Finally, we found a specific functional link between prefoldin and histone dynamics after nucleosome remodeling, which is consistent with the extensive network of genetic interactions between this factor and the machinery regulating chromatin function. This study establishes the involvement of prefoldin in transcription elongation, and supports a role for this complex in cotranscriptional histone eviction.Transcriptional elongation requires the concerted action of several factors that allow RNA polymerase II to advance through chromatin in a highly processive manner. In order to identify novel elongation factors, we performed systematic yeast genetic screening based on the GLAM (Gene Length-dependent Accumulation of mRNA) assay, which is used to detect defects in the expression of long transcription units. Apart from well-known transcription elongation factors, we identified mutants in the prefoldin complex subunits, which were among those that caused the most dramatic phenotype. We found that prefoldin, so far involved in the cytoplasmic co-translational assembly of protein complexes, is also present in the nucleus and that a subset of its subunits are recruited to chromatin in a transcription-dependent manner. Prefoldin influences RNA polymerase II the elongation rate  Transcription is the biological process that allows genes to be copied into RNA; the molecule that can be read by the cell in order to fabricate its structural components, proteins. Transcription is carried out by RNA polymerases, but these molecular machines need auxiliary factors to guide them through the genome and to help them during the RNA synthesis process. We searched for novel auxiliary factors using a genetic procedure and found a set of potential novel transcriptional players. Among them, we encountered a highly unexpected result: a factor, called prefoldin, so far exclusively involved in the folding of proteins during their fabrication. We confirmed that prefoldin binds transcribed genes and plays an important role during gene transcription. We also further investigated this transcriptional role and found that prefoldin is important for unpacking genes, thus facilitating the advance of the RNA polymerases along them. RNA polymerase II transcribes nuclear protein-coding genes in eukaryotes. After the assembly of the preinitiation complex onto the promoter and the subsequent initiation of transcription, the enzymatic activity of RNA polymerase II enables the synthesis of considerably long RNA molecules in the elongation phase of transcription. During this process, nascent RNA is modified by capping and polyadenylation, exons are defined for splicing, while hnRNP components are recruited, thus ensuring the production of mature mRNAs that can be exported to the cytoplasm This complexity explains the tight regulation of transcription elongation, which involves a large number of auxiliary factors. Some of these factors contribute to transcription elongation by controlling the cotranscriptional processes affecting nascent RNA Since the physiological substrate of transcription is chromatin, transcription elongation is one of the main processes that contribute to shape the nucleosomal landscape of the genome One last group of elongation factors controls the phosphorylation state of RNA polymerase II, particularly the carboxyterminal domain of its largest subunit. This CTD domain is the docking platform that allows some of the other above-described elements to act during transcription elongation. Accordingly, the factors affecting CTD phosphorylation control the overall integration of the molecular functions required to complete the transcription cycle Saccharomyces cerevisiae viable deletions. Using an assay based on the comparison of transcription units whose transcribed region length differs, we detected novel genes that have not been previously linked to transcription elongation. Among them, we found PFD1 encoding a subunit of the prefoldin complex.The number of protein complexes in the literature playing specific roles during transcription elongation has increased in the last few years, suggesting that our knowledge on this field is still partial. In order to detect novel factors involved in transcriptional elongation, we performed a systematic genetic screening of a collection of Caenorhabditis elegans and produces severe cytoskeletal defects in mice that promote their early death Prefoldin acts as a co-factor of group II chaperonins in eukaryotes and archaea Saccharomyces cerevisiae. To do this, we introduced plasmid pGAL1-PHO5 and pGAL1-PHO5::lacZ (−3) and in “gene expression”  , soh1Δ (Mediator), ccr4Δ (Ccr4-Not complex) and thp2Δ (THO complex), lacked general transcriptional machinery elements that are directly or indirectly involved in transcription elongation The GLAM (gene length-dependent accumulation of mRNA) assay is a useful tool to detect defects in mRNA biogenesis that occur in a gene length-dependent manner. By comparing two transcription units, both encoding the acid phosphatase Pho5 and whose only difference lies in the length of their 3′ UTR, we can easily detect gene mutations that specifically decrease the expression level of the long unit without affecting the short one O5::lacZ  into allO5::lacZ . After cO5::lacZ . In all,O5::lacZ . Values O5::lacZ . No spec · 10−3) . We repePFD1, which provided the third most significant effect in the GLAM assay when deleted (pfd1Δ by Northern blot. The levels of the full-length mRNAs transcribed from GAL1p-PHO5::lacZ were significantly reduced in pfd1Δ as compared to the short mRNA from GAL1p-PHO5 (GAL1p-PHO5::LAC4) We decided to focus on  deleted . We confL1p-PHO5 . The samPFD1 encodes one of the components of prefoldin, a heterohexameric complex composed of two alpha subunits (Gim2 and Gim5) and four beta subunits  gim1Δ, gim3Δ and gim5Δ also gave GLAM ratios that were significantly below the wild type  is excluded from the nucleus at the permissive temperature and accumulates in the nucleus at the restrictive temperature Although it was first described as a cytoplasmic complex ADH1 and PMA1 (To address whether prefoldin directly regulates transcription by binding to chromatin we constructed a Pfd1-Myc fusion  and carr−10 after Mann-Whitney test) than in genes exhibiting poor RNA polymerase II occupancy .Occupancy was stronger in the 3′ end of the genes tested . In ordeGAL1, under active and inactive conditions. We found that Pfd1 binding to GAL1 was transcription-dependent since the ChIP signal in the coding region was found exclusively under culture conditions where GAL1 transcription was active  and not under repressive conditions  that become transiently induced upon salt treatment The results above suggest that prefoldin recruitment is influenced by transcriptional activity. To confirm this, we analyzed Pfd1 binding to inducible genes like nditions . Similardetected . We alsoPMA1 and ADH1 diminished . In light of the GLAM phenotype of the prefoldin mutants, we compared long genes (>4 kbp) with shorter genes. We found that long genes exhibited significantly higher 3′/5′ ratios of total RNA polymerase II (Rpb3 ChIP) in pfd1Δ, but this relative enrichment in 3′ did not involve any global variation in their average 3′/5′ run-on ratio  because this modification is closely associated with transcription elongation pfd1Δ than in the wild type  of GAL1p-YLR454w to micrococcal nuclease (MNase). We found typical nucleosomal peaks in 5′ and 3′ under repressive conditions  of GAL1p-YLR454w at different times after adding glucose to cells exponentially growing in galactose medium. We found no significant difference in the kinetics of H3 occupancy when comparing pfd1Δ and the wild type  displays clear transcriptional defects without exhibiting any significant cytoskeletal dysfunction. Likewise, we detected no impairment in transcription elongation upon treatment with the microtubule-destabilizing drug benomyl. We, therefore, consider that the transcriptional defects exhibited by prefoldin mutants reflect a relevant nuclear role of prefoldin in transcription elongation.The involvement of prefoldin in the cytoplasmic folding of cytoskeletal components is well established pfd1Δ is not hypersensitive to latrunculin A, which is able to disrupt actin filaments. This suggests that, even if prefoldin acts on nuclear actin, there is another nuclear role of prefoldin in transcription elongation.Nuclear prefoldin might work together with nuclear actin, which contributes to transcription elongation in metazoa Prefoldin subunits have been connected to other nuclear events where actin does not seem to be involved, like the inhibition of the c-Myc transactivation domain A direct action of prefoldin on the elongating form of RNA polymerase II or in RNA polymerase II clearance after termination might explain the recruitment of prefoldin to gene bodies and the accumulation of hypophosphorylated RNA polymerase II at 3′ in its absence. According to this hypothesis, the effect of prefoldin on histone eviction would be indirectly mediated by RNA polymerase II. This would be supported by the described interactions of prefoldin-like complexes with human RNA polymerase II during their cytoplasmic assembly pfd1Δ and we showed that they were not merely due to suboptimal transcriptional activity. In fact, we detected optimal accessibility of micrococcal nuclease to transcribed DNA in the prefoldin mutant, suggesting that the initial steps of cotranscriptional chromatin dynamics are not impaired in the absence of prefoldin  exhibit clearly different levels of bound histones . The transformants obtained were then mated to the deletion library (MAT a) and sporulated for 7 days. The haploid spores carrying the plasmid (pSCh202 or pSCh212) and the deletion mutation were selected in the appropriate media.We introduced the plasmids of the GLAM system The resulting strains were then inoculated in duplicates into 96-well plates containing 200 µl of SGal-Ura medium per well, and were incubated for 48 h at 30°C before assaying acid phosphatase activity as described elsewhere S. cerevisiae strains utilized were in the BY4741 background, except for the xpo1-1 mex67-5 strain, which was in the W303 background. Strains were grown at 30°C in the indicated media, except for the xpo1-1 mex67-5 mutant, which was shifted from 24°C to 37°C for 4 h.The strains and plasmids used in this study are listed in To generate pGMZ2, a PCR product containing the PFD1 ORF lacking the termination codon and an additional 1 kb upstream of the ORF was obtained by PCR using the oligonucleotides listed in To generate Pfd1-Myc5, the Y07202 yeast strain was transformed with a PCR product obtained using the oligonucleotides listed in 600 0.5 and serial dilutions (1∶10) were spotted onto plates. The Latrunculin A-sensitivity assay was performed as previously described For the growth assays, yeast cultures were diluted to ODCells were grown to the mid-log phase in selective SD liquid medium, and were washed and resuspended in TBS buffer. Image acquisition was done either with a Leica DMR microscope equipped with a differential contrast (DC) camera, or with a Zeiss LSM 710 laser scanning confocal microscope at 2 µm pinhole aperture. Digital images were processed with Adobe Photoshop CS.32P]dCTP-labeled DNA probe. Membranes were exposed in Fuji BAS screens and developed with a FUJIX FLA5100 device. All the values were normalized in relation to the present amount of 18S rRNA.Six micrograms of total RNA prepared from yeast cells were subjected to electrophoresis on formaldehyde agarose gels, transferred to Hybond-N filters  and UV cross-linked prior to hybridization at 65°C with a [Laemmli-boiled crude extracts were run on a 12% SDS-polyacrylamide gel and transferred to nylon membranes (Hybond-ECL). After blocking with Tris-buffered saline containing 0.1% Tween 20 and 5% milk, the following primary antibodies were used: mouse monoclonal anti-Myc , rabbit polyclonal anti-L1 Yeast nuclear extracts were obtained as previously described in ChIP experiments were performed as previously described 2 values of the median intensities of the chosen different group of genes were represented.For Pfd1-Myc ChIP on chip experiments, chromatin immunoprecipitation was performed as above and, after crosslinking reversal, the obtained fragments (300 bp approximately) of enriched DNA were amplified unspecificly and labeled following Affymetrix Chromatin Immunoprecipitation Assay Protocol P/N 702238. Genomic DNA controls were processed in parallel. After PCR amplification with dUTP, the samples were purified using Qiagen QIAquick PCR Purification Kit (50) (Cat.No. 28104). DNA quality and quantity were checked using a NanoDrop ND-1000 Spectrophotometer. 