ChIP-exo analysis highlights Fkh1 and Fkh2 transcription factors as hubs that integrate multi-scale networks in budding yeast

The understanding of the multi-scale nature of molecular networks represents a major challenge. For example, regulation of a timely cell cycle must be coordinated with growth, during which changes in metabolism occur, and integrate information from the extracellular environment, e.g. signal transduction. Forkhead transcription factors are evolutionarily conserved among eukaryotes, and coordinate a timely cell cycle progression in budding yeast. Specifically, Fkh1 and Fkh2 are expressed during a lengthy window of the cell cycle, thus are potentially able to function as hubs in the multi-scale cellular environment that interlocks various biochemical networks. Here we report on a novel ChIP-exo dataset for Fkh1 and Fkh2 in both logarithmic and stationary phases, which is analyzed by novel and existing software tools. Our analysis confirms known Forkhead targets from available ChIP-chip studies and highlights novel ones involved in the cell cycle, metabolism and signal transduction. Target genes are analyzed with respect to their function, temporal expression during the cell cycle, correlation with Fkh1 and Fkh2 as well as signaling and metabolic pathways they occur in. Furthermore, differences in targets between Fkh1 and Fkh2 are presented. Our work highlights Forkhead transcription factors as hubs that integrate multi-scale networks to achieve proper timing of cell division in budding yeast

Predictive models of eukaryotic transcriptional regulation reveals changes in transcription factor roles and promoter usage between metabolic conditions

Transcription factors (TF) are central to transcriptional regulation, but they are often studied in relative isolation and without close control of the metabolic state of the cell. Here, we describe genome-wide binding (by ChIP-exo) of 15 yeast TFs in four chemostat conditions that cover a range of metabolic states. We integrate this data with transcriptomics and six additional recently mapped TFs to identify predictive models describing how TFs control gene expression in different metabolic conditions. Contributions by TFs to gene regulation are predicted to be mostly activating, additive and well approximated by assuming linear effects from TF binding signal. Notably, using TF binding peaks from peak finding algorithms gave distinctly worse predictions than simply summing the low-noise and high-resolution TF ChIP-exo reads on promoters. Finally, we discover indications of a novel functional role for three TFs; Gcn4, Ert1 and Sut1 during nitrogen limited aerobic fermentation. In only this condition, the three TFs have correlated binding to a large number of genes (enriched for glycolytic and translation processes) and a negative correlation to target gene transcript levels

Rab7b modulates autophagic flux by interacting with Atg4B

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co‐localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B

RAB24 facilitates clearance of autophagic compartments during basal conditions

Officially accepted for publication August 21, 2015.RAB24 belongs to a family of membrane traffic controlling RAB proteins and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy showed an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicated that this was due to a hindrance in their clearance. Formation of autophagosomes was shown to be unaffected by RAB24 silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 was also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.Peer reviewe

Epigenome-wide meta-analysis of blood DNA methylation in newborns and children identifies numerous loci related to gestational age

Background Preterm birth and shorter duration of pregnancy are associated with increased morbidity in neonatal and later life. As the epigenome is known to have an important role during fetal development, we investigated associations between gestational age and blood DNA methylation in children. Methods We performed meta-analysis of Illumina's HumanMethylation450-array associations between gestational age and cord blood DNA methylation in 3648 newborns from 17 cohorts without common pregnancy complications, induced delivery or caesarean section. We also explored associations of gestational age with DNA methylation measured at 4-18 years in additional pediatric cohorts. Follow-up analyses of DNA methylation and gene expression correlations were performed in cord blood. DNA methylation profiles were also explored in tissues relevant for gestational age health effects: fetal brain and lung. Results We identified 8899 CpGs in cord blood that were associated with gestational age (range 27-42 weeks), at Bonferroni significance, P <1.06 x 10(- 7), of which 3343 were novel. These were annotated to 4966 genes. After restricting findings to at least three significant adjacent CpGs, we identified 1276 CpGs annotated to 325 genes. Results were generally consistent when analyses were restricted to term births. Cord blood findings tended not to persist into childhood and adolescence. Pathway analyses identified enrichment for biological processes critical to embryonic development. Follow-up of identified genes showed correlations between gestational age and DNA methylation levels in fetal brain and lung tissue, as well as correlation with expression levels. Conclusions We identified numerous CpGs differentially methylated in relation to gestational age at birth that appear to reflect fetal developmental processes across tissues. These findings may contribute to understanding mechanisms linking gestational age to health effects.Peer reviewe

