Methylation Patterns of the FKBP5 Gene in Association with Childhood Maltreatment and Depressive Disorders.

Childhood maltreatment is an important risk factor for adult depression and has been associated with changes in the hypothalamic pituitary adrenal (HPA) axis, including cortisol secretion and methylation of the FKBP5 gene. Furthermore, associations between depression and HPA changes have been reported. This study investigated the associations of whole-blood FKBP5 mRNA levels, serum cortisol levels, childhood maltreatment, and depressive symptoms with the whole-blood methylation status (assessed via target bisulfite sequencing) of 105 CpGs at the FKBP5 locus using data from the general population-based Study of Health in Pomerania (SHIP) (N = 203). Both direct and interaction effects with the rs1360780 single-nucleotide polymorphism were investigated. Nominally significant associations of main effects on methylation of a single CpG site were observed at intron 3, intron 7, and the 3'-end of the gene. Additionally, methylation at two clusters at the 3'-end and intron 7 were nominally associated with childhood maltreatment × rs1360780 and depressive symptoms × rs1360780, respectively. The results add to the understanding of molecular mechanisms underlying the emergence of depression and could aid the development of personalised depression therapy and drug development.

High-throughput screening of glucocorticoid-induced enhancer activity reveals mechanisms of stress-related psychiatric disorders.

Exposure to stressful life events increases the risk for psychiatric disorders. Mechanistic insight into the genetic factors moderating the impact of stress can increase our understanding of disease processes. Here, we test 3,662 single nucleotide polymorphisms (SNPs) from preselected expression quantitative trait loci in massively parallel reporter assays to identify genetic variants that modulate the activity of regulatory elements sensitive to glucocorticoids, important mediators of the stress response. Of the tested SNP sequences, 547 were located in glucocorticoid-responsive regulatory elements of which 233 showed allele-dependent activity. Transcripts regulated by these functional variants were enriched for those differentially expressed in psychiatric disorders in the postmortem brain. Phenome-wide Mendelian randomization analysis in 4,439 phenotypes revealed potentially causal associations specifically in neurobehavioral traits, including major depression and other psychiatric disorders. Finally, a functional gene score derived from these variants was significantly associated with differences in the physiological stress response, suggesting that these variants may alter disease risk by moderating the individual set point of the stress response.

Extensive evaluation of DNA methylation of functional elements in the murine Fkbp5 locus using high-accuracy DNA methylation measurement via targeted bisulfite sequencing.

FKBP5 is an important stress-regulatory gene implicated in stress-related psychiatric diseases. Single nucleotide polymorphisms of the FKBP5 gene were shown to interact with early life stress to alter the glucocorticoid-related stress response and moderate disease risk. Demethylation of cytosine-phosphate-guanine-dinucleotides (CpGs) in regulatory glucocorticoid-responsive elements was suggested to be the mediating epigenetic mechanism for long-term stress effects, but studies on Fkbp5 DNA methylation (DNAm) in rodents are so far limited. We evaluated the applicability of high-accuracy DNA methylation measurement via targeted bisulfite sequencing (HAM-TBS), a next-generation sequencing-based technology, to allow a more in-depth characterisation of the DNA methylation of the murine Fkbp5 locus in three different tissues (blood, frontal cortex and hippocampus). In this study, we not only increased the number of evaluated sites in previously described regulatory regions (in introns 1 and 5), but also extended the evaluation to novel, possibly relevant regulatory regions of the gene (in intron 8, the transcriptional start site, the proximal enhancer and CTCF-binding sites within the 5'UTR). We here describe the assessment of HAM-TBS assays for a panel of 157 CpGs with possible functional relevance in the murine Fkbp5 gene. DNAm profiles were tissue-specific, with lesser differences between the two brain regions than between the brain and blood. Moreover, we identified DNAm changes in the Fkbp5 locus after early life stress exposure in the frontal cortex and blood. Our findings indicate that HAM-TBS is a valuable tool for broader exploration of the DNAm of the murine Fkbp5 locus and its involvement in the stress response.