0.5 µg of each were used to hybridize GeneChip S.Cerevisiae Tiling 1.0R custom arrays. This step was carried out in the Multigenic Analysis Service of the University of Valencia. The obtained CEL archives were normalized and the intensities of the signal were extracted using the TAS  developed by Affymetrix. The resulting text files were read using R scripts to adjudicate probe intensities to genes. The log600 0.5 by centrifugation. Cells were washed in 5 ml of 0.5% sarkosyl solution. Cells were centrifuged and the supernatant was completely removed. The transcription reaction was incubated for 5 min at 30°C. RNA was immediately extracted following the acid-phenol protocol.Run-on assays were performed as previously described Slot-blotted membranes were performed as formerly described DNA arrays were produced in the Sección de Chips de DNA-S.C.S.I.E of the University of Valencia as previously described Hybridization with labeled RNA (Run-on) or DNA (Rpb3 ChIP) samples was performed as described 2 of the 3′/5′ratio was obtained. At least three replicas of each experiment were performed.Data analysis and normalizations were performed according to For Rpb3 ChIP on chip purposes, the immunoprecipitated DNA was amplified and radiolabeled following the procedure described in CDC10 probe described in Yeast spheroplasts and micrococcal nuclease digestions were performed as previously described Figure S1A. Relevant information of the two transcription units utilized to obtain the GLAM ratios. B. Schematic description of the process followed during screening.SGA screening based on the GLAM assay. (TIF)Click here for additional data file.Figure S2A. GLAM ratios obtained after incubating wild-type cells in the presence of benomyl for the indicated times. 15 µg/ml benomyl was added to wild-type cells growing exponentially in selective medium. GLAM assay was performed at the indicated times. Mean and standard deviation of 3 biological replicates are shown. B. Drop assay showing that pfd1Δ exhibits much weaker sensitivity to benomyl than the other prefoldin mutants. Serial dilutions of exponentially growing cultures of the indicated strains were plated on YPD and YPD plus 15 µg/ml benomyl and incubated during 3 days at 30°C. C. Plate assay showing that gim2Δ is hypersensitive to latrunculin A, whereas pfd1Δ is not. The concentration of latrunculin A soaking each position is shown. Histograms represent the diameter of the inhibition halo of the 0.5 mM position for each strain after 2 days of incubation.Transcriptional phenotypes of prefoldin do not correlate with sensitivity to benomyl or latrunculin A. (TIF)Click here for additional data file.Figure S3A. The Pfd1-GFP fusion protein is functional. GLAM assays were performed with cells from a pfd1Δ transformant expressing Pfd1-GFP, from the non-transformed mutant and from an isogenic wild-type strain. The results shown represent the mean GLAM ratios and the standard deviations of three biological replicates. B. Analysis of cells expressing the indicated GFP fusions by confocal microscopy. The results reveal nucleo-cytoplasmic distribution for these proteins. Confocal microscopy was performed as described in the Nucleo-cytoplasmic localization of prefoldin. (TIF)Click here for additional data file.Figure S4A. The Pfd1-Myc fusion protein is functional. GLAM assays were performed with cells from a pfd1Δ transformant expressing Pfd1-Myc, from the non-transformed mutant and from an isogenic wild-type strain. The results shown represent the mean GLAM ratios and the standard deviations of three biological replicates. B. and C. Binding of Myc fusions of the indicated prefoldin subunits along GAL1 under repressed (YPD) and activating (YPGAL) conditions. Cells exponentially growing in glucose- (YPD) or in galactose-containing medium (YPGAL) were analyzed by anti-Myc ChIP, as described in the GAL1 positions. The results shown represent the means and the standard deviations of three biological replicates.Prefoldin binds chromatin. (TIF)Click here for additional data file.Figure S5A.ctk1Δ diminishes Ser2 phosphorylation of Rpb1 CTD. Occupancy of Ser2-phosphorylated RNA polymerase II was analyzed by ChIP using the same wild-type and ctk1Δ extracts of the Pfd1-Myc experiments shown in B. Co-immunoprecipitation experiments do not support a direct interaction of Pfd1 to Ser2-phosphorylated RNA polymerase II. 6 mg of concentrated yeast nuclear extract from Pfd1-TAP cells were incubated either with IgG-Sepharose or with Protein-A sepharose (negative control) during 4 h at 4°C on rotation. After washing extensively, half of the IP material was loaded in a 12% polyacrylamide SDS gel and analyzed for the presence of Pfd1-TAP by Western blot, and the other half was loaded in a 7% polyacrylamide SDS gel and analyzed for the presence of Ser2-phosphorylated Rpb1. In both cases 1% of the input material and 1% of the flow-through material were also analyzed. C. The Pfd1-TAP fusion protein used in the co-immunoprecipitation experiments shown above is functional. GLAM assays were performed with cells from a pfd1Δ transformant expressing Pfd1-TAP from the non-transformed mutant and from an isogenic wild-type strain. The results shown represent the mean GLAM ratios and the standard deviations of three biological replicates.Recruitment of prefoldin to transcribed genes. (TIF)Click here for additional data file.Figure S6A. Time courses reflecting the induction of the GAL1 gene by the addition of galactose to cells grown in glycerol-lactate medium. Cells of the indicated isogenic strains were grown in glycerol-lactate medium for two generations and then 2% galactose was added to the cultures. RNA samples were then taken at the indicated times and used for Northern experiments. Mean and standard deviation of the quantitated Northern signal of three biological replicates is shown. B. RNA polymerase II distribution across GAL1p-YLR454w. Occupancy of RNA polymerase II was measured by anti-Rpb3 ChIP in the indicated isogenic strains. Points represents the amplicons described in C. Distribution of transcriptionally active RNA polymerase II across GAL1p-YLR454w, as measured by run-on. The same data of D. TFIIS occupancy of GAL1p-YLR454w does not increase in the absence of prefoldin, as shown by Rpb3 and TFIIS-HA ChIP experiments. Wild-type and pfd1Δ cells expressing the HA-tagged version of TFIIS in its N-terminus and growing exponentially in YPGAL were processed for ChIP. The same extract was used to perform Anti-Rpb3 and anti-HA ChIP, in order to calculate TFIIS/Rpb3 ratios for each amplicon. Means and standard deviations of three biological replicates are shown. E. Northern blots of GAL1p-YLR454w in the indicated isogenic strains, showing the absence of the 5.3 kb cryptic transcript that these genes expressed in other genetic backgrounds F. The elongation rate of RNA polymerase II in GAL1p-YLR454w is affected by the absence of Pfd1. RNA polymerase II distribution is shown, 4 min after adding 2% glucose to cells of the indicated isogenic strains that were exponentially growing in galactose medium. RNA polymerase II levels were measured by anti-Rpb3 ChIP. Values were normalized to time 0 and correspond to the mean and the standard deviation of three biological replicates.Transcriptional function of prefoldin. (TIF)Click here for additional data file.Figure S7A. The absence of Pfd1 impairs the characteristic difference in histone occupancy along the transcribed region that occurs between active and inactive genes. The levels of H2B bound to transcribed (YPGAL) and untranscribed (YPD) GAL1p-YLR454w were measured by ChIP in wild-type (left) and pfd1Δ (right) exponentially growing cells. B.pfd1Δ also impairs transcriptional histone dynamics in GAL1. H3 and H2B binding to GAL1 were measured in the same experiment described in A.C. and D. The chromatin of the GAL1p-YLR454w transcribed region is enriched in trimethylated H3K36, although most H3 histones accumulated in transcribed GAL1p-YLR454w in pfd1Δ are not methylated in H3K36. The levels of H3K36Me3 bound to transcribed GAL1p-YLR454w was measured by ChIP in wild-type and pfd1Δ cells exponentially growing in YPGAL (C). Levels of total H3 bound to the same gene were also measured in the same extracts and used to calculate H3K36Me3/H3 ratios. Means and standard deviation of three biological replicates are shown. E. Overall H3K36 trimethylation does not change in the absence of Pfd1. Whole cell extracts of wild-type and pfd1Δ were used to analyze the cellular levels H3K36Me3 and total H3, as described in the F. Sensitivity of GAL1 chromatin to micrococcal nuclease, under activating (YPGAL) and non-activating (YPD) conditions is not affected by the absence of Pfd1. The experiments were performed as described in G. Transcriptional eviction of H2B in GALp-YLR454w, expressed by the ratio between the ChIP signals of the cells grown in YPGAL and YPD, in the wild type and in the indicated isogenic mutants.Prefoldin contribution to chromatin dynamics. (TIF)Click here for additional data file.Figure S8A model describing the potential role of prefoldin in histone dynamics during transcription elongation. Green ovals depict active RNA polymerase II; the pink one indicates backtracked RNA polymerase II. The other figures represent histones.(TIF)Click here for additional data file.Table S1Saccharomyces cerevisiae deletion mutants. calculated as described in GLAM ratios of the (XLS)Click here for additional data file.Table S2Strains and plasmids utilized in this work.(DOC)Click here for additional data file.Table S3DNA oligonucleotides utilized in this work.(DOC)Click here for additional data file.


