Coordinate Regulation Of Genes Research Articles

Abstract A27: Coordinate posttranscriptional regulation of breast cancer metastasis genes by RNA binding protein HuR

Abstract Breast cancer is one of the most prevalent cancers worldwide. Distance metastasis is responsible for patient mortality. Therefore, understanding the mechanisms underlying tumor pathogenesis and metastasis is crucial for the development of novel therapies as well as preventive strategies for those who are prone to breast cancer. In contrast to transcriptional gene regulation, posttranscriptional control mechanisms of gene expression are poorly understood. Yet, many metastasis genes are regulated by RNA binding proteins (RBPs) at the levels of mRNA stability and translation. The paraneoplastic antigen, HuR, is an RBP shown to regulate multiple genes that are significantly related to breast cancer metastasis by stabilizing target mRNAs and facilitating translation into proteins. Using novel techniques developed in our lab of RNA immunoprecipitation applied to microarrays (RIP-Chip) we identified novel discrete HuR-associated mRNAs in triple negative breast cancer and estrogen receptor positive breast cancer. Many of these HuR-associated mRNA transcripts were metastasis related. We investigated the role of HuR in an aggressive triple-negative breast cancer metastatic cell line, LM2. The LM2 cells were retrovirally transduced with triple fusion reporter construct encoding thymidine kinase1, GFP and firefly luciferase to obtain in vivo and in vitro tracking capabilities. The cells were further transfected with plasmid containing HA-HuR or empty vector control to demonstrate the function of HuR in LM2. HuR over-expressing clones showed greater metastatic capability by in vitro matrigel invasion assay. Furthermore, athymic mice that were intravenously injected with HuR over-expressing LM2 cells had greater (335-fold more) tumor metastasis to the lungs than empty vector control injected group as measured by IVIS imaging. Mice injected with HuR over-expressing LM2 cells were more moribund and had greater mortality as compared with mice injected with empty vector control cells. These results suggest that HuR may play a role in breast cancer metastasis by stabilizing various pro-metastatic genes. The implications of this work are twofold. First, HuR RIP-Chip can be used to identify novel cancer relevant genes and second, interference with HuR function may ameliorate distant metastasis in breast cancer, potentially providing new therapeutic approaches for treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A27.

Histone Crosstalk Directed by H2B Ubiquitination Is Required for Chromatin Boundary Integrity

Genomic maps of chromatin modifications have provided evidence for the partitioning of genomes into domains of distinct chromatin states, which assist coordinated gene regulation. The maintenance of chromatin domain integrity can require the setting of boundaries. The HS4 insulator element marks the 3′ boundary of a heterochromatin region located upstream of the chicken β-globin gene cluster. Here we show that HS4 recruits the E3 ligase RNF20/BRE1A to mediate H2B mono-ubiquitination (H2Bub1) at this insulator. Knockdown experiments show that RNF20 is required for H2Bub1 and processive H3K4 methylation. Depletion of RNF20 results in a collapse of the active histone modification signature at the HS4 chromatin boundary, where H2Bub1, H3K4 methylation, and hyperacetylation of H3, H4, and H2A.Z are rapidly lost. A remarkably similar set of events occurs at the HSA/HSB regulatory elements of the FOLR1 gene, which mark the 5′ boundary of the same heterochromatin region. We find that persistent H2Bub1 at the HSA/HSB and HS4 elements is required for chromatin boundary integrity. The loss of boundary function leads to the sequential spreading of H3K9me2, H3K9me3, and H4K20me3 over the entire 50 kb FOLR1 and β-globin region and silencing of FOLR1 expression. These findings show that the HSA/HSB and HS4 boundary elements direct a cascade of active histone modifications that defend the FOLR1 and β-globin gene loci from the pervasive encroachment of an adjacent heterochromatin domain. We propose that many gene loci employ H2Bub1-dependent boundaries to prevent heterochromatin spreading.

RUNX Transcription Factor-Mediated Association of Cd4 and Cd8 Enables Coordinate Gene Regulation

SummaryT cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.

