Chromatin in sperm is different from that in other cells, with most of the genome packaged by protamines not nucleosomes. Nucleosomes are, however, retained at some genomic sites, where they have the potential to transmit paternal epigenetic information. It is not understood how this retention is specified. Here we show that base composition is the major determinant of nucleosome retention in human sperm, predicting retention very well in both genic and non-genic regions of the genome. The retention of nucleosomes at GC-rich sequences with high intrinsic nucleosome affinity accounts for the previously reported retention at transcription start sites and at genes that regulate development. It also means that nucleosomes are retained at the start sites of most housekeeping genes. We also report a striking link between the retention of nucleosomes in sperm and the establishment of DNA methylation-free regions in the early embryo. Taken together, this suggests that paternal nucleosome transmission may facilitate robust gene regulation in the early embryo. We propose that chromatin organization in the male germline, rather than in somatic cells, is the major functional consequence of fine-scale base composition variation in the human genome. The selective pressure driving base composition evolution in mammals could, therefore, be the need to transmit paternal epigenetic information to the zygote. In most cells, DNA is packaged by protein complexes called nucleosomes. In sperm, however, nucleosomes are only retained at a small fraction of the genome, particularly at the start sites of genes. In this work, we show that the sites at which nucleosomes are retained in sperm are specified by variation in the base composition of the human genome. At a fine scale, the human genome varies extensively in the content of GC versus AT base pairs, and we find that in both genic and non-genic regions this predicts very well where nucleosomes are retained in mature sperm. These regions include transcription start sites, especially for genes that are expressed in all cells and for genes that regulate development. We also report that regions that retain nucleosomes in sperm are likely to be protected from DNA methylation in the early embryo, suggesting a further connection between the presence of nucleosomes on the paternal genome and the establishment of gene regulation in the embryo. Based on these results, we propose that an important selective pressure on base composition evolution in mammalian genomes may be the requirement to organize chromatin in sperm in a way that facilitates gene regulation in the early embryo. The chromatin of mature sperm differs dramatically from that of other cell types. Most of the sperm genome is packaged by small basic proteins called protamines, with only a few genomic sites remaining bound by nucleosomes In human sperm about 4% of the genome remains bound by nucleosomes Although they are transcriptionally inactive, mature spermatozoa do contain nucleosomes containing histones marked by post-translational modifications, including both activation (e.g. methylation of histone H3 lysine 4) and repression marks (e.g. tri-methylation of H3 at lysine 27) in vitroIn somatic cells and in lower eukaryotes several important influences on nucleosome occupancy and positioning have been demonstrated. First, many nucleosomes are located ‘statistically’ In vitro, nucleosomes bind preferentially to GC-rich DNA in vivo analysis of a number of mouse GC-rich promoters reached the opposite conclusion: GC-rich promoters were depleted of nucleosomes in vivoGC-content peaks are found at the promoters of many human genes, where they are termed CpG islands because of the elevated frequency of CpG bases de novo establishment of methylation patterns In the early embryo, genome-wide erasure of DNA methylation is followed by the Given the lack of transcription, we reasoned that the major influence of GC-content on chromatin organization might occur in the male germline rather than in somatic cells. Here we test this idea, and show that nucleosome retention in human sperm is indeed strikingly related to fine-scale base composition variation. Across both genic and non-genic regions of the genome, nucleosome retention sites are extremely well predicted by GC-composition. The retention of nucleosomes at GC-rich sequences with high intrinsic nucleosome affinity accounts for the previously reported enrichment of nucleosomes both at transcription start sites and at genes that regulate development. It also means that nucleosomes are retained at the start sites of most universally expressed genes, which may be important for their activation in the early embryo. Further, we report a striking association at CpG islands between nucleosome retention in sperm, and the establishment of unmethylated regions in the early embryo. This suggests that paternal nucleosome retention may assist in the establishment of these regions, possibly through the retention of H3K4me3-marked histones. Our findings suggest that chromatin organization in the male germline, rather than that in somatic cells, is the major functional consequence of fine-scale base composition variation in the human genome. We suggest that the selective pressure on this may be the requirement to propagate paternal epigenetic information to the embryo.Sites of nucleosome retention in mature human sperm were identified genome-wide by Hammoud and co-workers using micrococcal nuclease (MNase) digestion followed by deep sequencing. Comparing mononucleosome