Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell morphologies, revealing a novel function of a conserved, co-transcriptional histone modification.Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb), in the fission yeast  opposition on elongating RNA polymerase. We suggest that the positive feedback linking P-TEFb and H2Bub1 helps to maintain a balance between their opposing actions. These results highlight a novel regulatory role for a conserved histone modification during transcription elongation.Modification of histone proteins is an important transcriptional regulatory mechanism in eukaryotic cells. Although various histone modifications are found primarily within the coding regions of transcribed genes, how they influence transcription elongation remains unclear. Among these modifications is mono-ubiquitylation of histone H2B (H2Bub1), which is needed for co-transcriptional methylation of histone H3 at specific sites. Here we show that H2Bub1 and Cdk9, the kinase component of positive transcription elongation factor b (P-TEFb), are jointly regulated by a positive feedback loop: Cdk9 activity is needed for co-transcriptional H2Bub1, and H2Bub1 in turn stimulates Cdk9 activity toward one of its major substrates, the conserved elongation factor Spt5. We provide genetic evidence that the combined action of H2Bub1 on Spt5 phosphorylation and histone methylation accounts for the gene-regulatory effects of this modification. Surprisingly, our genetic and genome-wide studies indicate that P-TEFb and H2Bub1 act in  The elongation phase of transcription is a point of regulation for many genes transcribed by RNAPII in eukaryotes, and control of elongation is critical for coupling of transcription to downstream steps in gene expression H2Bub1, which appears to play a central role in the interplay between chromatin and the RNAPII elongation complex, is catalyzed in budding yeast by the ubiquitin-conjugating enzyme Rad6 and the E3 ubiquitin ligase Bre1 S. pombe revealed that the Set1-independent pathway is important for normal cell growth and morphology, and for elongation by RNAPII at select target genes S. cerevisiae and mammalian cells H2Bub1 also acts independently of histone methylation; our work in the fission yeast Co-transcriptional histone modifications are regulated by a conserved subset of cyclin-dependent kinases (CDKs) associated with the transcription machinery Drosophila cells P-TEFb also regulates histone modification. In mammalian cells, levels of H2Bub1, H3K4me, and H3K36me decrease after depletion of Cdk9 by RNA interference (RNAi), or treatment with flavopiridol, an inhibitor of Cdk9 and related kinases BUR1 deletion is suppressed by deletion of the H3K36 methyltransferase Set2 CDKs are themselves regulated by histone modification. In mammals, P-TEFb binds the bromodomain protein Brd4, whose recruitment to promoter-proximal sites is stimulated by acetylation of histone H4 S. pombe Spt5 spt5 mutations that prevent this phosphorylation cause slow growth, defects in transcript elongation and, when combined with partial truncations of the Rpb1 CTD, synthetic lethality In fission yeast, the P-TEFb ortholog is the essential Cdk9/Pch1 complex. In vitro, Cdk9 phosphorylates the Rpb1 CTD at both Ser5 and Ser2 S. pombe: mutations that impair phosphorylation of Spt5 by Cdk9 diminish levels of H2Bub1 and, reciprocally, mutants unable to generate H2Bub1 have reductions in chromatin-associated Cdk9 and phosphorylated Spt5. Ablation of the preferred phosphoacceptor site in Spt5 combined with deletion of +set1 phenocopies morphological defects of htb1-K119R mutants, suggesting that Cdk9 and Set1 govern separate, partially redundant pathways downstream of H2Bub1. Despite the dependencies between H2Bub1 and Spt5 phosphorylation, mutations that impair Cdk9 function suppress abnormal cell morphologies and reverse an RNAPII distribution defect, both due to loss of H2Bub1. Conversely, growth of htb1-K119R cells is resistant to selective Cdk9 inhibition, relative to that of +htb1 cells. Taken together, biochemical and genetic results suggest that P-TEFb and H2Bub1 oppose one another to regulate RNAPII elongation, but that proper balance between the two is ensured via a positive feedback loop, in which phosphorylation by Cdk9 stimulates H2Bub1 and vice versa.Here we uncover a mutual dependence of H2Bub1 and P-TEFb function in S. pombe genome at 200 base-pair intervals (ChIP-chip). We first mapped genome-wide distribution of H2Bub1 in wild-type cells with an antibody that specifically recognizes the ubiquitylated form of histone H2B [S. pombe, similar to its distribution in S. cerevisiae and metazoans r = 0.58).Our previous results suggested that H2Bub1 promotes expression of a subset of genes tone H2B . A FLAG-htb1-K119R mutant cells. This analysis revealed alterations in RNAPII occupancy within gene coding regions in the htb1-K119R mutant: a global, downstream shift in average RNAPII density, reflecting both a decrease in occupancy within the 5′ halves of genes and a more pronounced increase near the 3′ ends (P<10exp-5) decrease in RNAPII 5′-end occupancy in the absence of H2Bub1, whereas the increase at 3′ ends failed to reach significance (P>0.01). In the other three classes of genes the increases in RNAPII occupancy at the 3′ end were the most significant changes  terms that were significantly enriched among the 500 genes with the largest increases or decreases in RNAPII occupancy in the htb1-K119R mutant. The genes exhibiting loss of RNAPII occupancy were enriched for ribosomal protein genes , consistent with a role for H2Bub1 in promoting RNAPII occupancy at heavily transcribed genes.To ascertain whether genes affected by the P = 7.96exp-5). Thus, the ChIP-chip results and our previous gene-expression data argue that loss of H2Bub1 has a particularly strong negative impact on steady-state levels of poly(A+) mRNA produced from highly transcribed genes. However, the global redistribution of RNAPII in htb1-K119R cells  mutant versions, each of which was sensitive to the inhibitory analog 3-MB-PP1 ascdk9 cells, caused diminution of H2Bub1 signals in whole-cell extracts  responsible for that phosphorylation cdk9-T212E mutation, which substitutes a Glu residue for Thr212 to mimic constitutive phosphorylation mcs6-S165A, a T-loop mutation that renders Mcs6 refractory to CAK +lsk1 or +lsc1 [which encodes the cyclin partner of Lsk1 S. pombe CDKs implicated in transcript elongation, Cdk9 is uniquely required for H2Bub1.To further probe the dependence of H2Bub1 on P-TEFb, we tested effects of other mutations that perturb Cdk9 function . Two mutspt5 truncation mutants: spt5ΔC, in which the entire CTD is deleted; or variants containing seven repeats, either of wild-type sequence (spt5-WT), or with every Thr1 position mutated to Ala (spt5-T1A) or Glu (spt5-T1E) spt5ΔC and spt5-T1A mutants, but not in spt5-WT or an spt5-T1E mutant that mimics constitutive phosphorylation , which recognized Spt5 only after it had been phosphorylated by Cdk9 in vitro . In whol strains . Spt5-Ths 300 nM . In contto 40 µM . We conchtb1-K119R and brl2Δ  spt5-T1A set1Δ double mutant; the combination, which abolished two protein modifications shown to depend on H2Bub1, phenocopied the increased and aberrant septation produced by H2Bub1 loss  Togethhtb1-K119R mutant by ChIP. Control experiments confirmed that the Spt5-P antibody was suitable for immunoprecipitation and ChIP  that, according to our RNAPII ChIP-chip analysis, displayed a 3′ shift in RNAPII distribution in the absence of H2Bub1  of Spt5 during the transition from initiation to elongation . Therefore, lack of H2Bub1 specifically reduced dependency on active Cdk9, consistent with the two pathways acting antagonistically. Absence of H2Bub1 did not fully bypass the requirement for Cdk9 activity, however, because higher doses of 3-MB-PP1 were still capable of arresting proliferation of as htb1-K119Rcdk9 cells.Biochemical analyses revealed a reciprocal relationship between Spt5-P and H2Bub1, suggestive of a positive feedback loop. The synthetic phenotype of ckground . In contas htb1-K119Rcdk9 cells displayed hyperseptated and branched morphologies characteristic of H2Bub1-defective mutants. After treatment with 10 µM 3-MB-PP1 for 7 hr, however, cell morphology reverted to normal  besides Rpb1-Ser2.Cdk9 also contributes to phosphorylation of Ser2 and Ser5 of RNAPII CTD repeats ckground . Combinemutation . Therefo+ spt5-T1A htb1-K119Rcdk9 cells and suppressed as spt5-T1A htb1-K119Rcdk9 cells grew at similar rates in liquid culture. Moreover, the non-suppressed, as rpb1-S2A htb1-K119Rcdk9 strain grew more slowly than did the suppressed strain . Understanding the relative contributions of the Rpb1 CTD and other Cdk9 substrates in directing H2Bub1 will probably require better tools for study of metazoan P-TEFb and its targets in vivo.Cdk9-dependent phosphorylation of the Rpb1 CTD has been implicated in formation of H2Bub1 in mammalian cells S. pombe ortholog of Rad6)-Brl1/2 complex by PAF, to generate H2Bub1; and 4) Cdk9 recruitment, leading to reiteration of the cycle  did not suppress cdk9-T212A eliminated aberrant cell morphologies associated with htb1-K119R, the growth rate of double mutant cells was slower than that of either single mutant (data not shown). These results imply that other pathways can modulate the functions of Cdk9 and H2Bub1 in transcript elongation.The antagonism we observe between Cdk9 activity and H2Bub1 is incomplete—loss of H2Bub1 did not entirely bypass the essential function of Cdk9, and led to altered RNAPII occupancy in the 5′ portions of genes even when Cdk9 activity was reduced. Conversely, although the presence of S. cerevisiae, the instructive genetic interactions occurred between BUR1 and the H3K36 methyltransferase Set2; lethality of a bur1 null mutation was suppressed by deletion of SET2set2 deletion did not suppress growth arrest due to inhibition of Cdk9as . Interestingly, bur1Δ was also weakly suppressed by deletion of RAD6Our results strengthen the notion that P-TEFb activity is required to overcome repressive effects of histone modifications, first suggested by work in budding yeast. In In mammalian cells, the Bre1/Brl2 ortholog RNF20 selectively represses transcription of genes implicated in tumorigenesis; its knockdown reduces H2Bub1, enhances the transcriptional response to growth factors and promotes cellular