Global deficits in development, function, and gene expression in the endocrine pancreas in a deletion mouse model of Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a multisystem disorder caused by genetic loss of function of a cluster of imprinted, paternally expressed genes. Neonatal failure to thrive in PWS is followed by childhood-onset hyperphagia and obesity among other endocrine and behavioral abnormalities. PWS is typically assumed to be caused by an unknown hypothalamic-pituitary dysfunction, but the underlying pathogenesis remains unknown. A transgenic deletion mouse model (TgPWS) has severe failure to thrive, with very low levels of plasma insulin and glucagon in fetal and neonatal life prior to and following onset of progressive hypoglycemia. In this study, we tested the hypothesis that primary deficits in pancreatic islet development or function may play a fundamental role in the TgPWS neonatal phenotype. Major pancreatic islet hormones (insulin, glucagon) were decreased in TgPWS mice, consistent with plasma levels. Immunohistochemical analysis of the pancreas demonstrated disrupted morphology of TgPWS islets, with reduced α- and β-cell mass arising from an increase in apoptosis. Furthermore, in vivo and in vitro studies show that the rate of insulin secretion is significantly impaired in TgPWS β-cells. In TgPWS pancreas, mRNA levels for genes encoding all pancreatic hormones, other secretory factors, and the ISL1 transcription factor are upregulated by either a compensatory response to plasma hormone deficiencies or a primary effect of a deleted gene. Our findings identify a cluster of imprinted genes required for the development, survival, coordinate regulation of genes encoding hormones, and secretory function of pancreatic endocrine cells, which may underlie the neonatal phenotype of the TgPWS mouse model.

Identification of a GATA Switch In Megakaryocytic Development.

AbstractAbstract 2605GATA family transcription factors play critical roles in various mammalian developmental processes, including hematopoiesis. In particular, GATA-1 expression is necessary for proper terminal differentiation of mast cells, red blood cells, eosinophils, and megakaryocytes. GATA-2 is required for proliferation and survival of hematopoietic stem and progenitor cells, and is also expressed in erythroid precursors, mast cells, and early megakaryocytes. In developing erythrocytes, GATA-2 and GATA-1 are responsible for temporal control of a multi-factor transcriptional regulatory network that involves (a) GATA-2 positively regulating its own gene transcription, (b) GATA-2 positively regulating the expression of the Gata1 gene, (c) GATA-1 positively regulating its own gene transcription, and (d) GATA-1 negatively regulating Gata2 gene transcription. During this sequence of events, a “GATA switch” occurs, wherein GATA-1 replaces GATA-2 at canonical GATA binding sites within the regulatory regions of the Gata2 and Gata1 genes, as well as at many other genomic loci that encode genes responsible for proliferation or differentiation of erythroid progenitors. Similarly, in early megakaryocytic progenitors, GATA-2 promotes proliferation and suppresses expression of alternative-lineage genes; subsequent activation of GATA-1 precipitates terminal differentiation with concomitant downregulation of proliferative genes and activation of megakaryocyte-specific genes. The presence or role of a GATA switch in megakaryocytes has not yet been formally investigated. To address the role of the GATA switch in megakaryocytic differentiation, we performed massively parallel sequencing of chromatin immunoprecipitation (ChIP-Seq) material for GATA-2 and GATA-1 before or after GATA-1 restoration in the GATA1-null megakaryocytic progenitor cell line, G1ME. We obtained 22 million unique GATA-2 tags and 10 million unique GATA-1 tags and identified 14985 and 5102 high-confidence GATA-2 and GATA-1 binding sites, respectively. Additionally, we used 13 million tags from ChIP for H3K4me3 to identify 24909 genomic sites enriched for the presence of trimethylated lysine-4 on histone H3. Trimethylated H3K4 marks nearly half of all GATA-1 bound sites and one-third of GATA-2 bound sites. Over 40% of the sites bound by GATA-1 in differentiating G1ME cells were also bound by GATA-2 in proliferating G1ME cells, indicating that a GATA switch does indeed occur during megakaryocyte development. Coordinated analyses of these occupancy data with previously published gene expression datasets show that the lists of bound genes are significantly enriched for differentially expressed genes and the data depict a generally antagonistic relationship between GATA-2 and GATA-1. Interestingly, we find that even among genes that don't contain GATA switch sites, greater than 40% of those bound by GATA-1 were also occupied by GATA-2 at distinct sites. To further characterize the occupied loci, we surveyed the genomic regions bound by GATA-1 and GATA-2 to detect motifs enriched in the sequences surrounding the peak calls. As expected, we found that over 80% contained the canonical WGATAR binding motif. In contrast to reports of motifs enriched in GATA-1 ChIP studies in erythroid cells, we failed to observe significant enrichment of LRF binding motifs. Rather, the GATA-1 and GATA-2 bound regions in megakaryocytes are strongly enriched for motifs that match the binding sites for Ets family transcription factors. Finally, we have found that these genomic regions are indeed occupied by one or more Ets factors in proliferating G1ME cells. Together, these data establish the presence of a GATA switch in megakaryocyte development and provide novel insights into coordinated gene regulation by GATA factors and the differences between the closely related erythroid and megakaryocyte lineages. Disclosures:No relevant conflicts of interest to declare.

