Tet controls axon guidance in early brain development through glutamatergic signaling.

Mutations in ten-eleven translocation (TET) proteins are associated with human neurodevelopmental disorders. We find a function of Tet in regulating Drosophila early brain development. The Tet DNA-binding domain (TetAXXC) is required for axon guidance in the mushroom body (MB). Glutamine synthetase 2 (Gs2), a key enzyme in glutamatergic signaling, is significantly down-regulated in the TetAXXC brains. Loss of Gs2 recapitulates the TetAXXC phenotype. Surprisingly, Tet and Gs2 act in the insulin-producing cells (IPCs) to control MB axon guidance, and overexpression of Gs2 in IPCs rescues the defects of TetAXXC. Feeding TetAXXC with metabotropic glutamate receptor antagonist MPEP rescues the phenotype while glutamate enhances it. Mutants in Tet and Drosophila Fmr1, the homolog of human FMR1, have similar defects, and overexpression of Gs2 in IPCs also rescues the Fmr1 phenotype. We provide the first evidence that Tet controls the guidance of developing brain axons by modulating glutamatergic signaling.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

Tet Controls Axon Guidance in Early Brain Development through Glutamatergic Signaling.

Mutations in human TET proteins have been found in individuals with neurodevelopmental disorders. Here we report a new function of Tet in regulating Drosophila early brain development. We found that mutation in the Tet DNA-binding domain ( Tet AXXC ) resulted in axon guidance defects in the mushroom body (MB). Tet is required in early brain development during the outgrowth of MB ß axons. Transcriptomic study shows that glutamine synthetase 2 (Gs2), a key enzyme in glutamatergic signaling, is significantly downregulated in the Tet AXXC mutant brains. CRISPR/Cas9 mutagenesis or RNAi knockdown of Gs2 recapitulates the Tet AXXC mutant phenotype. Surprisingly, Tet and Gs2 act in the insulin-producing cells (IPCs) to control MB axon guidance, and overexpression of Gs2 in these cells rescues the axon guidance defects of Tet AXXC . Treating Tet AXXC with the metabotropic glutamate receptor antagonist MPEP can rescue while treating with glutamate enhances the phenotype confirming Tet function in regulating glutamatergic signaling. Tet AXXC and the Drosophila homolog of Fragile X Messenger Ribonucleoprotein protein mutant ( Fmr1 3 ) have similar axon guidance defects and reduction in Gs2 mRNA levels. Interestingly, overexpression of Gs2 in the IPCs also rescues the Fmr1 3 phenotype, suggesting functional overlapping of the two genes. Our studies provide the first evidence that Tet can control the guidance of axons in the developing brain by modulating glutamatergic signaling and the function is mediated by its DNA-binding domain.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates the axon guidance genes robo2 and slit in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases enzymes catalyzing the transition of 5mC to 5hmC in DNA and have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila because Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by determining Tet DNA-binding sites throughout the genome and by mapping the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC-modified sites can be found along the entire transcript and are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are frequently involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and are sensitized to reduced levels of slit. Both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs, primarily in developing nerve cells.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

Expression and functional analysis of various structural domains of tobacco topoisomerase II: To understand the mechanistic insights of plant type II topoisomerases.

In contrast to bacterial, yeast and animal systems, topoisomerases (topo) from plants have not been well studied. In this report, we generated four truncated topoisomerase II (Topo II) cDNA fragments encoding different functional domains of Nicotiana tabacum topo II (NtTopoII). Each of these recombinant polypeptides was expressed alone or in combination in temperature-sensitive topoisomerase II yeast mutants. Recombinant NtTopoII with truncated polypeptides fails to target the yeast nuclei and does not rescue the temperature-sensitive phenotype. In contrast complementation was achieved with the full-length NtTopoII, which localized to the yeast nucleus. These observations suggested the presence of a potent nuclear localization signal (NLS) in the extreme C-terminal 314 amino acid residues of NtTopoII that functioned effectively in the heterologous yeast system. Biochemical characterization of purified recombinant full-length and the partial NtTopoII polypeptides revealed that the ATP-binding and hydrolysis region of NtTopoIIwas located at 413 amino acid N-terminal region and this ATPase domain is functional both when it is expressed as a separate polypeptide or as part of the holoenzyme. The present findings also revealed that all NtTopoII truncated polypeptides were detrimental for in vitro supercoiled DNA relaxation and/or DNA nicking and ligation activity. Further, we discuss the possible disruption of coordinated macromolecular interface movements and the dimer interactions in truncated NtTopoII that are required for functional topoisomerase activity.

Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation.

KEY MESSAGE: The topoisomerase II expression varies as a function of cell proliferation. Maximal topoisomerase II expression was tightly coupled to S phase and G2/M phase via both transcriptional and post-transcriptional regulation. Investigation in meiosis using pollen mother cells also revealed that it is not the major component of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed. Synchronized tobacco BY-2 cell cultures were used to study the role of topoisomerase II in various stages of the cell cycle. Topoisomerase II transcript accumulation was observed during the S- and G2/M- phase of cell cycle. This biphasic expression pattern indicates the active requirement of topoisomerase II during these stages of the cell cycle. Through immuno-localization of topoisomerase II was observed diffusely throughout the nucleoplasm in interphase nuclei, whereas, the nucleolus region exhibited a more prominent immuno-positive staining that correlated with rRNA transcription, as shown by propidium iodide staining and BrUTP incorporation. The immuno-staining analysis also showed that topoisomerase II is the major component of mitotic chromosomes and remain attached to the chromosomes during cell division. The inhibition of topoisomerase II activity using specific inhibitors revealed quite dramatic effect on condensation of chromatin and chromosome individualization from prophase to metaphase transition. Partially condensed chromosomes were not arranged on metaphase plate and chromosomal perturbations were observed when advance to anaphase, suggesting the importance of topoisomerase II activity for proper chromosome condensation and segregation during mitosis. Contrary, topoisomerase II is not the major component of meiotic chromosomes, even though mitosis and meiosis share many processes, including the DNA replication, chromosome condensation and precisely regulated partitioning of chromosomes into daughter cells. Even if topoisomerase II is required for individualization and condensation of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed.

Promoter-Terminator Gene Loops Affect Alternative 3'-End Processing in Yeast.

Many eukaryotic genes undergo alternative 3'-end poly(A)-site selection producing transcript isoforms with 3'-UTRs of different lengths and post-transcriptional fates. Gene loops are dynamic structures that juxtapose the 3'-ends of genes with their promoters. Several functions have been attributed to looping, including memory of recent transcriptional activity and polarity of transcription initiation. In this study, we investigated the relationship between gene loops and alternative poly(A)-site. Using the KlCYC1 gene of the yeast Kluyveromyces lactis, which includes a single promoter and two poly(A) sites separated by 394 nucleotides, we demonstrate in two yeast species the formation of alternative gene loops (L1 and L2) that juxtapose the KlCYC1 promoter with either proximal or distal 3'-end processing sites, resulting in the synthesis of short and long forms of KlCYC1 mRNA. Furthermore, synthesis of short and long mRNAs and formation of the L1 and L2 loops are growth phase-dependent. Chromatin immunoprecipitation experiments revealed that the Ssu72 RNA polymerase II carboxyl-terminal domain phosphatase, a critical determinant of looping, peaks in early log phase at the proximal poly(A) site, but as growth phase advances, it extends to the distal site. These results define a cause-and-effect relationship between gene loops and alternative poly(A) site selection that responds to different physiological signals manifested by RNA polymerase II carboxyl-terminal domain phosphorylation status.

Cell cycle stage-specific differential expression of topoisomerase I in tobacco BY-2 cells and its ectopic overexpression and knockdown unravels its crucial role in plant morphogenesis and development.