fragments to a sonicated input control, 25,121 genomic regions were identified with statistically significant enrichment for sperm nucleosomes −16). This is not accounted for by the known GC-bias of Solexa sequencing −16). Further, GC-content also correlates with nucleosome enrichment as quantified by microarray hybridization in a second study using two different extraction protocols  Considering the whole genome, there is indeed a striking correlation between GC-content and the number of sequenced mononucleosome fragments isolated from sperm . This suggests that other factors such as competition with protamines or transition proteins, CpG-binding proteins High GC-content is associated with an increased binding affinity for nucleosomes The transmission of paternal nucleosomes The retention of nucleosomes at distal regulatory regions defined by DNase I hypersensitive sites is also consistent with a model in which the transmission of paternal nucleosomes and their modifications influences gene expression in the early embryo. Further, we suggest that the need to retain nucleosomes in sperm may explain why widely and highly expressed genes have high nucleosome occupancy encoded at their transcription start sites in the human genome, in contrast to the nucleosome-free regions encoded in the yeast genome de novo methylation, and these islands are accurately predicted by their nucleosome retention in sperm (74% of regions are identified with a precision of 86%). This suggests the interesting model that paternal nucleosome inheritance might aid in the establishment of DNA methylation-free regions  We also analyzed two additional nucleosome retention scores along the human genome from data generated by a second laboratory et alWe used the H3K4me3 and H3K27me3 ChIP-Seq data generated by Hammoud et alWe classified genes as overlapping retained nucleosomes at their transcription start sites when one or more start site is within 50 bp of a nucleosome enrichment region defined by sequencing from Hammoud et al using MNase digestion and deep sequencing We used the nucleosome occupancy data generated by Schones et alNucleosome binding preferences were predicted using the model of Kaplan et alet alWe retrieved gene expression data for ten tissues  quantified by Solexa sequencing from Wang et alThe abundance of mRNA in mature sperm from 13 different fertile donors was measured using Affymetrix gene expression microarrays by Platts Gene ontology (GO) annotations of genes were obtained from Ensembl. Genes coding for developmental transcription factors were defined as genes annotated with the Biological Process term “DNA-dependent regulation of cellular transcription” (GO:0006355) and also with a term that contains the word “development.”et al. This analysis was also performed separately for genic and non-genic windows (here 1 kb upstream of each start site was included in the genic portion of the genome). Receiver operating characteristic (ROC) analysis was used to assess how well we can predict the regions of nucleosome retention enrichment in sperm from GC- and CpG-content. ROC analysis was performed in R using the ROCR package et alTo test the performance of GC-content as a predictor of nucleosome retention throughout the human genome we used all non-repetitive 150 bp windows of the genome. We further excluded windows that had in total less than 5 sequenced reads from the nucleosome and the genomic control datasets, as low-read count windows were also excluded from the nucleosome retention peak finding algorithm used by Hammoud We retrieved the locations of DNase I hypersensitive sites for H1 human embryonic stem cells et alet al. Average signals were calculated for 4 kb regions centered at the middle position of the islands.We used the DNA methylation status annotation of CpG islands reported in Straussman Figure S1et al; micrococcal nuclease digestion  and salt-extraction followed by restriction digestion . In both cases the probe value corresponds to the ratio of the nucleosome to protamine normalized signal intensity. The GC-content shown corresponds to the window ±147 bp from the microarray probe.Correlations between GC-content and nucleosome to protamine normalized probe signal intensity ratio. The two plots show the correlation for the data generated by two different experimental methods of identifying nucleosome enriched regions from Arpanahi (EPS)Click here for additional data file.Figure S2et al. (B) The plot shows the percentage of transcription start and end sites that are within 50 bp of a nucleosome retention region. The total number of human (Ensembl) transcripts considered here is 52,312. Error bars indicate 95% confidence intervals.Nucleosomes are preferentially retained in sperm at transcription start sites. (A) Genome-wide distribution of nucleosome retention sites. Nucleosome enrichment regions are classified according to whether they are within 50 bp of a transcription start site , or end site , overlapping other parts of a gene  or other parts of the genome . The coordinates of regions of nucleosome retention defined by sequencing were retrieved from Hammoud (EPS)Click here for additional data file.Figure S32 -test for trend in proportions = 626.1, p-value<2.2×10−16). (B) However, there is also a strong positive correlation between expression breadth (the number of different tissues in which a gene is expressed) and nucleosome retention at the start site . The total number of genes (and the number of genes with nucleosomes retained at the start) in each bin are