Drosophila TET acts with PRC1 to activate gene expression independently of its catalytic activity.

Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS.

Adenine methylation is very scarce in the Drosophila genome and not erased by the ten-eleven translocation dioxygenase.

N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in Drosophila, for which the erasure of this epigenetic mark has been ascribed to the ten-eleven translocation (TET) enzyme. Here, we re-evaluated 6mA presence and TET impact on the Drosophila genome. Using axenic or conventional breeding conditions, we found traces of 6mA by LC-MS/MS and no significant increase in 6mA levels in the absence of TET, suggesting that this modification is present at very low levels in the Drosophila genome but not regulated by TET. Consistent with this latter hypothesis, further molecular and genetic analyses showed that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. Our results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans and underline the importance of TET non-enzymatic activity for fly development.

Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution.

NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10's essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation 'machinery' that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.

Patient-matched analysis identifies deregulated networks in prostate cancer to guide personalized therapeutic intervention.

Prostate cancer (PrCa) is the second most common malignancy in men. More than 50% of advanced prostate cancers display the TMPRSS2-ERG fusion. Despite extensive cancer genome/transcriptome data, little is known about the impact of mutations and altered transcription on regulatory networks in the PrCa of individual patients. Using patient-matched normal and tumor samples, we established somatic variations and differential transcriptome profiles of primary ERG-positive prostate cancers. Integration of protein-protein interaction and gene-regulatory network databases defined highly diverse patient-specific network alterations. Different components of a given regulatory pathway were altered by novel and known mutations and/or aberrant gene expression, including deregulated ERG targets, and were validated by using a novel in silico methodology. Consequently, different sets of pathways were altered in each individual PrCa. In a given PrCa, several deregulated pathways share common factors, predicting synergistic effects on cancer progression. Our integrated analysis provides a paradigm to identify druggable key deregulated factors within regulatory networks to guide personalized therapies.

Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential.

While many studies have described Drosophila embryonic and larval blood cells, the hematopoietic system of the imago remains poorly characterized and conflicting data have been published concerning adult hematopoiesis. Using a combination of blood cell markers, we show that the adult hematopoietic system is essentially composed of a few distinct mature blood cell types. In addition, our transcriptomics results indicate that adult and larval blood cells have both common and specific features and it appears that adult hemocytes reactivate many genes expressed in embryonic blood cells. Interestingly, we identify a small set of blood cells that does not express differentiation markers but rather maintains the expression of the progenitor marker domeMeso. Yet, we show that these cells are derived from the posterior signaling center, a specialized population of cells present in the larval lymph gland, rather than from larval blood cell progenitors, and that their maintenance depends on the EBF transcription factor Collier. Furthermore, while these cells are normally quiescent, we find that some of them can differentiate and proliferate in response to bacterial infection. In sum, our results indicate that adult flies harbor a small population of specialized cells with limited hematopoietic potential and further support the idea that no substantial hematopoiesis takes place during adulthood.

Prmt5 promotes vascular morphogenesis independently of its methyltransferase activity.

During development, the vertebrate vasculature undergoes major growth and remodeling. While the transcriptional cascade underlying blood vessel formation starts to be better characterized, little is known concerning the role and mode of action of epigenetic enzymes during this process. Here, we explored the role of the Protein Arginine Methyl Transferase Prmt5 in blood vessel formation as well as hematopoiesis using zebrafish as a model system. Through the combination of different prmt5 loss-of-function approaches we highlighted a key role of Prmt5 in both processes. Notably, we showed that Prmt5 promotes vascular morphogenesis through the transcriptional control of ETS transcription factors and adhesion proteins in endothelial cells. Interestingly, using a catalytic dead mutant of Prmt5 and a specific drug inhibitor, we found that while Prmt5 methyltransferase activity was required for blood cell formation, it was dispensable for vessel formation. Analyses of chromatin architecture impact on reporter genes expression and chromatin immunoprecipitation experiments led us to propose that Prmt5 regulates transcription by acting as a scaffold protein that facilitates chromatin looping to promote vascular morphogenesis.

CBP and P300 regulate distinct gene networks required for human primary myoblast differentiation and muscle integrity.