Investigating BEACH domain containing proteins for a role in autophagy

The BEACH domain is common for a protein family that has expanded through eukaryotic evolution and contains eight human proteins. These proteins are generally very large and at their C-terminal ends contain a conserved set of domains, a PH-like domain directly followed by a BEACH domain and a WD40 domain. Outside the recognized C-terminal domains, the rest of the proteins are weakly conserved and few functional domains have been recognized. According to their phylogenetic relationship they can be divided into four subfamilies with two closely related proteins in each. ALFY and WDFY4 make up one of these subfamilies where ALFY has been shown to be involved in degradation of protein aggregates, suggesting misregulation of ALFY might contribute to neurodegeneration. NBEAL1 and NBEAL2 form another closely related pair and little is known about these proteins, but recently mutations in NBEAL2 was shown to be causative for the bleeding disorder grey platelets syndrome. LRBA and NBEA are the most studied BEACH proteins and whereas LRBA is implicated in cancers of various types, NBEA is essential for neuromuscular transmission and correlated to autism. LYST and FAN form the final subclass of BEACH proteins. FAN is exceptional in being much smaller than the others, and LYST is well known because mutations in this protein causes Chediak-Higashi syndrome. In summary, the eight BEACH proteins are correlated to a varied set of diseases and studies of their cellular functions so far suggests they have little in common, although some BEACH proteins have been poorly studied. The BEACH domain is highly conserved through the protein family, but the function of the domain is not known. For ALFY, a region of the protein containing the PH-like and BEACH domains has been shown to interact with the ubiquitin-binding protein p62. p62 associates with misfolded and mutant aggregating proteins to aid in their controlled aggregation to form ubiquitinated p62 bodies, which can be targeted for degradation in the acidic interior of the lysosome. Lysosomal degradation in this case starts with de novo formation of a double-membrane structure that surrounds p62 bodies, sequestering them from the rest of the cell and eventually fusing with a lysosome where they are exposed to degrading hydrolases. This process is called macroautophagy and ALFY is linked to this process by its interaction with p62, but also by interacting with other components of the autophagic machinery through its WD40 and FYVE domains. Through this project we have found that the PH-BEACH domains of several BEACH proteins co-localize and interact directly with p62. The results indicate that BEACH proteins of all subfamilies interact with p62 through a groove formed between the PH-like and BEACH domains where residues from both domains seem to contribute to the interaction. Furthermore, autophagy assays were performed to study a possible role for the BEACH proteins in autophagy. Knockdown of the proteins seems to not influence the rate of starvation-induced autophagy, but might influence the turnover of p62 under conditions of basal selective autophagy. Another screen for involvement in autophagy was performed by investigating localization of endogenous BEACH proteins in cell culture upon autophagy-promoting stressors. The results showed that puromycin-induced aggregation of misfolded proteins causes a clear recruitment of several of the BEACH proteins to the p62 bodies, indicating that similar to ALFY, other BEACH proteins may play a role as scaffolding proteins involved in selective autophagy

HS1BP3 inhibits autophagy by regulation of PLD1.

Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1- and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3’-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization. This is an Accepted Manuscript of an article published by Taylor & Francis Group in Autophagy on 20 Mar 2017, available online: http://www.tandfonline.com/10.1080/12345678.1234.123456

Reconstruction of a Global Transcriptional Regulatory Network for Control of Lipid Metabolism in Yeast by Using Chromatin Immunoprecipitation with Lambda Exonuclease Digestion

Transcription factors play a crucial role in the regulation of gene expression and adaptation to different environments. To better understand the underlying roles of these adaptations, we performed experiments that give us high-resolution binding of transcription factors to their targets. We investigated five transcription factors involved in lipid metabolism in yeast, and we discovered multiple novel targets and condition-specific responses that allow us to draw a better regulatory map of the lipid metabolism.To build transcription regulatory networks, transcription factor binding must be analyzed in cells grown under different conditions because their responses and targets differ depending on environmental conditions. We performed whole-genome analysis of the DNA binding of five Saccharomyces cerevisiae transcription factors involved in lipid metabolism, Ino2, Ino4, Hap1, Oaf1, and Pip2, in response to four different environmental conditions in chemostat cultures, which allowed us to keep the specific growth rate constant. Chromatin immunoprecipitation with lambda exonuclease digestion (ChIP-exo) enabled the detection of binding events at a high resolution. We discovered a large number of unidentified targets and thus expanded functions for each transcription factor (e.g., glutamate biosynthesis as a target of Oaf1 and Pip2). Moreover, condition-dependent binding of transcription factors in response to cell metabolic state (e.g., differential binding of Ino2 between fermentative and respiratory metabolic conditions) was clearly suggested. Combining the new binding data with previously published data from transcription factor deletion studies revealed the high complexity of the transcriptional regulatory network for lipid metabolism in yeast, which involves the combinatorial and complementary regulation by multiple transcription factors. We anticipate that our work will provide insights into transcription factor binding dynamics that will prove useful for the understanding of transcription regulatory networks

A bioinformatic pipeline to analyze ChIP-exo datasets

The decrease of sequencing cost in the recent years has made genome-wide studies of transcription factor (TF) binding through chromatin immunoprecipitation methods like ChIP-seq and chromatin immunoprecipitation with lambda exonuclease (ChIP-exo) more accessible to a broader group of users. Especially with ChIP-exo, it is now possible to map TF binding sites in more detail and with less noise than previously possible. These improvements came at the cost of making the analysis of the data more challenging, which is further complicated by the fact that to this date no complete pipeline is publicly available. Here we present a workflow developed specifically for ChIP-exo data and demonstrate its capabilities for data analysis. The pipeline, which is completely publicly available on GitHub, includes all necessary analytical steps to obtain a high confidence list of TF targets starting from raw sequencing reads. During the pipeline development, we emphasized the inclusion of different quality control measurements and we show how to use these so users can have confidence in their obtained results