Cohort profile: InTraUterine sampling in early pregnancy (ITU), a prospective pregnancy cohort study in Finland: study design and baseline characteristics.

PURPOSE: The InTraUterine sampling in early pregnancy (ITU) is a prospective pregnancy cohort study. The overarching aim of ITU is to unravel genomic, epigenomic, transcriptomic, endocrine, inflammatory and metabolic maternal-placental-fetal mechanisms involved in the programming of health and disease after exposure to prenatal environmental adversity, such as maternal malnutrition, cardiometabolic disorders, infections, medical interventions, mental disorders and psychosocial stress. This paper describes the study protocol, design and baseline characteristics of the cohort. PARTICIPANTS: We included 944 pregnant Finnish women, their partners and children born alive between April 2012 and December 2017. The women were recruited through the national, voluntary trisomy 21 screening between 9+0 and 21+6 gestational weeks. Of the participating women, 543 were screen positive and underwent fetal chromosomal testing. Test result of these women suggested no fetal chromosomal abnormality. Further, we recruited 401 women who were screen negative and who did not undergo fetal chromosomal testing. FINDINGS TO DATE: We have collected chorionic villi and amniotic fluid from the screen-positive women; blood, urine, buccal swabs and diurnal salivary samples from all women; blood and buccal swabs from all partners; and placenta, cord blood and buccal swabs from all newborns for analyses of the genome, epigenome, transcriptome, and endocrine, inflammatory and metabolic markers. These data are coupled with comprehensive phenotypes, including questions on demographic characteristics, health and well-being of the women and their partners during pregnancy and of the women and their children at the child's age of 1.7 and 3 years. Data also come from patient records and nationwide registers covering health, lifestyle and medication data. FUTURE PLANS: Multiple layers of ITU data allow integrative data analyses, which translate to biomarker identification and allow risk stratification and understanding of the biological mechanisms involved in prenatal programming of health and disease.

Betamethasone administration during pregnancy is associated with placental epigenetic changes with implications for inflammation.

BACKGROUND: Glucocorticoids (GCs) play a pivotal role in fetal programming. Antenatal treatment with synthetic GCs (sGCs) in individuals in danger of preterm labor is common practice. Adverse short- and long-term effects of antenatal sGCs have been reported, but their effects on placental epigenetic characteristics have never been systematically studied in humans. RESULTS: We tested the association between exposure to the sGC betamethasone (BET) and placental DNA methylation (DNAm) in 52 exposed cases and 84 gestational-age-matched controls. We fine-mapped associated loci using targeted bisulfite sequencing. The association of placental DNAm with gene expression and co-expression analysis on implicated genes was performed in an independent cohort including 494 placentas. Exposure to BET was significantly associated with lower placenta DNAm at an enhancer of FKBP5. FKBP5 (FK506-binding protein 51) is a co-chaperone that modulates glucocorticoid receptor activity. Lower DNAm at this enhancer site was associated with higher expression of FKBP5 and a co-expressed gene module. This module is enriched for genes associated with preeclampsia and involved in inflammation and immune response. CONCLUSIONS: Our findings suggest that BET exposure during pregnancy associates with few but lasting changes in placental DNAm and may promote a gene expression profile associated with placental dysfunction and increased inflammation. This may represent a pathway mediating GC-associated negative long-term consequences and health outcomes in offspring.

Glucocorticoid exposure during hippocampal neurogenesis primes future stress response by inducing changes in DNA methylation.

