Neurog2 enhancer that requires direct input from Otx2, Foxa1, Foxa2 and an E-box transcription factor for its transcriptional activity. Furthermore, the chromatin remodelling factor Smarca1 was shown to function downstream of Foxa1 and Foxa2 to regulate differentiation from immature to mature midbrain dopaminergic neurons. Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons.Midbrain dopamine neuronal progenitors develop into heterogeneous subgroups of neurons, such as substantia nigra pars compacta, ventral tegmental area and retrorubal field, that regulate motor control, motivated and addictive behaviours. The development of midbrain dopamine neurons has been extensively studied, and these studies indicate that complex cross-regulatory interactions between extrinsic and intrinsic molecules regulate a precise temporal and spatial programme of neurogenesis in midbrain dopamine progenitors. To elucidate direct molecular interactions between multiple regulatory factors during neuronal differentiation in mice, we characterised genome-wide binding sites of the forkhead/winged helix transcription factor Foxa1, which functions redundantly with Foxa2 to regulate the differentiation of mDA neurons. Interestingly, our studies identified a rostral brain floor plate  Summary: ChIP-Seq and RNA-Seq experiments identify novel molecular mechanisms underlying midbrain dopaminergic neuron production downstream of Foxa1 and Foxa2 during mouse neurogenesis. The development of midbrain dopaminergic (mDA) neurons from progenitors in the caudal diencephalon and midbrain floor plate is a multistep process involving proliferation, specification, differentiation and axon pathfinding, which ultimately result in the formation of functional dopamine circuits. These brain circuits govern motivated and voluntary motor behaviour and are implicated in neurological disorders such as Parkinson's disease  and schiNeurog2 and Ascl1 exhibit complete loss of all mDA neurons in mice (Foxa1 and Foxa2 (Foxa1/2) have overlapping roles in the specification and differentiation of mDA neurons . Previou in mice . DespiteNeurog2 enhancer that requires direct input from Otx2, Foxa1/2 and an E-box transcription factor for its transcriptional activity. In addition, we found that Foxa1/2 directly regulate the expression of an epigenetic regulator, Smarca1 . Smarca1, an ISWI chromatin remodelling protein with multiple functions including nucleosome assembly/spacing during replication and transcriptional regulation, is known to regulate neuronal differentiation through repression of expression of the transcription factor Foxg1 (We therefore set out to discover the molecular mechanisms of how Foxa1/2 regulate neurogenesis in mDA cells by performing ChIP-Seq for these transcription factors on embryonic day (E) 12.5 mDA progenitors and neurons. Our results identified a rostral brain floor plate or Foxg1 . Recentlor Foxg1 . HoweverIn summary, our genome-wide studies of Foxa1 in embryonic mDA progenitors and neurons successfully identified an important regulatory enhancer and a novel chromatin factor facilitating mDA neurogenesis. These results attest to the high quality of our genome-wide data set that will serve as an excellent resource for mechanistic insights into the process of mDA neuronal differentiation.supplementary material Table S1) with FDR <5%, whereas the Foxa2-specific antibody identified only 2105 regions , we decided to use the Foxa1 ChIP-Seq data set for downstream analysis. 1427 of the 2105 (68%) Foxa2-bound regions overlapped with Foxa1-bound regions from the in vivo ChIP-Seq experiments with Foxa2 and Foxa1 antibodies, respectively, confirming high-quality reproducible datasets . 2171 Foxa1-bound regions overlapped with Foxa2-bound regions in mDA progenitors from in vitro generated mDA progenitors by differentiation of mouse embryonic stem cells (ESCs), as published previously    identified 7348 Foxa1-bound regions (able S3) . In addi regions C. Since  regions . Interesable S3) .Fig. 1.flox/flox;Foxa2flox/floxNestinCre;Foxa1 double-mutant embryos  at E12.5. In contrast to previously published work in which the lacZ/lacZ;NestinCre;Foxa2flox/floxFoxa1 mutants analysed .To identify functionally relevant target genes, we next overlapped the ChIP-Seq data set with a list of genes differentially expressed in E12.5 mDA cells in the presence or absence of Foxa1/2. The latter gene list was derived by using RNA-Seq to compare the transcriptome of mDA cells in the ventral midbrain of control versus analysed  had a nuNestinCre;Foxa1/2 cko embryos revealed 164 differentially expressed and Foxa1-bound genes . ChIP-Seq and RNA-Seq data of Smarca1 and Ferd3l are shown in Combining the associated Foxa1-bound genes and RNA-Seq data from wild-type versus supplementary material Table S5).We then surveyed the functional annotations of these putative Foxa1/2 targets using Gene Ontology (GO). A wide spectrum of biological processes were significantly enriched, including neuron differentiation, neuron projection morphogenesis and axon guidance, that are consistent with established roles for Foxa1/2 in mDA neurons  raised the possibility that Foxa2 might directly activate Neurog2 expression through this putative enhancer region in embryonic mDA progenitors  revealed, as expected, one putative forkhead binding site; however, the presence of highly conserved binding sites for homeodomain and bHLH transcription factors within this enhancer , five previously known Foxa1/2 downstream target genes, namely Ferd3l , Foxa1, Foxa2, Lmx1b and Nurr1 , already have defined roles in mDA neurogenesis , we also analysed Neurog2 expression by in situ hybridisation. Neurog2 expression was reduced in mDA progenitors in the floor plate and almost completely abolished in the basal progenitors of mutant embryos . Nurr1 labels both immature and mature mDA neurons, whereas Th specifically labels mature mDA neurons. Although the total numbers of Nurr1at E12.5 A. We con embryos B. By conat E16.5 .Fig. 6.Smarca1 mutants at E12.5, which appears to be rescued by E16.5. The defect probably occurs during the differentiation from immature to mature mDA neurons, as no obvious changes in the expression of progenitor markers, such as Sox2, Lmx1a, Lmx1b, Neurog2 and Corin, could be detected in Smarca1 mutants as compared with control embryos .These results indicate that there is a partial delay in the maturation of mDA neurons in Smarca1 expression construct. These Smarca1 gain-of-function ESCs were differentiated first into epiblast cells. These cells were then grown in differentiation medium alone or in the presence of doxycycline so that Smarca1 expression was induced in postmitotic mDA neurons at day 5 of monolayer differentiation, and the cells were then cultured to day 14 to generate βIII-tubulin+ Th+ neurons (+ neurons (37%) were observed in the parental or non-induced control cultures (+ neurons (62%) differentiated in monolayer cultures overexpressing Smarca1  and Vmat2 (Slc18a2), was also enhanced in cultures with induced Smarca1 expression compared with control cultures  that were associated with 4556 genes were discovered in the ChIP-Seq experiments, while 164 differentially expressed candidate Foxa1/2 target genes were identified by RNA-Seq experiments. As far as we know, these are the first ChIP-Seq and RNA-Seq data sets from embryonic mDA cells and therefore they provide unique and useful resources for probing molecular interactions during the differentiation of mDA cells in embryos and also from ESCs. We focused on two Foxa1/2-bound targets, namely Neurog2 and Smarca1, that are unique to the in vivo data set. Our results have provided new mechanistic insights into how Foxa1/2 regulate the differentiation of mDA neurons.We have previously published a list of candidate Foxa1/2-bound and regulated genes in mDA progenitors generated by use ESCs . Here, wNeurog2. However, it is not known whether these genes directly regulate Neurog2 expression. Our studies show that Otx2, Foxa2 and an E-box transcription factor directly bind to a Neurog2 enhancer and are required for its transcriptional activity in the midbrain floor plate in mouse embryos at 12.5. These results strongly suggest that these transcription factors cooperate on cis-regulatory sequences to regulate the differentiation of mDA progenitors in the ventral midbrain. Whether the E-box transcription factor corresponds to Ferd3l remains to be determined owing to the lack of a ChIP grade Ferd3l-specific antibody.Previous studies have shown that the floor plate progenitors rostral to the mid-hindbrain boundary undergo neurogenesis following the activation of Neurog2 and Ascl1 in these progenitors . Experiments are currently on-going to determine whether Smarca1 and Smarca5 share redundant roles in the differentiation of mDA neurons. Independent support for the role of Smarca1 in neuronal differentiation was obtained using gain-of-function experiments in mouse epiblast stem cells. The effect of Smarca1 on neuronal differentiation in vitro could be due to the induction of neuronal differentiation and/or the induction of a dopaminergic (DA) phenotype in mDA neurons.The transient role of Th and Dat (Slc6a3), during the differentiation of mDA neurons  was used as a negative control for non-specific enrichment. The fold enrichment of each target site was calculated as 2 to the power of the cycle. The enrichment of target sequences in ChIP material was calculated relative to input.Foxa1 ChIP experiments were performed as described . Brieflysupplementary material Table S1. Foxa1-associated and Foxa2-associated genes were assigned by mapping peaks to the closest transcription start site of Ensembl genes. ChIP-Seq data are available at Array Express under accession number E-MTAB-3332.The ChIP-Seq experiments and analysis were performed using Foxa1 and Foxa2 antibodies  accordinE12.5 wild-type and mutant embryos were snap-frozen in dry ice-cooled isopentane. Then 10 µm cryostat coronal sections of the midbrain were cut and mounted on membrane glass slides (Zeiss 415101-4401-000) at −20°C. LCM of the floor plate was performed immediately after section mounting on slides. RNA was extracted using the Pico RNA Isolation Kit (Arcturus Engineering). The RNA-Seq library was prepared using the Ovation