transformation htb1-K119R mutant, the balance is disrupted, resulting in Cdk9-dependent accumulation of RNAPII in 3′ regions of genes. We suggest this pattern reflects unchecked elongation that is poorly responsive to mRNA processing signals, and may imply enhancement of the RNAPII pausing normally associated with mRNA 3′ end processing How can we reconcile biochemical evidence for mutual dependence of Cdk9 activity and H2Bub1 with genetic evidence for their antagonism? We propose a homeostatic mechanism whereby Cdk9-H2Bub1 interdependence ensures a balance between their opposing functions. In this scenario, Cdk9 promotes H2Bub1 to regulate its own effects on elongation . In an hIn cell division, faithful genome duplication and segregation are ensured by checkpoints—extrinsic signaling pathways that enforce dependency between intrinsically independent events Strains used in this study are listed in S. pombe whole-cell extracts were prepared as previously described To detect Spt5 phosphorylation by immunoblotting, ChIP was carried out as described previously S. pombe cells were fixed and stained with diamino-phenylindole (DAPI) and calcofluor as described previously 600∼0.1 in YES and treated with 10 µM 3-MB-PP1 or DMSO for 7 hr, then fixed and stained for microscopy immediately or after return to growth in drug-free YES for 7 hr.Cultures were treated with 3-MB-PP1 at indicated doses, or with vehicle (DMSO), for indicated times at 30°C prior to extract preparation. Dose-response to 3-MB-PP1 was determined as previously described Assays were carried out as described previously Dataset S1htb1-K119R).Microarray data for ChIP-chip performed with a H2Bub1 antibody (wild-type) and a RNAPII antibody (wild-type and (ZIP)Click here for additional data file.Dataset S2Microarray data for ChIP-chip performed with a histone H3 antibody (wild-type).(ZIP)Click here for additional data file.Dataset S3htb1-K119R).Microarray data for ChIP-chip performed with a histone H3 antibody ((ZIP)Click here for additional data file.Dataset S4cdk9-T212A).Microarray data for ChIP-chip performed with a RNAPII antibody ((ZIP)Click here for additional data file.Dataset S5cdk9-T212A htb1-K119R).BED file containing microarray data for ChIP-chip performed with a RNAPII antibody ((ZIP)Click here for additional data file.Figure S1S. pombe. (A) H2Bub1 occupancy was measured by ChIP in wild-type (JTB62-1) and htb1-K119R (JTB67-1) strains and quantified at the inactive hsp16+ gene and constitutive act1+ gene. Values were normalized to H2B-FLAG occupancy measured in parallel to control for differences in nucleosome density. Positions of primer pairs used for qPCR are indicated in schematic at top. (B) Immunoblots of whole-cell extracts from untagged (JTB204), htb1-FLAG (JTB62-1), and htb1-K119R-FLAG (JTB67-1) strains using the indicated antibodies.Specificity of H2Bub1 antibody for ChIP in (PDF)Click here for additional data file.Figure S2S. pombe. (A) Average distribution of histone H3 at 540 S. pombe genes, as determined by ChIP-chip in a wild-type strain (JTB62-1). Genes were grouped according to total levels of RNAPII enrichment (see key at top). The grey box in the “average gene” representation at bottom denotes the gene coding region; 5′ and 3′ untranslated regions are denoted by thin black lines. The arrow denotes the transcription start site. (B) As in (A), determined in an htb1-K119R mutant strain (JTB67-1). Gene groupings were created using wild-type RNAPII enrichment values. (C) Average distributions of differences between mutant and wild-type histone H3 enrichment grouped according to RNAPII enrichment in wild-type cells. The key below the graph illustrates the statistical significance of the differences for each group at 50 positions along the average gene. The rows of the key are color-coded according to the graph. Open squares denote p>0.01; light shading denotes 0.01>p>10exp-5 .Loss of H2Bub1 does not significantly alter nucleosome density in (PDF)Click here for additional data file.Figure S3+eng1 gene by qPCR. Enrichment is plotted as percentage of the input signal for each primer pair. Positions of PCR primer pairs are indicated in the schematic at top. (B) Rtf1-TAP occupancy was measured by ChIP in the indicated strains and quantified at the +act1 gene by qPCR. Enrichment is plotted as percentage of the input signal for each primer pair. Positions of PCR primer pairs are indicated in the schematic at top. In all cases, treatment of the ascdk9 strains was with either DMSO (-) or 20 µM 3-MB-PP1 for 3 hr prior to harvesting. Error bars denote standard deviations from three independent experiments.Chromatin association of Rhp6 and the PAF protein Rtf1 requires Cdk9 activity. (A) Rhp6-TAP occupancy was measured by ChIP in the indicated strains and quantified at the (PDF)Click here for additional data file.Figure S4spt5-myc strains after treatment with increasing concentrations of 3-MB-PP1. Antibodies are indicated at right. (D) Both phospho-isoform- and pan-specific total Spt5 antibodies are specific for the wild-type nonapeptide repeat sequence of the Spt5 CTD. Whole-cell extracts from the indicated strains were probed with affinity-purified antibody  or crude serum  from a rabbit immunized with an Spt5-CTD phosphopeptide  Kinase reactions containing recombinant GST-Spt5 substrate and indicated combinations of purified, recombinant Cdk9/Pch1 and Csk1 were analyzed by immunoblotting with: affinity-purified antibody specific for phosphorylated Spt5 (Spt5-P); crude serum specific for wild-type Spt5 CTD repeats, regardless of phosphorylation state Click here for additional data file.Figure S5brl2Δ (JTB331), cdk9-T212A (HD7-24), and pob3Δ (JTB281-1) strains using the indicated antibodies. (B) Quantification of abnormal septation in strains of indicated genotypes . Error bars represent standard deviations from 2 independent experiments; at least 200 cells were counted in each.The FACT subunit Pob3 has functions distinct from those of H2Bub1. (A) Immunoblots of whole-cell extracts from wild-type (JTB204), (PDF)Click here for additional data file.Figure S6spt5-myc strains. (A) Spt5-P immunoprecipitation from whole-cell extracts. Extracts from indicated strains (bottom) were incubated with or without anti-Spt5-P (top), and immunoprecipitates were analyzed by immunoblotting with antibodies indicated at right. The ascdk9 strain was treated prior to extract preparation with either DMSO or 5 µM 3-MB-PP1, as indicated at bottom, for 20 min. For each treatment we analyzed 10% of input extract (“input”), 10% of supernatant after precipitation (“sup”) and 50% of immunoprecipitated material (“IP”). Antibodies are indicated at right. (B) ChIP of Spt5-P and Spt5-myc at eng1+. Patterns of Spt5-P occupancy on +eng1 are similar in wild-type  and spt5-myc  strains. Distribution of total Spt5 on +eng1 was measured in the spt5-myc strain (bottom). (C) Spt5-myc (top) and Spt5-P (middle) occupancies were measured by ChIP in a as spt5-myccdk9 strain and quantified at indicated positions within eng1+ by qPCR. Cells were treated prior to lysis with either DMSO or 5 µM 3-MB-PP1 for 20 min. Enrichment is plotted as percentage of input signal for each primer pair. The ratios of Spt5-P/Spt-myc at each position are plotted at bottom.Anti-Spt5-P specifically immunoprecipitates Spt5-P from wild-type and (PDF)Click here for additional data file.Figure S7+nup189 and +SPB354.10.  Spt5 phosphorylation was measured by ChIP using anti-Spt5-P in spt5-myc  and spt5-myc htb1-K119R  strains and quantified at the indicated genes by qPCR. In all graphs of this figure the enrichment is plotted as percentage of the input signal for each primer pair, positions of PCR primer pairs within coding regions are indicated in the schematic at top, error bars denote standard deviations from 3 independent experiments and asterisks denote a significant difference between wild-type and mutant .  Spt5-myc occupancy was measured by ChIP as in C and D.  Spt5-P enrichment normalized to total Spt5-myc occupancy.  Cdk9-myc occupancy was measured by ChIP in cdk9-myc  and cdk9-myc htb1-K119R  strains.Impact of H2Bub1 on Spt5 phosphorylation and Cdk9 recruitment at (PDF)Click here for additional data file.Figure S8eng1 (eng1-1) and an intergenic region as control (control). In all graphs of this figure the enrichment is plotted as percentage of the input signal for each primer pair and error bars denote standard deviations from 3 independent experiments. (B) Same experiment as in (A) in htb1-K119R strains .ChIP of Spt5-P, Spt5-myc, and Cdk9-myc at an intergenic sequence. (A) Occupancy of total Spt5, phospho-Spt5 and Cdk9 was measured by ChIP using anti-myc or anti-Spt5-P in wild-type strains , followed by qPCR using primers covering the 5′ end of (PDF)Click here for additional data file.Figure S9htb1-K119R  strains and quantified at the indicated genes by qPCR. Enrichment is plotted as percentage of the input signal for each primer pair, and positions of PCR primer pairs within coding regions are indicated in the schematic at top of each graph. (E) Levels of mRNA from the indicated genes were quantified by qRT-PCR and normalized to +act1. Throughout this figure error bars denote standard deviations from 3 independent experiments and asterisks denote a significant difference between wild-type and mutant .Impact of H2Bub1 on RNAPII occupancy and mRNA expression at individual genes. (A-D) RNAPII occupancy was measured by ChIP in wild-type  and (PDF)Click here for additional data file.Figure S10as complexes purified from S. pombe. Lysates from pcm1-myc (CS145) or as pcm1-myccdk9 (CS165) strains were incubated either with anti-myc-bound protein G beads (α-myc-beads) to co-immunoprecipitate Cdk9 bound to Pcm1-myc or with protein G alone (mock). Input lysate amounts in each immunoprecipitation were varied as indicated. Immunoprecipitates were analyzed by immunoblotting (top panels) and for Rpb1-CTD kinase activity (middle panels). Phosphorylation signals were visualized by autoradiography and quantified with a phosphorimager (bottom).Kinase activity of Cdk9 and Cdk9(PDF)Click here for additional data file.Table S1htb1-K119R strain.RNAPII distributions at H2Bub1-stimulated genes in the (DOC)Click here for additional data file.Table S2S. pombe strains used in this study.(DOC)Click here for additional data file.Text S1Supplemental materials and methods.(DOC)Click here for additional data file.


