCR and RT-qPCR analyses. Moreover,ChIRP-qPCR experiments have shown that snoRNA:U3:9B associates with the immune responsegenes in vivo, which suggests that the defects observed on chromatin compaction and gene expressionare due to direct regulatory events. In summary, our results reveal the existence of an epigeneticmechanism that requires snoRNA:U3:9B to modulate local chromatin accessibility and enable theinduction of immune response genes.

Skeletal muscle is one of the most abundant tissues in the body, and its main function is to generate the force for movement. The mature muscle cell is a giant, elongated, multinucleated cell surrounded by a specialized, extracellular matrix (ECM), the basement membrane (BM). The BM in muscle, as in other tissues, is composed of laminin, type IV collagen, entactin/nidogen and heparan sulphate proteoglycan. One major component of the BM in muscle is laminin-2, which is composed of a heavy chain laminina2 and two light chains,b1 and lamining1. Laminin-2 is predominantly expressed in skeletal muscle and peripheral nerve but is also found in other tissues.

Cell adhesion to the basement membrane is mediated by cell surface receptors, which thereby link the BM to the cytoskeleton. This linkage is thought to be important for generating the force required for movement. Mutations in adhesion molecules in muscle cause muscular dystrophy, proving the importance of cell adhesion in muscle.

In order to analyze the molecular mechanisms of cell adhesion in muscle, we have analyzed laminin-2 and two other muscle adhesion proteins, laminina-sarcoglycan and tetranectin, in muscle development and regeneration. Most importantly, we have developed in vitro and in vivo models for laminin-2 deficient muscular dystrophy.

We generated several lines of mutant embryonic stem (ES) cell with disruption of the laminin- laminina2 chain gene. We found that homozygous null mutant ES cells differentiate normally in vitro, giving rise to cardiomyocytes, myotubes, and smooth muscle cells in addition to many other cell types. However, the myotubes that are formed are unstable. They detach, collapse, and degenerate, a process which is initiated at the appearance of the mature, contractile phenotype of the cells. We propose that the detachment and death of contracting myotubes in vitro has its counterpart in vivo, and that contraction-induced myofiber damage, along with the lack of survival cues provided by laminin-2/merosin, is a significant contribution to muscle degeneration in merosin-deficient muscular dystrophy.

We used laminin laminina2 mutant mice to study the expression of laminin-2 in development and regeneration using the lacZ gene as a reporter for the lama2 gene. We found that the lacZ/lama2 gene is highly expressed in the early stages of myogenesis and is down regulated when myogenesis is completed. Most importantly, the gene is up-regulated early in muscle regeneration, suggesting that laminin-2 plays an important role in this process. Despite the prominent expression of lama2 in normal development, laminina2 null mutant mice have no obvious developmental defect. Instead, they develop muscular dystrophy two weeks after birth. We found extensive apoptosis in null mutant mice, and this cell death is dramatically reduced in mice in which laminin-2 expression is restored in skeletal muscle by expression of a wild type LAMA2 transgene. Most of the apoptotic cells in null mutant mice are newly formed myofibers, suggesting that laminin-2 is needed for maturation and survival of regenerated myotubes. The apparent abortive muscle regeneration in laminin-2 deficiency suggests that the severe disease of MCMD is caused by insufficient regeneration after muscle damage.

We have expressed a human LAMA2 transgene under the regulation of a muscle-specific creatine kinase promoter in mice with complete or partial deficiency of merosin. The transgene restored the synthesis and localization of laminin-2 in skeletal muscle, and greatly improved muscle morphology and integrity and the health and longevity of the mice. However, the transgenic mice share with the non-transgenic dystrophic mice a progressive lameness of hind legs, suggesting a nerve defect. These results indicate that the absence of merosin in tissues other than the muscle, such as nervous tissue, is a critical component of MCMD.

We have cloned and characterized, a-sarcoglycan/adhalin, a member of the dystrophin associated sarcoglycan complex in muscle. We showed that a-sarcoglycan is expressed very late in myogenic differentiation both in vitro and in vivo. In fact, the expression is associated with the capacity of muscle cells to contract. The sarcoglycans may therefore have a role in muscle contraction. We also analyzed an ECM-associated molecule, tetranectin. We showed that expression of tetranectin is closely associated with skeletal muscle development and regeneration, and with muscle cell differentiation in vitro.