RNA-Seq system (Nugen). RNA-Seq libraries were sequenced on an Illumina GAIIx and analysis was performed using CLC Genomics Workbench version 1 (CLC BIO). RNA-seq data are available at Array Express under accession number E-MTAB 3333.x is the number of genes shared by two gene lists (91), n is the number of genes identified by ChIP-Seq shared by Foxa1 and Foxa2 (1824), M is the number of genes of the second list (444), and N is the number of all Ensembl genes  . Note that the number of genes was used rather than the probe number (entities) as provided by the microarray from http://genome.crg.es/GOToolBox), using the MGI identities of the list of genes. Gene lists and GO term results are provided in supplementary material Table S4.Significance of enrichment between any two sets of genes was determined by applying the hypergeometric distribution as described by lacZ primers (5′-3′): forward, CGAGTGTGATCATCTGGTCG; and reverse, TTACCTTGTGGAGCGACATC. E12-12.5 embryos were harvested in cold PBS and fixed for 30 min at room temperature in 4% formaldehyde in PBS (pH 7.2). Whole-mount β-galactosidase staining and analysis of the embryos were performed as described . Transgenic mice were generated by standard procedures  using feescribed . Reportet-test was used to calculate statistical significance. The antibodies and dilutions used are described by Immunohistochemistry was performed as described . Quantithttp://genome.ucsc.edu/). All regulatory sequences assayed were cloned into the XbaI site of a reporter vector comprising the β-globin promoter, lacZ cDNA, SV40 large T-antigen poly(A) site and sequences mediating Neurog2 enhancer activity in an arrangement that retains the native orientation. The DNA sequence tested was in chromosome position chr3:127,349,185- 127,349,631 and was generated by PCR amplification using primers (5′-3′): forward, TCTAGAAATCTGTAAACCCATCACATG; reverse, TCTAGAAGAGGCCACCCTCTCCCCTC. Point mutations designed to disrupt DNA binding at the recognition sequences for Otx2, Foxa1 and the E-box were performed using the QuikChange Mutagenesis Kit (Agilent Technologies) with the following primers (5′-3′): Otx2, GGGCGGGGGCTTAGAAAAAGTCTCGGCTCTTCATGAATAG; Foxa1, GATGAGTGCCGACGGCGTACCCACCTTCATGAGCTAAAAG; E-box, CGTCATCTCAAAGCCGCATCTTTGCCATTGAGCCGAAAAG (mutated residues are underlined). The integrity and orientation of all constructs containing PCR-generated fragments was confirmed by DNA sequencing.Regulatory sequences beneath Foxa1 peak regions called by MACS were selected according to the limits defined by the highest sequence conservation among multiple vertebrate species from the UCSC genome browser  and mouse anti-Tuj1 . For quantitative analysis, DAPI-stained colonies were chosen randomly from three independent experiments of each condition and the percentage of cells expressing Th was determined. Two-tailed Student's in situ hybridisation of E12.5 mouse brains was performed as described .Section escribed . The fol


Zfy1 and Zfy2 as the Yp genes promoting the second meiotic division. Using the same mouse models we here show that Zfy2 (Zfy1but not ) contributes to the restructuring of the sperm head and is required for the development of the sperm tail. The preferential involvement of Zfy2 is consistent with the presence of an additional strong spermatid-specific promotor that has been acquired by this gene. This is further supported by the fact that promotion of sperm morphogenesis is also seen in one of the two markedly Yp gene deficient models in which a Yp deletion has created a Zfy2/1 fusion gene that is driven by the strong Zfy2 spermatid-specific promotor, but encodes a protein almost identical to that encoded by Zfy1. Our results point to there being further genetic information on Yp that also has a role in restructuring the sperm head.A previous study indicated that genetic information encoded on the mouse Y chromosome short arm (Yp) is required for efficient completion of the second meiotic division (that generates haploid round spermatids), restructuring of the sperm head, and development of the sperm tail. Using mouse models lacking a Y chromosome but with varying Yp gene complements provided by Yp chromosomal derivatives or transgenes, we recently identified the Y-encoded zinc finger transcription factors  Zfy, which together with the closely related X chromosome linked Zfx, encode zinc finger transcription factors [Zfy1 and Zfy2) is restricted to spermatogenic cells [Zfy2 encodes a much more potent transcription factor than Zfy1 [Zf’ genes were identified in 1989–1990 [The mouse Y chromosome has two copies of a gene,  factors –3. They  factors , 5. Postic cells –8. Zfy2 han Zfy1 , 10 and han Zfy1  suggesti989–1990  only rec989–1990 –14.Zfy with respect to the apoptotic elimination of spermatocytes with univalent chromosomes at the first meiotic metaphase [Zfy transgene additions to Yp gene deficient mice; this will be unfamiliar to most readers and can be accessed via the link http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=24967676.This study is a sequel to our two earlier papers documenting roles for mouse etaphase  and for etaphase . The latEif2s3yOSry males  [X  whether the Y*X addition per se had any discernible effect on spermiogenic progression by comparing XEOSry with XEY*XSry; and (2) whether the markedly increased haploid frequency associated with the Y*X addition in the context of Sxrb had any discernible effect on spermiogenic progression by comparing XESxrbO with XEY*XSxrb.For our published study showing that ermatids  we addedtids [X) , 17) to s [X) [X  comprisee MI SAC . We abbreference ). TherefEY*XSry males proved to be equivalent to that in XEOSry males  there is a clear-cut and unique arrest of spermiogenesis at step 7 of spermatid development [EY*XSry males that have the same Y gene complement, but also have the minute Y*X chromosome that enables the formation of a sex bivalent, thus avoiding MI SAC responses. The transition from spermatid step 7 to step 8 is pivotal in spermiogenesis: at step 7 the spermatids are round and randomly oriented with respect to the basal membrane, whereas at step 8 the spermatids have re-orientated so that the developing acrosomal cap is now facing the tubule periphery. This re-orientation heralds the dramatic restructuring of the round spermatids to form spermatozoa (‘sperm morphogenesis’). We hypothesized that the arrest at step 7 was a consequence of a lack of Zfy2-dependent transcriptional changes, and that the expression of Zfy2 at the critical stage was dependent on the alternative, strong, Cypt-derived, spermatid-specific promotor acquired by Zfy2 during the evolution of mice (muridae) [Zfy2 supports sperm morphogenesis, while Zfy1, which lacks this promotor, does not.The starting point for the present study was our finding that in Xelopment . In the muridae) . Our preZfy1 and Zfy2, together with their X-linked homologue Zfx, are predicted to bind the same DNA sequence [Zfx and Zfy are widely expressed, and Zfx is exempt from X dosage compensation, suggesting a constraining dosage requirement in somatic tissues [Zfx became subject to X-dosage compensation and the Zfy-encoded proteins diverged [Zfy with postnatal transcription restricted to spermatogenic cells [Cypt-derived promotor in the muridae [Sly and Slx being key protagonists [Sly and Slx  with Slermatids . ProgresCypt-derived Zfy2 promotor was triggered by our finding of substantial sperm morphogenesis in XEif2s3yY*XSxrb males in which a 1.3 Mb Yp deletion .Our original focus on the alternative n domain . Our prify2 see , Fig 4B)Cypt-deriEif2s3y,Zfy2Y*XSry males is inferior to that in XY*XSxra males points to there being other Yp genes that contribute to sperm morphogenesis. We have generated transgenic lines that express other Yp-located genes  that may have functions during spermiogenesis. Since our current study was completed two more male germ-line expressed genes, Prssly and Teyorf1, have been mapped to Yp [Sxra  males carry an X-located transgene encoding the spermatogonial proliferation factor Eif2s3y [Sry [X Eif2s3y  and an as3y [Sry , 42.Eif2s3yY*XSry (abbreviated as XEY*XSry) males have the same Y gene complement as XEOSry but carry a minute X chromosome derivative (Y*X) with a complete pseudoautosomal region (PAR) [Xon (PAR) –20.Eif2s3ySxrbO (abbreviated as XESxrbO) males have the X chromosome carrying an Eif2s3y transgene [Sxr-b attached distal to the X PAR. Tp(Y)1CtSxr-b is an Sxra derivative with a 1.3 Mb deletion that has removed the majority of the Yp gene complement and created a Zfy2/1 fusion gene [Xransgene  togetherion gene , 43.Eif2s3yY*XSxrb (abbreviated as XEY*XSxrb) have the same Y gene complement as XESxrbO but also carry Y*X.XSxraO have the X chromosome carrying the Yp derivative Tp(Y)1CtSxr-a [X1CtSxr-a  attachedXSxra males have the same Y gene complement as XSxraO but also carry Y*X.XY*Eif2s3y,Zfy1Y*XSry  males are genotype 2 males to which a single copy, X chromosome-located, Zfy1-Uba1y BAC (RP24-327G6) transgene [Xransgene  has beenEif2s3y,Zfy2Y*XSry  males are genotype 2 to which a single copy Zfy2 BAC transgene inserted by cassette mediated exchange (CME) into the Hprt locus on the X chromosome [Xromosome , 13 has The mice were fed ad libitum with a standard diet and maintained in a temperature and light-controlled room . The protocols for animal handling and treatment procedures were reviewed and approved by the Crick Biological Research Facility Strategic Oversight Committee (BRF-SOC) and Animal Welfare and Ethical Review Body(AWERB), in accordance with the United Kingdom Animal Scientific Procedures Act 1986 (Procedure Project Licence: 80/2186).For histology analysis, part of the testes were fixed in Bouin overnight and then stored in 70% ethanol prior to embedding in paraffin wax, sectioning at 5 μm, and staining with hematoxylin-eosin (H&E) or periodic acid Schiff and hematoxylin (PAS-H). The stages of seminiferous tubules were identified based on the composition of cells near the basal membrane according to the method described by Ahmed . This waSperm smears obtained from the testis were silver stained and analyzed as described previously .Mice were perfused, through the left ventricle , with icLemd1 in each sample. For further details see Band intensity was quantified using ImageJ, background subtracted and intensity normalised to Prssly and Teyorf1. The controls are Tspy-ps, present in the Sxrb deletion interval, and the autosomal gene Atr. For primers and annealing temperatures see Standard PCR on genomic DNA was used to map the Y chromosome genes S1 Appendix(DOCX)Click here for additional data file.S1 Fig(A) Genomic structure of the mouse Zfy1, Zfy2 and Zfy2/1 fusion genes are shown. Exons are represented by boxes (black for Zfy2 and white for Zfy1) and are not to scale. The position of the primers used for amplifying Cypt promotor dependent transcripts with exon 6 is depicted below each gene (see (B) Gel picture of the RT-PCR assays showing amplification Cypt-dependent transcripts for XYSxrb, XY, XE,Z2Y*XSry and XEY*XSxrb. Lemd1 is a round spermatids expressed gene used as a control.(TIF)Click here for additional data file.S2 FigLemd1 and loading control Hmbs were performed separately with standard Taq polymerase and mixed for migration. Primers used are in Lemd1 transcription begins between 20 and 27 dpp. RT-PCR for (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