Prenatal stress exposure is associated with risk for psychiatric disorders later in life. This may be mediated in part via enhanced exposure to glucocorticoids (GCs), which are known to impact neurogenesis. We aimed to identify molecular mediators of these effects, focusing on long-lasting epigenetic changes. In a human hippocampal progenitor cell (HPC) line, we assessed the short- and long-term effects of GC exposure during neurogenesis on messenger RNA (mRNA) expression and DNA methylation (DNAm) profiles. GC exposure induced changes in DNAm at 27,812 CpG dinucleotides and in the expression of 3,857 transcripts (false discovery rate [FDR] ≤ 0.1 and absolute fold change [FC] expression ≥ 1.15). HPC expression and GC-affected DNAm profiles were enriched for changes observed during human fetal brain development. Differentially methylated sites (DMSs) with GC exposure clustered into 4 trajectories over HPC differentiation, with transient as well as long-lasting DNAm changes. Lasting DMSs mapped to distinct functional pathways and were selectively enriched for poised and bivalent enhancer marks. Lasting DMSs had little correlation with lasting expression changes but were associated with a significantly enhanced transcriptional response to a second acute GC challenge. A significant subset of lasting DMSs was also responsive to an acute GC challenge in peripheral blood. These tissue-overlapping DMSs were used to compute a polyepigenetic score that predicted exposure to conditions associated with altered prenatal GCs in newborn's cord blood DNA. Overall, our data suggest that early exposure to GCs can change the set point of future transcriptional responses to stress by inducing lasting DNAm changes. Such altered set points may relate to differential vulnerability to stress exposure later in life.

The Atherosclerosis Risk Variant rs2107595 Mediates Allele-Specific Transcriptional Regulation of HDAC9 via E2F3 and Rb1.

Background and Purpose- Genome-wide association studies have identified the HDAC9 (histone deacetylase 9) gene region as a major risk locus for atherosclerotic stroke and coronary artery disease in humans. Previous results suggest a role of altered HDAC9 expression levels as the underlying disease mechanism. rs2107595, the lead single nucleotide polymorphism for stroke and coronary artery disease resides in noncoding DNA and colocalizes with histone modification marks suggestive of enhancer elements. Methods- To determine the mechanisms by which genetic variation at rs2107595 regulates HDAC9 expression and thus vascular risk we employed targeted resequencing, proteome-wide search for allele-specific nuclear binding partners, chromatin immunoprecipitation, genome-editing, reporter assays, circularized chromosome conformation capture, and gain- and loss-of-function experiments in cultured human cell lines and primary immune cells. Results- Targeted resequencing of the HDAC9 locus in patients with atherosclerotic stroke and controls supported candidacy of rs2107595 as the causative single nucleotide polymorphism. A proteomic search for nuclear binding partners revealed preferential binding of the E2F3/TFDP1/Rb1 complex (E2F transcription factor 3/transcription factor Dp-1/Retinoblastoma 1) to the rs2107595 common allele, consistent with the disruption of an E2F3 consensus site by the risk allele. Gain- and loss-of-function studies showed a regulatory effect of E2F/Rb proteins on HDAC9 expression. Compared with the common allele, the rs2107595 risk allele exhibited higher transcriptional capacity in luciferase assays and was associated with higher HDAC9 mRNA levels in primary macrophages and genome-edited Jurkat cells. Circularized chromosome conformation capture revealed a genomic interaction of the rs2107595 region with the HDAC9 promoter, which was stronger for the common allele as was the in vivo interaction with E2F3 and Rb1 determined by chromatin immunoprecipitation. Gain-of-function experiments in isogenic Jurkat cells demonstrated a key role of E2F3 in mediating rs2107595-dependent transcriptional regulation of HDAC9. Conclusions- Collectively, our findings imply allele-specific transcriptional regulation of HDAC9 via E2F3 and Rb1 as a major mechanism mediating vascular risk at rs2107595.

Identification of dynamic glucocorticoid-induced methylation changes at the FKBP5 locus.