Mus musculus), in which rDNA is similar in structure but highly divergent in sequence compared with human rDNA. Comparison of rDNA histone marks between mouse embryonic stem cells (mESCs) and more differentiated mouse cell types revealed differences between pluripotent and differentiated states. We also observed substantial divergence in rDNA histone modification patterns between mESCs and human embryonic stem cells (hESCs). Surprisingly, we found that the pluripotency factor OCT4 was bound to rDNA in similar patterns in mESCs and hESCs. Extending this analysis, we found that an additional 17 pluripotency-associated factors were bound to rDNA in mESCs, suggesting novel modes of rDNA regulation in pluripotent cells. Taken together, our results provide a detailed view of rDNA chromatin structure in an important model system and enable high-resolution comparison of rDNA regulation between mouse and human.The transcription of rRNA is critical to all living cells and is tightly controlled at the level of chromatin structure. Although the widespread adoption of genomic technologies including chromatin immunoprecipitation with massively parallel short-read sequencing (ChIP-seq) has allowed for the interrogation of chromatin structure on a genome-wide scale, until recently rDNA has not been analyzed by this technique. We extended genomic analysis of rDNA to mouse ( Its transcription accounts for more than half of all RNA synthesis in a growing cell  and low levels of CpG methylation. The remainder of the repeats adopt a heterochromatic structure containing histone modifications associated with transcriptional repression  and hypermethylation of CpG dinucleotides   added to chromosome 12 (MM8_plus_rDNA)  . The following antibodies were used for ChIP: rabbit anti-H3K4me1  and goat anti-OCT4 . Primers used for ChIP-PCR are detailed in Table S2.The mESCs and mNPCs were cultured as previously described (GAPDH was used for endogenous control qRT-PCR reactions. Primers used for qRT-PCR are detailed in Table S3.RNA was extracted from mESCs and mNPCs using TRIzol (Invitrogen), and cDNA was prepared using the High-Capacity cDNA Archive Kit (ABI). Triplicate PCR reactions using Sybr Green (ABI) on an ABI 7300 real-time thermal cycler. To analyze mouse rDNA by ChIP-seq, we constructed a build of the mouse genome containing a single copy of the mouse rDNA repeat. The mouse rDNA repeat was added to the proximal end of chromosome 12, on which rDNA is located endogenously, to allow comparison of rDNA signals to those on nucleoplasmic chromatin. This genome build was designated “MM8_plus_rDNA.” We discarded duplicate reads and reads aligning to multiple locations in the genome to reduce false-positives. After alignment, experimental data were normalized to input genomic DNA to correct for systematic biases.We also analyzed the mappability of mouse rDNA because there are many rDNA pseudogenes throughout the genome that might complicate analysis . MappabiWe first examined the distribution of seven histone modifications on rDNA . Histone modification signals were ∼3.5–13.6 times as intense as those seen along chromosome 12 . We therefore expanded the scale of our RNA-seq data to determine the presence of pRNA and any other low-abundance RNA species originating from rDNA. We observed RNA-seq signal at ∼43 kb and ∼45 kb into the repeat, indicating the presence of pRNA corresponding to the spacer and core promoter regions  , and rRN regions . Severalvs. mNPC: 2R = 0.04, P = 2.08 × 10−5; mESC vs. MEF: 2R = 0.005, P = 0.271). Visually, H3K4me2 also seemed to be lost at this region, but the major site of enrichment, at the spacer promoter, was unaffected, and the locus-wide correlation remained robust and highly significant . H3K4me3 also appeared to decrease at the spacer promoter region, but its locus-wide distribution was not affected . The locus-wide distributions of H3K9me3 and H3K27me3 were largely correlated between cell types, with the exception of MEF H3K27me3 . The distributions of H3K36me3 were not correlated between mESCs, mNPCs, and mESCs . Normalized tag densities for mNPC and MEF histone marks ranged from ∼2.5 to 9.2  in mNPCs vs. mESCs . Notably, we also found additional experimental evidence of nucleolar localization for nearly all of the tested factors , but closer inspection revealed that this region showed an irregular, jagged distribution of tags rather than the smooth gradation of tags generally seen with legitimate peak; therefore, we speculate that this is an artifact. We also analyzed ChIP-seq datasets for the CDX2, TBX3, SOX17, and ZC3H11A transcription factors . Given that this peak is precisely overlapping between each of the negative control datasets and does not overlap with binding sites for pluripotency factors, we speculate that it is an artifact. From these analyses, we are confident that the association of pluripotency factors with rDNA is legitimate.To ensure that the observed binding of pluripotency factors to rDNA was not simply attributable to the highly accessible chromatin architecture and high number of active rDNA repeats, we first analyzed ChIP-seq data for nuclear GFP (nGFP)  . We were2R = 0.39−0.58, P < 2.2 × 10−16 for all), further suggesting that Polycomb silencing is involved in the maintenance of transcriptional silencing at rDNA and indicating that the results of the H3K27me3 ChIP-seq analysis are legitimate. The normalized tag densities for Polycomb proteins were ∼6.0–14.6 , the clusters of factors we observed at rDNA (“rDNA MTLs”) were remarkably similar to those in the nucleoplasm; for instance, OCT4 and SOX2 were strongly correlated, as were C-MYC, N-MYC, and ZFX. This suggests that pluripotency factors function to regulate transcription in similar combinations in both the nucleoplasm and nucleolus.The association of pluripotency factors, including OCT4 and SOX2, with rDNA is particularly interesting. Factors such as MYOD and RUNX2, important for myogenic and osteogenic differentiation, respectively, also associate with rDNA and downregulate rRNA expression during differentiation. Therefore, it has been suggested that a general property of lineage-specific transcription factors is regulation of rRNA transcription as a means to coordinate cell growth and phenotype   (It has been appreciated for some time that the cell uses common epigenetic mechanisms to regulate both Pol I and Pol II transcription (odeling) . However CHD4/7) ; however CHD4/7) . Thus, i CHD4/7) . Thus, t


Drosophila melanogaster protein Corto's chromodomain. The Enhancer of Trithorax and Polycomb Corto is involved in both silencing and activation of gene expression. Over-expression of the Corto chromodomain (CortoCD) in transgenic flies shows that it is a chromatin-targeting module, critical for Corto function. Unexpectedly, mass spectrometry analysis reveals that polypeptides pulled down by CortoCD from nuclear extracts correspond to ribosomal proteins. Furthermore, real-time interaction analyses demonstrate that CortoCD binds with high affinity RPL12 tri-methylated on lysine 3. Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes in open chromatin. RNA–seq based transcriptomes of wing imaginal discs over-expressing either CortoCD or RPL12 reveal that both factors deregulate large sets of common genes, which are enriched in heat-response and ribosomal protein genes, suggesting that they could be implicated in dynamic coordination of ribosome biogenesis. Chromatin immunoprecipitation experiments show that Corto and RPL12 bind hsp70 and are similarly recruited on gene body after heat shock. Hence, Corto and RPL12 could be involved together in regulation of gene transcription. We discuss whether pseudo-ribosomal complexes composed of various ribosomal proteins might participate in regulation of gene expression in connection with chromatin regulators.Chromodomains are found in many regulators of chromatin structure, and most of them recognize methylated lysines on histones. Here, we investigate the role of the  Drosophila melanogaster chromatin factor Corto, we found that it interacts with methylated ribosomal protein L12 rather than with methylated histones. This is the first time that such an interaction is shown. Moreover, Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes. Our results represent a major breakthrough in the understanding of mechanisms by which ribosomal proteins achieve extra-ribosomal functions such as transcriptional regulation. Genome-wide analysis of larval tissue transcripts reveals that Corto and RPL12 deregulate large sets of common genes, which are enriched in ribosomal protein genes, suggesting that both proteins are implicated in dynamic coordination of ribosome biogenesis.Chromatin, the combination of DNA and histones, strongly impacts transcriptional regulation of genes. This is achieved thanks to various protein complexes that bind chromatin and remodel its structure. These complexes bind specific motifs, also called epigenetic marks, through specific protein domains. Among these domains, chromodomains are well known to bind methylated histones. Investigating