Global analysis reveals SRp20- and SRp75-specific mRNPs in cycling and neural cells

Members of the SR protein family of RNA-binding proteins have numerous roles in mRNA metabolism, from transcription to translation. To understand how SR proteins coordinate gene regulation, comprehensive knowledge of endogenous mRNA targets is needed. Here we establish physiological expression of GFP-tagged SR proteins from stable transgenes. Using the GFP tag for immunopurification of mRNPs, mRNA targets of SRp20 and SRp75 were identified in cycling and neurally induced P19 cells. Genome-wide analysis showed that SRp20 and SRp75 associate with hundreds of distinct, functionally related groups of transcripts that change in response to neural differentiation. Knockdown of either SRp20 or SRp75 led to up- or downregulation of specific transcripts, including identified targets, and rescue by the GFP-tagged SR proteins proved their functionality. Thus, SR proteins contribute to the execution of gene-expression programs through their association with distinct endogenous mRNAs.

Direct and Coordinate Regulation of ATP-binding Cassette Transporter Genes by Myc Factors Generates Specific Transcription Signatures That Significantly Affect the Chemoresistance Phenotype of Cancer Cells

Increased expression of specific ATP-binding cassette (ABC) transporters is known to mediate the efflux of chemotherapeutic agents from cancer cells. Therefore, establishing how ABC transporter genes are controlled at their transcription level may help provide insight into the role of these multifaceted transporters in the malignant phenotype. We have investigated ABC transporter gene expression in a large neuroblastoma data set of 251 tumor samples. Clustering analysis demonstrated a strong association between differential ABC gene expression patterns in tumor samples and amplification of the MYCN oncogene, suggesting a correlation with MYCN function. Using expression profiling and chromatin immunoprecipitation studies, we show that MYCN oncoprotein coordinately regulates transcription of specific ABC transporter genes, by acting as either an activator or a repressor. Finally, we extend these notions to c-MYC showing that it can also regulate the same set of ABC transporter genes in other tumor cells through similar dynamics. Overall our findings provide insight into MYC-driven molecular mechanisms that contribute to coordinate transcriptional regulation of a large set of ABC transporter genes, thus affecting global drug efflux.

Global Gene Expression Analysis Reveals Evidence for Decreased Lipid Biosynthesis and Increased Innate Immunity in Uninvolved Psoriatic Skin