as follows: 0-tissues = 792 (125), 1-tissue = 1472 (313), 2-tissues = 835 (284), 3-tissues = 742 (316), 4-tissues = 653 (264), 5-tissues = 562 (251), 6-tissues = 617 (287), 7-tissues = 657 (324), 8-tissues = 817 (433), 9-tissues = 1248 (711), 10-tissues = 7502 (4555). (C) The correlation between nucleosome retention at the start and expression level is largely explained by expression breadth. Here, we test the correlation between expression level and nucleosome retention at the start of genes, when controlling for expression breadth. In C, each panel corresponds to the genes in each expression breadth shown in panel B. Genes are sorted according to expression level and split in ten equally sized bins. The x-axis shows the average expression level of the genes in each bin. The y-axis shows the percentage of genes in a bin that have a start site within 50 bp from a region of nucleosome retention in sperm. From this figure it is also apparent that when controlling for expression level, the expression breadth of a gene correlates with the likelihood of nucleosome retention at the gene start (e.g. compare high expression level bins for genes expressed in one versus ten tissues).Nucleosome retention at transcription start sites correlates with the expression breadth of a gene rather than the expression level in the germline. (A) There is a positive trend in the proportion of genes that have retained nucleosomes at the start site and the expression level of the gene in testes Click here for additional data file.Figure S42 -test for trend in proportions = 158.9, p-value<2.2×10−16). (B) Expression breadth shows strong correlation with nucleosome retention at the gene start . The number of genes per bin are; 166 (0 tissues), 632 (1 tissue), 293 (2 tissues), 304 (3 tissues), 273 (4 tissues), 233 (5 tissues), 283 (6 tissues), 318 (7 tissues), 419 (8 tissues), 763 (9 tissues), 5,222 (10 tissues). (C) When considering genes expressed in one tissue only, i.e. tissue-specific genes, the trend between nucleosome retention and expression level in the germline is greatly reduced . (D) When considering genes expressed in all tissues, i.e. housekeeping genes, the trend between nucleosome retention and expression level in the germline is also greatly reduced . All genes with testes expression >0 and with present mRNA in at least seven out of thirteen healthy sperm donors are considered here . Genes are considered to be expressed in a tissue when their expression level is > = 0.5 RPKM. Genes are ranked according to increasing expression level in testes and split in bins with the same number of genes. The x-axis shows the average expression level of the genes in each bin. The y-axis shows the percentage of genes in a bin that have a start site within 50 bp from a region of nucleosome retention in sperm.Relationship between nucleosome retention at transcription start sites and the expression level or breadth of mRNAs detected as present in the mature sperm of at least seven out of thirteen healthy sperm donors. (A) The percentage of genes that retain nucleosomes at their start increases with gene expression level Click here for additional data file.Figure S5et al and Brykczynska et al. (G–I) Histone variant H2A.Z shows no enriched at gene start sites. This agrees with the observation that this histone variant is only enriched in pericentric heterochromatin.Sperm H3K4me3, H3K27me3 and H2A.Z retention at the start sites of housekeeping, tissue-specific and developmental regulatory genes. (A–C) Retention of H3K4me3 at the start sites of the three gene classes is similar to nucleosome retention, shown in main (EPS)Click here for additional data file.Figure S6Correlations between sperm nucleosome retention and GC- or CpG-content. Each point shows the correlation for all unique 150-bp windows in the human genome with the same number of G+C nucleotides (A) or CpG dinucleotides (B). GC-content correlates robustly with nucleosome retention over a wide range of window CpG-contents, whereas CpG content only correlates strongly with retention at high GC-contents. The analysis was carried out on windows with at least 5 reads from the histone and the input control dataset, as in (EPS)Click here for additional data file.


Aspergillus nidulans LAMMER kinase, LkhA, and report its involvement in multiple developmental processes. The gene for LkhA was highly expressed during reproductive organ development, such as that of conidiophores and cleistothecia. During vegetative growth, the patterns of germ tube emergence and hyphal polarity were changed and septation was increased by lkhA deletion. Northern analyses showed that lkhA regulated the transcription of brlA, csnD, and ppoA, which supported the detrimental effect of lkhA-deletion on asexual and sexual differentiation. LkhA also affected expression of cyclin-dependent kinase NimXcdc2, a multiple cell cycle regulator, and StuA, an APSES family of fungal transcription factors that play pivotal roles in multiple differentiation processes. Here, for the first time, we present molecular evidence showing that LAMMER kinase is involved in A. nidulans development by modulating the expression of key regulators of developmental processes.LAMMER kinase plays pivotal roles in various physiological processes in eukaryotes; however, its function in filamentous fungi is not known. We performed molecular studies on the function of the  LAMMERILG”, which is important for the kinase activity and substrate recognition Drosophila melanogaster, results in recessive lethality