It has been shown that TLP is indispensable for p53-activated transcription from an upstream TATA-less promoter of the p21 gene. We found that mutant TLPs having decreased TFIIA-binding ability exhibited weakened transcriptional activation function for the upstream promoter. Activity of the upstream promoter was enhanced considerably by an increased amount of TFIIA in a p53-dependent manner, whereas activity of the TATA-containing downstream promoter was enhanced only slightly. TFIIA potentiated the upstream promoter additively with TLP. Although TFIIA is recruited to both promoters, activity of the upstream promoter was much more dependent on TFIIA. Recruitment of TFIIA and TLP to the upstream promoter was augmented in etoposide-treated cells, in which the amount of TFIIA–TLP complex is increased, and TFIIA-reactive TLP was required for the recruitment of both factors. It was confirmed that etoposide-stimulated transcription depends on TLP. We also found that TFIIA-reactive TLP acts to decrease cell growth rate, which can be explained by interaction of the p21 promoter with the transcription factors that we examined. The results of the present study suggest that the upstream TATA-less promoter of p21 needs TFIIA and TFIIA-reactive TLP for p53-dependent transcriptional enhancement.TATA-binding protein-like protein (TLP) binds to transcription factor IIA (TFIIA) with high affinity, although the significance of this binding is poorly understood. In this study, we investigated the role of TFIIA in transcriptional regulation of the TLP physically interacts with TFIIA beta and TFIIA alpha by anti tag coimmunoprecipitation (View interaction)TFIIA alpha/beta physically interacts with TLP by anti bait coip (View interaction) Transcriptional regulation of an RNA polymerase II driven gene is governed by a particular set of gene-specific DNA-reactive transcription regulatory factors and their associating transcriptional cofactors. Moreover, general transcription factors such as TFIID (transcription factor IID) and TFIIA are assembled at a promoter region to conduct transcriptional initiation –3, whichNF-1, Cyclin-G2, TAp63 and Wee1 [Drosophila PCNA gene, TLP is engaged in transcriptional activation as a cofactor for a transcription regulatory factor called DREF [p21 (p21Waf1/Cip1) as one of the TLP-target genes [TATA-binding protein (TBP) is an essential component in TFIID that binds to the TATA-box promoter element ,4–6. TBPand Wee1 –12. TLP led DREF . Althouget genes .1 or G2 phase [p21 is regarded as a major gene for cell growth regulation. The amount of intracellular p21 is regulated at the transcription level, and its expression level is enhanced by multiple transcription factors [p21 contains several binding sites for p53, which enhances the promoter activity. p53, which works for genome homeostasis, is a typical tumor suppressor and major regulator of the p21 gene [p21 gene has two major promoters: a TATA-containing downstream promoter and an upstream TATA-less promoter [p21 is a CDK inhibitor and causes cell-cycle arrest at GG2 phase ,16. SincG2 phase , p21 is  factors ,19. The p21 gene –22. The promoter ,24. TFIIpromoter , and thepromoter ,25.The most attractive property of TLP is its potent TFIIA-binding ability. TFIIA is another member of the family of general transcription factors . Althougp21 gene regulation, and we found that mutant TLPs with weakened TFIIA-binding ability exhibit decreased transcription stimulation activity. Moreover, etoposide, which stimulates p21 gene expression, facilitated binding of the upstream promoter to TFIIA and TFIIA-reactive TLP. One reason why TLP possesses a strong TFIIA-binding ability may be elucidated through this study.In this study, we investigated the contribution of TLP–TFIIA interaction to We previously constructed various kinds of mutant TLPs . Among tp21 gene produces mainly alt-a and variant-1 transcripts from the upstream and downstream promoters, respectively  as well as the downstream promoter region (TATA-box)  were also decreased in TFIIA-depressed cells.It has been reported that TFIIA can be recruited to some TATA-less promoters as well as TFIID-dependent TATA-containing promoters ,27,30,31 region p3RE as we region p3RE as we region p3RE as we region p3RE as wep21 gene in etoposide-treated cells [Previously, we demonstrated that p53 and TLP are recruited to the same region of the ed cells . In thisp21 upstream promoter [p21 upstream promoter.Lastly, we investigated whether activity of the upstream promoter is modulated by TLP in etoposide-treated cells. TLP knockdown resulted in a decrease in upstream promoter-driven mRNA production .Human HCT116 cells (wild-type and p53-deficient mutant cells)  and HeLapCIneo-FH-TLP, which is an expression plasmid of flag/oligohistidine (FH) tagged mouse TLP, was described previously . Mouse ap21 promoter region encompassing from −2266 to −1875 was described previously [p21 downstream promoter, respectively. These constructs were generated by a PCR-based strategy using a reporter plasmid encompassing from −2677 to +66, which has been named p21luc1 as previously described [pGL4.10 vector  was used for construction of luciferase reporter plasmids. A reporter plasmid (designated p21up/GL4) containing a human eviously . The +1 escribed . Primer m of siRNA and cultured for an appropriate period.siRNAs were prepared by a Silencer siRNA Construction Kit . Sequences for target human TFIIAαβ were 5′-GATGGGCAGGTGGAAGAAG (sense) and 5′-CTTCTTCCACCTGCCCATC (antisense). The sequence for human TLP was described previously . A scramTotal cellular RNAs were prepared using an RNeasy Mini Kit , and RT-PCR was performed as described previously . Amplifi4cells·well−1). Twenty-four hours later, cells were transfected with the indicated amount of a reporter plasmid and an effector plasmid and cultured for 24 h. Total amounts of transfected DNA were adjusted with pRL-TK (Promega). Cells were disrupted with a Passive Lysis Buffer (Promega). Luciferase activity in lysates was determined by a Dual Luciferase Reporter Assay System (Promega).Cells were inoculated into a 24-well plate  membrane  and detected by an ECL Prime  as previously described  by usingm etoposide for an appropriate time. After fixation of cells, ChIP assay was performed as described previously [p21 promoter-specific primer sets. Primer sets for ChIP analysis were as follows: p53RE forward, 5′-CACCTTTCACCATTCCCCTA; reverse, 5′-GCAGCCCAAGGACAAAATAG; TATA-box forward, 5′-TGCTGGAACTCGGCCAGGCTCAGCTG; reverse, 5′-CCAGCTCCGGCTCCACAAGGAACTG; control forward, 5′-TGGTAGGCCTCTCCAAGGTA; reverse, 5′-ACACATGTGACTTGGGGTGA.Cells transfected with plasmids were treated with 50 μeviously . Endogenr console (ver. 3.0.3). The number of experiments used for statistical analysis was at least three (n = 3). P < 0.05 was considered to be statistically significant. Statistical significance of differences between samples is shown in the figures with asterisks: *P < 0.05; **P < 0.01; ***P < 0.001.Data in this study are shown as mean ± standard error of the mean obtained from at least three independent experiments. Statistical significance of quantitative data was determined using Bonferroni's method with


Post-translational modifications (PTMs) of transcription factors play a crucial role in regulating metabolic homeostasis. These modifications include phosphorylation, methylation, acetylation, ubiquitination, SUMOylation, and O-GlcNAcylation. Recent studies have shed light on the importance of lysine acetylation at nonhistone proteins including transcription factors. Acetylation of transcription factors affects subcellular distribution, DNA affinity, stability, transcriptional activity, and current investigations are aiming to further expand our understanding of the role of lysine acetylation of transcription factors. In this review, we summarize recent studies that provide new insights into the role of protein lysine-acetylation in the transcriptional regulation of metabolic homeostasis. Chronic metabolic disorder has become a worldwide health problem, in parallel with the increasing incidence of obesity and type 2 diabetes mellitus (T2DM)  and histone deacetylases (HDACs). HATs transfer acetyl groups to lysine residues of the substrate proteins, while HDACs catalyze the reverse reaction  family proteins including FoxO1, -3a, -4, and -6 are a subfamily of the forkhead group of transcription factors and play a pivotal role in energy metabolism in peripheral tissues  in the promoter regions of target genes  family  belong to a subfamily of the forkhead group containing a winged helix DNA-binding domain and play a crucial role in lipid metabolism   in transcription factors can trigger significant effects on whole-body energy homeostasis.Exploring the key features of non-histone protein acetylation, including that of transcription factors will be critical for understanding fine control of whole body metabolism. Thus understanding the molecular mechanisms and physiological relevance of acetylation is of great interest, considering the therapeutic potential of these processes in the development of drugs combating metabolic syndrome and T2DM.