the chromodomain of the  Indeed, post-translational histone modifications  and Heterochromatin Protein 1 (HP1) D. melanogaster corto gene encodes an Enhancer of Trithorax and Polycomb (ETP), i.e. a Polycomb (PcG) and Trithorax (TrxG) complex co-factor, involved in both silencing and activation of gene expression hsp70 occupancy by chromatin immunoprecipitation suggests that Corto and RPL12 cooperate in transcriptional regulation. Interestingly, the potential common targets of Corto and RPL12 are enriched in genes involved in heat response and ribosomal biogenesis.The in vivo, we used germline transformation and the binary UAS/Gal4 system to produce transgenic flies. These lines expressed either FLAG and HA double-tagged cortoCD fused to a nuclear localization signal coding sequence to force its entry into nuclei (FH-cortoCD), FLAG and HA double-tagged corto deleted of the chromodomain sequence (FH-cortoΔCD), or corto full-length . Whereas transgenic flies ubiquitously over-expressing cortoΔCD [using either Actin5C  or daughterless  drivers] were perfectly viable and had no visible phenotype, over-expression of corto using the same drivers was 100% lethal. Over-expression of cortoCD using again these drivers also induced high lethality at all developmental stages   To address the role of the Corto chromodomain m::Gal4) . Males ocortoCD in salivary glands  with anti-FLAG antibodies. FH-CortoCD bound polytene chromosomes at many discrete sites  of Corto chromodomain on chromatin. We incubated GST-CortoCD covalently bound on agarose beads with nuclear or cytoplasmic extracts from embryos and resolved retained polypeptides by SDS-PAGE. Four bands between 30 and 15 kDa  were consistently retained by GST-CortoCD and were enriched in peptide pull-down experiments performed with nuclear extracts extracts . The conextracts . Althougor RPS14 . Howeveror RPS14 . In a sior RPS14 . Howeveror RPS14 . These eD. melanogaster RPL12 was aligned with RPL12 from several other species to identify conserved residues described to be methylated in some of them Drosophila RPL12 cDNA, thus generating a series of mutants . These mutant cDNAs were introduced into a plasmid allowing their expression as mRFP-tagged proteins in Drosophila S2 cells. Similarly, the cortoCD cDNA, supplied with a nuclear localization signal, was introduced into a plasmid allowing its expression as an EGFP-tagged protein in S2 cells. When expressed in these cells, EGFP-CortoCD artificially entered the nucleus where it exhibited a punctuated pattern that recalled Polycomb bodies .To test whether Corto directly interacted with RPL12 lysine 3, we measured real-time binding between CortoCD and several RPL12 peptides using Biacore. GST-CortoCD and GST were immobilized on a CM5 sensor chip. Then, several RPL12 peptides  were assayed for their binding to GST-CortoCD or GST . None ofD>200 µM) (D = 0.4 µM). Surprisingly, the H3K27me3 peptide bound HP1CD with a similar affinity (KD = 0.7 µM), probably because sequences adjacent to the chromodomain (i.e. the hinge region) are required for selective targeting To investigate whether RPL12K3me3 could bind to other chromodomains, we repeated these experiments using that of HP1 (HP1CD). GST-HP1CD was immobilized on the sensor chip and binding of either RPL12, RPL12K3me3, RPL12K10me3 or RPL12K3A was tested. None of these peptides specifically interacted with HP1CD (K>200 µM) . Althoug>200 µM)  while, aAltogether these data demonstrate that the Corto chromodomain specifically recognizes RPL12 trimethylated on lysine 3 (RPL12K3me3).Drosophila larval salivary glands where it specifically associates with sites of transcription UAS::RpL12-Myc). Unlike corto or cortoCD, RpL12-Myc over-expression using ubiquitous Gal4 drivers  induced no lethality and adult flies presented no visible phenotype except a shortened development (data not shown). RpL12-Myc was then expressed in salivary glands with the esg driver  to test its binding to polytene chromosomes. RPL12-Myc bound polytene chromosomes at numerous sites, preferentially at DAPI interbands and puffs, suggesting that it mimics the binding of endogenous RPL12 esg::Gal4>UAS::FH-cortoCD,UAS::RpL12-Myc) established that CortoCD co-localized with RPL12 on a similar number of sites   and RNA Polymerase II  . In agreolIIS2p) . SimilarolIIS2p) . Corto, olIIS2p) . HoweverPolIIS2p , RPL12 sPolIIS2p , as prevTaken together, these data suggest that Corto and RPL12 mostly bind open, transcriptionally permissive chromatin.FH-cortoCD or RpL12-Myc under control of the wing-specific scalloped::Gal4 driver  . Total RNA from the assays, the sd::Gal4/+ control or a 1118w reference line were isolated from pools of wing imaginal discs and subjected to RNA-seq on an Illumina high throughput sequencer. Sequence reads were aligned with the D. melanogaster genome to generate global gene expression profiles. Sequence reads of the assays were compared to sequence reads of the sd::Gal4/+ control. Differential analyses were performed to obtain adjusted P-values associated to expression changes for the assays compared to the sd::Gal4/+ control. In addition, sequence reads from the 1118w reference line were compared to sequence reads of the sd::Gal4/+ control. This reference was used to fix the threshold of the adjusted P-value to get only 1% of transcripts as differentially expressed in this control experiment . By doing so, we obtained an adjusted P-value cutoff of 4.10−18. Using this threshold, we retrieved the highest expression variations from the two assays [with absolute log2(assay/control)>1]. 463 genes were upregulated when over-expressing cortoCD  (hsp70 that was one of the shared upregulated genes (hsp70The high correlation between genes deregulated when over-expressing either = 0.634)  as well ed genes . We analed genes . qPCR an1118w discs, higher RNAPolII occupancy of the promoter as compared to the gene body suggests that hsp70 was paused, corroborating previous results cortoCD or RpL12 , CortoCD and RPL12 bound hsp70 indicating that this gene was a direct target of both proteins. Simultaneously, RNAPolII binding was increased but kept the same profile suggesting that the gene was still paused but more loaded with RNAPolII. This could explain why more transcripts were generated. Thus, these data suggest that Corto, as well as RPL12, favors recruitment of RNAPolII on hsp70 in absence of heat shock.At 25°C, in control hsp70. Interestingly, CortoCD and RPL12 displayed the same binding profile i.e. increased binding from 5′ to 3′ of the gene body. CortoCD and RPL12 recruitment followed hsp70 transcription as revealed by enhancement of RNAPolII on gene body. Strikingly, recruitment of RNAPolII was higher in wing discs expressing cortoCD or RpL12 than in control wing discs, suggesting that Corto and RPL12 control transcriptional activation of hsp70.After a short heat shock (5 minutes), CortoCD, as well as RPL12, were massively recruited on cortoCD or RpL12 were over-expressed reveal that a large fraction of deregulated genes are common. Chromatin immunoprecipitation experiments reveal that CortoCD and RPL12 similarly bind one of these shared upregulated genes, hsp70, and are massively loaded on the promoter and gene body after heat shock. Hence, the ETP Corto and RPL12 might indeed be partners in regulation of some transcriptional targets.Chromodomains play a critical role in addressing transcriptional regulators to chromatin. Investigating the role of the ETP Corto's chromodomain, we found that it is a typical chromodomain, acting as a chromatin-targeting module. Surprisingly, the Corto chromodomain does not bind methylated histones, as most known chromodomains do, but Ribosomal Protein L12 trimethylated on lysine 3 (RPL12K3me3). In agreement, RPL12 and Corto share many sites on polytene chromosomes. Transcriptomic analyses of wing imaginal tissues in which either corto mutants show that Corto transcriptional targets are numerous and involved in many developmental pathways. The interaction reported here between Corto and RPL12 raises the interesting possibility of a connection between RPs and epigenetic regulation of gene expression. Our previous investigations into Corto partners have highlighted its interaction with several PcG proteins, leading to the conclusion that Corto might regulate PRC1 and PRC2 functions per se.The ETP Corto is a partner of Polycomb and Trithorax complexes and participates in epigenetic maintenance of gene expression, notably of homeotic genes E. coli where RPS10 is involved in anti-termination of transcription Apart from protein synthesis, RPs are involved in many cellular functions referred to as “extra-ribosomal” (reviewed in Polycomb (PcG) and trithorax (trxG) genes in flies have identified Minute mutants as PcG and trxG modifiers Minute mutations suppress the ectopic sex comb phenotype of Polycomb or polyhomeotic mutants D. melanogaster Minute loci are disseminated throughout the genome and many correspond to RP genes ; Actin5C ], expressed in salivary glands [escargot ], or wing-specific [scalloped ]. Five females bearing the Gal4 driver were crossed