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. Cell cycle coupled differential accumulation of topoisomerase I (Topo I) revealed biphasic expression maximum at S-phase and M/G1-phase of cultured synchronized tobacco BY-2 cells. This suggested its active role in resolving topological constrains during DNA replication (S-phase) and chromosome decondensation (M/G1 phase). Immuno-localization revealed high concentrations of Topo I in nucleolus. Propidium iodide staining and Br-UTP incorporation patterns revealed direct correlation between immunofluorescence intensity and rRNA transcription activity within nucleolus. Immuno-stained chromosomes during metaphase and anaphase suggested possible role of Topo I in resolving topological constrains during mitotic chromosome condensation. Inhibitor studies showed that in comparison to Topo I, Topo II was essential in resolving topological constrains during chromosome condensation. Probably, Topo II substituted Topo I functioning to certain extent during chromosome condensation, but not vice-versa. Transgenic Topo I tobacco lines revealed morphological abnormalities and highlighted its crucial role in plant morphogenesis and development.

Detection of short-range chromatin interactions by chromosome conformation capture (3C) in yeast.

We describe a modified 3C ("chromosome conformation capture") protocol for detection of transient, short-range chromatin interactions in the yeast Saccharomyces cerevisiae. 3C was initially described by Job Dekker and involves formaldehyde cross-linking to stabilize transient chromatin interactions, followed by restriction digestion, ligation, and locus-specific PCR. As such, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Using a modified version of Dekker's protocol, we are able to detect gene loops that juxtapose promoter and terminator regions of yeast genes with ORFs as short as 1 kb. We are using this technique to define the cis- and trans-acting requirements for the formation and maintenance of gene loops, and to elucidate their physiological consequences. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions, not limited to gene loops.

DNA looping facilitates targeting of a chromatin remodeling enzyme.

ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here, we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a mechanism for the recruitment of a chromatin remodeling enzyme and define a function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes.

The interaction of Pcf11 and Clp1 is needed for mRNA 3'-end formation and is modulated by amino acids in the ATP-binding site.

Polyadenylation of eukaryotic mRNAs contributes to stability, transport and translation, and is catalyzed by a large complex of conserved proteins. The Pcf11 subunit of the yeast CF IA factor functions as a scaffold for the processing machinery during the termination and polyadenylation of transcripts. Its partner, Clp1, is needed for mRNA processing, but its precise molecular role has remained enigmatic. We show that Clp1 interacts with the Cleavage-Polyadenylation Factor (CPF) through its N-terminal and central domains, and thus provides cross-factor connections within the processing complex. Clp1 is known to bind ATP, consistent with the reported RNA kinase activity of human Clp1. However, substitution of conserved amino acids in the ATP-binding site did not affect cell growth, suggesting that the essential function of yeast Clp1 does not involve ATP hydrolysis. Surprisingly, non-viable mutations predicted to displace ATP did not affect ATP binding but disturbed the Clp1-Pcf11 interaction. In support of the importance of this interaction, a mutation in Pcf11 that disrupts the Clp1 contact caused defects in growth, 3'-end processing and transcription termination. These results define Clp1 as a bridge between CF IA and CPF and indicate that the Clp1-Pcf11 interaction is modulated by amino acids in the conserved ATP-binding site of Clp1.

Control of eukaryotic gene expression: gene loops and transcriptional memory.

Gene loops are dynamic structures that juxtapose promoter­terminator regions of Pol II-transcribed genes. Although first described in yeast, gene loops have now been identified in yeast and mammalian cells. Looping requires components of the transcription preinitiation complex, the pre-mRNA 30-end processing machinery, and subunits of the nuclear pore complex. Loop formation is transcription-dependent, but neither basal nor activated transcription requires looping. Rather, looping appears to affect cellular memory of recent transcriptional activity, enabling a more rapid response to subsequent stimuli. The nuclear pore has been implicated in both memory and looping. Our working model is that loops are formed and/or maintained at the nuclear pore to facilitate hand-off of Pol II form the terminator to the promoter, thereby bypassing Pol II recruitment as the rate-limiting step in reactivation of transcription. Involvement of the nuclear pore also suggests that looping might facilitate mRNA export to the cytoplasm. The technology now exists to test these ideas.

Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase.

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge alpha-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in beta R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site "latch" assembly that includes a key trigger helix residue Tt beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.

A physiological role for gene loops in yeast.

DNA loops that juxtapose the promoter and terminator regions of RNA polymerase II-transcribed genes have been identified in yeast and mammalian cells. Loop formation is transcription-dependent and requires components of the pre-mRNA 3'-end processing machinery. Here we report that looping at the yeast GAL10 gene persists following a cycle of transcriptional activation and repression. Moreover, GAL10 and a GAL1p-SEN1 reporter undergo rapid reactivation kinetics following a cycle of activation and repression-a phenomenon defined as "transcriptional memory"-and this effect correlates with the persistence of looping. We propose that gene loops facilitate transcriptional memory in yeast.

Detection of gene loops by 3C in yeast.

"Chromosome conformation capture" (3C) is a powerful method to detect physical interaction between any two genomic loci. 3C involves formaldehyde crosslinking to stabilize transient interactions, followed by restriction digestion, ligation and locus-specific PCR. Accordingly, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Here, we describe a modified 3C protocol designed for detection of transient chromatin interactions in the yeast Saccharomyces cerevisiae. Using this protocol, we are able to detect juxtaposition of promoter and terminator regions of genes with ORFs as short as 1kb in length. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions and will facilitate the characterization of gene loops and their functional consequences.

The essential N terminus of the Pta1 scaffold protein is required for snoRNA transcription termination and Ssu72 function but is dispensable for pre-mRNA 3'-end processing.

Saccharomyces cerevisiae Pta1 is a component of the cleavage/polyadenylation factor (CPF) 3'-end processing complex and functions in pre-mRNA cleavage, poly(A) addition, and transcription termination. In this study, we investigated the role of the N-terminal region of Pta1 in transcription and processing. We report that a deletion of the first 75 amino acids (pta1-Delta75) causes thermosensitive growth, while the deletion of an additional 25 amino acids is lethal. The pta1-Delta75 mutant is defective for snoRNA termination, RNA polymerase II C-terminal domain Ser5-P dephosphorylation, and gene looping but is fully functional for mRNA 3'-end processing. Furthermore, different regions of Pta1 interact with the CPF subunits Ssu72, Pti1, and Ysh1, supporting the idea that Pta1 acts as a scaffold to organize CPF. The first 300 amino acids of Pta1 are sufficient for interactions with Ssu72, which is needed for pre-mRNA cleavage. By the degron-mediated depletion of Pta1, we show that the removal of this essential region leads to a loss of Ssu72, yet surprisingly, in vitro cleavage and polyadenylation remain efficient. In addition, a fragment containing amino acids 1 to 300 suppresses 3'-end processing in wild-type extracts. These findings suggest that the amino terminus of Pta1 has an inhibitory effect and that this effect can be neutralized through the interaction with Ssu72.

A transcription-independent role for TFIIB in gene looping.

Recent studies demonstrated the existence of gene loops that juxtapose the promoter and terminator regions of genes with exceptionally long ORFs in yeast. Here we report that looping is not idiosyncratic to long genes but occurs between the distal ends of genes with ORFs as short as 1 kb. Moreover, looping is dependent upon the general transcription factor TFIIB: the E62K (glutamic acid 62 --> lysine) form of TFIIB adversely affects looping at every gene tested, including BLM10, SAC3, GAL10, SEN1, and HEM3. TFIIB crosslinks to both the promoter and terminator regions of the PMA1 and BLM10 genes, and its association with the terminator, but not the promoter, is adversely affected by E62K and by depletion of the Ssu72 component of the CPF 3' end processing complex, and is independent of TBP. We propose a model suggesting that TFIIB binds RNAP II at the terminator, which in turn associates with the promoter scaffold.