The acetyltransferases CBP and P300 have been implicated in myogenesis in mouse immortalized cell lines but these studies focused only on the expression of a handful of myogenic factors. Hence, the respective role of these two related cofactors and their impact at global scale on gene expression rewiring during primary myoblast differentiation remain unknown. Here, we characterised the gene networks regulated by these two epigenetic enzymes during human primary myoblast differentiation (HPM). We found that CBP and p300 play a critical role in the activation of the myogenic program and mostly regulate distinct gene sets to control several aspects of HPM biology, even though they also exhibit some degree of redundancy. Moreover, CBP or P300 knockdown strongly impaired muscle cell adhesion and resulted in the activation of inflammation markers, two hallmarks of dystrophic disease. This was further validated in zebrafish where inhibition of CBP and P300 enzymatic activities led to cell adhesion defects and muscle fiber detachment. Our data highlight an unforeseen link between CBP/P300 activity and the emergence of dystrophic phenotypes. They thereby identify CBP and P300 as mediators of adult muscle integrity and suggest a new lead for intervention in muscular dystrophy.

From Drosophila Blood Cells to Human Leukemia.

The hematopoietic system plays a critical role in establishing the proper response against invading pathogens or in removing cancerous cells. Furthermore, deregulations of the hematopoietic differentiation program are at the origin of numerous diseases including leukemia. Importantly, many aspects of blood cell development have been conserved from human to Drosophila. Hence, Drosophila has emerged as a potent genetic model to study blood cell development and leukemia in vivo. In this chapter, we give a brief overview of the Drosophila hematopoietic system, and we provide a protocol for the dissection and the immunostaining of the larval lymph gland, the most studied hematopoietic organ in Drosophila. We then focus on the various paradigms that have been used in fly to investigate how conserved genes implicated in leukemogenesis control blood cell development. Specific examples of Drosophila models for leukemia are presented, with particular attention to the most translational ones. Finally, we discuss some limitations and potential improvements of Drosophila models for studying blood cell cancer.

Epigenetic Regulators Modulate Muscle Damage in Duchenne Muscular Dystrophy Model.

Histone acetyl transferases (HATs) and histone deacetylases (HDAC) control transcription during myogenesis. HDACs promote chromatin condensation, inhibiting gene transcription in muscle progenitor cells until myoblast differentiation is triggered and HDACs are released. HATs, namely CBP/p300, activate myogenic regulatory and elongation factors promoting myogenesis. HDAC inhibitors are known to improve regeneration in dystrophic muscles through follistatin upregulation. However, the potential of directly modulating HATs remains unexplored. We tested this possibility in a well-known zebrafish model of Duchenne muscular dystrophy. Interestingly, CBP/p300 transcripts were found downregulated in the absence of Dystrophin. While investigating CBP rescuing potential we observed that dystrophin-null embryos overexpressing CBP actually never show significant muscle damage, even before a first regeneration cycle could occur. We found that the pan-HDAC inhibitor trichostatin A (TSA) also prevents early muscle damage, however the single HAT CBP is as efficient even in low doses. The HAT domain of CBP is required for its full rescuing ability. Importantly, both CBP and TSA prevent early muscle damage without restoring endogenous CBP/p300 neither increasing follistatin transcripts. This suggests a new mechanism of action of epigenetic regulators protecting dystrophin-null muscle fibres from detaching, independent from the known improvement of regeneration upon damage of HDACs inhibitors. This study builds supporting evidence that epigenetic modulators may play a role in determining the severity of muscle dystrophy, controlling the ability to resist muscle damage. Determining the mode of action leading to muscle protection can potentially lead to new treatment options for muscular dystrophies in the future.

Expression patterns of CREB binding protein (CREBBP) and its methylated species during zebrafish development.

Proper embryonic development requires a fine-tuned control of gene expression, which is achieved in part through the activity of transcription coactivators or corepressors. The nuclear coactivator cAMP-response element-binding protein (CREB) binding protein (CREBBP or CBP) interacts with numerous transcription factors and thereby plays a key role in various signaling pathways. Interestingly, in cell-based studies CREBBP activity is modulated by post-translational modifications such as methylation on arginine residues which is catalyzed by coactivator-associated arginine methyltransferase 1 (CARM1). However, whether and where CREBBP, and in particular its methylated forms, are expressed during development in vertebrates has not been addressed so far. Here, we analyzed the expression of the two crebbp genes (crebbpa & crebbpb) during zebrafish development using both RT-qPCR and in situ hybridization. We found that while crebbpa expression is higher in posterior, caudal nascent somites during somitogenesis, crebbpb accumulates in anterior, rostral, and more mature somites. In addition, crebbpa mRNA is enriched in the central myotome at 24 hpf indicating that its expression is spatially and temporally controlled. We next characterized the expression of CREBBP protein from blastula to gastrula stages by immunohistochemistry. We found that while CREBBP is clearly cytoplasmic in the early blastula, it becomes both cytoplasmic and nuclear at 30% epiboly before turning mainly nuclear during gastrulation. Of interest, CREBBP methylated species appear to be mainly nuclear from 30% epiboly to 6-somite stage. This suggests that methylation may regulate CREBBP import to the nucleus during zebrafish development and could therefore participate in the control of early developmental processes.

