Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair.

Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.

Meg3 Non-coding RNA Expression Controls Imprinting by Preventing Transcriptional Upregulation in cis.

Although many long non-coding RNAs (lncRNAs) are imprinted, their roles often remain unknown. The Dlk1-Dio3 domain expresses the lncRNA Meg3 and multiple microRNAs and small nucleolar RNAs (snoRNAs) on the maternal chromosome and constitutes an epigenetic model for development. The domain's Dlk1 (Delta-like-1) gene encodes a ligand that inhibits Notch1 signaling and regulates diverse developmental processes. Using a hybrid embryonic stem cell (ESC) system, we find that Dlk1 becomes imprinted during neural differentiation and that this involves transcriptional upregulation on the paternal chromosome. The maternal Dlk1 gene remains poised. Its protection against activation is controlled in cis by Meg3 expression and also requires the H3-Lys-27 methyltransferase Ezh2. Maternal Meg3 expression additionally protects against de novo DNA methylation at its promoter. We find that Meg3 lncRNA is partially retained in cis and overlaps the maternal Dlk1 in embryonic cells. Combined, our data evoke an imprinting model in which allelic lncRNA expression prevents gene activation in cis.

Microarray screening for novel preeclampsia biomarker candidates.

INTRODUCTION: Our aim was to identify novel biomarker candidates for the near-term prediction of preeclampsia in a homogenous collective. In this study, we screened at the genome-wide level for gene expression in placental villous tissue from patients with severe preeclampsia in comparison to normal healthy pregnancies. MATERIAL AND METHODS: Total RNA was extracted from placental villous tissue from 9 preeclamptic patients and 7 normotensive controls after scheduled cesarean sections. After sample pooling, gene expression analysis was performed using six Affymetrix Human Gene 1.0 ST arrays, followed by quantitative RT-PCR and validation of selected markers in the serum of patients at the protein level. RESULTS: In total, 896 significantly differentially expressed genes were identified (p ≤ 0.05). After restricting these to molecules present in the circulation, 9 upregulated and 5 downregulated genes were selected. Four of them (ß-hCG, HTRA4, LHB1, all upregulated; and NOX4, downregulated) were validated by quantitative real-time RT-PCR. Finally, the maternal plasma protein levels of 2 of these genes (LHB and ß-hCG) were confirmed to be significantly different between preeclampsia cases and controls. DISCUSSION: We identified 14 potential new biomarker candidates for preeclampsia and validated 4 of them by quantitative RT-PCR and 2 of them with subsequent serum protein analyses. Further studies will assess the optimal marker combination for the imminent prediction of impending preeclampsia.

Genome-wide in silico identification of new conserved and functional retinoic acid receptor response elements (direct repeats separated by 5 bp).

The nuclear retinoic acid receptors interact with specific retinoic acid (RA) response elements (RAREs) located in the promoters of target genes to orchestrate transcriptional networks involved in cell growth and differentiation. Here we describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs. More than 15,000 DR5 RAREs were identified and analyzed for their localization and conservation in vertebrates. We selected 138 elements located ±10 kb from transcription start sites and gene ends and conserved across more than 6 species. We also validated the functionality of these RAREs by analyzing their ability to bind retinoic acid receptors (ChIP sequencing experiments) as well as the RA regulation of the corresponding genes (RNA sequencing and quantitative real time PCR experiments). Such a strategy provided a global set of high confidence RAREs expanding the known experimentally validated RAREs repertoire associated to a series of new genes involved in cell signaling, development, and tumor suppression. Finally, the present work provides a valuable knowledge base for the analysis of a wider range of RA-target genes in different species.

Phosphorylation control of nuclear receptors.