Azotobacter vinelandii produces the phenolic lipids alkylresorcinols (ARs) that are structural components of the cysts. The enzymes responsible for the ARs synthesis are encoded in the arsABCD operon, whose expression is activated by ArpR. The transcription of arpR is initiated from an RpoS dependent promoter. The nitrogen-related phosphotransferase system (PTSNtr) is a global regulatory system present in Gram negative bacteria. It comprises the EINtr, NPr and EIIANtr proteins encoded by ptsP, ptsO and ptsN genes respectively. These proteins participate in a phosphoryl-group transfer from phosphoenolpyruvate to protein EIIANtr via the phosphotransferases EINtr and NPr. In A. vinelandii, the non-phosphorylated form of EIIANtr was previously shown to repress the synthesis of poly-ß-hydroxybutyrate. In this work, we show that PTSNtr also regulates the synthesis of ARs. In a strain that carries unphosphorylated EIIANtr, the expression of arpR was reduced, while synthesis of ARs and transcription of arsA were almost abrogated. The expression of arpR from an RpoS-independent promoter in this strain restored the ARs synthesis. Taken together these results indicate that unphosphorylated EIIANtr negatively affects activation of arpR transcription by RpoS.Upon encystment induction,  Azotobacter vinelandii is a soil bacterium that undergoes a differentiation process resulting in the formation of a desiccation resistant cyst. A mature cyst consists of a contracted cell, known as the central body, which is surrounded by a capsule made up of a laminated outer layer called the exine and an inner layer called the intine [n-butanol or ß-hydroxybutyrate as the sole carbon source [e intine . The pole intine . Encystmn source .arsABCD gene cluster encodes the enzymes that synthesize these lipids [arsABCD operon, by direct binding to the arsA promoter region [rpoS impairs ARs synthesis [arpR [ARs play a structural role in the cyst, and strains carrying mutations in ARs biosynthetic genes produce cysts with a defective exine . The arse lipids . These ge lipids . The trar region . The mutynthesis  because is [arpR .ptsP, ptsO and ptsN genes encode EINtr, NPr and EIIANtr proteins, respectively, that are components of the nitrogen-related phosphotransferase system (PTSNtr), which is homologous to the carbohydrate transport PTS. The PTSNtr proteins participate in a phosphoryl transfer chain from phosphoenolpyruvate, where EIIANtr appears to be the terminal phosphoryl acceptor [Ntr regulates a wide variety of processes in bacteria; in Legionella pneumophila, a ptsP mutation, negatively affected its virulence in guinea pigs [Rhizobium species, the PTSNtr is associated to melanin synthesis, nitrogen fixation and regulation of ABC transport activation [Escherichia coli, the EIIANtr protein controls the potassium transport by interacting with the Trk transporter subunit TrkA and the sensor kinase KdpD (that controls the expression of high affinity potassium transporter system KdpFABC) [E. coli to phosphate starvation is also activated by EIIANtr due to an interaction with the sensor kinase PhoR [The acceptor . The PTSnea pigs ; in Rhiztivation ,11. In EKdpFABC) ,13. The ase PhoR .A. vinelandii UW136, the non-phosphorylated form of EIIANtr was shown to impair PHB production, by exerting a negative effect on expression of phbR, the gene encoding the transcriptional activator of the PHB biosynthetic operon phbBAC [In the n phbBAC .Ntr on alkylresorcinol synthesis and show that the non-phosphorylated EIIANtr protein has a negative effect on the transcriptional activation of arpR by RpoS.In this work we report the effect of mutations in the genes coding for the proteins of the PTSA. vinelandii was cultured at 30°C in Burk’s nitrogen-free salts medium [n-butanol (BBOH) for encystment induction. For determination of β-glucuronidase activity of transcriptional phbR-gusA and phbB-gusA fusions, the cells were grown in peptone yeast medium supplemented with 2% sucrose (PY). Liquid cultures were carried out in 250-mL or 125-ml flasks containing 50 or 25 ml of medium, respectively, in a rotatory shaker at 200 rpm and 30°C. Inocula for all experiments were grown on BS, washed three times with sterile 10mM MgSO4, and transferred to BBOH medium.Bacterial strains and plasmids used are listed in s medium  supplemeE. coli strain DH5α was grown in Luria-Bertani medium (LB) at 37°C. Transformation of A. vinelandii were carried out as previously described [escribed .DNA purification and cloning procedures were carried out as previously described . Total REcoRI and HindIII to clone the gusA reporter gene obtained from pAHFUTs-Tc [The pUMATc plasmid  was digeHFUTs-Tc , resultiA. vinelandii and used for transcriptional and translational fusions, respectively, were digested with SacI and KpnI restriction enzymes to remove the tetracycline cassette. The ends of the plasmids were made blunt by treatment with Klenow fragment and used for cloning a blunted MluI gentamicin cassette obtained from pBSL98 [The plasmids pUMATcgusAT and pUMATcgusAPT , unable m pBSL98 . The newarsA-gusA and arpR-gusA fusions, DNA fragments of 1.0 and 0.99 Kb, containing the promoter region of arsA and arpR, respectively, were amplified using the primers FwarsA and RvarsAtrans and FwarpR and RvarpRtrans (XbaI and PstI and ligated to XbaI-PstI pLM2 vector to construct the plasmids pLM4 (arsA-gusA) and pLM6 (arpR-gusA). These plasmids were digested with NdeI and ScaI, respectively, and used to transform A. vinelandii strains for the selection of transformants carrying transcriptional arsA-gusA or arpR-gusA fusions integrated into the melA gene by a double recombination event. Digestion of the plasmids was carried out in order to avoid the selection of strains with plasmids integrated into the chromosome generated by single recombination events. The melA gene has been previously used as a neutral site to introduce gene fusions [For the construction of transcriptional rpRtrans . The fra fusions . These sarsA´-´gusA and arpR´-´gusA fusions, DNA fragments of 1.3 and 1.1 Kb  were amplified with FwarsA and RvarsAtrad and FwarpR and RvarpRtrad primers for arsA and arpR, respectively. The PCR products were purified, digested with XbaI and PstI enzymes and ligated to XbaI-PstI pLM3 resulting in the plasmids pLM5 (arsA´-´gusA) and pLM7 (arpR´-´gusA). The plasmids pLM5 and pLM7 were digested with NdeI and ScaI, respectively, and used to transform A. vinelandii strains for the selection of the transformants carrying translational arsA´-´gusA or arpR´-´gusA fusions described in For the construction of translational gyrA cloning a 0.3 Kb DNA fragment containing the promoter region of gyrA gene (pgyrA) into vector pJET1.2/blunt (Thermo Scientific). A DNA fragment of 1.0 Kb containing the encoding region of arpR was amplified using the oligonucleotides arpRFw2 and arpRRv2  restored the ARs synthesis , which produces a non-phosphorylatable EIIANtr, on ARs production and on transcription of arpR and arsA. For this experiment we used the strain UW136::pALA8a, which carries the ptsN-H68A mutation [ptsP and ptsO mutants  carrying the plasmid pBpgyrA-arpR, which expresses the arpR gene from an RpoS-independent promoter (gyrA promoter). As shown in the arpR increased the AR levels in the strain UW136::pALA8a  showed a negative ARs production phenotype  as coinducer [Ntr also reduced the arpR transcription  [Ntr and the activity of sigma factors RpoS and RpoD has been previously described in E. coli [Ntr (in a ptsN mutant), the potassium levels increase (by derepression of activity of K+ Trk transporter) resulting in preferential binding of the core RNA polymerase to RpoS instead of RpoD, and therefore, affecting the transcription of sigma regulons [A. vinelandii the negative effect of the unphosphorylated EIIANtr on arpR transcription is through RpoS, since the expression of arpR from an RpoS-independent promoter was sufficient to restore ARs synthesis in the presence of unphosphorylated EIIANtr  . Additio E. coli . In the regulons . Here, w EIIANtr . Furtherependent ,31 and rent [Ntr . We carrromoters .. IndeedP mutant .. The meNtr also seems to control the expression of arpR at a posttranscriptional level since the ptsN mutation increased the activity of the translational arpR-gusA fusion in the wild type and ptsP strains  were more drastically reduced [Ntr negatively affects the arpR expression at posttranscriptional level remains to be determined. However, as the translational arpR fusion contains the 5’ untranslated region of arpR mRNA , this mechanism could be related to a reduction of mRNA stability and/or to a blockage of translation.Nonphosphorylated EIIA strains . AdditioA fusion , while t mutants . A simil reduced . The mecNtr is proposed  for 72 h at 30°C.(TIF)Click here for additional data file.S2 FigphbR-gusA(A) and phbB-gusA(B) fusions in UW136 and ptsP strains. The cells were grown in PY solid medium for 48 h at 30°C. The data represent the mean of two independent experiments. Error bars, SD.