The methyltransferases PRMT4/CARM1 and PRMT5 control differentially myogenesis in zebrafish.

In vertebrates, skeletal myogenesis involves the sequential activation of myogenic factors to lead ultimately to the differentiation into slow and fast muscle fibers. How transcriptional co-regulators such as arginine methyltransferases PRMT4/CARM1 and PRMT5 control myogenesis in vivo remains poorly understood. Loss-of-function experiments using morpholinos against PRMT4/CARM1 and PRMT5 combined with in situ hybridization, quantitative polymerase chain reaction, as well as immunohistochemistry indicate a positive, but differential, role of these enzymes during myogenesis in vivo. While PRMT5 regulates myod, myf5 and myogenin expression and thereby slow and fast fiber formation, PRMT4/CARM1 regulates myogenin expression, fast fiber formation and does not affect slow fiber formation. However, our results show that PRMT4/CARM1 is required for proper slow myosin heavy chain localization. Altogether, our results reveal a combinatorial role of PRMT4/CARM1 and PRMT5 for proper myogenesis in zebrafish.

Methylation specifies distinct estrogen-induced binding site repertoires of CBP to chromatin.

Multiple signaling pathways ultimately modulate the epigenetic information embedded in the chromatin of gene promoters by recruiting epigenetic enzymes. We found that, in estrogen-regulated gene programming, the acetyltransferase CREB-binding protein (CBP) is specifically and exclusively methylated by the coactivator-associated arginine methyltransferase (CARM1) in vivo. CARM1-dependent CBP methylation and p160 coactivators were required for estrogen-induced recruitment to chromatin targets. Notably, methylation increased the histone acetyltransferase (HAT) activity of CBP and stimulated its autoacetylation. Comparative genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) studies revealed a variety of patterns by which p160, CBP, and methyl-CBP (meCBP) are recruited (or not) by estrogen to chromatin targets. Moreover, significant target gene-specific variation in the recruitment of (1) the p160 RAC3 protein, (2) the fraction of a given meCBP species within the total CBP, and (3) the relative recruitment of different meCBP species suggests the existence of a target gene-specific "fingerprint" for coregulator recruitment. Crossing ChIP-seq and transcriptomics profiles revealed the existence of meCBP "hubs" within the network of estrogen-regulated genes. Together, our data provide evidence for an unprecedented mechanism by which CARM1-dependent CBP methylation results in gene-selective association of estrogen-recruited meCBP species with different HAT activities and specifies distinct target gene hubs, thus diversifying estrogen receptor programming.

Dual role of the arginine methyltransferase CARM1 in the regulation of c-Fos target genes.

Fos proteins, the prototypic members of basic region-leucine zipper (bZIP) transcription factors, bind to other bZIP proteins to form the activator protein-1 (AP-1) complex, which regulates the expression of a plethora of target genes. Notably, c-Fos target genes include members of the matrix metalloproteinase (MMP) gene family and c-fos is overexpressed in a number of metastatic cancers, suggesting its direct involvement in this process. Here, we reveal that c-Fos-mediated transcriptional activation is regulated by the protein arginine methyltransferase CARM1 and by all three members of the p160 protein family of coactivators. Carm1-deficient cells showed a dramatic reduction in the expression level of c-Fos target genes MMP-1b, -3, and -13, indicating a major role for CARM1 in regulating the expression of these genes. RNA interference combined with quantitative polymerase chain reaction demonstrated that CARM1 and p160 proteins synergize to activate expression of MMP-1b, -3, and -13 in vivo. Furthermore, we show that CARM1 also regulates MMP expression at the post-transcriptional level, either positively or negatively. Our data indicate that CARM1 can play a dual role in the expression of AP-1 target genes involved in cancer or other diseases by acting at the transcriptional as well as at the post-transcriptional levels.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene.

The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation/methylation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1/DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1, along with their transcriptional target CCNE1. All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.

The Ca2+-induced methyltransferase xPRMT1b controls neural fate in amphibian embryo.