BACKGROUND: Epigenetic mechanisms may play a major role in the biological embedding of early-life stress (ELS). One proposed mechanism is that glucocorticoid (GC) release following ELS exposure induces long-lasting alterations in DNA methylation (DNAm) of important regulatory genes of the stress response. Here, we investigate the dynamics of GC-dependent methylation changes in key regulatory regions of the FKBP5 locus in which ELS-associated DNAm changes have been reported. RESULTS: We repeatedly measured DNAm in human peripheral blood samples from 2 independent cohorts exposed to the GC agonist dexamethasone (DEX) using a targeted bisulfite sequencing approach, complemented by data from Illumina 450K arrays. We detected differentially methylated CpGs in enhancers co-localizing with GC receptor binding sites after acute DEX treatment (1 h, 3 h, 6 h), which returned to baseline levels within 23 h. These changes withstood correction for immune cell count differences. While we observed main effects of sex, age, body mass index, smoking, and depression symptoms on FKBP5 methylation levels, only the functional FKBP5 SNP (rs1360780) moderated the dynamic changes following DEX. This genotype effect was observed in both cohorts and included sites previously shown to be associated with ELS. CONCLUSION: Our study highlights that DNAm levels within regulatory regions of the FKBP5 locus show dynamic changes following a GC challenge and suggest that factors influencing the dynamics of this regulation may contribute to the previously reported alterations in DNAm associated with current and past ELS exposure.

Epigenetic upregulation of FKBP5 by aging and stress contributes to NF-κB-driven inflammation and cardiovascular risk.

Aging and psychosocial stress are associated with increased inflammation and disease risk, but the underlying molecular mechanisms are unclear. Because both aging and stress are also associated with lasting epigenetic changes, a plausible hypothesis is that stress along the lifespan could confer disease risk through epigenetic effects on molecules involved in inflammatory processes. Here, by combining large-scale analyses in human cohorts with experiments in cells, we report that FKBP5, a protein implicated in stress physiology, contributes to these relations. Across independent human cohorts (total n > 3,000), aging synergized with stress-related phenotypes, measured with childhood trauma and major depression questionnaires, to epigenetically up-regulate FKBP5 expression. These age/stress-related epigenetic effects were recapitulated in a cellular model of replicative senescence, whereby we exposed replicating human fibroblasts to stress (glucocorticoid) hormones. Unbiased genome-wide analyses in human blood linked higher FKBP5 mRNA with a proinflammatory profile and altered NF-κB-related gene networks. Accordingly, experiments in immune cells showed that higher FKBP5 promotes inflammation by strengthening the interactions of NF-κB regulatory kinases, whereas opposing FKBP5 either by genetic deletion (CRISPR/Cas9-mediated) or selective pharmacological inhibition prevented the effects on NF-κB. Further, the age/stress-related epigenetic signature enhanced FKBP5 response to NF-κB through a positive feedback loop and was present in individuals with a history of acute myocardial infarction, a disease state linked to peripheral inflammation. These findings suggest that aging/stress-driven FKBP5-NF-κB signaling mediates inflammation, potentially contributing to cardiovascular risk, and may thus point to novel biomarker and treatment possibilities.

Sex dependent impact of gestational stress on predisposition to eating disorders and metabolic disease.

OBJECTIVE: Vulnerability to eating disorders (EDs) is broadly assumed to be associated with early life stress. However, a careful examination of the literature shows that susceptibility to EDs may depend on the type, severity and timing of the stressor and the sex of the individual. We aimed at exploring the link between chronic prenatal stress and predisposition to EDs and metabolic disease. METHODS: We used a chronic variable stress protocol during gestation to explore the metabolic response of male and female offspring to food restriction (FR), activity-based anorexia (ABA), binge eating (BE) and exposure to high fat (HF) diet. RESULTS: Contrary to controls, prenatally stressed (PNS) female offspring showed resistance to ABA and BE and displayed a lower metabolic rate leading to hyperadiposity and obesity on HF diet. Male PNS offspring showed healthy responses to FR and ABA, increased propensity to binge and improved coping with HF compared to controls. We found that long-lasting abnormal responses to metabolic challenge are linked to fetal programming and adult hypothalamic dysregulation in PNS females, resulting from sexually dimorphic adaptations in placental methylation and gene expression. CONCLUSIONS: Our results show that maternal stress may have variable and even opposing effects on ED risk, depending on the ED and the sex of the offspring.