Using a genetic model, we present a high-resolution chromatin fiber analysis of transcriptionally active (Xa) and inactive (Xi) X chromosomes packaged into euchromatin and facultative heterochromatin. Our results show that gene promoters have an open chromatin structure that is enhanced upon transcriptional activation but the Xa and the Xi have similar overall 30 nm chromatin fiber structures. Therefore, the formation of facultative heterochromatin is dependent on factors that act at a level above the 30 nm fiber and transcription does not alter bulk chromatin fiber structures. However, large-scale chromatin structures on Xa are decondensed compared with the Xi and transcription inhibition is sufficient to promote large-scale chromatin compaction. We show a link between transcription and large-scale chromatin packaging independent of the bulk 30 nm chromatin fiber and propose that transcription, not the global compaction of 30 nm chromatin fibers, determines the cytological appearance of large-scale chromatin structures. ► High-resolution chromatin analysis of active and inactive X chromosomes ► Gene promoters have a transcription-dependent open chromatin structure ► Active and inactive X chromosomes have similar bulk 30 nm chromatin fiber structures ► Transcription promotes the decompaction of large-scale chromatin structures Chromatin structure modulation is central to the control of gene expression . This isUsing microscopy, a large proportion of the mammalian genome can be seen to be packaged at levels beyond the 30 nm chromatin fiber, sometimes visualized as 60–130 nm “chromonema” fibers . These aStudies show that transcription correlates with the reorganization of large-scale chromatin structures, for example, at the level of chromosome territories transcriptionally inactive gene-poor chromosome 18 has a more compact organization that the transcriptionally active gene-rich chromosome 19 . ChromosTo understand the relationship between chromatin structure and transcription the chromatin fiber needs to be characterized at active and inactive genes. As chemical inhibition of transcription will directly influence the 30 nm chromatin fiber structure, we have utilized the transcriptionally active and inactive X chromosomes as a genetic model. We present a genome-wide analysis of 30 nm chromatin fiber structures using a biophysical technique  in conjuGene-rich regions of the human genome are enriched in “open” (disrupted) 30 nm chromatin fibers . To map 5 by random permutation)  A and S1Butation) C. Althouutation)  on our eAs gene-rich chromosomes  are very enriched in open chromatin A and S1BTo investigate the relationship between chromatin fiber structure and transcription, we have used the X chromosome as a model. In female cells, the active (Xa) and the inactive X (Xi) chromosomes have almost identical DNA sequences so we can directly analyze the effect of transcription on chromatin structures. As described, we fractionated chromatin fibers and mapped them to the X chromosome by hybridization to genomic SNP arrays A. SATO3 It is unknown whether transcription-dependent changes in chromatin structure B–1D are Although chromosome-wide analysis indicated that the two X chromosomes had similar euchromatin-like 30 nm chromatin fiber structures A, our pr2 open/input units. This corresponds to a disruption smaller than the disruption found at transcriptionally inactive genes  enrichment of chromatin that can be related to the sedimentation properties of differently shaped chromatin fibers  extraction and isopropanol precipitation. RNA was hybridized to expression arrays .4 cells) were cytospun on to glass slides at 600 rpm for 10 min and processed using standard techniques with minor modifications (Nonadherent lymphoblastoid cells (2 × 10ications . Slides ications .The method used to determine chromosome haplotypes is described in detail in the


Histone post-translational modifications (PTMs) constitute a branch of epigenetic mechanisms that can control the expression of eukaryotic genes in a heritable manner. Recent studies have identified several PTM-binding proteins containing diverse specialized domains whose recognition of specific PTM sites leads to gene activation or repression. Here, we present a high-throughput proteogenomic platform designed to characterize the nucleosomal make-up of chromatin enriched with a set of histone PTM binding proteins known as histone PTM readers. We support our findings with gene expression data correlating to PTM distribution.We isolated human mononucleosomes bound by the bromodomain-containing proteins Brd2, Brd3 and Brd4, and by the chromodomain-containing heterochromatin proteins HP1β and HP1α. Histone PTMs were quantified by mass spectrometry (ChIP-qMS), and their associated DNAs were mapped using deep sequencing. Our results reveal that Brd- and HP1-bound nucleosomes are enriched in histone PTMs consistent with actively transcribed euchromatin and silent heterochromatin, respectively. Data collected using RNA-Seq show that Brd-bound sites correlate with highly expressed genes. In particular, Brd3 and Brd4 are most enriched on nucleosomes located within HOX gene clusters, whose expression is reduced upon Brd4 depletion by short hairpin RNA.Proteogenomic mapping of histone PTM readers, alongside the characterization of their local chromatin environments and transcriptional information, should prove useful for determining how histone PTMs are bound by these readers and how they contribute to distinct transcriptional states. Chromatin modifications and the gene expression patterns that accompany them are maintained when a cell divides and are thus known as 'epigenetic' [A cell's transcriptional program is governed not only by genetic' ,2. Histogenetic' ,4. Howevgenetic' ,5.Drosophila trithorax Fsh1 and Fsh2 proteins [In vitro studies have demonstrated that Brd2 and Brd3 possess nucleosome chaperone activities that allow RNA polymerase II to elongate transcripts through hyperacetylated nucleosomes, implicating their direct role in transcription [The 'histone code' hypothesis proposes that specific histone PTMs encode regulatory information that is read by the binding of accessory proteins. These accessory proteins, termed 'readers', bind via specialized histone-PTM-binding domains such as bromodomains, chromodomains and plant homeodomains (PHDs) . The broproteins . The Brdproteins . In vitrcription . Severalcription ,10. Thuscription ,11.In contrast to bromodomains, chromodomains are methyl-lysine binding domains found in proteins such as the heterochromatic proteins HP1α and HP1β that bind to H3K9me3 . ChromodHere, we report experiments that elucidate the combinations of histone PTMs on nucleosomes associated with the histone code reading proteins Brd2, Brd3, Brd4, HP1α and HP1β Figure , applyind0- or d5-propionyl groups such that two samples (each labeled with a different isotope) can be analyzed together in a single nanoLC-MS/MS experiment  as depicted in Figures To gain insight into the local chromatin make-up of histone PTM binding proteins, we optimized a ChIP technique to purify mononucleosomes from cells expressing tagged chromodomain- and bromodomain-containing proteins. Specifically, we generated HEK293 cell lines that express FLAG-tagged fusions of Brd2, 3, 4 and HP1α and β Figure , as thest Figure  or samplPTMs observed on the H4 molecules from Brd-bound nucleosomes contained marks consistent with gene activation. Most striking was the enrichment in the overall degree of acetylation. All the potentially acetylated lysines  occur on the same propionylated tryptic peptide, so we first quantified H4 tail peptides that were unmodified, mono, di-, tri- and tetra-acetylated, irrespective of the residue modified Figure , table. t-Tests performed with data from three independent experiments for each ChIP revealed that these fold changes are indeed significant as the P-values were all <1 × 10-2  accurately deconvoluted the mixed tandem mass spectra resulting from isobaric acetylation combinations that occur on H4 . When compared to whole genome chromatin, nucleosomes bound by all three Brd proteins were enriched with the H4 diacetylated forms , while only Brd3- and Brd4-bound nucleosomes were enriched in the K5acK8unK12acK16un species  for H3K4me2 and 23.6-fold  for H3K4me3 , but was highly enriched on both HP1s  and H3K18acK23ac  . Figure At the gene level Figure , approxiHOXB5, HOXB3, HOXC5 and HOXC11) by ChIP, using a monoclonal antibody raised against Brd4  and the Drosophila Brd protein (FSH-S) are required for the expression of the HOX gene Ubx and the overexpression of FSH-S can induce the ectopic expression of several HOX genes in Drosophila [To more fully probe the genes bound by each reader, we focused on those genes whose promoters had the most significant binding. As shown in Figure up genes . Trithorsegments ,32. The segments . Therefosegments -36. In fosophila . These rAs stated above, the HP1s were found at the promoter regions of 1 to 2% of the genes they bind and most of these are transcriptionally silent Figure . MoreoveIn order to determine whether Brds directly regulate the expression of the genes they bind, we performed gene expression microarrays on cells depleted of Brd4 by short hairpin RNA (shRNA). We choose to