Schizosaccharomyces pombe LAMMER kinase, Lkh1, acts in filamentous adhesion growth on agar surfaces and negative regulation for a divalent cation-dependent asexual flocculation The LAMMER kinases are found throughout eukaryotes and are known to conserve the motif “EHAspergillus nidulans has a complex life cycle. The duplication cycle is initiated after breaking the dormancy of an asexual spore The homothallic ascomycete brlA and abaAbrlA is expressed in the form of two overlapping transcriptional units, brlAα and brlAβ, that are required for normal conidiophore production upstream developmental activators (UDAs), which are encoded by six genes  that are responsible for vegetative growth, conidiation, sterigmatocystin biosynthesis, and trehalose biogenesis During the asexual cycle, conidia (mitospores) reproduction is spatiotemporally controlled by a central regulatory pathway, which includes A. nidulans. The fungal CSN complex is not essential for viability, but is involved in cell cycle progression and development. In addition, the CSN complex regulates the transcription level of ppoA, a psi  factor β, psiα, and psiγ) influence sexual and asexual sporulation, especially spatial modulation and balance between conidiation and ascosporogenesis Sexual reproduction is influenced by various environmental and endogenous factors; however, this process is not well characterized compare to the asexual cycle. Several factors affecting the maturation process of cleistothecia have been identified, including VeA, NsdD, COP9 signalosome (CSN), and psi factors  A. nidulans is controlled by complicated signal transduction and genetic regulatory pathways. Various developmental regulatory proteins are also involved in the formation of conidia during mitosis and ascospores during karyogamy (nuclear fusion) and meiosis. Cdc2, a mitosis-promoting factor (MPF), is universally conserved and central to the timing and initiation of mitosis in all eukaryotic cells A. nidulans, the cdc2AFnimX mutant, which is mutated at the inhibitory phosphorylation sites Thr14 and Tyr15 into non-phosphorylatable residues, shows a deficiency in the S-phase and G2/M DNA damage checkpoint controls, resulting in defects in septation, sporulation, and morphogenesis during conidiation cdc2nimX mRNA expression is induced by ectopic expression of brlAA. nidulans.As previously mentioned, differentiation in A. nidulans LAMMER kinase, LkhA, was not essential for viability, but its deletion revealed pleiotropic phenotypes that show changes in the expression of a fungal specific APSES family consisting of a basic helix-loop-helix transcription factor, StuA. To our knowledge, this is the first study on the involvement of LAMMER kinase in developmental regulation and cell cycle progression in filamentous fungi.In this paper, we identified LAMMER kinase, a novel regulator of development, in filamentous fungi. A. nidulans strains employed in this study are listed in Escherichia coli strain (DH5α) was applied as described elsewhere The et al. niiA promoter or pQa harboring an alcA promoter and pyroA marker for selection. The niiA promoter was induced using 0.6% sodium nitrate and repressed by adding 0.2% ammonium tartrate. The alcA promoter was induced by adding 2% threonine and repressed by adding 1% glucose.Fungal genomic DNA was isolated as described by Kim Aspergillus nidulans argB gene from FGSC4 was used as a selectable marker for transformation. The information of primers for fusion products are listed in lkhA  were amplified using primer sets, LkhA-A1/LkhA-A2, LkhA-B1/LkhA B2, and argB-For/argB-Rev. The full deletion cassette obtained by double joint PCR using nested primers LkhA-C1/LkhA -C2  was added to the cell powder. After vortexing, the debris was removed by centrifugation. The supernatant was then overlaid onto 2 ml of 5.7 M CsCl solution . The RNA pellet was isolated by centrifugation at 10°C, 125,000×g for 16 h using an SW 55Ti rotor . The pellet was resuspended in 400 ul TES buffer  and extracted with phenol/chloroform (pH 4.5). RNAs were enriched by ethanol precipitation, dissolved in DEPC-treated water, and stored at −70°C. Ten to twenty micrograms of total RNAs preparations was separated on an 1% agarose gel containing formaldehyde and transferred onto a Hybond-N+ membrane . Gene-specific probes were prepared from PCR-generated fragments and labeled with [α-32P] dCTP using a random primer DNA labeling kit . Signal was visualized by exposing the filter to X-ray film . The band intensity was calculated by computer program Image J.Total RNAs were prepared by a modified guanidine thiocyanate/CsCl density gradient ultracentrifugation method, as described by Glisin A. nidulans cells were harvested after an appropriate time and immediately ground using liquid nitrogen. The cells were resuspended in protein extraction buffer  with 2 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail , 10 mM sodium fluoride and 1 mM sodium vanadate and centrifuged at 16,600×g, 4°C for 30 min to obtain the supernatant. One hundred and fifty micrograms of total protein was solubilized by boiling in 5x SDS-PAGE sample buffer  for 5 min and resolved on 12% SDS-PAGE gels. The proteins were electroblotted onto a PVDF membrane with transfer buffer  using a Semiphor transfer unit . The membrane was saturated with a blocking buffer [5% skim milk in TBST ] for at least 1 h at room temperature. The primary antibodies against the Cdc2 PSTAIRE motif (Santa Cruz Biotechnology) and α-tubulin , and secondary antibody against the goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) were used. Immunoblotting was carried out by the enhanced chemiluminescent method using the WEST-oneTM Western Blot Detection System  as described in the manufacturer's manual.Protein extraction was performed as described previously 2. Finally, the samples were sputtered with gold in a SC502 ‘mini’ sputter coater  and observed using the scanning electron microscope . For transmission electron microscopy, the cells were fixed in 2.5% paraformaldehyde-glutaraldehyde mixture buffered with 0.1 M phosphate (pH 7.2) for 2 h, post-fixed in 1% osmium tetroxide in the same buffer for 1 h, dehydrated in graded ethanol and propylene oxide, and embedded in Epon-812. Ultra-thin sections, prepared using a Leica ULTRACUT E ultramicrotome , were stained with uranyl acetate and lead citrate and examined under a CM-20 electron microscope .Calcofluor white and DAPI staining were performed as previously described Aspergillus nidulans, a homologous gene was identified by searching through the A. nidulans genome database for its conserved motif, EHLAMMERILG, and we named this LkhA . The ORF of lkhA, which was 2,253 bp in length, was divided into six exons and 5 introns  and sexual development . At least two isoforms of the lkhA transcript were detected during vegetative growth, suggesting that lkhA can influence fungal development, possibly through alternative splicing, which was earlier observed in Drosophila Doa.To analyze the temporal expression pattern of analysis . NortherlkhA, a gene-deletion assay was performed by homologous recombination using lkhA knock-out cassette and confirmed by Southern analysis (data not shown). The lkhAΔ strain was viable, but showed abnormalities in growth such as reduced radial growth  in liquid medium .To understand the functions of l growth  and depo mutants , suggest at 37°C . Becaused medium , suggesttion See . The hyp culture . To obselkhAΔ strain displayed a reduced number of primary sterigmatae, the emergency of secondary conidiophores from its vesicle, and irregularity in the length of sterigmata (non-separated)  of conidia produced in the wild type.To investigate the function of LkhA in asexual development, the morphology of the asexual reproductive organ, the conidiophore, was examined using light (LM) and scanning electron microscopy (SEM). In contrast to normal conidiophores, which are highly symmetric structures consisting of a stalk, vesicle, double layer of sterigmatae , and a chain of dormant spores (conidia), those of the parated) . When thparated) , the lkhlkhA deletion, the expression of transcription factors for asexual development was analyzed. As shown in flbB, flbC, and flbD, showed no dramatic changes during the vegetative stage following by lkhA deletion. After the initiation of asexual development, however, the level of brlAβ and brlAα expression was dramatically decreased in the lkhA deletion mutants. The level of abaA expression was also reduced significantly in the lkhA deletion mutants approximately 12 h after asexual development. These results are consistent with abnormal conidiophore morphogenesis, particularly defects in sterigmata formation and reduced production of conidia in the lkhAΔ strain. These results suggest that the lkhA may play a role in conidiophore development through transcriptional modulation of brlA expression.To understand changes in asexual development at the molecular level that may be associated with the lkhA deletion  and abnormal conidiophores morphogenesis , whereas over-expression of nimX in the lkhA-deletion strain, a partial reversion was observed in that of the mutant phenotype, such as disappearance of septa in conidiophore stalk (th panel). Over-expression of cdc2AFnimX in the lkhAΔ strain resulted in the exacerbation of irregularities in conidiophores, including thickened and multi-septated stalks, lack of sterigmata  . CleistolkhA deletion, the transcription level of csnD, core component of the COP9 signalosome (CSN), which is particularly related with maturation of primordium to cleistothecium csnD transcription was lower, particularly during the early sexual stage (S24) of the lkhAΔ strain, whereas in the wild type a significant level of expression from the early stages to the late stages was observed . Because lkhAΔ showed a decrease in ascospore production, transcription of ppoA, which is involved in production of the psi factor, psiBα, was tested. The psiBα stimulates sexual spore production, but represses asexual spore production ppoA transcription was observed at the vegetative stage (V12), whereas an upregulation of transcription was observed during sexual development in the wild type. These results suggest that lkhA influences ascosporogenesis through the transcriptional regulation of ppoA.To confirm whether the failure in formation of sexual reproductive organs was due to the changes in expression of the sexual reproductive organ-related genes, the transcription of sexual genes was analyzed. Because the developmental immaturity of cleistothecial primordia was observed by observed . Decreaselopment . To testcospores . These rlkhAΔ strain cultured under normoxia showed an earlier appearance of Hülle cells  and produces isoforms during the vegetative growth  of NimX was reduced the lkhA deletion mutants . This result suggests that LkhA affects the cell division cycle by modulating expression of the NimXcdc2. On the other hand, conidiophores of the lkhAΔ strain with forced expression of cdc2AFnimX (non-tyrosine phosphorylated form) showed an exacerbation