The Role of m6A/m-RNA Methylation in Stress Response Regulation.

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed m6A/m, in the stress response in the adult brain in vivo is currently unknown. Here, we provide a detailed analysis of the stress epitranscriptome using m6A/m-seq, global and gene-specific m6A/m measurements. We show that stress exposure and glucocorticoids region and time specifically alter m6A/m and its regulatory network. We demonstrate that deletion of the methyltransferase Mettl3 or the demethylase Fto in adult neurons alters the m6A/m epitranscriptome, increases fear memory, and changes the transcriptome response to fear and synaptic plasticity. Moreover, we report that regulation of m6A/m is impaired in major depressive disorder patients following glucocorticoid stimulation. Our findings indicate that brain m6A/m represents a novel layer of complexity in gene expression regulation after stress and that dysregulation of the m6A/m response may contribute to the pathophysiology of stress-related psychiatric disorders.

Correction to: Lifetime stress accelerates epigenetic aging in an urban, African American cohort: relevance of glucocorticoid signaling.

Upon publication of the original article [1] it was highlighted by the authors that a transposition error affected Additional file 1, causing the misplacement of several columns and rendering the table difficult to read. This transposition does not influence any of the results nor analyses presented in the paper and has since been formally noted in this correction article; the corrected file is available here as an Additional File. The publisher apologizes for this error.

Placental miR-340 mediates vulnerability to activity based anorexia in mice.

Anorexia nervosa (AN) is a devastating eating disorder characterized by self-starvation that mainly affects women. Its etiology is unknown, which impedes successful treatment options leading to a limited chance of full recovery. Here, we show that gestation is a vulnerable window that can influence the predisposition to AN. By screening placental microRNA expression of naive and prenatally stressed (PNS) fetuses and assessing vulnerability to activity-based anorexia (ABA), we identify miR-340 as a sexually dimorphic regulator involved in prenatal programming of ABA. PNS caused gene-body hypermethylation of placental miR-340, which is associated with reduced miR-340 expression and increased protein levels of several target transcripts, GR, Cry2 and H3F3b. MiR-340 is linked to the expression of several nutrient transporters both in mice and human placentas. Using placenta-specific lentiviral transgenes and embryo transfer, we demonstrate the key role miR-340 plays in the mechanism involved in early life programming of ABA.

Functional Impact of An ADHD-Associated DIRAS2 Promoter Polymorphism.

The DIRAS2 gene is coding for a small Ras GTPase with so far unknown function. In a previous study, we described the association of DIRAS2 rs1412005, as well as a haplotype containing this polymorphism and located in the promoter region of this gene with attention-deficit/hyperactivity disorder (ADHD). In the present study, we searched for rare variants within or near the DIRAS2 gene that might be associated with ADHD using next-generation sequencing. As we were not able to detect any rare variants associated with the disease, we sought to establish a functional role of DIRAS2 rs1412005 on the molecular or systems level. First, we investigated whether it has an influence on gene expression by means of a luciferase-based promoter assay. We could demonstrate that the minor risk allele goes along with the increased expression of the reporter gene. Next, we aimed to identify differences in response inhibition between risk-allele and non-risk allele carriers in children suffering from ADHD and healthy control individuals by analyzing event-related potentials in the electroencephalogram during a Go/NoGo task. Risk-allele carriers showed a changed NoGo anteriorization. Therefore, our results suggest an impact of the investigated polymorphism on the prefrontal response control in children with ADHD. These results imply that the promoter polymorphism is indeed the associated as well as in itself a causal variant. Further research is thus warranted to clarify the mechanisms linking DIRAS2 to ADHD.