knockdown Brd4 because it shared the least overall overlap of bound promoters with the other Brd ChIPs. We achieved a three-fold knockdown of Brd4 with a custom shRNA in HEK293 cells as determined by RT-PCR. The knockdown was also verified at the protein level by western blot  outlined the need for a project to provide high-resolution epigenome maps and suggested guidelines for the first pass of a human epigenome reference . At the Two different models have been proposed for the function of Brd proteins in transcription. In one model Brd4 recruits the transcription elongation factor pTefb to the transcription complex at the transcription start site, which then phosphorylates the CTD of RNA polymerase II, leading to more processive transcript elongation . In the HP1-bound nucleosomes were most enriched with histones containing the repressive PTMs, H3K9me2/3 (approximately 85% combined) and H4K20me2/3 (approximately 75% combined). In addition to binding the ZNF clusters of chromosome 19, our ChIP-seq results have revealed that the HP1 proteins reside on centromeric heterochromatin and inactive × chromatin, both of which harbor these marks . Also inSilent Hox genes are a defining hallmark of pluripotent stem cells and their expression is tightly regulated during differentiation . The dif0d- or 'heavy' 10d-propionic anhydride, trypsinized and prepared for mass spectrometry as previously described in [Mononucleosomes were purified after  Mnase digestion with FLAG-Brd or FLAG-HP1 proteins as described in  from HEKribed in  with minHistone peptides were separated by reverse phase nanospray liquid chromatography on C18 resin with an Agilent 1200 series HPLC. Mass spectrometry was performed on a LTQ-Orbitrap mass spectrometer .Nucleosomal DNAs were treated with alkaline phosphatase to remove the 3' phosphates left after MNase digestion and purified by phenol extraction. The DNA fragments (approximately 1 μg) were further prepared using the SolexaThe raw sequence data for the RNA-Seq in HEK293 cells were obtained from   with the negative values set to zero. A clustering approach described in [For the chromosomal binding patterns of Brd and HP1 proteins Figures  and 5c, ribed in  was usedThe sequence read density (per 100-bp window and 5 million non-redundant reads) was determined along the gene body and in the upstream/downstream 2-kb regions in the sample and control libraries. The signal level in each window was computed as the difference between the read densities in the sample and control libraries, with the negative values set to zero. Genes were ranked based on their expression levels estimated as reads per kilobase per million mapped reads  overlaps the promoter. Genes with a particular combinatorial promoter-binding pattern of the five proteins  shRNA vectors (pLKO.1-puro) harboring shRNAs to Brd4 (clone TRCN0000062223), HP1β (clone TRCN0000021427) or control (empty pLKO.1-puro vector). After 72 hours the infected cells were selected with puromycin according to the manufacturer's protocols (Sigma Aldrich). Knockdowns of target RNAs were analyzed by qRT-PCR. For qRT-PCR, RNA from cells was purified with an RNeasy kit (Qiagen) and prepared with a cDNA synthesis kit . qRT-PCR reactions were performed with SYBR FAST qPCR supermix .HEK293 cells were infected with lentiviral MISSION® software . More detailed methods are described in Additional file RNA was extracted and purified with an RNeasy kit (Qiagen) from Brd4, HP1β and control shRNA knockdown HEK293 cells. For each sample 400 ng RNA was linearly amplified and labeled with Cy5-CTP using Low RNA Linear Amplification reagents . Reference RNA from wild-type HEK293 cells was purified, amplified and labeled with Cy3 in the same manner. Equal quantities of the Cy5- and Cy3-labeled cRNAs were mixed and competitively hybridized to human GE 4x44K microarrays  for 18 h using an Agilent hybridization kit. Slides were washed according to the manufacturer's protocols and scanned using an Agilent G2565BA scanner. Raw image data were extracted and subjected to standard background subtraction and linear and lowess normalization using Agilent Feature extraction software v9.5. Heatmaps were generated using MATLABbp: base pair; ChIP: chromatin immunoprecipitation; ChIP-seq: chromatin immunoprecipitation-high throughput deep sequencing; DamID: DNA adenine methyltransferase identification; nanoLC-MS/MS: nano scale liquid chromatography-tandem mass spectrometry; PCR: polymerase chain reaction; PHD: plant homeodomain; PTM: post-translational modification; qMS: quantitative mass spectrometry; RNA-Seq: RNA-high-throughput deep sequencing; RPKM: reads per kilobase per million mapped reads; shRNA: short hairpin RNA.The authors declare that they have no competing interests.GL performed most of the experimental work. IC carried out the analysis of the deep sequencing data. PAD helped analyze the mass spectrometry data. MAB helped with the analysis of microarray data. BMZ assisted with experimental work. KZ and BAG supervised and participated in the design and coordination of the project. GL and BAG wrote the manuscript. All authors read and approved the final manuscript.Western blots of whole cell extracts from cell lines expressing FLAG-Brd4, FLAG-HP1β and control cell line (empty vector). Blots were probed with anti-Brd4 , anti-HP1β  and β-actin control .Click here for fileTable of relative PTM abundances determined by quantitative mass spectrometry on histones H3 and H4 averaged from three independent ChIP experiments with each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin.Click here for fileTable of relative PTM abundances determined by quantitative mass spectrometry on the histone H4 peptide (amino acids 4 to 17) averaged from three independent ChIP experiments with each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin.Click here for fileP-values from t-tests performed on the fold changes (ChIP/Genomic) from the histone H4 data presented in Additional file t-Tests were performed with data from three independent ChIP experiments for each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin. P-values were adjusted using the Benjamini-Hochberg correction method to control the false discovery rate (FDR).Click here for fileP-values from t-tests performed on the fold changes (ChIP/Genomic) from the histone data presented in Additional file t-Tests were performed with data from three independent ChIP experiments for each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin. P-values were adjusted using the Benjamini-Hochberg correction method to control the false discovery rate (FDR).Click here for fileTable of relative combinatorial PTM abundances determined by quantitative mass spectrometry on the histone H3 peptides (amino acids 9 to 17), (amino acids 18 to 26) and (amino acids 27 to 40) averaged from three independent ChIP experiments with each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin.Click here for fileP-values from t-tests performed on the fold changes (ChIP/Genomic) from the histone H3 data presented in Additional file t-Tests were performed with data from three independent ChIP experiments for each Brd and HP1 protein and data from three experiments with HEK293 genomic chromatin. P-values were adjusted using the Benjamini-Hochberg correction method to control the false discovery rate (FDR).Click here for fileSpreadsheets of all promoters bound by the Brd and HP1 proteins. Promoters are ranked by P-values.Click here for fileHeatmap of motifs enriched in the HP1 and Brd ChIPs. Lists of consensus sequences (motifs) are found in the matrix used to create the heatmap .Click here for fileSpreadsheets containing Gene Ontology terms enriched in Brd and HP1 ChIPs. Gene Ontology terms are ranked by false discovery rates (FDRs).Click here for file® Hercules, CA, USA)Products from PCR reactions were run on 2% agarose gels stained with ethidium bromide and visualized on a Gel Doc XR system , Brd4 ChIP and ChIP input DNA.Click here for fileWestern blots of whole cell extracts from Brd4 shRNA knockdown, HP1β shRNA knockdown and control shRNA knockdown cell lines. Blots were probed with anti-Brd4 , anti-HP1β  and β-actin control .Click here for fileSupplemental Materials and methods.Click here for file