Psoriasis is a genetically determined inflammatory skin disease. Although the transition from uninvolved into lesional skin is accompanied by changes in the expression of multiple genes, much less is known about the difference between uninvolved skin from psoriatic patients as opposed to skin from normal individuals. Multiple biochemical and morphological changes were reported decades ago in uninvolved psoriatic skin but remain poorly understood. Here, we show dysregulation of 223 transcripts representing 179 unique genes in uninvolved psoriatic skin, 178 of which were not previously known to be altered in their expression. The proteins encoded by these transcripts are involved in lipid metabolism, antimicrobial defenses, epidermal differentiation, and control of cutaneous vasculature. Cluster analysis of transcripts with significantly altered expression identified a group of genes involved in lipid metabolism with highly correlated gene expression. Promoter analysis showed enrichment for binding sites of three transcription factors; peroxisome proliferator-activator receptor alpha (PPARA), sterol regulatory element-binding protein (SREBF), and estrogen receptor 2 (ESR2), suggesting that the coordinate regulation of lipid metabolic genes may be related to the action of these factors. Taken together, our results identify a "pre-psoriatic" gene expression signature, suggesting decreased lipid biosynthesis and increased innate immunity in uninvolved psoriatic skin.

Dynamics of Genomic Organization.

Abstract SCI-17We have investigated the relationships between lineage-specific gene expression, and total genomic organization during hematopoiesis. First, we determined the linear chromosomal distribution of genes that are co-regulated (identified via microarray analysis) when murine hematopoietic progenitor cells (FDCP-mixA) are differentiated to the erythroid and neutrophil lineages, as well as the organization of all chromosomes (in the form of rosettes) in the three cell types. Our analysis revealed a significant tendency for co-regulated genes to be proximal, which is related to the association of homologous chromosomes and the spatial juxtaposition of lineage-specific gene domains. This led us to hypothesize that the genome—at the level of chromosomes—may self-organize to facilitate coordinate gene regulation during cellular differentiation. We tested this hypothesis by applying the approaches of distance matrices and coupled oscillators to our datasets of gene expression and chromosomal associations from the differentiation of the progenitor to the erythroid and neutrophil lineages. Our analysis revealed that coordinate gene expression undergoes a phase transition—characterized by an increase in entropy—upon commitment of the progenitor. As differentiation continues, there is a gradual loss of entropy, culminating in a highly ordered state in the differentiated cell types. The coregulated gene sets of the semi-ordered progenitor and ordered erythroid and neutrophil lineages are significantly correlated with lineage-specific chromosomal association patterns. Furthermore, by transforming the gene expression networks along the time course to corresponding chromosomal association matrices, we found that chromosomal topologies change dynamically during differentiation but, as with gene expression, result in a more highly ordered state in the differentiated cell types. Our analysis demonstrates that the networks of co-regulated gene expression and chromosomal association are mutually related during differentiation, resulting in the self-organization of lineage-specific chromosomal topologies. DisclosuresNo relevant conflicts of interest to declare.

AMP-activated protein kinase and carbohydrate response element binding protein: A study of two potential regulatory factors in the hepatic lipogenic program of broiler chickens

This study investigated the effects of fasting and refeeding on AMP-activated protein kinase (AMPK) and carbohydrate response element binding protein (ChREBP) mRNA, protein and activity levels; as well as the expression of lipogenic genes involved in regulating lipid synthesis in broiler chicken (Gallus gallus) liver. Fasting for 24 or 48 h produced significant declines in plasma glucose (at 24 h), insulin and thyroid hormone (T3) levels that were accompanied by changes in mRNA expression levels of hepatic lipogenic genes. The mRNA levels of malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase alpha (ACCalpha), fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1) and thyroid hormone responsive Spot 14 (Spot 14) declined in response to fasting. Refeeding for 24 h increased mRNA levels for each of these genes, characterized by a significant increase ('overshoot') above fed control values. No change in mRNA expression of the two AMPK alpha subunit genes was observed in response to fasting or refeeding. In contrast, ChREBP and sterol regulatory element binding protein-1 (SREBP-1) mRNA levels decreased during fasting and increased with refeeding. Phosphorylation of AMPK alpha subunits increased modestly after a 48 h fast. However, there was no corresponding change in the phosphorylation of ACC, a major downstream target of AMPK. Protein level and DNA-binding activity of ChREBP increased during fasting and declined upon refeeding as measured in whole liver tissue extracts. In general, evidence was found for coordinate transcriptional regulation of lipogenic program genes in broiler chicken liver, but specific regulatory roles for AMPK and ChREBP in that process remain to be further characterized.