of abnormalities, such as an increase in septa in the stalk and irregular formation of reproductive organs  is associated with transcriptional regulation, protein phosphorylation, deubiquitination, development, DNA-damage checkpoint control by direct interaction with ataxia-telangiectasia mutated (ATM), and the cell cycle in mammals al stage . Howeverdeletion . The def of csnD . AlthougppoA is involved in the production of a psi factor, psiBα, which balances the production of conidia and ascospores, including the initiation of asexual and sexual reproductive organs. The transcript level of ppoA in the lkhAΔ strain was reduced during acquisition of developmental competence  was changed in lkhA deletion mutants  and sexual stages (S48), indicating that LkhA may have regulated the factors that activate the transcription of stuAα and stuAβ at the upstream activation sequences (UASs) of stuA. These results suggest that LkhA directly and/or indirectly controls the expression of stuA, which is required for temporal regulation of the entire life cycle of A. nidulans.Deletion of ild type , suggest mutants . StuA, alkhAΔ strain showed pleiotropic defects in germination and formation of developmental organs. Northern blot analyses revealed that LkhA affected fungal differentiation through the transcriptional regulation of various developmental genes. Among these, stuA has recently been associated with morphogenesis, sporogenesis, pathogenicity, and the production of secondary metabolites in many filamentous fungi stuAβ may regulate stuA expression, stuAα is expressed in a brlA-dependent manner during development by binding to BrlA at the BrlA response elements found in the stuAα promoter stuA expression through the transcriptional control of BrlA, particularly during mid- to late asexual development , A/TCGCGT/ANA/C, is identical to the MCB (Mlu I-cell cycle box) binding motif, ACGCGTNA, and the MBF (MCB-binding factor) complex that regulates the cell cycle in S. cerevisiae and S. pombe. In addition, the DNA-binding motif of StREs is enriched with promoter sequences of genes belonging to the functional MIPS category “Cell cycle and DNA processing” in the three ascomycetes, Fusarium graminearum, Neurospora crassa, and S. cerevisiaeA. nidulans cell cycle genes nimE and nimO was reported nimX promoter region also revealed two StREs located at −434 bp and −444 bp (data not shown). These results suggest that the transcription of nimX is modulated by StuA. Although further investigation is needed, it may be postulated that LkhA modulates the regulatory circuit, which consists of BrlA, StuA, and cell cycle genes, including NimX Click here for additional data file.Figure S2qRT-PCR analysis of mRNA level for csnE and csnD, which are components of the COP9 signalosome. Relative mRNA levels were determined by quantitative real-time (qRT)-PCR. Gene expression levels were normalized with tubC amplified RNAs extracted from 14 h cultured mycelia. The X-axis indicates the culture time after transfer of mycelia onto the MM to allow development.(TIF)Click here for additional data file.Table S1List of A. nidulans strains used in this study.(DOCX)Click here for additional data file.Table S2List of oligonucleotides used in this study.(DOCX)Click here for additional data file.


The T-box transcription factor Brachyury (T) is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos. Work in the frog and the zebrafish has identified some direct genomic targets of Brachyury, but little is known about Brachyury targets in the mouse.Axin2, Fgf8 and Wnt3a, is down regulated in Brachyury mutant embryos and we demonstrate that they are also Brachyury targets in the human. Surprisingly, we do not observe enrichment of the canonical T-domain DNA binding sequence 5′-TCACACCT-3′ in the vicinity of most Brachyury target genes. Rather, we have identified an (AC)n repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus. We do not understand the significance of this sequence, but speculate that it enhances transcription factor binding in the regulatory regions of Brachyury target genes in rodents.Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells. The targets we identify are enriched for sequence-specific DNA binding proteins and include components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Expression of some of these targets, such as Our work identifies the genomic targets of a key regulator of mesoderm formation in the early mouse embryo, thereby providing insights into the Brachyury-driven genetic regulatory network and allowing us to compare the function of Brachyury in different species. Brachyury (T) is expressed in the primitive streak, tailbud and notochord of the early mouse embryo XenopusBrachyury orthologues resemble those of the mouse, and these genes play similar roles in early development Brachyury function has been conserved throughout evolution.Xenopus embryos we have used differential screening approaches to isolate target genes such as eFGFBix family Wnt11In an effort to understand how Brachyury exerts its effects, we have searched for genomic targets of this transcription factor. In previous work using 5′-TCACACCT-3′Axin2 (Axil/Conductin), which encodes a negative regulator of Wnt signalling, as well as Wnt3a and Fgf8. Significantly, expression of all three genes is down regulated in homozygous Brachyury mutant embryos and we show by ChIP-qPCR that these are also genomic targets of BRACHYURY in differentiating human ES cells. These results are consistent with work in the zebrafish