CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder with unclear underlying mechanisms. Here, the authors unravel the contribution of a stress-responsive pathway to RSTS where impaired HSF2 acetylation, due to RSTS-associated CBP/EP300 mutations, alters the expression of neurodevelopmental players, in keeping with hallmarks of cell-cell adhesion defects.Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.</p

Fluorodeoxyuridine enhances the heat shock response and decreases polyglutamine aggregation in an HSF-1-dependent manner in Caenorhabditis elegans.

The heat shock response (HSR) protects cells from protein-denaturing stress through the induction of chaperones. The HSR is conserved in all organisms and is mediated by the transcription factor HSF-1. We show here that a compound commonly used to prevent larval development in Caenorhabditis elegans, 5-fluoro-2\u27-deoxyuridine (FUdR), can enhance heat shock induction of hsp mRNA in an HSF-1-dependent manner. Treatment with FUdR can also decrease age-dependent polyglutamine aggregation in a Huntington\u27s disease model, and this effect depends on HSF-1 as well. Therefore, FUdR treatment can modulate the HSR and proteostasis, and should be used with caution when used to inhibit reproduction

Networks and biological processes impacted by HSF-1-regulated miRNAs during HS.

<p>(<b>A</b>) Schematic depicting integrated miRNA/mRNA network generation. The miRNAs determined via miRNA-seq to be regulated by HSF-1 during HS were run through the target prediction tools mirWIP or TargetScan to uncover predicted mRNA targets. Due to the inhibitory nature of miRNAs, predicted mRNA targets were inversely correlated to mRNAs found to be regulated by HSF-1 during HS via mRNA-seq performed in parallel to this study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183445#pone.0183445.ref007" target="_blank">7</a>]. (<b>B</b>) Predicted network suppressed by HSF-1-regulated miRNAs upon HS. The miRNAs that we found to normally be induced by HSF-1 during HS via miRNA-seq (rectangles) were compared to mRNAs previously determined to normally be suppressed by HSF-1 during HS via mRNA-seq (circles). (<b>C</b>) Biological processes predicted to be suppressed by HSF-1-regulated miRNAs during HS. DAVID was used to uncover biological processes predicted to be suppressed by HSF-1 upon HS using the network in (B). (<b>D</b>) Predicted network induced by HSF-1-regulated miRNAs upon HS. The miRNAs that we found to normally be suppressed by HSF-1 during HS via miRNA-seq (rectangles) were compared to mRNAs previously determined to normally be induced by HSF-1 via mRNA-seq (circles). (<b>E</b>) Biological processes predicted to be induced by HSF-1-regulated miRNAs during HS. DAVID was used to uncover biological processes predicted to be induced by HSF-1 upon HS using the network in (D).</p

HSF-1 is a regulator of miRNA expression in <i>Caenorhabditis elegans</i>

<div><p>The ability of an organism to sense and adapt to environmental stressors is essential for proteome maintenance and survival. The highly conserved heat shock response is a survival mechanism employed by all organisms, including the nematode <i>Caenorhabditis elegans</i>, upon exposure to environmental extremes. Transcriptional control of the metazoan heat shock response is mediated by the heat shock transcription factor HSF-1. In addition to regulating global stress-responsive genes to promote stress-resistance and survival, HSF-1 has recently been shown to regulate stress-independent functions in controlling development, metabolism, and longevity. However, the indirect role of HSF-1 in coordinating stress-dependent and -independent processes through post-transcriptional regulation is largely unknown. MicroRNAs (miRNAs) have emerged as a class of post-transcriptional regulators that control gene expression through translational repression or mRNA degradation. To determine the role of HSF-1 in regulating miRNA expression, we have performed high-throughput small RNA-sequencing in <i>C</i>. <i>elegans</i> grown in the presence and absence of <i>hsf-1</i> RNAi followed by treatment with or without heat shock. This has allowed us to uncover the miRNAs regulated by HSF-1 via heat-dependent and -independent mechanisms. Integrated miRNA/mRNA target-prediction analyses suggest HSF-1 as a post-transcriptional regulator of development, metabolism, and longevity through regulating miRNA expression. This provides new insight into the possible mechanism by which HSF-1 controls these processes. We have also uncovered oxidative stress response factors and insulin-like signaling factors as a common link between processes affected by HSF-1-regulated miRNAs in stress-dependent and -independent mechanisms, respectively. This may provide a role for miRNAs in regulating cross-talk between various stress responses. Our work therefore uncovers an interesting potential role for HSF-1 in post-transcriptionally controlling gene expression in <i>C</i>. <i>elegans</i>, and suggests a mechanism for cross-talk between stress responses.</p></div

miRNAs normally regulated by HSF-1 independently of HS.