Sonic Hedgehog Induces Notch Target Gene Expression in Vascular Smooth Muscle Cells via VEGF-A

Notch, VEGF, and components of the Hedgehog (Hh) signaling pathway have been implicated in vascular morphogenesis. The role of Notch in mediating hedgehog control of adult vascular smooth muscle cell (SMC) growth and survival remains unexplored. In cultured SMCs, activation of Hh signaling with recombinant rShh (3.5 mug/mL) or plasmid encoded Shh increased Ptc1 expression, enhanced SMC growth and survival and promoted Hairy-related transcription factor (Hrt) expression while concomitantly increasing VEGF-A levels. These effects were significantly reversed after Hh inhibition with cyclopamine. Shh-induced stimulation of Hrt-3 mRNA and SMC growth and survival was attenuated after inhibition of Notch-mediated CBF-1/RBP-Jk-dependent signaling with RPMS-1 while siRNA knockdown of Hrt-3 inhibited SMC growth and survival. Recombinant VEGF-A increased Hrt-3 mRNA levels while siRNA knockdown abolished rShh stimulated VEGF-A expression while concomitantly inhibiting Shh-induced increases in Hrt-3 mRNA levels, proliferating cell nuclear antigen (PCNA), and Notch 1 IC expression, respectively. Hedgehog components were expressed within intimal SMCs of murine carotid arteries after vascular injury concomitant with a significant increase in mRNA for Ptc1, Gli(2), VEGF-A, Notch 1, and Hrts. Hedgehog promotes a coordinate regulation of Notch target genes in adult SMCs via VEGF-A.

The emergence of lineage-specific chromosomal topologies from coordinate gene regulation

Although the importance of chromosome organization during mitosis is clear, it remains to be determined whether the nucleus assumes other functionally relevant chromosomal topologies. We have previously shown that homologous chromosomes have a tendency to associate during hematopoiesis according to their distribution of coregulated genes, suggesting cell-specific nuclear organization. Here, using the mathematical approaches of distance matrices and coupled oscillators, we model the dynamic relationship between gene expression and chromosomal associations during the differentiation of a multipotential hematopoietic progenitor. Our analysis reveals dramatic changes in total genomic order: Commitment of the progenitor results in an initial increase in entropy at both the level of gene coregulation and chromosomal organization, which we suggest represents a phase transition, followed by a progressive decline in entropy during differentiation. The stabilization of a highly ordered state in the differentiated cell types results in lineage-specific chromosomal topologies and is related to the emergence of coherence-or self-organization-between chromosomal associations and coordinate gene regulation. We discuss how these observations may be generally relevant to cell fate decisions encountered by progenitor/stem cells.

Coordinate posttranscriptional regulation of asthma genes by the RNA binding protein HuR (79.6)

Abstract Traditional microarray analysis has uncovered a variety of genes involved in diseases such as asthma. However, it may potentially miss important genes due to the discordance between steady state mRNA levels and protein products. Posttranscriptional events conferred by RNA binding proteins (RBPs) contribute to this phenomenon. We have developed novel techniques known as RIP-on-Chip (microchip) which allow for the immunoprecipitation and identification of in vivo mRNA targets bound to RBPs. We study the RBP HuR, which regulates many early response genes including cytokines and chemokines. Using RIP-on-Chip, HuR has been demonstrated to associate and regulate many asthma relevant genes including recently identified genes IL-4 and IL-13. We have extended this assay to solid organ tissues. Employing the standard ovalbumin model of allergic airway inflammation, coupled to genome-wide arrays (47,000 genes) we have identified in vivo HuR targets in lungs of naïve, alum only or ova challenged mice. Remarkably, there are only 12 HuR targets (2 fold or greater) up regulated in ova and alum immunized over alum alone mice. Some are known HuR target genes such as TLR4 and IL-13; others are potentially novel asthma genes. Furthermore, we have created a transgenic mouse which over expresses HuR in CD4+ T cells. In the allergic airway inflammation model these mice have an increase in lung lymphocyte infiltration when compared to wild-type littermates, further implicating coordinate posttranscriptional gene regulation and HuR in allergic airway inflammation.