Lifetime stress accelerates epigenetic aging in an urban, African American cohort: relevance of glucocorticoid signaling.

BACKGROUND: Chronic psychological stress is associated with accelerated aging and increased risk for aging-related diseases, but the underlying molecular mechanisms are unclear. RESULTS: We examined the effect of lifetime stressors on a DNA methylation-based age predictor, epigenetic clock. After controlling for blood cell-type composition and lifestyle parameters, cumulative lifetime stress, but not childhood maltreatment or current stress alone, predicted accelerated epigenetic aging in an urban, African American cohort (n = 392). This effect was primarily driven by personal life stressors, was more pronounced with advancing age, and was blunted in individuals with higher childhood abuse exposure. Hypothesizing that these epigenetic effects could be mediated by glucocorticoid signaling, we found that a high number (n = 85) of epigenetic clock CpG sites were located within glucocorticoid response elements. We further examined the functional effects of glucocorticoids on epigenetic clock CpGs in an independent sample with genome-wide DNA methylation (n = 124) and gene expression data (n = 297) before and after exposure to the glucocorticoid receptor agonist dexamethasone. Dexamethasone induced dynamic changes in methylation in 31.2 % (110/353) of these CpGs and transcription in 81.7 % (139/170) of genes neighboring epigenetic clock CpGs. Disease enrichment analysis of these dexamethasone-regulated genes showed enriched association for aging-related diseases, including coronary artery disease, arteriosclerosis, and leukemias. CONCLUSIONS: Cumulative lifetime stress may accelerate epigenetic aging, an effect that could be driven by glucocorticoid-induced epigenetic changes. These findings contribute to our understanding of mechanisms linking chronic stress with accelerated aging and heightened disease risk.

Re-Annotator: Annotation Pipeline for Microarray Probe Sequences.

Microarray technologies are established approaches for high throughput gene expression, methylation and genotyping analysis. An accurate mapping of the array probes is essential to generate reliable biological findings. However, manufacturers of the microarray platforms typically provide incomplete and outdated annotation tables, which often rely on older genome and transcriptome versions that differ substantially from up-to-date sequence databases. Here, we present the Re-Annotator, a re-annotation pipeline for microarray probe sequences. It is primarily designed for gene expression microarrays but can also be adapted to other types of microarrays. The Re-Annotator uses a custom-built mRNA reference database to identify the positions of gene expression array probe sequences. We applied Re-Annotator to the Illumina Human-HT12 v4 microarray platform and found that about one quarter (25%) of the probes differed from the manufacturer's annotation. In further computational experiments on experimental gene expression data, we compared Re-Annotator to another probe re-annotation tool, ReMOAT, and found that Re-Annotator provided an improved re-annotation of microarray probes. A thorough re-annotation of probe information is crucial to any microarray analysis. The Re-Annotator pipeline is freely available at http://sourceforge.net/projects/reannotator along with re-annotated files for Illumina microarrays HumanHT-12 v3/v4 and MouseRef-8 v2.

Functional coding variants in SLC6A15, a possible risk gene for major depression.

SLC6A15 is a neuron-specific neutral amino acid transporter that belongs to the solute carrier 6 gene family. This gene family is responsible for presynaptic re-uptake of the majority of neurotransmitters. Convergent data from human studies, animal models and pharmacological investigations suggest a possible role of SLC6A15 in major depressive disorder. In this work, we explored potential functional variants in this gene that could influence the activity of the amino acid transporter and thus downstream neuronal function and possibly the risk for stress-related psychiatric disorders. DNA from 400 depressed patients and 400 controls was screened for genetic variants using a pooled targeted re-sequencing approach. Results were verified by individual re-genotyping and validated non-synonymous coding variants were tested in an independent sample (N = 1934). Nine variants altering the amino acid sequence were then assessed for their functional effects by measuring SLC6A15 transporter activity in a cellular uptake assay. In total, we identified 405 genetic variants, including twelve non-synonymous variants. While none of the non-synonymous coding variants showed significant differences in case-control associations, two rare non-synonymous variants were associated with a significantly increased maximal (3)H proline uptake as compared to the wildtype sequence. Our data suggest that genetic variants in the SLC6A15 locus change the activity of the amino acid transporter and might thus influence its neuronal function and the risk for stress-related psychiatric disorders. As statistically significant association for rare variants might only be achieved in extremely large samples (N >70,000) functional exploration may shed light on putatively disease-relevant variants.