<p>miRNAs normally regulated by HSF-1 independently of HS.</p

Integrated target prediction analysis uncovers miRNA/mRNA interaction networks regulated by HSF-1 independently of HS.

<p>The miRNAs (rectangles) that we determined to be regulated by HSF-1 independently of HS were used for target prediction analysis carried out by mirWIP or TargetScan. The mRNA targets (circles) were consolidated by comparing the predicted mRNAs to those determined by mRNA-sequencing, performed in parallel to miRNA sequencing, to be regulated by HSF-1 independently of HS. Interactions were predicted using the Agilent literature search tool, and network generation was done with Cytoscape. Transcripts that are not colored were not affected in our dataset, but are neighbors shared by at least two transcripts that were affected. The color of each miRNA or mRNA corresponds to the degree of HSF-1 regulation, where red represents induction and blue represents suppression. Bold connecting lines represent connections between upregulated and downregulated clusters.</p

miRNAs normally regulated by HSF-1 upon HS.

<p>miRNAs normally regulated by HSF-1 upon HS.</p

Scheme for miRNA-sequencing experimental setup and data normalization.

<p>(<b>A</b>) Schematic depicting miRNA-sequencing conditions. miRNA samples were generated from wildtype (N2) <i>C</i>. <i>elegans</i> treated with the four indicated conditions, where “<i>hsf-1</i>(+)” refers to worms treated with control (empty vector) RNAi, and “<i>hsf-1</i>(-)” refers to worms treated with <i>hsf-1</i> RNAi. Synchronous worms were given RNAi from the L1 larval stage to the L4 larval stage. At the L4 larval stage, worms were left untreated (-HS) or given a 30 minute 33°C heat shock (+HS). miRNA-sequencing was performed in biological duplicates on the Illumina Hi-Seq 2000 platform and analyzed using miRDeep2. (<b>B</b>) Scheme for data normalization. Each treatment condition was compared relative to the empty vector control [<i>hsf-1</i>(+);-HS] to determine the relative fold change in expression of each miRNA. The Benjamini-Hochberg correction test was used to identify all differentially expressed genes relative to the <i>hsf-1</i>(+);-HS control, and also between treatment conditions. (<b>C</b>) Separating miRNAs regulated by HSF-1 during and independently of HS. The Venn diagram shows the total number of miRNAs that were found to be significantly altered (q-value<0.05), as compared to the <i>hsf-1</i>(+);-HS control, for each of the indicated comparisons between samples. The q-value is the FDR-adjusted p-value of the test statistic, as determined by the Benjamini-Hochberg correction for multiple testing. (<b>D</b>) Validation of miRNA-seq hits for HSF-1-regulated miRNAs. The log₂ fold change of miR-784, a miRNA normally upregulated by HSF-1 upon HS, in the <i>hsf-1</i>(+); +HS vs control condition. (<b>E</b>) The log₂ fold change of miR-239b, a miRNA normally downregulated by HSF-1 independently of HS, in the <i>hsf-1</i>(-);-HS vs control condition.</p

A model for heat stress-dependent and -independent processes controlled by HSF-1-regulated miRNAs.

<p>HSF-1 controls miRNA expression during and independently of HS. During HS, HSF-1 is predicted to post-transcriptionally regulate genes involved in cytoprotection, development, metabolism, and longevity. These processes may be connected through the oxidative stress response transcription factor SKN-1. Independently of HS, HSF-1 is predicted to post-transcriptionally regulate genes involved in development, metabolism, and longevity. These processes may be connected through the insulin-like signaling transcription factor DAF-16. This work highlights a possible role for HSF-1 in post-transcriptionally regulating gene expression and various biological processes, and provides a possible mechanism for cross-talk between stress responses.</p