β-glucuronidase activity of transcriptional (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Mammalian transcription occurs stochastically in short bursts interspersed by silent intervals showing a refractory period. However, the underlying processes and consequences on fluctuations in gene products are poorly understood. Here, we use single allele time-lapse recordings in mouse cells to identify minimal models of promoter cycles, which inform on the number and durations of rate-limiting steps responsible for refractory periods. The structure of promoter cycles is gene specific and independent of genomic location. Typically, five rate-limiting steps underlie the silent periods of endogenous promoters, while minimal synthetic promoters exhibit only one. Strikingly, endogenous or synthetic promoters with TATA boxes show simplified two-state promoter cycles. Since transcriptional bursting constrains intrinsic noise depending on the number of promoter steps, this explains why TATA box genes display increased intrinsic noise genome-wide in mammals, as revealed by single-cell RNA-seq. These findings have implications for basic transcription biology and shed light on interpreting single-cell RNA-counting experiments. Recent assays in single cells confirmed transcriptional bursting in many organisms  to validate genome-wide the prediction that TATA box promoters, owing to their reduced number of promoter steps, showed increased intrinsic noise.et al, et al, N + 1-state promoter cycle , modeling the scenario of promoter progression  are not known and will be estimated from data. The resulting stochastic gene expression model (The two-state promoter cycle (telegraph model) predicts exponentially distributed transcriptionally silent periods, yet evidence points toward peaked  durations (Harper on model  consistson model . AlthougT. A unique step (N = 1) yielded exponentially distributed off-times  followed a peaked (Gamma) distribution. For N = 1, we observed large variability in the silent periods. Due to the short active periods, the mRNA and protein time traces were irregular   algorithm . For the likelihood, we used calibrated luminescence signals   Fig. Similary one Fig and C. Tsions Fig and varim and τa . Remarkasions Fig. Finallyr biases . Thus, cet al, Bmal1 promoter (B clones); seven clones obtained by lentiviral trapping  of endogenous promoters (gene names in Dbp gene (including its promoter) or minimal synthetic promoters combining a TATA box and one (H1) or two (H2) CCAAT boxes with multiple mutations. Additionally, we generated two more H1 clones that used new FRT sites in different genomic locations  stably driving a short-lived luciferase reporter from a single allele , independent of the fraction of time spent in the active state, which remained under 10%  than the burst sizes, which was the most varying kinetic parameter among the clones . Although it is difficult to gain further insights on the nature of these rate-limiting steps, their timescales of 10 min were more consistent with the dynamics of histone modifications than the interactions of transcription factor with DNA (Discussion). Supporting this, for the Bmal1 promoter treated with the histone deacetylation inhibitor (TSA), which renders the chromatin more permissive for transcription, N reduced from 7 to 3 and T reduced from 60 to 40 min  and ones with markedly more steps (Group II: N ∼ 6) , while the endogenous promoters  had shorter cycles (50 min). Moreover, all promoters with large N were endogenous. As shown for representative cells for the H1 (Group I synthetic), Prl2C2 (Group I endogenous), and Gls promoters (Group II), the distinct kinetics are visible in individual cells, based on the raw signals as well as the mRNA counts and gene activities  Fig. In addiities Fig–C.Ctgf and Prl2C2) was alleviated by more frequent activations. Complex promoter cycles (Group II) involved several transitions and short silent periods, thus leading to more regular activation patterns constrained by a refractory period.In summary, the analyzed promoter cycles suggested two distinct groups, simple promoter cycles and complex promoter cycles Fig. Simple H1a, H2, H2 1M, H2 2M) were inserted into the same genomic location, the low number of states and long promoter cycle observed might reflect a property of the insertion site, for example, the chromatin state, rather than the promoter architecture. We therefore generated additional clones  by integrating the minimal promoter H1 at distinct genomic locations. Remarkably, the three H1 insertions retained very similar promoter cycles  in two distinct locations also showed very similar cycles  contained a canonical TATA box element  was non-random . Promoter architecture and, in particular, the presence of TATA boxes seemed to influence the promoter cycles. Further evidence that this holds genome-wide is presented below.Interestingly, the synthetic (Group I) and the two endogenous promoters with small et al, η2 (intrinsic noise) arises from gene-specific fluctuations whereas NcKap1 or Ctgf  was on the order of 100% (η2 between 0.3 and 2.1), independent of expression levels. As shown below, this a direct consequence of transcriptional bursting. In comparison, ηe was in the range of 70%  plus the variance in the means across cells (extrinsic variance) , which sets a lower bound on intrinsic noise, and c refers to “cycle”) corresponds to the promoter noise. In the clones, η2 was larger than b (from 1 to 80) explains most of the variation in expression /τm is the duration of the promoter cycle, or intervals between bursts, normalized by the lifetime of the transcript τm, and sets the scale of the intrinsic noise. The coefficient C, originating from promoter fluctuations, approaches zero in a constitutive regime (τa ≫ T) and is always between 1/2 (large N) and 1 (N = 1) in a bursting regime (τa ≪ T). This shows that b > 2, which is the case for most clones , or by increasing N. Although C is a complicated function in general (τa ≪ T), when the inactive sub-steps have equal durations (τi = T/N) and T ≪ τm. This limit links with noise N . It turns out that the different clones are well approximated by C(N)  Fig, with a Thus, we showed that since mammalian genes are typically transcribed as short and large bursts, the intrinsic mRNA noise was on the order of the normalized promoter cycle duration.N . In yeast, TATA box promoters are known to exhibit increased noise , estimated to be around 10%. Indeed, at low counts, non-biological sampling noise (showing Poisson statistics) dominated in both the split controls and the single cells , the difference in promoter noise between TATA  and TATA-less promoters  amounts to f/4 . The same quantitative difference is predicted for genes on the autosomal vs. X chromosomes (assuming C ∼ 1/2 for endogenous genes). Gene-specific values of f are not known but estimated between 0.01 and 0.5, based on promoter cycle times in the range of 1–2 h , either across cells or over time  would be nearly insensitive. Moreover, the reduction of noise from a larger number of rate-limiting steps in the promoter cycle would be close to optimal for long-lived transcripts  affected promoter noise, with effect sizes that were in the expected range  stands for the probability to measure s gray levels given p protein copies and follows from our previous microscope calibration  are derived from the master equation, where the discrete state pmg stands for the protein (p), mRNA (m) copy numbers, and the state of the promoter g. P0(s0p0m0g0) corresponds to the stationary distribution, and the sum runs over all possible state trajectories Λ = {pmg}.To perform inference, we computed the exact likelihood that bioluminescence time traces were generated by a stochastic gene expression model describing the promoter progression toward activation, followed by synthesis and degradation of mRNAs and proteins. The promoter cycle constituted of P, P is the prior distribution and P using a reversible jump Markov chain Monte Carlo (RJ-MCMC) algorithm , and the kinetic parameters are estimated from the marginal P(θ¦D).Using the likelihood, the optimal model was inferred from the joint posterior distribution D, using the optimal model as a prior P. Thus, we sampled from P. given by From the optimal model and kinetic parameters, the bioluminescent time traces are deconvolved using Gibbs sampling , while the modeled distributions can be analytically calculated from the promoter cycle model P. All details on the modeling are given in The empirical distribution of silent periods and the mRNA steady-state distribution can be estimated directly from m¦i> in cell i across the population. σ2 approximates the intrinsic variance of an idealized cell with parameters close to the population mean , The intrinsic noise can be further separated as q varies from cell to cell, the expected population noise n) relates to the total biological noise q> and q, respectively. For low q, the first term  typically dominates over the biological noise (Figq> and et al, Considering that the recovery rate noise Fig and B. E


A high-resolution hydroxymethylome map is required to fully understand the function of hmC in the human brain.We present genome-wide and single-base resolution maps of hmC and mC in the human brain by combined application of Tet-assisted bisulfite sequencing and bisulfite sequencing. We demonstrate that hmCs increase markedly from the fetal to the adult stage, and in the adult brain, 13% of all CpGs are highly hydroxymethylated with strong enrichment at genic regions and distal regulatory elements. Notably, hmC peaks are identified at the 5′splicing sites at the exon-intron boundary, suggesting a mechanistic link between hmC and splicing. We report a surprising transcription-correlated hmC bias toward the sense strand and an mC bias toward the antisense strand of gene bodies. Furthermore, hmC is negatively correlated with H3K27me3-marked and H3K9me3-marked repressive genomic regions, and is more enriched at poised enhancers than active enhancers.We provide single-base resolution hmC and mC maps in the human brain and our data imply novel roles of hmC in regulating splicing and gene expression. Hydroxymethylation is the main modification status for a large portion of CpGs situated at poised enhancers and actively transcribed regions, suggesting its roles in epigenetic tuning at these regions. Methylation of cytosine (mC) plays a role in many crucial cellular processes. In 2009, it was shown that mC can be oxidized to 5-hydroxymethylcytosine (hmC) by tet methylcytosine dioxygenase (Tet) family of enzyme, and that embryonic stem cells (ESCs) and mouse brain tissues contain high levels of hmC ,2. TheseSoon after the discovery, a series of genome-wide mapping studies of hmC were performed using affinity or enzyme-based approaches by us and others -16. ThesThe content of hmC in the mammalian brain is typically five to ten times higher than in any other tissues, suggesting a potential role for hmC in the brain ,8,19. hmet al. reported comprehensive genome-wide DNA methylation maps in the human and mouse brain using BS-Seq, which also include hmC maps in the mouse brain using TAB-Seq [However, the exact function of hmC in the mammalian brain remains to be understood. Recently, Lister  TAB-Seq . Here, wvs 0.154)  and mC 1.18%), and a high protection rate of hmC (97.6%). We also applied TAB-Seq to a DNA sample isolated from the prefrontal cortex of a fetal brain and sequenced it to an average depth of 11× per strand, with the non-conversion rates of unmodified cytosine and mC being 0.25% and 1.51%, respectively. The sequencing details are summarized in Additional file .18%, andTET2 transcription start site (TSS) further supports the need for TAB-Seq. For many individual CpG sites, the hmC level is higher than the mC level. In particular, the highly modified CpGs immediately upstream the TSS