We have previously shown that an increase in intracellular Ca2+ is both necessary and sufficient to commit ectoderm to a neural fate in Xenopus embryos. However, the relationship between this Ca2+ increase and the expression of early neural genes has yet to be defined. Using a subtractive cDNA library between untreated and caffeine-treated animal caps, i.e., control ectoderm and ectoderm induced toward a neural fate by a release of Ca2+, we have isolated the arginine N-methyltransferase, xPRMT1b, a Ca2+-induced target gene, which plays a pivotal role in this process. First, we show in embryo and in animal cap that xPRMT1b expression is Ca2+-regulated. Second, overexpression of xPRMT1b induces the expression of early neural genes such as Zic3. Finally, in the whole embryo, antisense approach with morpholino oligonucleotide against xPRMT1b impairs neural development and in animal caps blocks the expression of neural markers induced by a release of internal Ca2+. Our results implicate an instructive role of an enzyme, an arginine methyltransferase protein, in the embryonic choice of determination between epidermal and neural fate. The results presented provide insights by which a Ca2+ increase induces neural fate.

Interplay among coactivator-associated arginine methyltransferase 1, CBP, and CIITA in IFN-gamma-inducible MHC-II gene expression.

Class II major histocompatibility (MHC-II) genes are prototype targets of IFN-gamma. IFN-gamma activates the expression of the non-DNA-binding master regulator of MHC-II, class II transactivator (CIITA), which is crucial for enhanceosome formation and gene activation. This report shows the importance of the histone methyltransferase, coactivator-associated arginine methyltransferase (CARM1/PRMT4), during IFN-gamma-induced MHC-II gene activation. It also demonstrates the coordinated regulation of CIITA, CARM1, and the acetyltransferase cyclic-AMP response element binding (CREB)-binding protein (CBP) during this process. CARM1 synergizes with CIITA in activating MHC-II transcription and synergy is abrogated when an arginine methyltransferase-defective CARM1 mutant is used. Protein-arginine methyltransferase 1 has much less effect on MHC-II transcription. Specific RNA interference reduced CARM1 expression as well as MHC-II expression. The recruitment of CARM1 to the promoter requires endogenous CIITA and results in methylation of histone H3-R17; hence, CIITA is an upstream regulator of histone methylation. Previous work has shown that CARM1 can methylate CBP at three arginine residues. Using wild-type CBP and a mutant of CBP lacking the CARM1-targeted arginine residues (R3A), we show that arginine methylation of CBP is required for IFN-gamma induction of MHC-II. A kinetic analysis shows that CIITA, CARM1, and H3-R17 methylation all precede CBP loading on the MHC-II promoter during IFN-gamma treatment. These results suggest functional and temporal relationships among CIITA, CARM1, and CBP for IFN-gamma induction of MHC-II.

Control of CBP co-activating activity by arginine methylation.

The histone acetyltransferases CREB binding protein (CBP) and the related p300 protein function as key transcriptional co-activators in multiple pathways. In the case of transcriptional activation by nuclear receptors, ligand promotes the recruitment of co-activators of the p160 family, such as GRIP-1. Subsequently, the p160 co-activators recruit other co-activators via two activation domains, AD1 and AD2. AD1 binds CBP or p300, whereas AD2 has been shown to activate transcription through the recruitment of the arginine methyltransferase CARM1. Recently, the KIX domain of CBP has been shown to be methylated by CARM1 in vitro. Here, we report that another domain of CBP is specifically methylated by CARM1 on conserved arginine residues in vitro and in vivo. We also provide functional evidence that arginine residues methylated by CARM1 play a critical role in GRIP-1-dependent transcriptional activation and in hormone-induced gene activation. Altogether, our data provide strong evidence that arginine methylation represents an important mechanism for modulating co-activator transcriptional activity.

Functional and physical interaction between the histone methyl transferase Suv39H1 and histone deacetylases.

The histone methyl transferase Suv39H1 is involved in silencing by pericentric heterochromatin. It specifically methylates K9 of histone H3, thereby creating a high affinity binding site for HP1 proteins. We and others have shown recently that it is also involved in transcriptional repression by the retinoblastoma protein Rb. Strikingly, both HP1 localisation and repression by Rb also require, at least in part, histone deacetylases. We found here that repression of a heterologous promoter by Suv39H1 is dependent on histone deacetylase activity. However, the enzymatic activity of Suv39H1 is not required, since the N-terminal part is by itself a transcriptional repression domain. Coimmunoprecipitation experiments indicated that Suv39H1 can physically interact with HDAC1, -2 and -3, therefore suggesting that transcriptional repression by Suv39H1 could be the consequence of histone deacetylases recruitment. Consistent with this interpretation, the N-terminal transcriptional repression domain of Suv39H1 bound the so-called 'core histone deacetylase complex', composed of HDAC1, HDAC2 and the Rb-associated proteins RbAp48 and RbAp46. Taken together, our results suggest that a complex containing both the Suv39H1 histone methyl transferase and histone deacetylases could be involved in heterochromatin silencing or transcriptional repression by Rb.