In summary, our studies show the importance of laminin-2 in skeletal muscle. We have provided new information on three markers for different stages of myogenic differentiation, laminin laminina2, laminina-sarcoglycan, and tetranectin. In addition, our studies contribute to a better understanding of the mechanism of human disease caused by laminin-2 deficiency.

Neuropeptide Y (NPY) has been implicated in depression, emotional processing and stress response. Part of this evidence originates from human single-nucleotide polymorphism (SNP) studies. In the present study, we report that a SNP in the rat Npy promoter (C/T; rs105431668) affects in vitro transcription and DNA-protein interactions. Genotyping studies showed that the C-allele of rs105431668 is present in a genetic rat model of depression (Flinders sensitive line; FSL), while the SNP's T-allele is present in its controls (Flinders resistant line; FRL). In vivo experiments revealed binding of a transcription factor (CREB2) and a histone acetyltransferase (Ep300) only at the SNP locus of the FRL. Accordingly, the FRL had increased hippocampal levels of Npy mRNA and H3K18 acetylation; a gene-activating histone modification maintained by Ep300. Next, based on previous studies showing antidepressant-like effects of physical activity in the FSL, we hypothesized that physical activity may affect Npy's epigenetic status. In line with this assumption, physical activity was associated with increased levels of Npy mRNA and H3K18 acetylation. Physical activity was also associated with reduced mRNA levels of a histone deacetylase (Hdac5). Conclusively, the rat rs105431668 appears to be a functional Npy SNP that may underlie depression-like characteristics. In addition, the achieved epigenetic reprogramming of Npy provides molecular support for the putative effectiveness of physical activity as a non-pharmacological antidepressant.

Neuropeptide Y (NPY) is known to influence emotional processing and decreased NPY levels have been associated with mood and anxiety disorders. Alternative splicing of pre-messenger RNA is a cellular mechanism that allows for transcriptome diversity, yet there is limited knowledge in this respect with regard to Npy. Since the hippocampus and the prefrontal cortex play an important role in affective disorders, we investigated alternative splicing of Npy in these regions of a rat model of depression (Flinders Sensitive Line, FSL) and its controls (Flinders Resistant Line, FRL). The existence of different Npy messenger RNA (mRNA) variants was examined using 5' and 3' RACE. In addition to the Npy mRNA species annotated in GenBank and Ensembl, we identified a novel short mRNA splice variant. Immunoblotting results argued against a putative translation of this short mRNA into protein in brain tissue. Compared to the FRL, the FSL had reduced short Npy mRNA levels in the HIP (P = 0.00014) and the PFC (P = 0.016). Gene expression analyses in five brain regions of an outbred rat strain supported the presence of the short Npy transcript in all examined regions and showed that it is expressed in similar to 2.4-fold lower levels than the long Npy mRNA. Finally, sequencing of the 5' RACE products revealed a transcription start site of Npy that is different from the currently annotated position. These data add to the characterization of the rat Npy mRNA and demonstrate the presence of a novel transcript with a so far unknown function.

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide(1,2) has successfully identified specific subtypes of regulatory elements(3). In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements(4), chromatin states(5), transcription factor binding sites(6-9), RNA polymerase II regulation(8) and insulator elements(10); however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.

Background: Tissue development and organ growth require constant remodeling of cell-cell contacts formed between epithelial cells. The Hippo signaling cascade curtails organ growth by excluding the transcriptional co-activator Yes Associated Protein 1 (YAP1) from the nucleus. Angiomotin family members recruit YAP1 to tight junctions [1], but whether YAP1 plays a specific role outside of the nucleus is currently unknown. Methodology/Principal Findings: The present study demonstrates that the E3 ubiquitin ligase Nedd4.2 targets Angiomotin-like 1 (AMOTL1), a family member that promotes the formation of epithelial tight junctions, for ubiquitin-dependent degradation. Unexpectedly, YAP1 antagonizes the function of Nedd4.2, and protects AMOTL1 against Nedd4.2-mediated degradation. YAP1 recruits c-Abl, a tyrosine kinase that binds and phosphorylates Nedd4.2 on tyrosine residues, thereby modifying its ubiquitin-ligase activity. Conclusions/Significance: Our results uncover a novel function for cytoplasmic YAP1. YAP1 recruits c-Abl to protect AMOTL1 against Nedd4.2-mediated degradation. Thus, YAP1, excluded from the nucleus, contributes to the maintenance of tight junctions.