are highly hydroxymethylated and could be mistaken as highly methylated sites when applying traditional BS-Seq alone. These results highlight the need to apply TAB-Seq to distinguish hmC and mC unequivocally for obtaining accurate hydroxymethylation and methylation maps in the brain. The hmC map of the fetal brain is also provided and suggested that patterns of hmCs in the adult brain have been partially established in the fetal stage.Examples of the hmC and mC maps were shown in Figure high, modC ≥ 50%), 6.9% are unmodified , and 3.3%  are modified at low levels   hmChigh: hmC > mC; (2) hmClow: hmC < mC; and (3) hmCno: no hmC. We determined that hmChigh  accounts for a notable proportion (13.4%) of all captured CpGs. The hmClow  and hmCno  categories comprised 49% and 27.4%, respectively. Since further analysis revealed that hmClow and hmCno have similar characters, they were grouped into mChigh referring to highly-methylated cytosines , whereas most mCs occur at high frequency (median 59.7% in the adult brain)  occur at genic regions   represent a large proportion of human genes  Figure b. The Cp) Figure a and b. ) Figure a. Overaln = 12,980), and found that they have higher CpG occurrence and much lower hmC and mC levels than the internal exons  into three groups of identical size of high, middle and low expression levels  and compared their inclusion rates . We also examined the number of the alternatively spliced (AS) exon, which was defined as the inclusion rate being less than 0.8. The results demonstrated that the hmChigh and mChigh exons comprised a similar fraction of AS exons  vs. 5.63% , Figure vs. 0.9908, P = 0.13, two-tailed Mann-Whitney-Wilcoxon (MWW) test, Figure high and mChigh exons with the internal exons put together ). These results suggested that hmC and mC at the 5′ss CpG site are similarly related to splicing. Interestingly, the modClow and modCno groups involved markedly more AS exons (9.67% (59 out of 610 exons) and 18.76% (103 out of 549 exons), respectively, Figure low and modCno exons were slightly but significantly reduced comparing with the hmChigh and mChigh groups . Both hmC and mC also displayed a slight difference in genes that are classified as not expressed , but these differences were not statistically significant (P = 0.39 for hmC and P = 0.44 for mC). No strand difference was found for randomly-selected intergenic regions  based on their expression levels and the gene-body mC and hmC levels were calculated for each strand of the genes. The results revealed that, from the lowest to highest expressed genes, an average seven-fold increase in the hmC bias  and an average five-fold increase in the mC bias  were observed Figure b. Also, s Figure b. Also, s Figure , explainvs. 0.28% in SAS region) and a 66% reduction in the mC bias , astrocytes  and oligodendrocytes  [n = 2,402) [P <0.001, Figure P = 2.4 × 10-7 for expression level 4, Figure P >0.05 for expression levels 1 and 2, Figure To determine whether the hmC and mC strand biases are cell type-specific, we examined genes enriched in different brain cell types including neurons  . We foun= 2,402)  exhibitehigh subgroup at the core region of the poised enhancer  and Neurexin 1 (NRXN1) gene loci as examples  at the exon-intron boundary in human embryonic stem cells and fibroblast . Since Breported . Furtherreported . The resreported ,33.Second, our data revealed a surprising transcription-correlated hmC bias toward the sense strand and an mC bias toward the antisense strand of the gene body in the adult brain tissues of both human and mouse. In contrast to the well-established role of DNA methylation in CpG island promoter regions for transcription repression, the function of gene body methylation is still largely unknown -41. Prevn = 5,692,354) are highly-hydroxymethylated (the hmChigh category). Since hydroxymethylation is the main modification status of these CpG sites, it points to a regulatory role of these CpG sites when hydroxymethylated. Indeed, we found that this CpG category is strongly enriched at enhancers and genic regions, which is distinct from the highly-methylated (mChigh), as well as the lowly-hydroxymethylated  categories. The hmChigh is also more enriched at poised enhancers, which is distinct from enrichment of modClow at active enhancers. Finally, we showed that the hmC level is greatly reduced in H3K27me3-marked regions, which is different from its enrichment on the H3K27me3-associated bivalent promoters in ESCs [Third, we demonstrated that, in the adult human brain, 13.4% of all CpGs n = ,692,354 In summary, we present the genome-wide and single-base-resolution maps of hmC and mC in the human brain. The results imply novel roles for hmC in gene splicing and gene expression regulation. The identified highly-hydroxymethylated CpGs, which comprise 13.4% of all CpGs in human genome and are strongly enriched at poised enhancers and actively transcribed regions, also serve as a starting point for future investigations on the mechanisms that establish hydroxymethylation patterns and their associated functions in the human brain.This project was approved by the Reproductive Ethics Committee of Peking University Third Hospital (2012SZ010). Brain tissues for research were obtained with the written informed consent. The methods applied in this study are complied with the Helsinki Declaration. For TAB-Seq and BS-Seq, the adult human brain samples were obtained from a postmortem 42-year-old woman, and the fetal brain samples were obtained from an aborted 22-week-old male fetus. For LC-MS/MS, another adult human brain sample was obtained from a postmortem 28-year-old man, and another fetal sample was obtained from an aborted 22-week-old female fetus. All individuals had no sign of brain diseases. Genomic DNAs were extracted using Blood & Cell Culture DNA Kits (Qiagen) following the manufacturer’s instructions.For TAB-Seq, genomic DNAs and spike-in control DNAs were sheared to an average size of 200 bp using a Covaris S2 instrument. Glycosylation and oxidation of genomic DNAs was performed following a previous protocol with small modifications . BrieflyFor Tet-assisted reduced representation bisulfite sequencing (TA-RRBS), genomic DNAs extracted from purified neuron nucleus were first digested with MspI for 3 h at 37°C and purified using the QIAquick Nucleotide Removal Kit as described . Then, gThe DNA sample for BS-Seq was the same batch of sheared genomic DNA with spike-in controls that was used for TAB-Seq but without glycosylation and oxidation preparation. DNA samples for TAB-Seq and BS-Seq (0.5 to 1 μg) were end-repaired, A-tailed, and ligated to methylated adaptors using Illumina TruSeq Sample Preparation Kits following the manufacturer’s instructions. The ligated fragments were then bisulfite converted using the MethylCode Kit (Invitrogen). Bisulfite-treated DNA was separated into three reactions for PCR amplification using PfuTurbo Cx Hotstart DNA polymerase (Agilent Technologies) for seven cycles, yielding three independent libraries for the same biological sample. Final libraries were purified with 1× AMPure XP beads and sequenced using an Illumina Hiseq 2000. For Tet-assisted reduced representation bisulfite sequencing, an additional size selection of 180 to 600 bp fragments on a 2% agarose gel was performed after the adapter ligation.Fully methylated Lambda DNA was added to the human brain genomic DNAs in a ratio of 1 to 200 as a spike-in DNA control to calculate the non-conversion rates of unmodified cytosine and mC. For the spike-in control to calculate the non-conversion rates of hmC, a DNA fragment of approximately 1.6 kb was amplified from a pUC19 vector using a primer pair (5′-GCAGATTGTACTGAGAGTGC-3′ and 5′-TGCTGATAAATCTGGAGCCG-3′) and a cocktail of dATP/dGTP/dTTP and dhmCTP . The control was then added to genomic DNA at a ratio of 1 to 400.Total RNA was extracted from the tissue sample using QIAGEN RNeasy Mini Kits (Qiagen), and mRNA was isolated by Sera-Mag Oligo (dT) beads (Thermo Scientific). Libraries were prepared using the NEB Next mRNA Sample PreP Master Mix Set 1 according to the manufacturer’s protocol and sequenced using an Illumina Hiseq 2000.Genomic DNA (2 μg) was digested by nuclease P1, venom phosphodiesterase I (Type VI), and alkaline phosphatase (Sigma). After desalting and filtration, 10 μL of the solution was injected into HPLC-MS/MS for analysis. HPLC-MS/MS was carried out by reverse-phase ultra-performance liquid chromatography on an Agilent ZORBAX Eclipse XDB-C18 column , equipped with a ZORBAX Eclipse XDB-C8 guard column , eluted with buffer A (0.1% formic acid in H2O) and buffer B (0.1% formic acid in methanol) with a flow rate of 0.5 mL min-1 at 35°C with a 2% to 25% gradient in 4.5 min, with online mass spectrometry detection using Agilent 6410 triple-quadrupole (QQQ) LC mass spectrometer in multiple reaction monitoring (MRM) positive electrospray ionization (ESI) mode. The nucleosides were quantified using the nucleoside-to-base ion mass transitions of 258 to 142 with collision energy of 1 eV (hmC) and 228 to 112 with collision energy of 5 eV (C). Quantification and detection limits were determined by comparison with the standard curves obtained from nucleoside standards running at the same volume and time.Nuclei extraction was processed as described . BrieflyImmunostaining mix was prepared by combining 300 μL PBS, 1.2 μg NeuN antibody , 200 μL Blocking Mix ( 2.5% BSA and 10% Goat Serum in PBS), and 2 μg of Alexa Fluor 488 conjugated secondary antibody  together and rotated for 5 min at room temperature in the dark. An isotype antibody control was processed in parallel by adding the same amount of mouse IgG instead of NeuN antibody. Then 1 mL nuclei suspension was added, and the mixture was incubated by rotating in the cold room for 45 min. After incubation, samples were retrieved and stained with Hoechst 33342 for another 2 min. The immunostaining result was checked under the microscope.Immumotagged samples were diluted 10 times in PBS and filtered through a 40 μm filter before loaded to the FACS machine. A preliminary run was performed to gate out the proper nuclei size and the fluorescence bright population before the sort which separate the NeuN + nuclei. Once the sort was done, a small amount of sorted sample was run again through the instrument to confirm the purity.After FACS, PBS was added to raise the volume of the sort to 10 mL. Then the sorted sample was mixed with 2 mL Sucrose