with three males bearing the UAS transgene or 1118w as a control. Crosses were transferred to new vials every third day. The sd::Gal4, UAS::FH-cortoCD and UAS::RpL12-Myc lines were isogenized for six rounds with the isogenic 1118w line, prior to deep-sequencing, as described 2, 0.05% Igepal, 20 mM Hepes, 300 mM KCl, 10% glycerol, protease inhibitor cocktail (Roche)] for 1 h at 25°C. After 5 washes in binding buffer, bound polypeptides were resolved on a large 15% SDS–polyacrylamide gel and stained either with EZblue (Sigma) or with SilverQuest staining kit (Invitrogen). Bands were excised from the gel and were analyzed by LC-MS/MS mass spectrometry.Cytoplasmic and nuclear extracts were prepared from 0–16 h embryos as described in Drosophila medium (Lonza) supplemented with 10% heat inactivated fetal bovine serum and 100 units.mL−1 of penicillin and streptomycin. Cells were transfected using Effecten (Qiagen) as described S2 cells were cultured at 25°C in Schneider's Co-immunoprecipitations were performed as described S2 cells were harvested 24 h after transfection and treated as described via its N-terminal amino acid. The carboxymethylated dextran surface was activated by injecting a mixture of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) and 0.05 M N-hydroxysuccinimide. GST was immobilized on the chip by injecting a 30 µg.mL−1 solution in NaAc pH5 buffer. GST-CortoCD and GST-HP1CD were immobilized by injecting a 100 µg.mL−1 solution in the same buffer. Binding tests were performed by injecting peptides at 1 or 10 µM in running buffer at a flow rate of 5 µL.min−1 during 5 min. Kinetic assays were performed only when the binding test was positive. Real-time monitoring was displayed in a sensorgram as the optical response (RU) versus time (s). To calculate association constants, peptides were diluted in series from 1 to 10 µM in running buffer and dilutions were injected sequentially at a flow rate of 5 µL.mn−1 during 5 min. Dissociation kinetics were then run during 10 min to calculate dissociation constants. Between assays, the chip was regenerated with 10 mM glycine pH2.0. Kinetic constants were calculated with BIAevaluation Software (Biacore) using the Fit kinetic simultaneous ka/kd . RPL12 peptides were synthesized at the IFR83 Peptide synthesis facility  in running buffer  . Real-time protein interaction assays were performed using a Biacore 3000. Kinetics and binding tests were first performed on empty surfaces. Data presented here result from substraction of empty surface RU (1 to 5 depending on the experiment) from active surface RU. GST was covalently coupled to a CM5 sensor chip  facility . HistonePolytene chromosome immunostainings were performed as described −1 . Total RNA were extracted using RNeasy kit (Qiagen). Library preparation and Illumina sequencing (multiplexed 50 bp paired-end sequencing on HiSeq 2000) were performed at the BC Cancer Agency Genome Sciences Center (Canada). Messenger (polyA+) RNAs were purified from 4 µg of total RNA with oligo(dT). Libraries were prepared using the bi-directional RNA-Seq library preparation kit (Illumina). A mean of 46±11 million reads was obtained for each of the 4 samples . Detailed informations on Paired-End read counts at each step of the analysis workflow are available in D. melanogaster genome  using Bowtie mapper (version 0.12.7) D. melanogaster GFF3 genome annotation from FlyBase (version 5.38) was used. All overlapping regions between alignments and referenced exons were counted.Wing imaginal discs of third instar female larvae (one disc per larva) were dissected by batches of 50 in ice-cold PBS and frozen in liquid nitrogen. 300 discs (6 batches) were pooled and homogenized in lysis buffer using a FastPrep-24 during 20 s at 4 m.sP-value adjustment was made. The RNA-Seq gene expression data and raw fastq files are available at the GEO repository (www.ncbi.nlm.nih.gov/geo/) under accession number: GSE38435.Technical replicates coming from paired-end reads were first summed. Then, all samples were normalized together. Data were normalized according to the scaling normalization proposed by Robinson and Oshlack and implemented in the edgeR package version 1.6.10 Wing imaginal discs of third instar larvae were dissected by batches of 100 in serum-free Schneider medium at room temperature. They were fixed in 500 µL of paraformaldehyde 1% in PBS for 10 minutes at room temperature under gentle agitation. Cross-link reaction was stopped by adding 50 µL of glycine 1.25 M. Fixed wing discs were washed 3 times with PBS, dried, flash-freezed in liquid nitrogen and stored at −80°C. Cell lysis was performed by adding 100 µL of lysis buffer  complemented with 1% SDS, and sonicated in a Bioruptor sonifier (Diagenode). Conditions were established to obtain chromatin fragments from 200 to 1000 bp in length . Pooled chromatin was centrifuged for 10 min at 13000 g at 4°C. The supernatant (soluble chromatin) was recovered and 5 µL were kept as input sample. For each IP, 10 µl of 50% (v/v) protein A or G coated paramagnetic beads (Diagenode) were washed once in lysis buffer, 1 µg of antibody was added, and beads were incubated for 2 h at 4°C on a rotating wheel. After washing, antibody coated beads were resuspended in 450 µL of lysis buffer and 50 µl of chromatin were added. After incubation on a rotating wheel overnight at 4°C, beads were washed at 4°C five times for 10 min each in lysis buffer, once in LiCl buffer  and twice in TE . Immunoprecipitated as well as input DNAs were purified with the IPure kit following the manufacturer's instructions (Diagenode). Elution was performed twice with 35 µl of water. 2 µl of DNA were used per PCR. Real-time PCR data were normalized against Input sample and depicted as percentage of Input , anti-HA tag  and anti-Myc tag . Mouse IgGs were used as a negative control .Heat shock treatments were performed as previously described Figure S1CortoCD does not co-immunoprecipitate with nuclear ribosomal proteins RPL7, RPS10 and RPS14. (A) Transfection of FLAG-CortoCD and RPL7-Myc in S2 cells. (B) Transfection of FLAG-CortoCD and RPS10-Myc in S2 cells. (C) Transfection of FLAG-CortoCD and RPS14-Myc in S2 cells. Immunoprecipitations were performed with anti-Myc and revealed by Western blot with either anti-Myc (α-Myc) or anti-FLAG (α-FLAG). Spnt: supernatant, IP: immunoprecipitation.(TIF)Click here for additional data file.Figure S2Corto overlaps with transcriptional factories. (A) Immunostaining of S2 cells with anti-Corto (green) and anti-PH (red) antibodies showing that Corto bodies and Polycomb bodies did not overlap. Blue: DAPI. Close-up of a nucleus. (B) Immunostaining of S2 cells with anti-Corto (green) and anti-RNAPolII (red) antibodies showing that Corto bodies and transcriptional factories overlapped. Blue: DAPI. Close-up of a nucleus.(TIF)Click here for additional data file.Figure S3Real-time interaction binding assays. (A) Biacore sensorgram showing binding of H3K9me3 peptide (1 µg.mL-1) to GST-HP1CD, GST-CortoCD or GST. Binding  is expressed in Resonance Unit (RU). Note that H3K9me3 bound GST-HP1CD as expected but did not bind GST-CortoCD or GST. (B) Biacore sensorgram showing binding of RpL12K3me3 or RpL12K3A peptides (10 µg.mL-1) to GST-HP1CD, GST-CortoCD or GST. Binding  is expressed in resonance unit (RU).(TIF)Click here for additional data file.Figure S4hsp70 genes are upregulated when either RpL12-Myc or FH-CortoCD are expressed in wing imaginal discs. (A) Read count data and log2 fold change (FC) analysis relative to sd::Gal4/+ for each hsp70 transcript. (B) IGV (Integrative Genomics Viewer) screenshot showing read alignments along the hsp70Ab locus  in control 1118w, sd::Gal4>UAS-RpL12-Myc and sd::Gal4>UAS-FH-cortoCD wing imaginal discs. Sense reads appear in pink and reverse reads in blue.(TIF)Click here for additional data file.Table S1cortoCD using ubiquitous Gal4 drivers. Three different insertions of the cortoCD transgene  and one full-length corto transgene were analysed. nd: not determined. *: difference with driver alone highly significant . **: difference with driver alone significant ; ***: difference with driver alone highly significant .Phenotypes of flies overexpressing (PDF)Click here for additional data file.Table S2Mass spectrometry analysis of peptides pulled down by CortoCD. Four bands  were excised from the gel see  and anal(PDF)Click here for additional data file.Table S3sd::Gal4>UAS::FH-cortoCD vs sd::Gal4/+. FC: Fold Change.Genes up-regulated in (PDF)Click here for additional data file.Table S4sd::Gal4>UAS::RpL12-Myc vs sd::Gal4/+. FC: Fold Change.Genes up-regulated in (PDF)Click here for additional data file.Table S5sd::Gal4>UAS::FH-cortoCD vs sd::Gal4/+. FC: Fold Change.Genes down-regulated in (PDF)Click here for additional data file.Table S6sd::Gal4>UAS::RpL12-Myc vs sd::Gal4/+. FC: Fold Change.Genes down-regulated in (PDF)Click here for additional data file.Table S7vs sd::Gal4/+ and sd::Gal4>UAS::RpL12-Myc vs sd::Gal4/+.Genes de-regulated in sd::Gal4>UAS::FH-cortoCD (PDF)Click here for additional data file.Table S8sd::Gal4>UAS::FH-cortoCD vs sd::Gal4/+.Ontology of genes up-regulated in (PDF)Click here for additional data file.Table S9sd::Gal4>UAS::FH-cortoCD vs sd::Gal4/+.Ontology of genes down-regulated in (PDF)Click here for additional data file.Table S10sd::Gal4>UAS:RpL12-Myc vs sd::Gal4/+.Ontology of genes up-regulated in (PDF)Click here for additional data file.Table S11sd::Gal4>UAS::RpL12-Myc vs sd::Gal4/+.Ontology of genes down-regulated in (PDF)Click here for additional data file.Table S12cortoCD, respectively. For RpL12 mutants, the mutated triplet is underlined.Primers used for constructs and qPCRs. Note that an ATG and two nuclear localization signals (underlined) were added to the forward and reverse primers of (PDF)Click here for additional data file.Table S13Sequences of the RpL12 peptides used in this study.(PDF)Click here for additional data file.Table S14Read count data for sequencing experiments. For each edge of Paired-End sequenced samples (PE1 and PE2), the number of raw reads (raw), reads passing quality control filters (QC filter), uniq alignments  and the number of alignment used to estimate transcript abundance (transcript) are given.(PDF)Click here for additional data file.Text S1Supporting methods.(DOC)Click here for additional data file.


FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections.Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human  Foxp2 codes for an intriguing regulatory protein that provides a window into unusual aspects of brain function in multiple species. For example, the gene is implicated in speech and language disorders in humans, song learning in songbirds, and learning of rapid movement sequences in mice. Foxp2 acts by tuning the expression levels of other genes (its downstream targets). In this study we used genome-wide techniques to comprehensively identify the major targets of Foxp2 in the embryonic brain, in order to understand its roles in fundamental biological pathways during neurodevelopment, which we followed up through functional analyses of neurons. Most notably, we found that Foxp2 directly and indirectly regulates networks of genes that alter the length and branching of neuronal projections, an important route for modulating the wiring of neural connections in the developing brain. Overall, our findings shed light on how Foxp2 directs particular features of nervous system development, helping us to build bridges between genes and complex aspects of brain function. Forkhead-box protein P2 is a highly conserved vertebrate protein, belonging to an important group of transcription factors FOXP2 gene causes a rare neurodevelopmental disorder, characterised by difficulties mastering sequences of mouth movements during speech, as well as impaired language processing Foxp2) yield dramatic reductions in synaptic plasticity of cortico-striatal brain circuits, associated with deficits in learning of rapid motor skills Foxp2 mutations show more severe neural effects – gross motor impairments, delayed postnatal maturation of the cerebellum and dramatic reductions in emission of ultrasonic vocalisations – against a background of reduced weight-gain and postnatal lethality FoxP2) is required for normal vocal learning in songbirds Functional evidence from multiple species implicates Forkhead-box protein P2 in particularly intriguing aspects of brain development and function FOXP2 and mouse Foxp2 identified similarly strong CNS  expression during embryogenesis, which is confined to neurons  and enriched in various brain structures, including deep layers of the developing cortical plate, and parts of the striatum, thalamus and cerebellum FOXP2 mutations Studies of both human The above observations of well-conserved and specific CNS expression patterns in vivo ChIP-chip screen of the embryonic mouse brain, coupling Foxp2-ChIP to high-density arrays with oligonucleotides tiled across >17,000 promoters. We robustly established the empirical significance of our ChIP results in wild-type brains by determining the null distribution of signals generated by matched control tissue from littermates that expressed no Foxp2 protein. Under strict empirical thresholds that minimised false positive signals, we isolated a set of 264 high-confidence in vivo targets. Gene ontology (GO) analyses of the ChIP-chip data, as well as genome-wide expression profiling and in situ hybridisations of wild-type and mutant mice, converged on neurite outgrowth as one of the most prominent biological themes associated with Foxp2 function in the embryonic CNS. We went on to directly demonstrate, using neuronal cell models and primary neurons from the embryonic mouse brain, that Foxp2 alters expression of neurite-outgrowth targets and thereby influences neurite process length and branch number.In the present study, we performed a systematic large-scale In vivo Foxp2-ChIP screening was carried out using brains harvested from embryonic mice. Experiments were performed with mice that were wild-type for Foxp2, as well as homozygous littermates that do not express any Foxp2 protein  excluded 99% of the data-points in the mutant null control experiments. When we applied this threshold to data from wild-type experiments, we identified a set of 1,217 promoter regions that were consistently enriched by Foxp2-ChIP over 3 replicates in wild-type mouse brains . On inspPigt gene), indicating an extremely low rate of false positives under these stricter selection criteria. We excluded two genes from the strict wild-type shortlist (Pigt and Zfp496) since they contained probes that exceeded the 99% threshold (i.e. window score of >0.974) in mutant null controls  and with progressively less enrichment observed as the distance to the binding site increases on either side. in silico for any over-represented sequence motifs  were found to be significantly over-represented (p<0.05) in the shortlist of high confidence target promoter sequences , we assessed regulatory cascades further downstream (indirect targets) via an expression profiling approach. Again we focused on E16 mouse brain tissue, analysing the same genotypes  on the same genomic background as used for the ChIP experiments. While ChIP identifies DNA-binding events of Foxp2-positive cells, expression profiling is expected to be more sensitive to tissue heterogeneity. Therefore we selected a key site of high Foxp2 expression with considerable prior evidence of functional relevance Foxp2-S321X homozygous mutant samples , Nrn1 (neuritin), Cck (cholecystokinin), and Alcam (activated leukocyte cell adhesion molecule). Notably, the human orthologues of Nrn1 and Cck have been independently proposed as top direct targets in small-scale ChIP screens of human foetal tissue In addition to the use of  samples . 180 of  samples . Of thes samples , includiWe went on to determine whether any biological themes were over-represented within the direct targets (promoter bound by Foxp2) and indirect targets , using unbiased GO analyses. The Foxp2-ChIP and expression profiling datasets were each assessed independently using the WebGestalt program in situ hybridisation on brains from wild-type and Foxp2-S321X E16 embryos, to further assess major targets suggested by the ChIP and expression profiling screens. Consistent with previously published data in situ hybridisation data confirmed regulation of Shhrs , a transcript showing greater than 200-fold increased expression levels in S321X mice. This noncoding RNA is highly specific to the ganglionic eminences in the embryo and is known to play a vital role in the control of the homeodomain transcription factors Dlx5 and Dlx6 Foxp2 can influence transcripts central to key neurodevelopmental processes in vivo. We then focused on target genes common to both ChIP and expression profiling datasets, to determine whether expression changes could be observed, not only in the ganglionic eminences, but also elsewhere in the developing brain  and Wasf1 (WASP family 1), are ubiquitously expressed in the developing mouse brain Foxp2, further in situ hybridisation was carried out at E16. The results demonstrate that genes from the ChIP dataset with established CNS functions are found in a range of Foxp2-expressing brain regions . Interestingly, a number of different but interacting genes within this pathway were identified in the two datasets  in which the protein is expressed at normal levels, but is nevertheless dysfunctional R552H mice recapitulate an aetiological mutation that causes speech and language deficits in a large human family. This change yields a substitution in the DNA-binding domain which severely impairs the transcription factor function of the mutant protein R552H animals is very similar to that observed for mice which completely lack Foxp2 R552H homozygotes still express detectable levels of the protein, allowing us to clearly identify Foxp2-positive cells in our primary cultures via antibody staining. This represents an important measure, given the heterogeneous nature of the dissected material used to generate the primary culture.To further assess the Foxp2-R552H littermates  and striatopallidal (Drd2) MSNs continue to increase their