AP4 is a mediator of epithelial-mesenchymal transition and metastasis in colorectal cancer.

The basic helix-loop-helix transcription factor AP4/TFAP4/AP-4 is encoded by a c-MYC target gene and displays up-regulation concomitantly with c-MYC in colorectal cancer (CRC) and numerous other tumor types. Here a genome-wide characterization of AP4 DNA binding and mRNA expression was performed using a combination of microarray, genome-wide chromatin immunoprecipitation, next-generation sequencing, and bioinformatic analyses. Thereby, hundreds of induced and repressed AP4 target genes were identified. Besides many genes involved in the control of proliferation, the AP4 target genes included markers of stemness (LGR5 and CD44) and epithelial-mesenchymal transition (EMT) such as SNAIL, E-cadherin/CDH1, OCLN, VIM, FN1, and the Claudins 1, 4, and 7. Accordingly, activation of AP4 induced EMT and enhanced migration and invasion of CRC cells. Conversely, down-regulation of AP4 resulted in mesenchymal-epithelial transition and inhibited migration and invasion. In addition, AP4 induction was required for EMT, migration, and invasion caused by ectopic expression of c-MYC. Inhibition of AP4 in CRC cells resulted in decreased lung metastasis in mice. Elevated AP4 expression in primary CRC significantly correlated with liver metastasis and poor patient survival. These findings imply AP4 as a new regulator of EMT that contributes to metastatic processes in CRC and presumably other carcinomas.

Genome-wide characterization of miR-34a induced changes in protein and mRNA expression by a combined pulsed SILAC and microarray analysis.

The gene encoding the miR-34a microRNA is a transcriptional target of the p53 tumor suppressor protein and subject to epigenetic inactivation in colorectal cancer and numerous other tumor types. Here, we combined pulsed SILAC (pSILAC) and microarray analyses to identify miR-34a-induced changes in protein and mRNA expression. pSILAC allowed to quantify the de novo protein synthesis of 1206 proteins after activation of a conditional miR-34a allele in a colorectal cancer cell line. ∼19% of the detected proteins were differentially regulated, with 113 proteins being down- and 115 up-regulated. The proteins with a miR-34a seed-matching-sequence in the 3'-untranslated region (UTR) of the corresponding mRNA showed a clear bias toward translational repression. Proteins involved in DNA replication, e.g. the MCM proteins, and cell proliferation, were over-represented among indirectly down-regulated proteins lacking a miR-34a seed-match. The decrease in de novo protein synthesis of direct miR-34a targets correlated with reduced levels of the corresponding mRNA in most cases, indicating an interdependence of both types of regulation. In addition, 43 mRNAs encoding proteins not detected by pSILAC were down-regulated after miR-34a expression and contained miR-34a seed-matches. The direct regulation of selected miR-34a target-mRNAs was confirmed using reporter assays. Via down-regulation of the proteins encoded by these mRNAs miR-34a presumably inhibits glycolysis (LDHA), WNT-signaling (LEF1), invasion/migration (AXL) and lipid metabolism (ACSL1, ACSL4). Furthermore, miR-34a may activate p53 by inhibiting its acetylation (MTA2, HDAC1) and degradation (YY1). In summary, miR-34a presumably participates in multiple tumor suppressive pathways by directly and indirectly suppressing the expression of numerous, critical proteins.