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A genome-wide genetic interaction screen reveals metabolic roles of human histone deacetylase 1

ABSTRACT: Dynamic acetylation of metabolic proteins has emerged as a ubiquitous post-translational modification of human metabolic proteins. However, the corresponding modifying enzymes and the functions of the modification await exploration. Using a genome-wide synthetic lethality screen, we constructed a genetic interaction network of human histone deacetylases (HDACs) and discovered many metabolic substrates of these enzymes. We further confirmed that the adenosine monophosphate-activated protein kinase (AMPK) catalytic subunit is acetylated and deacetylated by EP300 and HDAC1, respectively. Deacetylation of AMPK catalytic subunit enhances physical interaction with the upstream kinase LKB1, and leads to AMPK phosphorylation and activation. These findings highlight the importance of genetic interaction profiling to identify specific substrates of individual HDACs and elucidate how cells use protein (de)acetylation to coordinate nutrient availability and cellular energy status.

ORGANISM(S): Homo sapiens

PROVIDER: GSE29662 | GEO | 2012/02/10

SECONDARY ACCESSION(S): PRJNA141139

REPOSITORIES: GEO

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Project description:Dynamic acetylation of metabolic proteins has emerged as a ubiquitous post-translational modification of human metabolic proteins. However, the corresponding modifying enzymes and the functions of the modification await exploration. Using a genome-wide synthetic lethality screen, we constructed a genetic interaction network of human histone deacetylases (HDACs) and discovered many metabolic substrates of these enzymes. We further confirmed that the adenosine monophosphate-activated protein kinase (AMPK) catalytic subunit is acetylated and deacetylated by EP300 and HDAC1, respectively. Deacetylation of AMPK catalytic subunit enhances physical interaction with the upstream kinase LKB1, and leads to AMPK phosphorylation and activation. These findings highlight the importance of genetic interaction profiling to identify specific substrates of individual HDACs and elucidate how cells use protein (de)acetylation to coordinate nutrient availability and cellular energy status. To study the functional specificity of individual HDAC, we developed a genome-wide genetic interaction profiling technology in cultured human cells by RNAi using pooled TRC (The RNAi Consortium) 75k human shRNA library and complexity deconvolution by high-density microarray with a half-hairpin barcode design. The HCT116 colon cancer cell line was chosen as the screen platform because of its quasinormal diploid karyotype. The performance of the customized microarray we designed was first evaluated by the receiver operating characteristic (ROC) curve showing high sensitivity (89%), specificity (94%) and area under curve (0.95), as well as a control hybridization without DNA sample showing low background signal. Both results suggest that the fluorescent signals were generated by specific hybridization reactions. The high correlation of signal between Cy5 and Cy3 channel as well as dye-swap experiments indicated that biases between the two labeling fluorophores during competitive hybridization were negligible. Moreover, correlation between technical and biological replicates confirmed the high reproducibility of the methodology. In the screen, we used stable polyclonal cells expressing shRNA constructs targeting firefly luciferase (shLuciferase) as control. We checked the knockdown efficiency in protein or RNA level of individual shRNA constructs for 12 human HDAC genes (HDAC1~4, HDAC6~9, SIRT1~3 and SIRT5) by immunoblotting or quantitative PCR, and generated stable polyclonal query cell lines expressing two shRNA constructs with the highest knockdown efficiency for each HDAC gene. After transduction by TRC shRNA lentiviral pools, benchmark samples were harvested prior to puromycin selection, and the remaining cells were propagated under selection and harvested again after 18 population doublings as the end samples. Half-hairpin barcode library of the benchmark and end samples was recovered from genomic DNA respectively by PCR with Cy5 and Cy3-labeled primers, gel purified, and hybridized to the microarray. For each shRNA construct, the Z-score of the log2(Cy5/Cy3) was computed, and Z-score difference was calculated by subtracting the Z-score of the control sample from that of the query sample. Z-score differences larger than 1.5 and less than -1.5 were used as arbitrary thresholds to define candidate negative and positive genetic interactions, respectively.

Project description:Reversible protein acetylation provides a central mechanism for controlling gene expression and cellular signaling events. It is governed by the antagonistic commitment of two enzymes families: the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). HDAC4, like its class IIa counterparts, is a potent transcriptional repressor through interactions with tissue-specific transcription factors via its N-terminal domain. Whilst the lysine deacetylase activity of the class IIa HDACs is much less potent than that of the class I enzymes, HDAC4 has been reported to influence protein deacetylation through its interaction with HDAC3. To investigate the influence of HDAC4 on protein acetylation, we employed the unbiased AcetylScan proteomic screen. We identified many proteins known to be modified by acetylation, but found that the absence of HDAC4 had no effect on the acetylation profile of the murine neonate brain. This is consistent with the biochemical data suggesting that HDAC4 may not function as a lysine deacetylase, but these in vivo data do not support the previous report showing that the enzymatic activity of HDAC3 might be modified by its interaction with HDAC4. To complement this work, we used Affymetrix arrays to investigate the effect of HDAC4 knock-out on the transcriptional profile of the postnatal murine brain. There was no effect on global transcription, consistent with the absence of a differential histone acetylation profile. Validation of the array data by Taq-man qPCR indicated that only protamine 1 and Igfbp6 mRNA levels were increased by more than one-fold and only CamK4 was decreased. The lack of a major effect on the transcriptional profile is consistent with the cytoplasmic location of HDAC4 in the P3 murine brain. mRNA expression analysis was performed by microarray in 3-day-old HDAC4 KO pups and WT littermates. Ten samples were analysed for each genotype. Microarray quality control was performed using the software package provided on RACE (http://race.unil.ch).

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A genome-wide genetic interaction screen reveals metabolic roles of human histone deacetylase 1

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