dendritic area well into adulthood, Drd1a MSNs develop significantly more dendrites These findings are strongly in agreement with differences in levels of Foxp2 expression, neurite outgrowth and correlated physiological properties between the two major subpopulations of striatal medium spiny neurons (MSNs) in vivo activities of Foxp2-dependent pathways in the developing CNS.Although early studies of Foxp2 orthologues in multiple species suggested that it may play crucial roles in neurodevelopment Foxp2 gene on neurite outgrowth phenotypes in genetically manipulated neuronal cell models and primary neurons from embryos of mutant mice. Our functional experiments confirmed regulation of the highlighted gene networks and indicated that wild-type Foxp2 thus enhances multiple facets of neurite development in vivo, including outgrowth process length and branch number. The data suggest that the mode of action may be predominantly cell autonomous, since the functional effects were mainly restricted to the subset of Foxp2-expressing cells within a mixed population of neurons. This possibility of cell-autonomous effects is an interesting hypothesis that could be clarified in further studies.Of note, among the biological themes that we identified, our comprehensive ChIP-chip and expression profiling in midgestation brain tissue independently and consistently highlighted gene networks underlying neurite development and morphogenesis, axon extension and axon guidance. These findings drove us to specifically assess the impact of the Foxp2 gene of mice, and observed that striatal neurons had significantly longer dendrites and increased synaptic plasticity Our neurite outgrowth findings are in line with new evidence regarding the functional impact of evolutionary differences between FOXP2 orthologues Foxp2 and neural connectivity may be informative for wider discussions regarding the evolution of vocal learning FOXP2 has been implicated in speech abilities FoxP2 is required for normal song-learning in songbirds Furthermore, the identification of potential regulatory links between in vivo characterisation of direct and indirect Foxp2 targets in the embryonic brain. It is of interest to consider how the present findings relate to published screens that used more limited ChIP surveys To our knowledge, the current report represents the first large-scale in vitro and in vivoNrn1 is a highly robust downstream target. The Nrn1 gene encodes neuritin, which is already expressed at embryonic stages of development and was initially identified as a downstream effector of neuronal activity and neurotrophin-induced neurite outgrowth Nrn1 not only showed one of the strongest enrichment signals in our in vivo ChIP experiments, but was independently detected as a target in our systematic expression profiling experiments of equivalent tissue and by in situ hybridisation – the corresponding human homologue was also one of the top direct targets reported in a small-scale ChIP screen of human foetal brain tissue Nevertheless, many important consistencies are observed between the different datasets, particularly in the biological themes and processes that they implicate. For example neurite outgrowth pathways and synaptic plasticity are over-represented in all FoxP2 ChIP-chip datasets across different species and neuronal cell-type, Cck gene, which showed convergent evidence in our embryonic ChIP experiments, expression profiling screens and in situ hybridisation analyses, was reported as a direct target in both prior published human ChIP-chip studies Lmo4 (Lim domain only 4) was found to be indirectly downregulated by Foxp2 in our analyses of embryonic brain tissue , a well-validated direct target  – encoding a synaptic glycoprotein involved both in development/maintenance of synaptic connections Wasf1 – a gene that regulates activity-induced changes in dendritic spine morphogenesis Sema4fSema6dYwhah , which encodes an adapter protein implicated in presynaptic plasticity Evf1/2 (Shhrs) showed such highly increased expression in Foxp2-S321X mice. It has been shown that the Evf2 RNA molecule co-operates with the Dlx2 protein to activate the Dlx5/6 enhancer element DLX1/2 and DLX5/6 loci have been implicated in autism via independent studies, including a common polymorphism in the DLX5/6 enhancer itself Given the substantially enhanced scope of ChIP screening in the present study, we were able to identify many interesting novel targets that could not be isolated in the earlier work. For example, our high-confidence shortlist of direct targets includes Sema3a. The promoter of this gene was bound by Foxp2 in our ChIP study  between mutant and wild-type ganglionic eminences, only 19 (∼5%) corresponded to putative direct targets of Foxp2 from the ChIP-chip screens. Thus, most of the expression differences observed in the transcriptional profiling experiments are unlikely to represent direct modulation due to Foxp2 binding, but could instead represent cascade effects further downstream . Discrepancies between the ChIP-chip and expression profiling datasets may also result from our experimental design: the former could potentially detect binding events of Foxp2-expressing neurons anywhere in the brain, while the latter was targeted specifically at the ganglionic eminences, a region showing particularly high Foxp2 levels. Foxp2 target genes that are not expressed in this structure could therefore be observed in the ChIP study, but would not be detected in the expression analysis. An example of such a target is IP study , but itsIP study . NeverthCNTNAP2 (contactin-associated-protein-like-2) a gene implicated in language impairments and autism CNTNAP2, this was not represented on the arrays used in this study, and hence it escaped detection. When we carried out ChIP-PCR experiments using the same mouse embryonic brain tissue as used for ChIP-chip we demonstrated clear Foxp2 occupancy of the orthologous region in mouse Cntnap2. Specific enrichment was observed in the wild-type brains; while no enrichment was found in equivalent tissue from the mutant null controls  noncoding molecules which provide extra layers of dynamic control in networks of gene expression Among the validated direct targets of Foxp2 identified in our study there were a number of microRNA (miRNA) molecules, including in vivo genomic screening strategies in the developing embryonic brain has proved to be a powerful approach for understanding the biology of Foxp2, one of the most intriguing transcription factors of the CNS. This starting point led us to functional characterisation of new mechanisms of Foxp2 action, in particular the modulation of networks involved in neurite outgrowth, axonogenesis and other core aspects of neural development. Future studies will define how these regulatory networks differ between distinct species, what role miRNAs play in Foxp2-related pathways and phenotypes and will investigate whether it is possible to rescue the established neurobiological effects associated with loss of Foxp2 function, through manipulation of key targets. Ultimately, such work promises to fully uncover the functional pathways that connect Foxp2 with plasticity of the developing CNS.In conclusion, the use of In vivo Foxp2-ChIP in embryonic mouse brain tissue was performed according to the protocol previously described by Vernes and colleagues Foxp2-S321X mutants as negative controls. S321X mutants carry an early nonsense mutation that disrupts Foxp2; the resulting combination of nonsense-mediated RNA decay and protein instability leads to a complete lack of detectable Foxp2 protein in the brain in vivo ChIP lysis buffers at room temperature for ten minutes each: Buffer 1 ; Buffer 2 . After collection via centrifugation, nuclei were resuspended in 5 ml sonication buffer . Samples underwent 15 rounds of 20-second sonication pulses at 30% power, with 60 seconds on ice between each round . Agarose gel electrophoresis was used to confirm that fragment size was 300–1000 bp. Cells were centrifuged at 10,000 g and 4°C for 10 minutes to remove cell debris. 