CREB3 subfamily transcription factors are not created equal: Recent insights from global analyses and animal models

<p>Abstract</p> <p>The CREB3 subfamily of membrane-bound bZIP transcription factors has five members in mammals known as CREB3 and CREB3L1-L4. One current model suggests that CREB3 subfamily transcription factors are similar to ATF6 in regulated intramembrane proteolysis and transcriptional activation. Particularly, they were all thought to be proteolytically activated in response to endoplasmic reticulum (ER) stress to stimulate genes that are involved in unfolded protein response (UPR). Although the physiological inducers of their proteolytic activation remain to be identified, recent findings from microarray analyses, RNAi screens and gene knockouts not only demonstrated their critical roles in regulating development, metabolism, secretion, survival and tumorigenesis, but also revealed cell type-specific patterns in the activation of their target genes. Members of the CREB3 subfamily show differential activity despite their structural similarity. The spectrum of their biological function expands beyond ER stress and UPR. Further analyses are required to elucidate the mechanism of their proteolytic activation and the molecular basis of their target recognition.</p

Data integration for microarrays: enhanced inference for gene regulatory networks

Microarray technologies have been the basis of numerous important findings regarding gene expression in the last decades. Studies have generated large amounts of data describing various processes, which, due to the existence of public databases, are widely available for further analysis. Given their lower cost and higher maturity compared to newer sequencing technologies, these data continue to be produced, even though data quality has been the subject of some debate. However, given the large volume of data generated, integration can help overcome some issues related e.g. to noise or reduced time resolution, while providing additional insight on features not directly addressed by sequencing methods. Here we present an integration test case based on public Drosophila melanogaster datasets (gene expression, binding site affinities, known interactions). Using an evolutionary computation framework, we show how integration can enhance the ability to recover transcriptional gene regulatory networks from these data, as well as indicating which data types are more important for quantitative and qualitative network inference. Our results show a clear improvement in performance when multiple data sets are integrated, indicating that microarray data will remain a valuable and viable resource for some time to come

EGIA–evolutionary optimisation of gene regulatory networks, an integrative approach

Quantitative modelling of gene regulatory networks (GRNs) is still limited by data issues such as noise and the restricted length of available time series, creating an under-determination problem. However, large amounts of other types of biological data and knowledge are available, such as knockout experiments, annotations and so on, and it has been postulated that integration of these can improve model quality. However, integration has not been fully explored, to date. Here, we present a novel integrative framework for different types of data that aims to enhance model inference. This is based on evolutionary computation and uses different types of knowledge to introduce a novel customised initialisation and mutation operator and complex evaluation criteria, used to distinguish between candidate models. Specifically, the algorithm uses information from (i) knockout experiments, (ii) annotations of transcription factors, (iii) binding site motifs (expressed as position weight matrices) and (iv) DNA sequence of gene promoters, to drive the algorithm towards more plausible network structures. Further, the evaluation basis is also extended to include structure information included in these additional data. This framework is applied to both synthetic and real gene expression data. Models obtained by data integration display both quantitative and qualitative improvement

Golgi Outpost Synthesis Impaired by Toxic Polyglutamine Proteins Contributes to Dendritic Pathology in Neurons

Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. Here, we show that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, our results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway. ? 2017 The Author(s)113Ysciescopu

The CrebA/Creb3-like transcription factors are major and direct regulators of secretory capacity

CrebA up-regulates expression of both the general protein machinery required in all cells for secretion and genes encoding cell type–specific secreted components

Genome-wide features of neuroendocrine regulation in Drosophila by the basic helix-loop-helix transcription factor DIMMED.

Neuroendocrine (NE) cells use large dense core vesi-cles (LDCVs) to traffic, process, store and secrete neuropeptide hormones through the regulated secre-tory pathway. The dimmed (DIMM) basic helix-loop-helix transcription factor of Drosophila controls the level of regulated secretory activity in NE cells. To pursue its mechanisms, we have performed two in-dependent genome-wide analyses of DIMM’s activi-ties: (i) in vivo chromatin immunoprecipitation (ChIP) to define genomic sites of DIMM occupancy and (ii) deep sequencing of purified DIMM neurons to char-acterize their transcriptional profile. By this com-bined approach, we showed that DIMM binds to con-served E-boxes in enhancers of 212 genes whose expression is enriched in DIMM-expressing NE cells. DIMM binds preferentially to certain E-boxes within first introns of specific gene isoforms. Statistical ma-chine learning revealed that flanking regions of puta-tive DIMM binding sites contribute to its DNA binding specificity. DIMM’s transcriptional repertoire features at least 20 LDCV constituents. In addition, DIMM no-tably targets the pro-secretory transcription factor, creb-A, but significantly, DIMM does not target any neuropeptide genes. DIMM therefore prescribes the scale of secretory activity in NE neurons, by a sys-tematic control of both proximal and distal points in the regulated secretory pathway