Saccharomyces cerevisiae, posing the question how most yeast promoters nucleate PIC assembly. Here we show that one third of all yeast promoters contain a novel conserved DNA element, the GA element (GAE), that generally does not co-occur with the TATA box. The distance of the GAE to the transcription start site (TSS) resembles the distance of the TATA box to the TSS. The TATA-less TMT1 core promoter contains a GAE, recruits TBP, and supports formation of a TBP-TFIIB-DNA-complex. Mutation of the promoter region surrounding the GAE abolishes transcription in vivo and in vitro. A 32-nucleotide promoter region containing the GAE can functionally substitute for the TATA box in a TATA-containing promoter. This identifies the GAE as a conserved promoter element in TATA-less promoters.Initiation of RNA polymerase (Pol) II transcription requires assembly of the pre-initiation complex (PIC) at the promoter. In the classical view, PIC assembly starts with binding of the TATA box-binding protein (TBP) to the TATA box. However, a TATA box occurs in only 15% of promoters in the yeast  In eukaryotes, transcription of protein-coding genes relies on RNA polymerase (Pol) II, the general transcription factors (GTFs) TFIIB, -D, -E, -F, and –H, and coactivators such as Mediator and the Spt-Ada-Gcn5 acetyltransferase complex (SAGA) Since TATA-less promoters require TBP for function in vivo reporter gene assays, and in vitro biochemistry to identify and functionally characterize a region in TATA-less yeast core promoters that is bound by TBP, required for Pol II transcription and contains a novel conserved promoter element, the GA element, or GAE.Based on these observations, we hypothesized that TATA-less core promoters contain DNA elements that are functionally similar to the TATA box in promoting PIC assembly and Pol II transcription. Here we used a combination of bioinformatics, In contrast to higher eukaryotes, where the distance between the TATA box and the TSS is fixed at around 30 nucleotides, a variable distance of 40−120 nucleotides is observed in yeast, apparently due to a TSS scanning mechanism Sc, S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus). We allowed for a shift of up to +/− 3 nucleotides between motifs in the multiple sequence alignments. Again, the GAE stands out as being the most highly conserved of all the more frequent 5-mers . This search identified a DNA element comprising one guanine followed by four adenines (GAAAA) as being most highly anti-correlated to the TATA box , only trSc promoters containing only a GAE are three times more abundant than promoters containing only a TATA box  stretches, which are proposed to occlude nucleosomes from promoters TMT1 promoter function is strongly dependent on the sequence context in the GAE-containing region.Activity of the adenines , lane 10adenines  in vitroHIS4 and TMT1 promoter DNA. TBP bound HIS4 DNA as expected, but also bound the TATA-less TMT1 DNA  and fluorescently labeled DNA encompassing 40 base pairs of TMT1 DNA . As expein vivo and in vitro. We found that the HIS4 TATA box can functionally replace the 12-nucleotide GAE-containing region in the TMT1 promoter  tract in the ILV1 promoter decreased transcription significantly without affecting nucleosome organization in vivoSeveral studies of poly(dA:dT) tracts in individual model promoters indicated a role in promoter function that is linked to nucleosome positioning Sc, S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus) were used. For calculating the number of mutations, the start position in a window of +/−3 bp with the least number of mutations to the motif in Sc in every species was used.The TSS used for our analysis were generated by merging the cDNA data set of Miura et. al HIS4 (428 bp upstream to 24 bp downstream of the start codon) and TMT1 (273 bp upstream to 24 bp downstream of the start codon) and their mutant variants were cloned between HindIII/BamHI into a pRS315 plasmid with the lacZ gene inserted between NotI/SalI. Reporter plasmids were transformed into BY4741 wild type yeast cells and grown in biological duplicates in SD (–leu) medium to an OD600 of 0.5–1.0. β-galactosidase levels were determined using the Yeast β-galactosidase Assay Kit .The native promoter sequences of www.fhcrc.org/science/basic/labs/hahn) 33]. In vitro transcription and analysis by primer extension were performed as described 33]. Primer extension for all constructs was done using the same 5’-Cy5-labeled oligonucleotide (5’-TTCACCAGTGAGACGGGCAAC). For activated transcription 200 ng of recombinant full-length Gcn4 was added. Template plasmids were generated by inserting the respective promoter sequence as described above in pBluescript KS+ with HindIII and BamHI. The Sc Gcn4 gene was inserted into vector pET21a with NcoI and SacI introducing an N-terminal hexahistidine tag. Transformed E. coli BL21 (DE3) RIL cells (Stratagene) were grown in LB medium at 37°C to an OD600 of 0.5. Expression was induced with 0.5 mM IPTG for 16 h at 18°C. For protein purification, cells were lysed by sonication in buffer A . After centrifugation, the supernatant was loaded twice onto a 2 ml Ni-NTA column (Qiagen) equilibrated with buffer A. The column was washed three times with 10 column volumes (CV) of buffer A containing 20 mM imidazole. Bound protein was eluted with 10 CV of buffer A containing 500 mM imidazole. Protein was further purified by cation exchange chromatography using a HiTrap SP column  equilibrated with buffer B . The protein was eluted with a linear gradient of 10 CVs from 0 to 1 M NaCl in buffer B. After concentration, the sample was applied to a Superdex 200 size exclusion column (Amersham) equilibrated with buffer C . The sample was concentrated to approximately 0.5 mg/ml, flash frozen in small aliquots in liquid nitrogen, and stored at −80°C. All buffers contained a protease inhibitor cocktail.Nuclear extracts were prepared from 3 l of BY4741 wild type yeast culture as described  in a total reaction volume of 20 µl. Recombinant TBPcore and TFIIB were expressed in E. coli and purified as described 35]. Proteins were diluted in dilution buffer . Samples were incubated for 30 min at 18°C and loaded on a 6% native polyacrylamide gel . The gel was pre-run for five min and then run at 160V for 15 min at room temperature in 190 mM glycine, 25 mM Tris pH 8.3 23°C. Gels were analyzed with a typhoon scanner FLA9400 and ImageQuant Software . The oligos used for the assay had the following sequence (only coding strand is shown):Electrophoretic mobility shift assays (EMSA) were performed as described 34], with minor modifications. Templates were generated by annealing the complimentary PAGE-purified oligonucleotides (coding strand was 5’-Cy5-labeled). The binding reaction contained 3 nM of 40 bp 5’-Cy-5-labeled dsDNA, 300 nM TBPHIS4 (TATA), 5’-ACAGTAGTATACTGTGTATATAATAGATATGGAACGTTAT;HIS4 (GC-8), 5’-ACAGTAGTATACTGTGGGCCGGCCAGATATGGAACGTTAT;HIS4 (GAE), 5’-TAGTATACTGTGAAAGGAAAAGGAAGATATGGAACGTTAT;TMT1 (GAE), 5’-ATTTTCATTTTTAAAGGAAAAGGAATGTAACTAATTTAGT;TMT1 (GC-12), 5’-ATTTTCATTTTTGGCCGGCCGGCCATGTAACTAATTTAGT;TMT1 (TATA), 5’-ATTTTCATTTTTTATATAATATGTAACTAATTTAGT;ATTTTCATTTTTAAAAAAAAAAAAATGTAACTAATTTAGTTMT1 (A-12), 5’- Figure S1The GAE can occur in multiple copies. Fraction of sequences with the TATA box (purple) and GAE (green) in at least a given number of multiple occurrences per sequences in 3711 Sc promoters aligned at the TSS.(EPS)Click here for additional data file.Figure S2Frequency of the GAE with different length of A-tracts in 3711 Sc core promoters.(EPS)Click here for additional data file.Figure S3Only double stranded templates are bound by TBP and TFIIB. Electrophoretic mobility shift assay. A 5’-Cy5-labeled 40 bp single (ssDNA) and double stranded (dsDNA) templates were incubated with recombinant TBPcore alone or TBP core and TFIIB. The protein-DNA complex was separated from free DNA by native polyacrylamide gel electrophoresis. Free DNA and bound protein-DNA complexes are indicated.(EPS)Click here for additional data file.Figure S4GAE function depends on promoter context. (A) and (B) Electrophoretic mobility shift assay. A 5’-Cy5-labeled 40 bp double stranded DNA of the respective promoter construct was incubated with recombinant TBPcore alone or TBP core and TFIIB. The protein-DNA complex was separated from free DNA by native polyacrylamide gel electrophoresis. Free DNA and bound protein-DNA complexes are indicated. (C) Promoter activity in an in vivo β-galactosidase reporter gene assay and in vitro transcription (txn) assay of the wild type and mutated Sc SER3 promoter. The 8-nucleotide TATA box of the SER3 promoter was substituted by the 12-nucleotide GAE-containing region of the TMT1 promoter.(EPS)Click here for additional data file.Figure S5The GAE-containing region can functionally replace the TATA box. (A) Schematic depiction of promoter substitution constructs of the TATA-less TMT1 and the TATA-containing HIS4 promoter. TATA box and bioinformatically defined GAE are marked by a black box. Solid and dashed lines represent HIS4 and TMT1 promoter sequences, respectively. TSS of HIS4 and TMT1 are labeled with a and b, respectively. Half-arrows mark primer annealing sites for primer extension reaction. (B) Promoter activity in an in vivo β-galactosidase reporter gene assay and in vitro transcription (txn) assay of the wild type and mutated HIS4 and TMT1 promoter. Negative control (lane 8) is background signal of the reporter plasmid without a promoter. Specific transcripts from the HIS4 TSS and TMT1 TSS are marked by a and b, respectively.(EPS)Click here for additional data file.Figure S6GAE has no inhibitory effect on the TATA-containing HIS4 promoter. Promoter activity in an in vivo β-galactosidase reporter gene assay of the wild type and mutated HIS4 promoter. Promoter substitution constructs as indicated on the right. Negative control is background signal of the reporter plasmid without a promoter.(EPS)Click here for additional data file.Figure S7The GAE anti-correlates with SAGA-dominated genes. Percentage of Sc core promoters containing TATA box, GAE, both elements or none of the elements in SAGA- and TFIID dominated Sc promoter classes (EPS)Click here for additional data file.Table S1All Sc promoters containing a bioinformatically defined GAE.(XLS)Click here for additional data file.


Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF  family members mTTF and mTerf5 in  Drosophila. These factors, mTTF and mTerf5, share common binding sites in the mitochondrial genome, which we found to coincide with sites of replication pausing. Knockdown of either factor by RNA interference resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in random stalling and regression of replication forks, with impaired synthesis of the lagging strand. This we attribute to random collisions with the transcriptional machinery. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.All genomes require a system for preventing collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of two proteins of the mTERF  family in  The mitochondrial genome and its expression are essential to maintain oxidative phosphorylation (OXPHOS), a central metabolic process in higher eukaryotes. OXPHOS failure during development leads to developmental arrest in a diverse range of metazoans, including both insects H) L). Synthesis of the two strands on this model is asynchronous, but continuous on both strands, i.e. without a need for Okazaki fragments.In metazoans, mtDNA replication has been most extensively studied in mammals, where several competing models have been proposed. The strand-displacement model RNA Incorporation Throughout the Lagging Strand), a provisional lagging-strand, consisting of RNA segments derived from processed transcripts, is established as the replication fork proceeds L. RITOLS shares many features with the strand-displacement model, the only major difference being that the latter postulates that the parental strand displaced during heavy-strand replication remains single-stranded until the light-strand initiates. The second type of replication intermediate detected by 2DNAGE is composed fully of dsDNA, whose structure implies bidirectional initiation of replication across a wider origin zone, stretching beyond the NCR. However, termination at OH means that this mode of replication is also effectively unidirectional 2DNAGE was developed almost three decades ago, to separate and characterize branched from linear DNA Drosophila melanogaster, based both on early TEM Mitochondrial DNA replication in E. coli, for example, transcription termination is essential for the maintenance of genome integrity The coding region of metazoan mtDNAs shows a highly compact organization, with little or no non-coding sequences between genes. Typically, genes are encoded on both strands, a type of organization that unavoidably risks encounters between the transcription and replication machineries, which compete for the same template. As in other genetic systems, these processes should be subject to regulation, in order to minimize and resolve potential conflicts, including both co-directional and anti-directional collisions between the two molecular machineries. Defects in this collision regulation have been shown to cause abortive DNA synthesis, mutagenesis and genomic instability in a wide range of organisms Mterf3 and Mterf4 are essential genes Mterf2 is not in vitro at its major binding site downstream of the rRNA genes The mitochondrial transcription termination factor (mTERF) family comprises a set of mitochondrial DNA-binding proteins with diverse, documented roles in mitochondrial gene expression Drosophila, of which the best characterized is mTTF (CG18124). mTTF binds two sequence elements in Drosophila mtDNA in vitro and is required for transcriptional attenuation in vivoFour proteins of this family have been identified in in vitro to have contrahelicase activity D. melanogaster mtDNA with the regions of replication pausing identified in our earlier study suggests that mTERF family proteins could be considered as candidates for a similar role.As DNA binding proteins with an established role in the regulation of transcription, mTERF family members are strong candidates for mediating conflicts between the replisome and transcription complexes. Moreover, MTERF proteins may have multiple roles in mtDNA metabolism, considering that alterations in the levels of MTERF1 or its homologs MTERF2 (MTERFD3) and MTERF3 (MTERFD1) were reported to modulate the levels of paused replication intermediates in cultured human cells in vivo. Here we show that both factors are required for normal mtDNA topology and maintenance. Lack of either (or both) resulted in developmental arrest at L3 larval stage. mTTF knockdown led to the accumulation of nicks, dsDNA breaks and recombination junctions. 2DNAGE demonstrated stalled and reversed replication forks over broad zones surrounding the mTTF binding sites, and an accumulation of aberrant replication intermediates with extended segments of RNA/DNA hybrid, indicating a failure to complete lagging-strand DNA synthesis. Knockdown of mTerf5 had an opposite effect on mtDNA replication intermediates, bringing about an increase in replication pause strength when compared to wild-type, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species with even the short segments of RNA/DNA hybrid that we were able to detect in wild-type cells.To test the possible involvement of mTTF and mTerf5 in mtDNA replication, we investigated their effects on mtDNA metabolism after manipulation of their expression by RNAi, both in cultured cells and Because of their opposing but essential roles in mtDNA expression and synthesis, we propose that the balance of these two mTERF family members facilitates the orderly and productive passage of oppositely moving replication and transcription complexes, preventing collisions that would otherwise result in abortive replication and loss of genome integrity.D. melanogaster mtDNA were previously localized to approximately 1/3 and 2/3 of genome length from the replication origin, located in the NCR Ser(UCN) gene boundary (here designated bs1) and the tRNAPhe/tRNAGlu gene boundary  or CG5924 . The relative amount of intact mtDNA detected by Southern blot was also diminished by mTTF knockdown (Drosophila mtDNA digested with restriction enzymes curring once in the genome). Recombination structures linking two whole copies of the genome following such linearization are most easily revealed in the Bsp1407I digest, where the characteristic X-arc that they form  driver. We confirmed that the parental RNAi line (itself homozygous viable) produced normal numbers of progeny with a wild-type phenotype when mated to flies not expressing da-GAL4. We also confirmed that RNA interference in vivo produced ∼90% knockdown of mTTF at the mRNA level at larval stage  dsRNA transgene targeted on mTTF, using the ubiquitous and constitutive al stage , which wal stage . mTTF knal stage . More thMitochondrial RNA levels were altered in a similar manner as in mTTF knockdown cells, e.g. COX2 mRNA was decreased, whereas cytochrome b mRNA was elevated . Mitocho1118w ; da-GAL4/+) displayed an identical pattern of replication intermediates to that described previously for the Oregon-R wild-type strain , single-strand binding protein (mtSSB) and CCDC56, required for the assembly of cytochrome c oxidase DrosophilaDrosophila must consist of two classes, as in vertebrates. One class appears to be composed entirely of dsDNA, and is represented by the standard Y-arcs seen both before and after RNase H treatment. The second class, akin to the RITOLS intermediates seen in vertebrates Although we previously found no evidence for RNA-containing mtDNA replication intermediates in These observations raise the issue of whether transcription and DNA replication can occur simultaneously on the same template and, if so, whether this association is obligatory. The existence of a population of mtDNA molecules only able to enter agarose gels after treatment with a ribonuclease strongly suggests that these are molecules engaged in active transcription. After RNA removal, they migrate along 2DNAGE arcs expected for an iterative set of replication intermediates, strongly supporting the idea that replication and transcription can take place on the same template. Those replication intermediates that can be resolved on 2D gels without ribonuclease treatment may represent a distinct subset of replicating molecules, in which transcription is prevented. Resolving these issues will require the development of novel methods for metabolic labeling and analysis of replication and transcription intermediates.Knockdown of mTTF or mTerf5 produced specific and reciprocal effects on mtDNA synthesis. Lack of mTTF caused random stalling and fork regression, whilst decreasing those molecules specifically paused at the binding site itself. RusA treatment confirmed the presence of Holliday-like junctions, a signature of fork reversal associated with stalling due to replication impediments Some of the effects of mTTF knockdown on mtDNA replication could be indirect, e.g. resulting from altered transcript levels. However, a failure of replication due to primer insufficiency would lead to the progressive disappearance of shorter replication bubbles, rather than the accumulation of broken termination intermediates. Evidence of a role for preformed transcripts in RITOLS replication of mammalian mtDNA, via the bootlace mechanism Drosophila, as illustrated in The effects of mTTF and mTerf5 knockdown imply that RNA incorporation, replication-fork pausing and lagging-strand synthesis are related phenomena. RNA incorporated via the bootlace mechanism is one possible source of primers for the synthesis of lagging-strand DNA, although other mechanisms of lagging-strand initiation are consistent with RITOLS S. cerevisiae, Reb1 in S. pombe and TTF1 in mammals) are unrelated to the mTERF family and to each other. Thus, there is both a precedent and a rationale for mTTF and mTerf5 to integrate transcription and DNA replication.The prevention and/or regulation of collisions between the transcription and replication machineries is indispensable for all genetic systems Saccharomyces cerevisiae rDNA However, the many cases of mitochondrial proteins having multiple functions, e.g. Ilv5, Aco1 and RNase P 6 cells/ml. Standard Drosophila strains, plus the mTTF RNAi line 101656 and mTerf5 RNAi lines 2899, 2900 and 107227 from the Vienna Drosophila RNAi Center (VDRC), were cultured as described previously S2 cells Gene-specific dsRNAs were synthesized from templates created from S2 cell cDNA by a nested PCR strategy, which introduced the T7 promoter sequence on both sides of each final amplicon .Q-RT-PCR to measure RNA levels was performed essentially as described earlier Drosophila mtDNA were generated by PCR, with [α-32P]-dCTP  in the reaction mix Click here for additional data file.Figure S2XhoI, run on a 0.35% agarose gel, and probed successively for mtDNA and nuclear rDNA using PCR-derived probes for nt 9363–9888 (ND4/ND4L region) of mtDNA (NCBI Accession U37541) and nt 1953–2446 of Drosophila rDNA (NCBI Accession M21017), labeled by random-primed synthesis in presence of α-32P-dCTP and hybridized under the standard conditions A. Q-RT-PCR analysis of mTTF transcript levels in S2 cells after 3 days of dsRNA treatment against mTTF. B. Q-RT-PCR analysis of S2 cell mitochondrial transcripts transcribed in the opposite direction to that of replication fork passage, in untreated cells. C. Analysis of mtDNA copy number by Southern hybridization, following 5 d of dsRNA treatment against mTTF or an inert dsRNA targeted against GFP, as shown. Biological replicate samples were digested with (PDF)Click here for additional data file.Figure S3Comparison of 2DNAGE patterns produced by NdeI digestion in control and mTTF knockdown cells. A. Top panels reproduced from (PDF)Click here for additional data file.Figure S4in vivo, at the RNA and protein levels. A. Q-RT-PCR of mTTF mRNA in control  and mTTF knockdown  larvae. B. Western blot analysis of mTTF knockdown at the protein level in vivo. Protein extracts (25 µg) from males and females of different control strains and mTTF knockdown larvae (KD). Red arrow denotes the polypeptide corresponding with mTTF.Verification of mtTTF knockdown (PDF)Click here for additional data file.Figure S52DNAGE analysis of larval mtDNA from mTTF RNAi and control strains. Note the spreading of the signal along the Y-arc (red arrows) in larvae knocked down for mTTF, compared with the more specific pause in control larvae, plus the increase in X-structures (blue arrows).(PDF)Click here for additional data file.Figure S6RusA distinguishes the products of random collisions of the replication and transcription machineries from those of protein-mediated replication pausing. A. Unregulated collisions of the replication (blue) and transcription (green) machineries result in fork reversal, creating chicken-foot structures containing a Holliday (4-way) junction, that require a restart pathway to resume DNA replication. Protein-mediated replication pausing does not lead to fork reversal, and the paused Y-intermediate does not contain a Holliday junction. The Holliday junction formed upon fork reversal is susceptible to digestion by RusA. B. RusA cuts symmetrically in either of two modes (blue or orange arrows), degrading the chicken-foot species into 1n and sub-1n linear fragments, consistent with (PDF)Click here for additional data file.Figure S7Comparison of RusA effect on replication intermediates in control and mTTF knockdown cells (3 days after start of dsRNA treatment). Equal amounts of material from single mtDNA preparations were cut with ClaI and then treated with 0, 25 and 100 nM concentrations of RusA see . Samples(PDF)Click here for additional data file.Figure S8Additional material is resolved by 2DNAGE following RNase H treatment, as indicated. A. HindIII digest hybridized to probe 3 (two different exposures). B. ClaI digest hybridized to probe 6. Panels from (PDF)Click here for additional data file.Figure S9Absence of RNAi cross-reaction between mTerf5 and mTTF. Q-RT-PCR analysis of mTerf5 and mTTF transcript in cells after 1–5 days of RNAi treatment against mTerf5. Knockdown of mTerf5 mRNA is effective within 24 h, whereas there is no knockdown of mTTF mRNA .(PDF)Click here for additional data file.Figure S10Comparable exposures of gels (ClaI fragments and probes as indicated) from control cells and mTerf5 knockdown cells, illustrating the increase in signal from the specific pause sites (red arrows), after mTerf5 knockdown. In particular, the relative strengths of the pause signals can be judged with reference to other features of the gels that are essentially invariant, such as the linear ‘partial’ species lying on the diagonal (orange arrows). These ‘partials’ are not the result of insufficient activity of the restriction enzyme, which is always present in excess, but are a constant feature of these gels and are seen in all digests. Note that the signals from these partials are actually slightly stronger in the control panels than in the corresponding gels from mTerf5 knockdown cells, in which the pauses are clearly stronger. The invariant part of the standard Y-arc may also be used for reference, for example the initial segment of the Y-arc, which is well separated from the pause region in the bs1-containing fragment (green arrow), or the apex of the Y-arc in the bs2-containing fragment (blue arrow).(PDF)Click here for additional data file.Table S1Oligonuclelotides.(PDF)Click here for additional data file.Text S1Supplemental materials and methods and reference.(DOC)Click here for additional data file.