Zebrafish reward mutants reveal novel transcripts mediating the behavioral effects of amphetamine

Transcriptome analysis of a zebrafish mutant that does not respond to amphetamine identifies a network of coordinated gene regulation that may underlie the susceptibility to addiction.

Frequency-modulated nuclear localization bursts coordinate gene regulation

In yeast, the transcription factor Crz1 is dephosphorylated and translocates into the nucleus in response to extracellular calcium. Here we show, using time-lapse microscopy, that Crz1 exhibits short bursts of nuclear localization (typically lasting 2 min) that occur stochastically in individual cells and propagate to the expression of downstream genes. Strikingly, calcium concentration controls the frequency, but not the duration, of localization bursts. Using an analytic model, we also show that this frequency modulation of bursts ensures proportional expression of multiple target genes across a wide dynamic range of expression levels, independent of promoter characteristics. We experimentally confirm this theory with natural and synthetic Crz1 target promoters. Another stress-response transcription factor, Msn2, exhibits similar, but largely uncorrelated, localization bursts under calcium stress suggesting that frequency-modulation regulation of localization bursts may be a general control strategy used by the cell to coordinate multi-gene responses to external signals.

Coordinating assembly of a bacterial macromolecular machine

The assembly of large and complex organelles, such as the bacterial flagellum, poses the formidable problem of coupling temporal gene expression to specific stages of the organelle-assembly process. The discovery that levels of the bacterial flagellar regulatory protein FlgM are controlled by its secretion from the cell in response to the completion of an intermediate flagellar structure (the hook-basal body) was only the first of several discoveries of unique mechanisms that coordinate flagellar gene expression with assembly. In this Review, we discuss this mechanism, together with others that also coordinate gene regulation and flagellar assembly in Gram-negative bacteria.

Histoplasma Requires SID1, a Member of an Iron-Regulated Siderophore Gene Cluster, for Host Colonization

The macrophage is the primary host cell for the fungal pathogen Histoplasma capsulatum during mammalian infections, yet little is known about fungal genes required for intracellular replication in the host. Since the ability to scavenge iron from the host is important for the virulence of most pathogens, we investigated the role of iron acquisition in H. capsulatum pathogenesis. H. capsulatum acquires iron through the action of ferric reductases and the production of siderophores, but the genes responsible for these activities and their role in virulence have not been determined. We identified a discrete set of co-regulated genes whose transcription is induced under low iron conditions. These genes all appeared to be involved in the synthesis, secretion, and utilization of siderophores. Surprisingly, the majority of these transcriptionally co-regulated genes were found clustered adjacent to each other in the genome of the three sequenced strains of H. capsulatum, suggesting that their proximity might foster coordinate gene regulation. Additionally, we identified a consensus sequence in the promoters of all of these genes that may contribute to iron-regulated gene expression. The gene set included L-ornithine monooxygenase (SID1), the enzyme that catalyzes the first committed step in siderophore production in other fungi. Disruption of SID1 by allelic replacement resulted in poor growth under low iron conditions, as well as a loss of siderophore production. Strains deficient in SID1 showed a significant growth defect in murine bone-marrow-derived macrophages and attenuation in the mouse model of infection. These data indicated that H. capsulatum utilizes siderophores in addition to other iron acquisition mechanisms for optimal growth during infection.

MITOCHIP assessment of differential gene expression in the skeletal muscle of Ant1 knockout mice: Coordinate regulation of OXPHOS, antioxidant, and apoptotic genes