Most transcription factors including nuclear receptors (NRs) act as sensors of the extracellular and intracellular compartments. As such, NRs serve as integrating platforms for a variety of stimuli and are targets for Post-translational modifications such as phosphorylations. During the last decade, knowledge of NRs phosphorylation advanced considerably because of the emergence of new technologies. Indeed, the development of a wide range of phosphorylation site databases, high accuracy mass spectrometry, and phospho-specific antibodies allowed the identification of multiple novel phosphorylation sites in NRs. New and improved methods also emerge to connect these data with the downstream consequences of phosphorylation on NRs structure (computational prediction, NMR), intracellular localization (FRAP), interaction with coregulators (proteomics, FRET, FLIM), and affinity for DNA (ChIP, ChIP-seq, FRAP). In the future, such integrated strategies should provide data with a treasure-trove of information about the integration of numerous signaling events by NRs.

Vinexinß, an atypical "sensor" of retinoic acid receptor gamma signaling: union and sequestration, separation, and phosphorylation.

The transcriptional activity of nuclear retinoic acid receptors (RARs) relies on the association/dissociation of coregulators at the ligand-binding domain. However, we determined that the N-terminal domain (NTD) also plays a role through its phosphorylation, and we isolated vinexinß, a cytoskeleton protein with three SH3 domains, as a new partner of the RARγ NTD. Here we deciphered the mechanism of the interaction and its role in RARγ-mediated transcription. By combining molecular and biophysical (surface plasmon resonance, NMR, and fluorescence resonance energy transfer) approaches, we demonstrated that the third SH3 domain of vinexinß interacts with a proline-rich domain (PRD) located in RARγ NTD and that phosphorylation at a serine located in the PRD abrogates the interaction. The affinity of the interaction was also evaluated. In vivo, vinexinß represses RARγ-mediated transcription and we dissected the underlying mechanism in chromatin immunoprecipitation experiments performed with F9 cells expressing RARγ wild type or mutated at the phosphorylation site. In the absence of retinoic acid (RA), vinexinß does not occupy RARγ target gene promoters and sequesters nonphosphorylated RARγ out of promoters. In response to RA, RARγ becomes phosphorylated and dissociates from vinexinß. This separation allows RARγ to occupy promoters. This is the first report of an RAR corepressor association/dissociation out of promoters and regulated by phosphorylation.

SUG-1 plays proteolytic and non-proteolytic roles in the control of retinoic acid target genes via its interaction with SRC-3.

Nuclear retinoic acid receptor alpha (RARalpha) activates gene expression through dynamic interactions with coregulatory protein complexes, the assembly of which is directed by the ligand and the AF-2 domain of RARalpha. Then RARalpha and its coactivator SRC-3 are degraded by the proteasome. Recently it has emerged that the proteasome also plays a key role in RARalpha-mediated transcription. Here we show that SUG-1, one of the six ATPases of the 19 S regulatory complex of the 26 S proteasome, interacts with SRC-3, is recruited at the promoters of retinoic acid (RA) target genes, and thereby participates to their transcription. In addition, SUG-1 also mediates the proteasomal degradation of SRC-3. However, when present in excess amounts, SUG-1 blocks the activation of RARalpha target genes and the degradation of RARalpha that occurs in response to RA, via its ability to interfere with the recruitment of SRC-3 and other coregulators at the AF-2 domain of RARalpha. We propose a model in which the ratio between SUG-1 and SRC-3 is crucial for the control of RARalpha functioning. This study provides new insights into how SUG-1 has a unique role in linking the transcription and degradation processes via its ability to interact with SRC-3.

Protein kinases and the proteasome join in the combinatorial control of transcription by nuclear retinoic acid receptors.

Nuclear retinoic acid receptors (RARs) are transcriptional transregulators that control the expression of specific subsets of genes in a ligand-dependent manner. The basic mechanism for switching on gene transcription by agonist-liganded RARs involves their binding at specific response elements located in target genes. It also involves interactions with coregulatory protein complexes, the assembly of which is directed by the C-terminal ligand-binding domain of RARs. In addition to this scenario, several recent studies highlighted a fundamental role for the N-terminal domain in the transcriptional activity of RARs, following phosphorylation by the CDK7 kinase of the general transcription factor TFIIH and by p38MAPK. It has also emerged that the ubiquitin-proteasome system has a key role in RAR-mediated transcription. Here, we review new insights into how N-terminal domain and the proteasome pathway can influence the dynamics of RAR transcriptional activity.