A Journey to Understand Glucose Homeostasis: Starting from Rat Glucose Transporter Type 2 Promoter Cloning to Hyperglycemia

My professional journey to understand the glucose homeostasis began in the 1990s, starting from cloning of the promoter region of glucose transporter type 2 (GLUT2) gene that led us to establish research foundation of my group. When I was a graduate student, I simply thought that hyperglycemia, a typical clinical manifestation of type 2 diabetes mellitus (T2DM), could be caused by a defect in the glucose transport system in the body. Thus, if a molecular mechanism controlling glucose transport system could be understood, treatment of T2DM could be possible. In the early 70s, hyperglycemia was thought to develop primarily due to a defect in the muscle and adipose tissue; thus, muscle/adipose tissue type glucose transporter (GLUT4) became a major research interest in the diabetology. However, glucose utilization occurs not only in muscle/adipose tissue but also in liver and brain. Thus, I was interested in the hepatic glucose transport system, where glucose storage and release are the most actively occurring.ope

Tissue-specific ChIP-seq of Drosophila Salivary Gland Transcription Factors Reveal that CrebA Directly Regulates Secretory Regulators and Effectors

Transcription factors (TFs) regulate the expression of tissue specific genes. In the Drosophila embryonic salivary gland (SG), four of these transcription factors include: CrebA, fork head (fkh), Sage, and sensless (sens). CrebA and its Creb3-like homologs are known for their regulation of secretory capacity. fkh is a regulator of SG morphogenesis and, with Sage and sens, controls SG survival. With the exception of Sage, all of these TFs are expressed in multiple tissues. To determine the SG-specific direct targets of these TFs, all four were tagged with GFP in preparation for Chromatin Immunoprecipitation and sequencing (ChIP-seq) in which GFP would be pulled down. The GFP-tagged forms were expressed using two drivers that have the SG in common, so by studying the intersection of these two datasets, SG-specific binding can be inferred. As controls, wild-type embryonic chromatin is immunoprecipitated with either CrebA or Sage antisera. For CrebA, which is ubiquitously expressed, this potentially permits us to distinguish tissue-specific targets of CrebA from CrebA targets bound in all tissues. Because Sage is SG specific, a comparison of the GFP datasets and the wild-type Sage pull down dataset controls for the accuracy ofgood the Sage-GFP dataset. All of the GFP tagged TFs are fully functional, and to date, we have obtained the results for CrebA binding. For the Fkh, Sage, and Sens data, we have sent the prepared chromatin to our collaborators at University of Minnesota, Duluth. The CrebA ChIP-seq datasets revealed that CrebA binds to open chromatin containing consensus CrebA binding sites; that the subset of genes that CrebA regulates are involved in secretion, including Xbp1, TSN, and tbc1; and that loss of CrebA and/or the binding sites leads to misregulation of Xbp1 and TSN. The highly conserved gene, tbc1 is known to be involved in the trans Golgi network from mammalian studies and we find that it is localized to the trans most portion of the TGN in Drosophila. Loss of tbc1 leads to apical membrane defects and secretion defects in the SG and cuticle secretion defects. This predicted Rab-GAP co-localizes with several Rabs but does not affect the steady-state levels two of these Rabs, Rab5 and Rab11

Identification of Creb3l4 as an essential negative regulator of adipogenesis

Understanding the molecular networks that regulate adipogenesis is crucial for combating obesity. However, the identity and molecular actions of negative regulators that regulate the early development of adipocytes remain poorly understood. In this study, we investigated the role of CREB3L4, a member of the CREB3-like family, in the regulation of adiposity. Constitutive overexpression of CREB3L4 resulted in the inhibition of adipocyte differentiation, whereas knockdown of Creb3l4 expression caused differentiation of preadipocytes into mature adipocytes, bypassing the mitotic clonal expansion step. In 3T3-L1 preadipocytes, Creb3l4 knockdown resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ2) and CCAAT/enhancer binding protein (C/EBPα), either by increasing the protein stability of C/EBPβ or by decreasing the expression of GATA3, a negative regulator of PPARγ2 expression. Consequently, increased PPARγ2 and C/EBPα levels induced adipocyte differentiation, even in the presence of minimal hormonal inducer. Thus, it can be speculated that CREB3L4 has a role as gatekeeper, inhibiting adipogenesis in 3T3-L1 preadipocytes. Moreover, adipocytes of Creb3l4-knockout mice showed hyperplasia caused by increased adipogenesis, and exhibited improved glucose tolerance and insulin sensitivity, as compared with littermate wild-type mice. These results raise the possibility that Creb3l4 could be a useful therapeutic target in the fight against obesity and metabolic syndrome.ope