NAGS gene. Reporter assays confirmed that these regions represent promoter and enhancer and that the enhancer is tissue specific. Within the promoter, we identified multiple transcription start sites that differed between liver and small intestine. Several transcription factor binding motifs were conserved within the promoter and enhancer regions while a TATA-box motif was absent. DNA-protein pull-down assays and chromatin immunoprecipitation confirmed binding of Sp1 and CREB, but not C/EBP in the promoter and HNF-1 and NF-Y, but not SMAD3 or AP-2 in the enhancer. The functional importance of these motifs was demonstrated by decreased transcription of reporter constructs following mutagenesis of each motif. The presented data strongly suggest that Sp1, CREB, HNF-1, and NF-Y, that are known to be responsive to hormones and diet, regulate NAGS transcription. This provides molecular mechanism of regulation of ureagenesis in response to hormonal and dietary changes.The urea cycle converts toxic ammonia to urea within the liver of mammals. At least 6 enzymes are required for ureagenesis, which correlates with dietary protein intake. The transcription of urea cycle genes is, at least in part, regulated by glucocorticoid and glucagon hormone signaling pathways. N-acetylglutamate synthase (NAGS) produces a unique cofactor, N-acetylglutamate (NAG), that is essential for the catalytic function of the first and rate-limiting enzyme of ureagenesis, carbamyl phosphate synthetase 1 (CPS1). However, despite the important role of NAGS in ammonia removal, little is known about the mechanisms of its regulation. We identified two regions of high conservation upstream of the translation start of the  Ammonia, the toxic product of protein catabolism, is converted to urea by the urea cycle in the liver of mammals. Incorporation of two nitrogen atoms into urea is catalyzed by six enzymes: three of them mitochondrial, N-acetylglutamate synthase , carbamylphosphate synthetase 1  and ornithine transcarbamylase , and the other three cytosolic, argininosuccinate synthetase , argininosuccinate lyase  and arginase 1 .cis-acting motifs regulating transcription of the urea cycle enzymes CPS1 NAGS gene was not identified until 2002 NAGS catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of CPS1, in the mitochondrial matrix of hepatocytes and small intestine epithelial cells CPS1 is activated by TATA-binding protein (TBP) while its proximal and distal enhancers contain binding sites for glucocorticoids and cAMP responsive factors including CCAAT-enhancer bind protein (C/EBP), activator protein-1 (AP-1), glucocorticoid receptor (GR) and cAMP response element binding (CREB). Sites for binding tissue specific factors including hepatic nuclear factor 3 (HNF-3) are also present OTC gene is induced in the intestine and liver by HNF-4, which binds in the promoter ASS transcription is regulated by cooperative binding of multiple specificity protein 1 (Sp1) ASL is regulated through Sp1 and the positive regulator, nuclear factor Y (NF-Y), which binds within the promoter of ASL to activate its transcription ARG1 transcription while two C/EBP factors and two unidentified proteins bind within an enhancer in intron 7 to confer glucocorticoid responsiveness Many studies have identified regulatory links between the urea cycle genes and glucocorticoids and glucagon NAGS could contribute to the understanding of such mechanism.Abundance of urea cycle enzymes correlates with dietary protein intake NAGS promoter and Hepatic Nuclear Factor 1 (HNF-1) and NF-Y within the enhancer regions. Chromatin immunoprecipitation (ChIP) and quantitative real-time PCR have been used to independently verify that Sp1 and CREB bind to the promoter region, and HNF-1 and NF-Y bind to the enhancer region. We also used 5′RACE analysis to identify multiple transcription start sites for NAGS that may be species and tissue specific. These findings provide new information on the regulation of the NAGS gene, and suggest possible mechanisms for coordinated regulation of the genes involved in ureagenesis.In this study, we identified two regulatory regions upstream of the NAGS translation start site that contain highly conserved protein-binding DNA motifs. We subsequently confirmed that these regions function as promoter and enhancer and that the enhancer is most effective in liver cells. Avidin-agarose protein-DNA pull-down assays have been used to confirm binding of Sp1 and CREB within the NAGS translational start site and sequence of intron one in 7 mammalian species including: human (NM_153006.2), chimpanzee (XM_001152480.1), dog (XM_548066.2), cow (XM_618194.4), horse (XM_001917302.1), mouse (NM_145829.1) and rat (NM_001107053.1). The highly conserved regulatory regions of CPS1 were identified by gathering 15 kilobases of genomic sequences 5′ of the translational start site from human (NM_001875), chimpanzee (XM_001146604), dog (XM_856862), mouse (NM_001080809), and rat (NM_017072). Genomic sequences were subject to pair-wise comparison using BLAST bl2seq tool Identification of highly conserved regions was conducted by gathering 15 kilobases of genomic sequence 5′ of the Cis-element OVERrepresentation (CLOVER) NAGS and CPS1. CLOVER analysis of these conserved regions identified known protein binding DNA motifs in the TRANSFAC Pro database by calculating over-representation of these sequences compared to a background of ppr_build_33.fa generated from NCBI build 33 The NAGS, were amplified from human genomic DNA with primer pairs hPromXH and hEnhXH or hPromHXrev and hEnhHXrev  was used for amplification with the following conditions: initial denaturation at 95°C for 2 min., followed by 35 cycles of denaturation at 95°C for 30 sec., annealing at 57°C for 30 sec. and extension at 68°C for 1 min., and final extension at 68°C for 6 min. Promoter and enhancer PCR products were ligated with TOPO-TA sequencing vector (Invitrogen) according to manufacturer's instructions and referred to as TOPOProm, TOPOEnh, TOPOPromRev, and TOPOEnhRev, respectively. Mouse Nags (mNags) promoter and enhancer were inserted into TOPO-TA vector following the same methods. Correct DNA sequences were confirmed using sequencing primers specified by Invitrogen.The promoter and enhancer of EnhHXrev , respectPhotinus pyralis) luciferase luc2, and pGL4.23 (Promega) vector containing a minimal TATA promoter with luc2 were cut with XhoI (New England Biolabs) and HindIII (New England Biolabs). The vectors were treated with Antarctic Alkaline Phosphatase (AAP) (New England Biolabs) according to manufacturer's instructions, and the NAGS regions were ligated with the vectors to form the plasmids in BamHI and SalI restriction enzyme sites at the 5′ and 3′ ends of the enhancer, respectively. The amplified enhancer product and 4.10Prom were cut with BamHI (New England Biolabs) and SalI (New England Biolabs), the vector was treated with AAP, and the enhancer was ligated with the vector  basic vector containing firefly  according to manufacturer's instructions. Primers hCREBm Fw and Rv .2 at 37°C. Human alveolar basal epithelial cells (A549)  were cultured in complete media containing Ham's F-12 medium (Invitrogen) supplemented with 10% FBS and 5% Penicillin/Streptomycin. Human colorectal adenocarcinoma cells (Caco-2) (ATCC) were cultured in Eagle's Minimum Essential Medium (Invitrogen) supplemented with 20% FBS. Cells were plated at a density of 5×105 cells/well on 24-well culture plates 24 hours prior to transfection. The cells  were then transfected using Lipofectamine 2000 reagent (Invitrogen) and cultured in transfection media containing medium and serum only. A total of 0.25 ug of DNA was transfected with 0.225 ug of vector expressing luc2 and 0.025 ug of pGL4.74 vector containing Renilla reniformis luciferase (hRluc) as an internal control (Promega). For co-transfections 0.225 ug of luc2 vector was combined with either 0.25 ug of expression vector or empty vector pUC19 (Invitrogen), and 0.025 ug of hRluc control vector.Human hepatoma cells (HepG2)  were cultured in complete media containing RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (ATCC) and 5% Penicillin/Streptomycin (Invitrogen) under 5% CORenilla luciferase activity using Dual-Luciferase Reporter Assay System (Promega) and Berthold Centro 960 luminometer (Berthold) according to the manufacturer's protocol. All reporter assay measurements were corrected for transfection efficiency by normalizing the firefly luciferase signal to the Renilla luciferase values. Expression level of each construct was determined relative to luciferase expression under control of the NAGS promoter in each cell line. All results are an average of three independent experiments that were each carried out in triplicate. Values were expressed as mean ± SEM and analyzed using Student's t-test.24 hours following transfection, cells were assayed for both firefly and NAGS specific primers complementary to sequence within Exon 1  was performed using RNA isolated from donated mouse livers by Trizol reagent (Invitrogen). RNA from mouse small intestine (Origene), human duodenum (Ambion), or human liver (Ambion) was commercially available. Products were synthesized with human or mouse n Exon 1 . All reaNags (mNags) promoter regions A and B . Probes were generated using biotinylated or non-biotinylated forward primer and non-biotinylated reverse primers with Platinum Taq PCRx DNA Polymerase (Invitrogen) and amplification conditions: initial denaturation at 95°C for 2 min., followed by 35 cycles of denaturation at 95°C for 30 sec., annealing at 60°C for 30 sec. and extension at 68°C for 1 min., and final extension at 68°C for 6 min. The mouse  A and B  were amp A and B . A regioNuclear extract was isolated from donated adult mouse livers of C57BL/6 mice using Nuclear Extraction Kit (Origene) according to manufacturer's instructions. The protein concentration of the nuclear extract was determined using bovine serum albumin as the protein standard with Bradford Assay dye concentrate reagents (Bio-Rad). On average, 10 mg of nuclear protein was obtained from mouse liver.For the avidin-agarose protein-DNA pull-down assay Donated livers from adult C57BL/6 mice were minced and chromatin was precipitated using SimpleChIP Enzyme Chromatin Kit (Origene) with the variation for whole tissue. Briefly, fresh tissue was minced and washed with PBS including Protease Inhibitor Complete tablets (Roche). Proteins and DNA were cross-linked with 1.5% formaldehyde, and tissue was disaggregated with dounce homogenizer. Chromatin was sheared to an approximate size of 100–1000 bp by micrococcal nuclease digestion followed by sonication. Immunoprecipitation was conducted using