Genetic inactivation of the nuclear-encoded mitochondrial heart-muscle adenine nucleotide translocator-1 (ANT1), which exports mitochondrial ATP to the cytosol in both humans (ANT1−/−) and mice (Ant1−/−), results in lactic acidosis and mitochondrial cardiomyopathy and myopathy, the latter involving hyper-proliferation of mitochondria, induction of oxidative phosphorylation (OXPHOS) enzymes, increased reactive oxygen species (ROS), and excessive mtDNA damage. To understand these manifestations, we analyzed Ant1−/− mouse skeletal muscle for changes in gene expression using our custom 644 and 1087 gene MITOCHIP microarrays and for changes in the protein levels of key mitochondrial transcription factors. Thirty-four mRNAs were found to be up-regulated and 29 mRNAs were down-regulated. Up-regulated mRNAs included the mitochondrial DNA (mtDNA) polypeptide and rRNA genes, selected nuclear-encoded OXPHOS genes, and stress-response genes including Mcl-1. Down-regulated mRNAs included glycolytic genes, pro-apoptotic genes, and c-Myc. The mitochondrial regulatory proteins Pgc-1α, Nrf-1, Tfam, and myogenin were up-regulated and could account for the induction of the OXPHOS and antioxidant enzymes. By contrast, c-Myc levels were reduced and might account for a reduction in apoptotic potential. Therefore, the Ant1−/− mouse skeletal muscle demonstrates that energy metabolism, antioxidant defenses, and apoptosis form an integrated metabolic network.

Rapid detection and curation of conserved DNA via enhanced-BLAT and EvoPrinterHD analysis

BackgroundMulti-genome comparative analysis has yielded important insights into the molecular details of gene regulation. We have developed EvoPrinter, a web-accessed genomics tool that provides a single uninterrupted view of conserved sequences as they appear in a species of interest. An EvoPrint reveals with near base-pair resolution those sequences that are essential for gene function.ResultsWe describe here EvoPrinterHD, a 2nd-generation comparative genomics tool that automatically generates from a single input sequence an enhanced view of sequence conservation between evolutionarily distant species. Currently available for 5 nematode, 3 mosquito, 12 Drosophila, 20 vertebrate, 17 Staphylococcus and 20 enteric bacteria genomes, EvoPrinterHD employs a modified BLAT algorithm [enhanced-BLAT (eBLAT)], which detects up to 75% more conserved bases than identified by the BLAT alignments used in the earlier EvoPrinter program. The new program also identifies conserved sequences within rearranged DNA, highlights repetitive DNA, and detects sequencing gaps. EvoPrinterHD currently holds over 112 billion bp of indexed genomes in memory and has the flexibility of selecting a subset of genomes for analysis. An EvoDifferences profile is also generated to portray conserved sequences that are uniquely lost in any one of the orthologs. Finally, EvoPrinterHD incorporates options that allow for (1) re-initiation of the analysis using a different genome's aligning region as the reference DNA to detect species-specific changes in less-conserved regions, (2) rapid extraction and curation of conserved sequences, and (3) for bacteria, identifies unique or uniquely shared sequences present in subsets of genomes.ConclusionEvoPrinterHD is a fast, high-resolution comparative genomics tool that automatically generates an uninterrupted species-centric view of sequence conservation and enables the discovery of conserved sequences within rearranged DNA. When combined with cis-Decoder, a program that discovers sequence elements shared among tissue specific enhancers, EvoPrinterHD facilitates the analysis of conserved sequences that are essential for coordinate gene regulation.

Object-Place Recognition Learning Triggers Rapid Induction of Plasticity-Related Immediate Early Genes and Synaptic Proteins in the Rat Dentate Gyrus

Long-term recognition memory requires protein synthesis, but little is known about the coordinate regulation of specific genes. Here, we examined expression of the plasticity-associated immediate early genes (Arc, Zif268, and Narp) in the dentate gyrus following long-term object-place recognition learning in rats. RT-PCR analysis from dentate gyrus tissue collected shortly after training did not reveal learning-specific changes in Arc mRNA expression. In situ hybridization and immunohistochemistry were therefore used to assess possible sparse effects on gene expression. Learning about objects increased the density of granule cells expressing Arc, and to a lesser extent Narp, specifically in the dorsal blade of the dentate gyrus, while Zif268 expression was elevated across both blades. Thus, object-place recognition triggers rapid, blade-specific upregulation of plasticity-associated immediate early genes. Furthermore, Western blot analysis of dentate gyrus homogenates demonstrated concomitant upregulation of three postsynaptic density proteins (Arc, PSD-95, and α-CaMKII) with key roles in long-term synaptic plasticity and long-term memory.