Identification et caractérisation de modulateurs naturels et synthétiques du facteur de transcription AIBZIP

Androgen-induced bZIP (AlbZIP) a été identifié au début des années 2000 dans le cadre d'une étude visant à identifier des gènes régulés par les androgènes. Chez l'homme, ce facteur est très abondant au niveau de la prostate et présente une expression beaucoup plus importante au niveau des cellules cancéreuses prostatiques. L'analyse de la structure d'AIbZIP a révélé la présence d'un domaine bZIP. Ce domaine constitue la signature de la famille ATF/CREB, d'où l'appartenance d'AIbZIP à cette famille. Durant la dernière décennie, 4 autres membres de la famille ATF/CREB ont été découverts et, avec AlbZIP, ils constituent la sous-famille de CREB3. Au-delà de l'homologie observée entre leurs domaines bZIP, les membres de cette sous-famille sont impliqués dans le stress du reticulum endoplasmique (RE). AlbZIP est une protéine transmembranaire de type II localisée au RE sous sa forme inactive. En effet, la protéine AlbZIP pleine longueur est ancrée à la membrane du reticulum endoplasmique via son domaine transmembranaire et orientée de sorte que son domaine C-terminal se trouve dans la lumière du RE alors que son domaine N-terminal est projeté dans le cytoplasme. Des expériences réalisées par notre équipe ont montré qu'AlbZIP est régulée par le mécanisme RIP. En effet, suite à l'altération des concentrations calciques, nous observons la migration de la forme pleine longueur d'AIbZIP vers l'appareil de golgi où elle va subir un clivage protéolytique par les proteases SIP et S2P. La forme active libérée migre au noyau où elle va réguler l'expression de ses gènes cible via les éléments de réponse UPRE et ERSEII. Lors de mes études doctorales, j'ai tenté de déterminer comment AlbZIP est impliquée dans le stress du RE des cellules prostatiques cancéreuses et quels sont les mécanismes impliqués dans son activation et dans la régulation de ses gènes cibles. Dans un premier temps, j'ai tenté d'inhiber l'activité transcriptionnelle d'AIbZIP. Pour ce faire, j'ai généré et caractérisé un dominant négatif en utilisant l'algorithme développé par Mason et collaborateurs. Ce dominant négatif est capable de lier la forme nucléaire d'AIbZIP de type sauvage, de l'empêcher de lier les éléments de réponse et par conséquent d'inhiber son activité transcriptionnelle. J'ai tenté, dans un deuxième temps, d'identifier les partenaires de la forme pleine longueur d'AIbZIP, quand celle-ci se trouve au RE. Dans le but de mieux comprendre le mécanisme d'action impliqué dans l'activation d'AIbZIP, il était nécessaire de connaître les partenaires d'AIbZIP au RE. Grâce à cette étude, plusieurs protéines ont été identifiées. Ces protéines sont localisées au RE ou à l'appareil de golgi. Elles sont transmembranaires ou solubles et peuvent être impliquées dans la rétention d'AIbZIP au RE ou dans son transport vers les autres organites. Dans un troisième temps, j'ai tenté d'identifier les partenaires de la forme nucléaire d'AIbZIP. WD repeat domain 5 (WDR5) est une des partenaires d'AIbZIP au noyau. WDR5 présente 7 répétitions WD40 formant une structure rigide nécessaire pour les interactions protéine-protéine. De plus, elle est impliquée dans la tri-méthylation de l'histone H3. En réponse au stress du RE déclenché par une depletion des concentrations calciques, AlbZIP est activée par le mécanisme RIP. Une fois au noyau, la forme active d'AIbZIP, sous forme de dimères, recrute WDR5 au niveau de l'ADN. Ce recrutement est nécessaire pour modifier la chromatine, la rendre accessible à la machinerie de la transcription et par conséquent activer l'expression des gènes cibles d'AIbZIP. Ensemble, toutes ces données permetteront de mieux comprendre la régulation d'AIbZIP et la régulation de ses gènes cibles et ainsi cerner son rôle dans la réponse au stress du RE dans les cellules cancéreuses prostatiques humaines