emphasising the importance of Wnt and Fgf signal transduction pathway components as Brachyury targets Axin2 in the early mouse embryo is regulated by Brachyury as well as by TCF/Lef proteins Our results show that Brachyury targets in differentiating ES cells are enriched for sequence-specific DNA binding proteins and components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Interestingly, most binding peaks were not enriched for the canonical T-box binding site Brachyury usually peaked at day 4 of differentiation , and then to parts of the brain and tail Axin2, Wnt3a and Fgf8, which are discussed below, genes that have been reported to be co-expressed with Brachyury include Msgn1, whose expression is down regulated in Brachyury mutants Meis1Trim 28Zic2Foxa2, present in the node and notochord Adam19 (meltrin beta), present in tailbud mesenchyme Brachyury (or are activated shortly after Brachyury) in embryoid bodies, and typical profiles of Msgn1, Meis1 and Foxa2 expression are shown in Brachyury during gastrulation The targets we identify include components of the WNT, MAPK, JNK, TGF-β, Hedgehog, FGF and G-protein coupled signal transduction pathways . AnalysiGdf5, Lmx1b and Dlx5 are involved in morphogenesis (−6), Gabra1, Gfra3 and Cbln3 are involved in anchoring the plasma membrane to cytoskeletal proteins (−3) and others encode proteins involved in cell adhesion, such as the glycosyltransferases B4galnt2 and Cml4/Nat8. This last observation is consistent with data showing that glycosyltransferases, and especially galactotransferases, are mis-expressed in T/T mutant mice Xenopus orthologue Camello (Xcml), inhibits gastrulation in XenopusIn particular, cellular component analysis highlights gene products involved in morphogenesis, cell adhesion and cell polarity. Targets such as Asap1 (Ddef1), which encodes an ADP-ribosylation factor GTPase-activating protein implicated in metastatic prostate cancer WT1Interestingly, another group of targets is associated with germ cells , includi−5). Several families of transcription factors are represented, including the Ets, paired box, homeobox, winged helix/forkhead, bZip and zinc finger families. Under our ES cell culture conditions, expression of many of these transcription factors peaks either at the same time as Brachyury  or just afterwards   are transcription factors , and indnd Slug) . As we d5′-TCACACCT-3′n n region is mutated. Together, these observations indicate that the (AC)n sequence interacts only weakly with Brachyury, if at all, and that its role may be restricted to stabilizing binding to an adjacent or even a distant T box site.To assess the significance of these observations we first performed electrophoretic mobility shift assays . As expeXenopus genomes shows that 38 of these AC-rich regions are unique to the mouse while 68 are also present in the rat. Sixteen of the AC repeats are present in the human genome, of which 11 are also present in rat. However, none of the repeats are conserved in zebrafish or Xenopus . By E8.5, when Axin2 is expressed in the headfold, tailbud and primitive streak of wild type embryos, its expression in the posterior region of Brachyury mutant embryos is very weak or absent  positioned 63 nucleotides from an (AC)19 repeat. The temporal expression profile of Fgf8 resembles that of Brachyury during embryoid body differentiation . Our experiments involving ChIP-qPCR with hESC-derived mesoderm cells indeed detected a strong enrichment for these sequences, thus indicating that BRACHYURY binds to the same genomic regions in the human  are expressed in the early mouse embryo during formation of the primitive streak, and their proper expression during development requires Brachyury function. We also note that several targets are expressed in primordial germ cells, perhaps the first differentiated population to emerge during early gastrulation Brachyury until E12.5, when the gene is down regulated in a non-migrating population Brachyury may not be involved directly in the specification of the germ cells We have identified genomic targets of Brachyury in differentiating mouse ES cells, demonstrating that embryoid bodies provide sufficient material for chromatin immunoprecipitation experiments and that they represent an effective model of early mouse development. Although they do not undergo proper morphogenesis, they do generate pattern, as illustrated by the formation of beating cardiomyocytes Brachyury mouse embryos is poorly developed Our work has identified 396 potential targets of Brachyury, and gene ontology analysis indicates that many of these encode sequence-specific DNA binding proteins and proteins involved in cell adhesion and embryonic morphogenesis. Analysis of the former category will help in the elucidation of the genetic regulatory network that underlies mesoderm formation (see below). The latter category includes cell junction proteins and glycosyltransferases wnt11, snail1a and blf and this analysis Wnt3a, Snail2 and genes such as Gdf5, Etv1, Krt5, Krt8, Lmx1b, Syk, and Gnaq); muscle specification (where both studies identified Msgn1 and Pax3); posterior identity  share little homology with those of their zebrafish orthologues. It is possible that Brachyury binds different locations in different genomes, which has been noted for other transcription factors et al., in preparation). We note that both the mouse and zebrafish arrays were based on promoter regions, so that enhancer binding is not available in these datasets, perhaps explaining why some targets seem to be unique to