10 µg of polyclonal rabbit anti-Foxp2 antibody  E16 mouse brains were extracted, snap frozen in liquid nitrogen and stored at −80°C until use. Each whole brain was weighed, then chopped finely with a razor on ice. Brains were pooled to achieve a total weight of between 0.3 and 0.5 g of tissue (between 5–6 brains per replicate) and resuspended in 5 ml PBS. A 1/10 volume (500 µl) of cross-linking buffer was added prior to 15 minutes incubation with agitation at room temperature. Formaldehyde was quenched via the addition of 125 mM glycine. Cross-linked tissue was washed in PBS before brief mechanical homogenisation. Pellets were then incubated in two 2 µg of amplified immunoprecipitated chromatin, or total input DNA was fluorescently labelled with Cy5 and Cy3 respectively using random primers provided in the BioPrime DNA labelling system (Invitrogen). The labelling reaction was allowed to proceed for 16 hours at 37°C, before purification by sodium acetate precipitation. Hybridisation to mouse promoter arrays  was carried out by the UCLA microarray core facility, according to the manufacturer's instructions. Arrays consisted of 60-mer oligonucleotides spanning ∼8 kb (5.5 kb upstream and 2.5 kb downstream of TSS) at each of ∼17,000 mouse promoter regions. Probes were spaced on average, between 100–300 bp apart, with approximately 25 probes for each promoter region. Three littermate matched sets of pooled wild-type or mutant control chromatin samples were applied to microarrays, each using its respective input DNA sample as the internal reference on the array. Thus, the three wild-type and three mutant control datasets represent signals obtained from a total of 34 individual mouse embryos.Array images were scanned using the Axon GenePix 4000B. Data were retrieved and initial quality control carried out using the Axon GenePix 4000B software package. All promoter coordinates and probes were mapped with reference to the NCBI m36 mouse assembly. Microarray data analysis was carried out using the mArray package for R The use of mutant null controls enabled us to robustly assess the empirical significance of wild-type ChIP results. The data from the mutant control experiments were used to estimate a null distribution of window scores; that is, the non-specific signals produced by the Foxp2-ChIP protocol even when there is no Foxp2 protein available for pulldown. , and its quality was assessed using RNA6000 Nano Assays on an Agilent Bioanalyzer 2100 .The ganglionic eminences, sites of particularly high embryonic Foxp2 expression in vitro transcription was used to synthesize biotin-labelled complementary RNA (cRNA). 1500 ng of biotin-labelled cRNA was hybridized to Mouse WG-6 V2 Expression BeadChip arrays  at 55°C for 18 h. The hybridized arrays were washed and labelled with streptavidin-Cy3 according to the manufacturer's protocols before being scanned with the Illumina Bead Array Scanner. Raw data were exported from the Illumina BeadStudio software (v3.4.0) for further processing and analysis using the R statistical software Gene expression profiling was performed using whole-genome mouse BeadChip arrays from Illumina , which include 45,281 probes representing 31,492 mouse transcripts. In brief, 500 ng of total RNA was reverse transcribed to synthesize first- and second-strand complementary DNA (cDNA). Following purification on spin columns, http://www.illumina.com).Statistical analysis was performed using the ‘Linear Models for Microarray Analysis’ (Limma) package 2. Stable Neuro2a cell-lines overexpressing Foxp2 protein or non-expressing controls, were generated via transfection with pcDNA3.1/Foxp2 (mouse isoform I - untagged) or the empty vector, using Genejuice (Novagen) according to the manufacturers' instructions. Cells were cultured in complete medium supplemented with 500 µg/ml G418  as a selective agent. Resistant single colonies were isolated 20 days after transfection, then cultured and expanded independently in the presence of G418 (500 µg/ml). Expression of recombinant Foxp2 was confirmed using qRT-PCR and Western blotting with two polyclonal antibodies recognizing different epitopes of the protein  cells were cultured in ‘Growth media’: Modified Eagles Medium (MEM) (Sigma) supplemented with 10% Foetal Calf Serum (Sigma), 2 mM L-glutamine (Sigma) and 2 mM Penicillin/Streptomycin (Sigma). Cells were grown at 37°C in the presence of 5% COFoxp2 or the empty control vector following culture in growth media or differentiation media (for 24 or 48 hours). Total RNA was extracted from cells harvested in TRIzol reagent using the RNeasy kit (QIAGEN) according to manufacturers' instructions. Reverse transcription was performed as described previously RNA was extracted from three independent clones of Neuro2a cells stably transfected either with murine GAPDH/Gapdh  and U6  were designed using PrimerBank GAPDH housekeeping gene (for gene expression) or U6 small nuclear RNA (for miRNA expression). Data are expressed as mean of values from three independent clones ± standard error of the mean (SEM). Statistical significance was assessed using Students t-tests (two-tailed).PCR reactions utilised SYBR Green supermix (BioRad) as described Foxp2-R552H E16 littermates R552H mice carry a missense mutation affecting a conserved arginine residue located in the Foxp2 DNA binding domain, matching an aetiological mutation found in a well-characterised multigenerational family with speech and language disorder (the KE family) R552H homozygous mice demonstrate comparable phenotypes to homozygous Foxp2 knockouts in vitro functional studies 3, 25 mM glucose in HBSS-CMF) and dissected regions were immediately chopped on ice and pelleted at 800 RPM and 4°C for 5 minutes. The pellet was incubated in papain solution  on ice for 5 minutes then at 37°C for 10 minutes, agitating regularly. The enzymatic reaction was halted by addition of Ovo-BSA solution . Cells were pelleted at 1000 RPM for 5 minutes at 4°C and the pellet was washed then re-suspended in complete medium  supplemented by 2 mM Glutamax (Sigma). 2 mM Penicillin/Streptomycin (Sigma) and 1X B27 supplement). Suspension was triturated using plastic and glass pipettes to dissociate any remaining cell clumps before passing the cell suspension through a 70 µm cell strainer. Single cell suspensions were seeded onto laminin and poly-D-lysine coated coverslips (BD Biosciences) at a density of 6.3×104 cells per well into 24 well plates and grown at 37°C in the presence of 5% CO2 in complete medium.Ganglionic eminences from both hemispheres were dissected from wild-type and homozygous After 4 days in culture, cells were fixed using 4% Paraformaldehyde solution for 15 minutes at room temperature and permeablised in wash solution (0.1% Triton X-100 in TBS). Antibodies were diluted in Blocking Solution . Cells were co-stained at 4°C overnight, using two primary antibodies; an anti-MAP2 rabbit polyclonal antibody (Chemicon) and an anti-Foxp2 mouse monoclonal antibody recognising an epitope near the C-terminal end of the protein (Gift from Prof. A. Banham). Detection was then facilitated via four rounds of antibody incubation, which allowed magnification of the Foxp2 signal. Cells were incubated with anti-rabbit TRITC  plus anti-mouse biotinylated  secondary antibodies, followed by incubation with anti-rabbit TRITC plus anti-biotin FITC  antibodies, each for 1 hour, shaking under limited light exposure. This secondary/tertiary antibody incubation was then repeated under the same conditions. Nuclei were visualised using mounting media containing a DAPI counterstain (VectaShield). Cells were viewed on a Nikon Eclipse TE2000U fluorescence inverted microscope. Images were captured using a Hamamatsu black and white C4742-95 Orca hi-sensitivity CCD camera with IPLab imaging software , and analysed using the neurite outgrowth function of Metamorph Version 7.6 (Molecular Devices). Statistical analyses were carried out using ANCOVA  for genotype and embryo, followed by post-hoc Sidak correction. Data are expressed as the mean ± standard error of the mean (SEM).In situ hybridization was carried out on 10 µM frozen sections of E16.5 embryo heads as previously described Foxp2-S321X mutant embryos and all slides were developed for 16 hours, or 6 hours in the case of Shhrs. In all cases a sense-strand negative control riboprobe gave no specific signal (data not shown).Figure S1Western blot analysis of endogenous Foxp2 protein.(TIF)Click here for additional data file.Figure S2Chromosome distribution of enriched probes.(TIF)Click here for additional data file.Figure S3Window scores for all probes that exceeded the strict threshold (enrichment score of >1.5) in wild-type Foxp2-chip.(TIF)Click here for additional data file.Figure S4EMSA analysis of a novel Foxp2 DNA-binding motif.(TIF)Click here for additional data file.Figure S5Differential expression of Foxp2 target genes in E16 developing mouse ganglionic eminences.(TIF)Click here for additional data file.Figure S6Co-expression of Foxp2 and putative target genes in the E16 mouse brain.(TIF)Click here for additional data file.Figure S7Expression of Foxp2 in medium spiny neurons (MSNs).(TIF)Click here for additional data file.Figure S8In vivo binding of mouse Foxp2 to intron 1 of Cntnap2 in embryonic brain tissue.(TIF)Click here for additional data file.Table S1Foxp2-ChIP (window score ≥0.97) complete dataset.(XLS)Click here for additional data file.Table S2Foxp2-ChIP putative target promoters .(XLS)Click here for additional data file.Table S3Foxp2-ChIP putative target promoters .(XLS)Click here for additional data file.Table S4Foxp2-dependent gene expression changes in the E16 mouse brain (p<0.01).(XLS)Click here for additional data file.Table S5Overlapping target genes from Foxp2-ChIP and expression analysis studies.(XLS)Click here for additional data file.Table S6Significant over-represented KEGG pathways identified in the Foxp2-ChIP and gene expression datasets.(XLS)Click here for additional data file.Table S7Cntnap2 semi-quantitative PCR primers.(XLS)Click here for additional data file.Text S1Supporting (DOC)Click here for additional data file.