Solution, 50 μL CaCl2 (1 M), and 30 μL Mg(Ac)2 (1 M), and incubated on ice for 15 min before centrifuged at 1,786 g for 15 min at 4°C. The NeuN + nuclei pellet was resuspended in PBS and went on with the Tet-assisted reduced representative bisulfite sequencing.FASTQ format reads generated by the Illumina HiSeq2000 platform were aligned to the human reference sequence (HG19) using the Bismark program. Briefly, first, whole or any subsets of adaptor sequences were trimmed on 3′ and 5′ of reads before alignment. Second, a read was removed if more than 10% bases were N, or more than 50% bases were of Phred quality lower than 5, or at least three unmethylated CHs were present, or PCR redundancy occurred, so that only high quality data were used for downstream analysis, as described previously ,29. ThirThe non-conversion rate of unmodified cytosine was calculated as the percentage of sequenced cytosines in non-CG contexts relative to all covered cytosines in non-CG contexts in the Lambda genome. The non-conversion rate of mC was calculated as the value in a CG context, and the non-conversion rate of hmC was measured as the value for all cytosines in the spike-in pUC19 sequences. The original data are shown in Additional file P value for each cytosine detected by TAB-Seq and BS-Seq was calculated via binomial distribution as previously described [P value cutoff, we applied the Benjamini-Hochberg method [P value of 1E-8 when applying it to the adult TAB-Seq data. This is likely because the number of bona fide hydroxymethylated cytosines of the adult brain is much greater than those of ESCs and the fetal brain. To be consistent, we applied the Benjamini-Hochberg method for both the adult and fetal TAB-Seq data. For calculating the FDR for the BS-Seq data, the Benjamini-Hochberg method yielded a similar result with the method described by Lister et al.[The escribed ,29. To mg method  and set g method  to calcuThe modC frequency was calculated as dividing modCG by the total coverage according to the BS-Seq data (modCG/CG). The hmCG frequency was similarly calculated according to the TAB-Seq data (hmCG/CG), and the mCG frequency was calculated by subtracting hmCG/CG from modCG/CG.The hmC level (%hmC) at a given genomic region or site is calculated by dividing the number of sequenced cytosines by the number of sequenced cytosines plus thymines at this region or site according to the TAB-Seq data, where the reference is in CG context. The mC level (%mC) is calculated by subtracting %hmC from the total modification level (%(hmC + mC)) according to the BS-Seq data.All genomic features were defined based on the HG19 genomic annotation downloaded from the UCSC database. Different genic elements, including transcription start sites (TSS), exons, introns, and transcription terminal sites (TTS), were defined based on the Reference Sequence (RefSeq) database. The CGIs were retrieved from the cpgIslandExt table in UCSC database, and promoter CGIs were defined as overlapping with ± 1 kb of TSS. Repetitive sequences  were acquired from the Repeat-Masker track in the UCSC database.low, mChigh, hmChigh, or Fetal > Adult hmCGs), we counted the fraction of the involved CpG sites at each genomic element as the ‘observed’ distribution value. We also counted the fraction of the overall captured genomic CpG sites at each genomic element as the ‘expected’ distribution value. The fold enrichment value for each genomic element was calculated by dividing the ‘observed’ distribution value by the ‘expected’ distribution value.For a given CpG group  were divided into two groups according to whether they are transcribed along the positive strand or the negative strand. The SAS gene paired region was identified as the overlap region between these two gene groups.Genes enriched in different brain cell types including neurons, astrocytes, and oligodendrocytes were defined according to a previous paper with exclusion of genes that are not expressed in our RNA-Seq data . The houTo profile the distribution of hmC and mC on genomic regions with different histone modifications, we used the recently published genome-wide maps of chromatin states in the adult brain midfrontal lobe , includihigh, mChigh, mCall, and modClow) relative to all CpGs were binned into 50-bp sliding windows in 25-bp steps.Enhancers were defined as H3K4me1 peaks that are distant from ± 2.5 kb around any TSS in the switchDbTss table downloaded from the UCSC database. To produce the profiles in Figure To profile hmC and mC in H3K27me3- and H3K9me3-marked regions, each region was divided into 100 equally sized bins, and average hmC, mC, and modC levels and the hmC/mC ratio were calculated for each bin, as well as in 1-kb windows throughout 10 kb upstream and downstream of the regions.The TAB-Seq and BS-Seq data have been deposited to the NCBI under accession number GSE46710, and the TA-RRBS data have been deposited under accession number GSE55249.The authors declare that they have no competing interests.LW, JQ, CH, RuiL, and FT conceived and designed the experiments. LW, XianL, LY, RongL, CS, MY, XiaoyuL, YH, HG, XW, JX, YangyuZ, YW, and YanZ performed the experiments. LW, YT, Xiaomeng L, and PZ conducted the bioinformatic analyses of the data. LW, CH, JQ, and FT wrote the paper . All authors have read and approved the manuscript for publication.Summary of sequencing details.Click here for fileQuantitative values of hmC relative to dC measured by LC-MS/MS. LC-MS/MS was performed to genomic DNAs isolated from several regions of two adult brain and two fetal brain samples. For each sample, the average value with the standard deviation from technical duplicates was shown.Click here for fileChromatograms of LC-MS/MS. The LC-MS/MS chromatograms of hmC (left panels) and C (right panels) for genomic DNAs extracted from the adult (a) and fetal (b) frontal lobes were shown with the standard curve (c). The peak area counts are marked. Please go to the figshare website  for all Click here for fileThe percentages of hmC (by TAB-Seq) or modC (by BS-Seq) in the fetal hippocampus in the contexts of CG, CHH, and CHG.Click here for file Distribution of all covered CpGs according to their hydroxymethylation and methylation frequencies in the adult (a) and the fetal (b) brains. (c) Distribution of the CpG categories  and all captured CpGs (All CpGs) on different genomic elements. (d) Average absolute levels of hmC at different genomic elements in the fetal brain.Features of hydroxymethylome in the human brain. Click here for filen = 176,455) in the sense strand.Prominent hmC changes at the exon-intron boundaries in the human brain. Profiles of hmC and mC for a 200-bp window (50 bp for exon and 150 bp for intron) around the exon-intron and intron-exon boundaries. Modification levels of hmC, mC, total DNA methylation (hmC + hmC), and the ratio of hmC to mC are shown for all internal exons  were shown for a 40-bp window around the exon-intron boundaries at single-nucleotide resolution of two types of exons, which have a CpG at 5′ss position +4 or +5, and are named +4CG and +5CG exons, respectively. Since a CpG at one position will lead to absence of CpG at the nearest neighboring position and thus no methylation value, we merged the data of the sense and the antisense strands for each type of exons.Click here for fileAll expressed genes in the adult human brain revealed by RNA-Seq.Click here for filehmC, mC, and modC levels on each strand of all individual genes.Click here for filet test. nd, no statistical difference (P >0.01).The average levels of modC on sense and antisense strands of genes expressed at different levels in the adult brain. One-tailed paired Student’s Click here for fileThe mC profiles across each strand of the exon. The profile across exons of sense (lined) and antisense (dotted) strands of highly-expressed genes (red) and no-expression genes (black) in the adult brain showed that a transcription-correlated mC bias toward the antisense strand of the exon.Click here for filen = 11,424, red) and genes with no expression  in the adult mouse brain showed that a transcription-correlated mC bias toward the antisense strand of the gene body in the mouse brain. The TAB-Seq, BS-Seq, and RNA-Seq data for analysis were obtained from Lister et al.[The mC profiles across each strand of the gene body in the mouse brain. The profile across genes of the sense (lined) and antisense (dotted) strands of expressed genes (er et al..Click here for fileFACS scatter plot for the control sample for isolation of neuronal nuclei, which was processed with IgG.Click here for filelow, mChigh, and hmChigh surrounding the midpoints of active or poised enhancers.Distribution of modCClick here for fileet al.[hmC and mC maps in a 2-mb genomic region on chromosome 11. This example shows depletion of hmC and enrichment of mC on the H3K9me3- and H3K27me3-marked repressive regions. ChIP-Seq data for H3K4me1, H3K4me3, H3K9me3, and H3K27me3 were obtained from Zhu et al..Click here for fileNRXN1 gene. This example shows enrichment of hmC and H3K4me1 within the genic region and enrichment of mC and H3K27me3 in the neighboring intergenic regions. ChIP-Seq data for H3K4me1, H3K4me3, H3K9me3, and H3K27me3 were obtained from Zhu et al.[hmC and mC maps in a 2.8-mb genomic region surrounding the hu et al..Click here for file