antibodies to transcription factors Sp1 (Millipore), CREB (Santa Cruz Biotech), C/EBP (Santa Cruz Biotech), HNF-1 (Santa Cruz Biotech), NF-Y (Santa Cruz Biotech), SMAD2 (Santa Cruz Biotech) and AP-2 (Santa Cruz Biotech) and control antibodies to histone H3 and non-specific rabbit IgG . Chromatin was eluted from protein G agarose beads, cross-linking was reversed, and DNA was purified according to manufacturer's instructions.t-test.ChIP enriched DNA samples included 2% input control and dilutions for a standard curve, positive control immunoprecipitate from anti-histone H3 antibody sample, negative control immunoprecipitation from anti-rabbit IgG antibody, no antibody control, water control, and test antibodies. Enriched DNA was subject to quantitative real-time PCR using iTaq SYBR Green Supermix with ROX (Bio-Rad) and gene specific primers  includinNAGS and sequence of the first intron from human, chimpanzee, dog, horse, cow, mouse and rat were aligned and compared using pair-wise BLAST. Comparisons showed three highly conserved regions upstream of human NAGS at −57 to −284, −498 to −576, and −2978 to −3344 relative to the start ATG, and no significant conservation within the intron or between −5 and −15 kb upstream (NAGS genes using phastCons (green) and phyloP (blue), which identified three non-coding regions of conservation located 3 kb upstream, immediately upstream, and within the first intron of NAGS, respectively , control reversed promoter (4.10PromRev), enhancer alone (4.10Enh), promoter and enhancer (4.10PromEnh), and enhancer in both orientations with the heterologous TATA-box promoter (4.23Enh and 4.23EnhRev) by measuring the expression of a luciferase reporter gene in cultured HepG2 cells , stimulated transcription of the luciferase gene while the upstream regulatory region (plasmid 4.10Enh) alone, did not (NAGS promoter and upstream regulatory region were both present (4.10PromEnh plasmid), transcription increased by 50% compared to the promoter alone confirming that the upstream conserved region can function as an enhancer of transcription. When the NAGS enhancer was paired with a heterologous promoter containing a TATA-box, in the 4.23Enh construct, the transcription of luciferase about three times higher compared to construct with minimal TATA-box. The backbone vector 4.10 did not stimulate expression of the luciferase gene. As expected, positive control vector 4.13, containing a strong promoter, activated transcription in this cell culture system (NAGS promoter acts in a direction dependent manner (NAGS enhancer (4.23EnhRev) to stimulate transcription with the heterologous promoter was orientation independent  in human and mouse liver and small intestine were identified using 5′ RACE  and expressed sequence tags (ESTs) from Genbank. Results suggest that NAGS has multiple TSS and that some may be species and tissue-specific. Combined 5′RACE, DBTSS, and Genbank results indicate that within human liver, the most frequently occurring TSS was at −42 bp upstream of the ATG codon, while in human small intestine it was at −146 bp  followed by Real-Time PCR. Measurement compared the enrichment of target DNA regions to the negative control locus MIP-2. ChIP with Sp1 and CREB antibodies significantly enriched the  p>0.05; . The NAGReporter assays in liver hepatoma cells with mutated transcription factor binding motifs demonstrate the functional importance of each site. Following these sequence substitutions, transcription factor binding motifs were no longer detected by CLOVER . Within NAGS promoter (4.10Prom) increases expression of luciferase more than 50%  increased expression of the reporter gene by 50% in liver derived cells  was selected to identify conserved regions that could support multiple binding sites where complexes of transcription factors may form In this study we used bioinformatic analyses to predict regulatory regions based on the hypothesis that non-coding DNA sequences that are highly conserved between species are important for gene regulation. Multiple pair-wise BLAST alignments and sequence alignment from the UCSC genome browser were used to identify two conserved regions within NAGS in vivo. Similarly, the enhancer in either orientation increases expression of luciferase by approximately 50% relative to the promoter alone, suggesting that it stimulates NAGS transcription as well. The relatively small but significant effect of the enhancer could be due to spacing differences between the genomic NAGS promoter and enhancer and their spacing in the reporter constructs. Alternatively, while HepG2 cells express transcription factors that we identified using bioinformatic tools, the NAGS enhancer may bind additional factors, absent in HepG2 cells, and have larger effect in vivo than in cultured cells. Another explanation for the relatively small effect of the NAGS enhancer is the possible presence of a proximal enhancer within the region we termed the promoter. Additional experiments are necessary to distinguish between these two possibilities.The reporter assay results confirm that the two highly conserved regions within 1 kb and 3 kb upstream of the translational start site function as promoter and enhancer, respectively. The promoter activates expression of the luciferase reporter gene and we therefore infer that it will activate transcription of NAGS transcriptional start sites identified multiple TSS that may be species and tissue specific. While the function of each TSS is unknown, these results are consistent with transcription initiation by Sp1 Our analysis of the NAGS in these regions. By filtering for the highly over-represented and spatially conserved binding sites, relative to the translational start codon, we identified Sp1, CREB, and C/EBP in the promoter and HNF-1 AP-2, NF-Y, and SMAD-3 in the enhancer as transcription factors that could bind to the NAGS upstream region. This filtering method was confirmed by analysis of the −6.3 kb enhancer of CPS1 in which binding sites for the previously published C/EBP, CREB, GR, and HNF-3 were identified.After we confirmed that the promoter and enhancer initiate and increase transcription, we looked for transcription factors that bind and regulate NAGS promoter and enhancer, while we could not detect binding of C/EBP, AP-2 and SMAD3  gene, which is on the reverse strand. This overlap was identified with a PYY cDNA isolated from a brain astrocytoma cDNA library that has an 80 nucleotide long exon located between regions A and B of the NAGS promoter PYY transcripts initiate about 500 bp upstream of the PYY coding region, which is located 51 kb upstream of the NAGS translation initiation codon. Recent analysis of human transcripts revealed that many protein coding loci are associated with at least one transcript that initiates from a distal site NAGS and PYY genes raises the interesting possibility that these two genes share cis-acting regulatory elements and might be co-regulated NAGS and PYY is likely to be complex because of their differing tissue expression patterns PYY is expressed in the intestinal neuroendocrine cells NAGSOTC and CPS1NAGS and PYY genes; they are located approximately 9.5 and 21 kb upstream of the NAGS coding region. The CTCF binding sites could act as chromatin insulators PYY by the NAGS enhancer or enable cell type specific regulation of each gene by the NAGS enhancer and promoter. Our results show that the NAGS promoter in the reverse orientation does not activate transcription of the reporter gene in liver derived cells  and phyloP (blue) from the UCSC genome browser indicate regions that are highly conserved across all mammals (A). Pair-wise blast analysis of human, chimpanzee, dog, mouse, and rat 5′ non-coding region of CPS1 were used to identify two known and three previously unknown regions of high conservation, referred to enhancer/repressor regions A, B, and C. Highly conserved regions within the CPS1 5′ non-coding sequence include the proximal promoter, region A, the -enhancer, region B, and region C.(TIF)Click here for additional data file.Figure S2Highly conserved regulatory regions, upstream of the mouse Nags gene, function as promoter and enhancer elements. Mouse promoter (m4.10Prom), promoter and enhancer (m4.10PromEnh), and enhancer with TATA promoter (m4.23Enh) stimulated transcription while enhancer lacking a promoter (m4.10Enh) did not in liver cells. Calculated results are an average of three independent experiments that were carried out in triplicate, normalized to Rluc expression, and expressed relative to the promoter for each experiment with error reported as ±SEM.(TIF)Click here for additional data file.Figure S3Novel transcription factor binding motifs, in the enhancer region of CPS1, were identified using CLOVER. Several highly conserved transcription factor binding sites were present in the enhancer region. An asterisk denotes an experimentally verified transcription factor binding site. All motifs were spatially conserved between mammalian species.(TIF)Click here for additional data file.Table S1Sequences of primers that were used to amplify human or mouse DNA by PCR for insertion of the promoter and enhancer regions into sequencing and reporter assay vectors.(DOCX)Click here for additional data file.Table S2Primer sequences used to determine transcription start sites of NAGS with 5′ RACE. Primers were designed according to manufacturer's instructions and used to determine transcription start sites of human and mouse NAGS in liver and small intestine RNA using 5′ RACE.(DOCX)Click here for additional data file.Table S3Primer sequences used to generate DNA probes of the specified regions of mNags. Primers were used to generate DNA probes, by PCR, of the promoter, enhancer, or non-specific specified regions of mNags.(DOCX)Click here for additional data file.Table S4Primer sequences used for quantitative real-time PCR analysis of chromatin immunoprecipitation samples.(DOCX)Click here for additional data file.Table S5Results of CLOVER analysis of the enhancer region with sequence information for human and mouse CPS1. Results were filtered to exclude motifs for transcription factors that are not expressed in the liver.(DOCX)Click here for additional data file.Table S6Results of CLOVER analysis of the promoter region with sequence information for human and mouse NAGS. Results were filtered to exclude motifs for transcription factors that are not expressed in liver.(DOCX)Click here for additional data file.Table S7Results of CLOVER analysis of the enhancer region with sequence information for human and mouse NAGS. Results were filtered to exclude motifs for transcription factors that are not expressed in the liver.(DOCX)Click here for additional data file.