The size and shape of epithelial tubes determine the transporting capacities of tubular organs. Here, we analyze two genes involved in airway tube size regulation in Drosophila. Obst-A and gasp encode secreted proteins with chitin binding domains that are conserved among insect species. mRNA in situ hybridizations show that both genes are strongly expressed during airway tube expansion. Gasp protein is secreted into the airway tubes and colocalizes with a chitin binding probe and other chitin binding proteins. Analysis of obst-A and gasp single mutants and obst-A; gasp double mutant shows that both genes are required for larval elongation and airway tube dilation. The assembly of the apical chitinous matrix of the airway tubes is defective in gasp and Obst-A mutants. The defects become exaggerated in double mutants indicating that the genes have partially redundant functions in chitin structure modification. The phenotypes in luminal chitin assembly in the airway tubes are accompanied with a corresponding reduction of tube diameter in the mutants. Conversely, overexpression of Obst-A or Gasp in the airways expands the tube circumference.  Our results indicate that the level of distinct matrix binding proteins in the tubes determines the extent of diametric growth. We propose that Obst-A and Gasp organize the assembly of the luminal matrix and thereby provide distending forces that stretch the apical cell membranes to expand tube diameter accordingly.

The Saccharomyces cerevisiae Dun1 protein kinase is a downstream target of the conserved Mec1-Rad53 checkpoint pathway. Dun1 regulates dNTP pools during an unperturbed cell cycle and after DNA damage by modulating the activity of ribonucleotide reductase (RNR) by multiple mechanisms, including phosphorylation of RNR inhibitors Sml1 and Dif1. Dun1 also activates DNA-damage-inducible genes by inhibiting the Crt1 transcriptional repressor. Among the genes repressed by Crt1 are three out of four RNR genes: RNR2, RNR3, and RNR4. The fourth RNR gene, RNR1, is also DNA damage-inducible, but is not controlled by Crt1. It has been shown that the deletion of DUN1 is synthetic lethal with the deletion of IXR1, encoding an HMG-box-containing DNA binding protein, but the reason for this lethality is not known. Here we demonstrate that the dun1 ixr1 synthetic lethality is caused by an inadequate RNR activity. The deletion of IXR1 results in decreased dNTP levels due to a reduced RNR1 expression. The ixr1 single mutants compensate for the reduced Rnr1 levels by the Mec1-Rad53-Dun1-Crt1-dependent elevation of Rnr3 and Rnr4 levels and downregulation of Sml1 levels, explaining why DUN1 is indispensible in ixr1 mutants. The dun1 ixr1 synthetic lethality is rescued by an artificial elevation of the dNTP pools. We show that Ixr1 is phosphorylated at several residues and that Ser366, a residue important for the interaction of HMG boxes with DNA, is required for Ixr1 phosphorylation. Ixr1 interacts with DNA at multiple loci, including the RNR1 promoter. Ixr1 levels are decreased in Rad53-deficient cells, which are known to have excessive histone levels. A reduction of the histone gene dosage in the rad53 mutant restores Ixr1 levels. Our results demonstrate that Ixr1, but not Dun1, is required for the proper RNR1 expression both during an unperturbed cell cycle and after DNA damage.

BMP signaling responses are refined by distinct secreted and intracellular antagonists in different cellular and temporal contexts. Here, we show that the nuclear LEM-domain protein MAN1 is a tissue-specific antagonist of BMP signaling in Drosophila. MAN1 contains two potential Mad-binding sites. We generated MAN1DeltaC mutants, harbouring a MAN1 protein that lacks part of the C-terminus including the RNA recognition motif, a putative Mad-binding domain. MAN1DeltaC mutants show wing crossvein (CV) patterning defects but no detectable alterations in nuclear morphology. MAN1(DeltaC) pupal wings display expanded phospho-Mad (pMad) accumulation and ectopic expression of the BMP-responsive gene crossveinless-2 (cv-2) indicating that MAN1 restricts BMP signaling. Conversely, MAN1 overexpression in wing imaginal discs inhibited crossvein development and BMP signaling responses. MAN1 is expressed at high levels in pupal wing veins and can be activated in intervein regions by ectopic BMP signaling. The specific upregulation of MAN1 in pupal wing veins may thus represent a negative feedback circuit that limits BMP signaling during CV formation. MAN1DeltaC flies also show reduced locomotor activity, and electrophysiology recordings in MAN1DeltaC larvae uncover a new presynaptic role of MAN1 at the neuromuscular junction (NMJ). Genetic interaction experiments suggest that MAN1 is a BMP signaling antagonist both at the NMJ and during CV formation.