The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process.A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM) was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation.Pparg which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving Klf4, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch  progressively decreases repression of Xbp1 gene transcription, in concert with decreased translational repression by miR-214. The transcription factor Xbp1 mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with Atf6. Transfection studies show that Xbp1Mist1 promoter. In addition, Xbp1 and Mist1 each activate the parotid secretory protein (Psp) gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation.Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for Pparg, Klf4, and Sox11 during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a genetic switch involving transcription factors and microRNAs, and transition to an Xbp1 driven differentiation network. This proposed network suggests key regulatory interactions in parotid gland terminal differentiation.This study identifies novel expression patterns of  Salivary gland dysfunction affects millions across the nation, and results in complications that can lead to a decline in oral health as well as overall quality of life . Whole sCurrent work has made headway towards this goal by focusing on gland progenitor cells. Insights from explant cultures have shown that parasympathetic nerves, which develop within the gland, are vital for maintaining epithelial progenitor cell populations during development . Within Differentiation of rat parotid salivary gland acinar cells occurs during the last week of gestation and the first postnatal month . Just beXbp1) or basic helix-loop-helix family, member a15  impairs both salivary gland and exocrine pancreas development, and while each gland still develops in knockout mice, the acinar cells are either disorganized or poorly formed. Mist1 is a basic helix-loop-helix transcription factor that is specific to serous exocrine tissue . Mi. MiXbp1 e points . Their ents (–5) . Transfe of Xbp1 . This exPsp or amylase. Psp expression increases dramatically during acinar differentiation, particularly after birth. The tissue specific promoter region of this gene has been identified [Xbp1 binding [Xbp1 (Xbp1-S) activated the 500 bp and 1kb Psp promoter in luciferase transfection experiments in ParC5 cells , nor did it activate a 1.5 kbp Psp promoter construct even in the presence of the Mist1 dimerization partner Tcf3/E2A [Psp gene. Since these are Mist1 consensus binding sites, a 1320 bp rat DNA fragment from the end of exon 2 through the start of exon 4 was cloned (Psp promoter-luciferase construct. This promoter+exon construct was activated 2.3-fold by co-transfection with Mist1 and Tcf3 cDNAs (p = 0.0227), whereas the 1.5 kbp Psp promoter was not activated . Tcf3 was included in these experiments as a heterodimerization partner with Mist1. Since Tcf3 mRNA was not differentially expressed (p = 0.25), it is not included in the proposed regulatory network, however, it is constitutively expressed throughout acinar cell differentiation. While Tcf3 is typically present in most cell types, it apparently has inadequate levels in the ParC5 cells used for transfections. Overall, these experiments indicate that the increase of Mist1 expression during acinar differentiation contributes to the increase of Psp gene expression through binding sites flanking exon 3.Regulation of the Tcf3/E2A Fig 8B)..Psp genes cloned  into theKlf4 mRNA. This includes miR-29c, which has been shown to regulate Klf4 expression in breast cancer cells. Loss of miR-29c in those cells results in dedifferentiation due to Klf4, leading to a population of stem-like cells [Klf4 mRNA. Klf4 does not decrease in expression until late in postnatal development when miR-29c increases. Expression of these two genes are negatively correlated across acinar differentiation . In order to test potential miRNA target genes, HEK293 cells were co-transfected with a miRNA mimic and a luciferase expressing plasmid containing a 3'UTR of interest. Transfection experiments confirmed repression of rat Klf4 by miR-29c  which inhibit differentiation, and subsequent switch to an Xbp1 pathway which drives and maintains markers of terminal differentiation. Our network suggests that Klf4 is initially regulated by Egr1 and, remarkably, subsequently repressed by 4 different miRNAs which increase strongly in the late stages. We demonstrate that either miR-200c or miR-29c can down-regulate the expression of Klf4. By affecting its expression, these miRNAs could be important drivers of terminal differentiation. This is one example of the broad observation that miRNAs have extensive roles driving parotid differentiation. Remarkably, 52 significantly increasing miRNAs target 524 significantly decreasing mRNAs, potentially contributing to direct regulation of 32% of the DE cluster #1 mRNAs.Terminal differentiation involves enduring changes in the gene expression patterns of a cell. We have defined profiles of expression of mRNAs and microRNAs across the month-long process of parotid acinar cell differentiation. Based on these profiles, we have generated a regulatory network which suggests transitions in gene expression driving parotid differentiation. The focus on acinar cell differentiation is supported by the use of laser-capture microdissection to isolate developing acinar cells at multiple time points. Incorporating time as a variable in this study allows comparison of the profile of expression of a transcription factor mRNA with the profile of a putative regulated gene. Co-variance supports inclusion of the transcription factor and its target gene in the regulatory network. Similarly, microRNAs were only included in the network when the profile of expression of the microRNA was inversely related to that of the target mRNA. This ensures that the network has both knowledge-based and expression level support which strengthens the results. This putative network provides a context for changes in transcription factors which regulate differentiation. The network identifies two main branches; initial expression of stemness factors , and only a 50% reduction of salivary amylase [Xbp1-null mice was not described, based on the proposed regulatory network we would predict a dramatic impairment of parotid differentiation, more similar to the pancreas. Xbp1 and Mist1 likely work as 'scaling' factors, a concept developed by Mills et al. [Parotid acinar expression of elopment . Pancrea amylase . While ts et al.  which coXbp1, the serous exocrine specific transcription factor Mist1 is up-regulated. We confirm direct regulation of the Mist1 promoter by Xbp1 in a parotid cell line (Mist1 target genes (Rab3D and Rab26) are also up-regulated, likely contributing to cell maturation [Psp, Bpifa2) as a marker of terminal differentiation since it is almost exclusively expressed in parotid acinar cells and is one of the most abundant salivary proteins. PSP is a lipid and LPS-binding salivary protein with anti-inflammatory and antimicrobial activity [Xbp1 or Mist1 can directly activate expression of the Psp gene, acting at binding sites either in the promoter (Xbp1) or in introns (Mist1). In the Mist1 knockout mice both pancreatic and parotid acinar cells are severely disorganized, although some secretory granules are present and Rab3D mRNA is expressed at 50% of normal [Xbp1 or Mist1 can activate Psp suggests that the incomplete disruption of the acinar phenotype in Mist1-null parotid may be due to the ability of Xbp1 to ‘work around’ Mist1 to activate terminal markers of differentiation. In this view, Xbp1 and Mist1 act in concert, rather than sequentially, to drive differentiation.Downstream of ell line . Severalturation . We alsoactivity , 65. Traf normal . Our obsPparg along with 18 of its downstream targets  mRNA is strongly up-regulated following the Pparg-related peak, potentially through Pparg and RXR binding sites within the Pgc1α gene. Pgc1α/β directs maturation of cardiac myocytes [Pparg and related factors in parotid differentiation remains unknown.Additional new insights from the mRNA profiles include transient up-regulation of  targets . Expressmyocytes , and couWe have used LCM to isolate developing parotid acinar cells in a nearly comprehensive approach to identify profiles of mRNAs and miRNAs which drive terminal differentiation. Cluster analysis shows 4 stages of development with strongly increased expression of key parotid genes, including several secreted cargo proteins, in the final stage . Data frS1 FigUsing LCM, parotid acini were able to be isolated in both embryonic and postnatal tissue. (A) H&E stained cryosections (5 μm) of rat parotid gland from embryonic day 20 (E20) and postnatal day 25 (P25). (B) Subsequent capture of cells on LCM cap, shows acinar cell isolation without appreciable contamination of ductal cells or connective tissue.(PDF)Click here for additional data file.S2 FigPsp, Xbp1, and Nupr1. Expression of Rplp2 was used for normalization. The expression profiles (plotted in Log base 10) replicate the increase in expression seen in the microarrays.Taqman qRT-PCR was run on triplicate RNA samples spanning nine time points of parotid acinar differentiation, using primers that amplify (PDF)Click here for additional data file.S3 FigQuadratic regression was used to identify mRNAs with expression profiles that significantly match a quadratic model.430 were identified as having a significant match  and a heatmap was generated using a dissimilarity distance matrix.(PDF)Click here for additional data file.S4 FigGenes with a significant cubic trend were clustered into the above 5 expression profiles. Only 18 genes were available for clustering and most clusters have only a few genes.(PDF)Click here for additional data file.S5 Fig69 miRNAs have a significant linear trend and are divided into two clusters. 55 increase in expression, while 14 decrease. Normalized CT values for each microRNA were scaled to a mean = 0 and stdev = 1 before plotting in gray. The red line traces the average expression for the cluster.(PDF)Click here for additional data file.S6 FigPsp promoter contains two CCACG boxes which is a consensus sequence for Xbp1 binding. (B) Two E-boxes flank exon 3 in the Psp gene which match the Mist1-binding consensus sequence.(A) the (PDF)Click here for additional data file.S7 Figtargetscan.org). (B) However the rat sequence contains a predicted miR-214 binding site about 600 bp upstream.(A) The human miR-214 binding site in the Xbp1 3'UTR is not conserved in rat ((PDF)Click here for additional data file.S1 TableCloning primers used for luciferase based assays.(PDF)Click here for additional data file.S2 TableGO enrichment analysis of DE cluster #1 .(XLSX)Click here for additional data file.S3 TableGO enrichment analysis of DE cluster #2 .(XLSX)Click here for additional data file.S4 TableEnrichment of transcription factor targets in DE cluster #2 .(PDF)Click here for additional data file.S5 TableGO enrichment analysis of DE cluster #4 .(XLSX)Click here for additional data file.S6 TableGO enrichment analysis of quadratic cluster #1 .(XLSX)Click here for additional data file.S7 TableGO enrichment analysis of quadratic cluster #7 .(XLSX)Click here for additional data file.S8 TablePredicted target genes were generated in Targetscan for differentially expressed miRNAs.(XLSX)Click here for additional data file.S9 TableTarget gene enrichment analysis. Enrichment of DE clusters in predicted miRNA target genes.(XLSX)Click here for additional data file.