Phosphorylation by PKA potentiates retinoic acid receptor alpha activity by means of increasing interaction with and phosphorylation by cyclin H/cdk7.

Nuclear retinoic acid receptors (RARs) work as ligand-dependent heterodimeric RAR/retinoid X receptor transcription activators, which are targets for phosphorylations. The N-terminal activation function (AF)-1 domain of RARalpha is phosphorylated by the cyclin-dependent kinase (cdk) 7/cyclin H complex of the general transcription factor TFIIH and the C-terminal AF-2 domain by the cAMP-dependent protein kinase A (PKA). Here, we report the identification of a molecular pathway by which phosphorylation by PKA propagates cAMP signaling from the AF-2 domain to the AF-1 domain. The first step is the phosphorylation of S369, located in loop 9-10 of the AF-2 domain. This signal is transferred to the cyclin H binding domain (at the N terminus of helix 9 and loop 8-9), resulting in enhanced cyclin H interaction and, thereby, greater amounts of RARalpha phosphorylated at S77 located in the AF-1 domain by the cdk7/cyclin H complex. This molecular mechanism relies on the integrity of the ligand-binding domain and the cyclin H binding surface. Finally, it results in higher DNA-binding efficiency, providing an explanation for how cAMP synergizes with retinoic acid for transcription.

Cyclin H binding to the RARalpha activation function (AF)-2 domain directs phosphorylation of the AF-1 domain by cyclin-dependent kinase 7.

The transcriptional activity of nuclear retinoic acid receptors (RARs), which act as RAR/retinoid X receptor (RXR) heterodimers, depends on two activation functions, AF-1 and AF-2, which are targets for phosphorylations and synergize for the activation of retinoic acid target genes. The N-terminal AF-1 domain of RARalpha is phosphorylated at S77 by the cyclin-dependent kinase (cdk)-activating kinase (CAK) subcomplex (cdk7/cyclin H/MAT1) of the general transcription factor TFIIH. Here, we show that phosphorylation of S77 governing the transcriptional activity of RARalpha depends on cyclin H binding at a RARalpha region that encompasses loop 8-9 and the N-terminal tip of helix 9 of the AF-2 domain. We propose a model in which the structural constraints of this region control the architecture of the RAR/RXR/TFIIH complex and therefore the efficiency of RARalpha phosphorylation by cdk7. To our knowledge, this study provides the first example of a cooperation between the AF-2 and AF-1 domains of RARs through a kinase complex.

Vinexin beta interacts with the non-phosphorylated AF-1 domain of retinoid receptor gamma (RARgamma) and represses RARgamma-mediated transcription.

Nuclear retinoic acid receptors (RARs) are ligand-dependent transcription factors that regulate the expression of retinoic acid target genes. Although the importance of RAR phosphorylation in their N-terminal domain is clearly established, the underlying mechanism for the phosphorylation-dependent transcriptional activity of the receptors had not been elucidated yet. Here, using a yeast two-hybrid system, we report the isolation of vinexin beta as a new cofactor that interacts with the N-terminal A/B domain of the RARgamma isotype. Vinexin beta is a multiple SH3 motif-containing protein associated with the cytoskeleton and also present in the nucleus. We demonstrate that vinexin beta colocalizes with RARgamma in the nucleus and interacts with the non-phosphorylated form of the AF-1 domain of RARgamma. We also show that this interaction is prevented upon phosphorylation of the AF-1 domain. Using F9 cells stably overexpressing vinexin beta or vinexin knockdown by RNA interference, we demonstrate that vinexin beta is an inhibitor of RARgamma-mediated transcription. We propose a model in which phosphorylation of the AF-1 domain controls RARgamma-mediated transcription through triggering the dissociation of vinexin beta.