each species.The fact that there are some differences between the mouse and zebrafish targets may derive from the presence of an additional  n repeat sequence that recognised, albeit weakly, the Brachyury T domain in electrophoretic mobility shift experiments. We do not yet fully understand the significance of this observation. Mouse Brachyury binds to an imperfect T-box site palindrome in the Nanog promoter Drosophila Brachyenteron, where modular variations on the T-box consensus binding sequence determine the degree of transcriptional activation Ciona, with regulatory motifs comprising Ci-Brachyury and Ci-foxA binding sites Our previous work searching for targets of zebrafish Ntl showed that the canonical T-box site TCACACCT was enriched in the vicinity of Ntl target genes et al., in preparation).Moreover, many transcription factors bind directly to DNA in distal enhancer elements It is also possible that the AC repeats cause the transient formation of left handed DNA helices and bends, changing the chromatin architecture and encouraging transcription factor binding Wnt3a and Axin2 are expressed normally in Brachyury homozygous mutant embryos, and found that although both genes are expressed at E7.5 , neither is expressed at E8.5 in mesodermal derivatives  Act 1986.3 units/ml Embryonic stem (ES) cell culture was as described ES cells were differentiated in spinner flasks to produce large numbers of embryoid bodies (EBs) undergoing synchronized differentiation 7 cells in 5 ml medium. After 24 h (day 1 of differentiation) a further 50 ml spinner medium was added to each flask. Each day thereafter EBs were allowed to sink and 50 ml medium was aspirated and replaced with 50 ml fresh pre-warmed spinner medium.Medium (45 ml) was pre-equilibrated in 100 ml silicon-coated  spinner flasks (Integra Biosciences). ES cells were recovered from the gelatin-coated differential sedimentation plates and centrifuged at 800 g for 5 min. Cells were fully dissociated to ensure that cultures were initiated from single cells, and each spinner flask was inoculated with 10Human ESCs  were maintained and differentiated as previously described beta actin primers were used as an endogenous control to express relative expression levels , digested with DNA-free DNAse I (Ambion), and checked for integrity using an Agilent Technologies 2100 Bioanalyser. cDNA was generated from l µg RNA using Superscript III Reverse Transcriptase , and this was followed by real-time PCR using the LightCycler 480 SYBR Green I master kit (Roche). Mouse n levels .Several anti-Brachyury antibodies were tested for use in this work. Of these, the goat polyclonal C19 antibody , raised against a C-terminal sequence of human Brachyury, performed best in chromatin immunoprecipitation. This antibody, raised against a divergent region of Brachyury that does not include the T box, has been well characterised in previous studies Brachyury mutant embryos from BTBR T+tT/J×BTBR T+tT/J heterozygote crosses Axin2 probe was generated from IMAGE clone 1361800 (Geneservice), a Wnt3a probe from IMAGE clone pENTR223.1 100015989 after subcloning into pCS2+ (Fgf8 probe from IMAGE clone 6513131 (Geneservice) in pCMV-SPORT 6.1. Digoxigenin labelled or fluorescein labelled antisense RNA probes were generated using T7 RNA polymerase from linearised templates and whole mount in situ hybridisation was performed as described Wild type mouse embryos were collected from MF1 or 129 strains, and to pCS2+ , and an Embryos were fixed as described above and rehydrated to PBS for staining. Free aldehyde groups were blocked using 1 M glycine, embryos were washed in PBS/0.1% Tween 20 (PBST), and endogenous peroxidases were blocked using 3% hydrogen peroxide in PBS. Embryos were incubated overnight at 4°C in 1∶400 C19 antibody in PBST supplemented with 0.2% bovine serum albumin (BSA) and 10% heat inactivated FBS. They were then washed, incubated with 1∶400 rabbit anti-goat biotinylated IgG , and stained using Vectastain Elite ABC substrate (Vector laboratories) with Sigma Fast Nickel Enhanced DAB chromagen (Sigma).Brachyury mRNA was synthesized using the pCS2+ construct described above and the Ambion mMessage mMachine (Applied Biosystems/Ambion). mRNA was translated in a rabbit reticulocyte lysate (Promega). In vitro translation products and embryoid body extracts were subjected to polyacrylamide gel electrophoresis (PAGE) and western blots were performed using CAPS transfer buffer . Membranes were blocked with 5% milk powder in PBST overnight, and antibodies were diluted in the same solution. Washes were in PBST. Primary antibodies were R&D Systems anti-T and SantaCruz anti-T (see above). Both were used at a dilution of 1∶250. Secondary antibodies were HRP-linked SantaCruz D anti-goat IgG  and HRP-linked Amersham NA934V anti-rabbit IgG . All antibody incubations were 1 hour at room temperature. Both endogenous and in vitro translated T proteins were immunoprecipitated for western blotting using the Santacruz Exactacruz D anti-goat system  to avoid detection of heavy and light chains of the IP antibody. Detection used the Pierce Supersignal West Dura Extended Duration Substrate (Thermo Scientific).9 cells) were fixed in 1 M formaldehyde for 20 min when Brachyury expression was at its highest level . This was followed by quenching and isolation of nuclei. Our protocol differs from a previously-published procedure Chromatin immunoprecipitation/location analysis was based on the Agilent Mammalian ChIP-chip Protocol, incorporating the Whole Genome Amplification GenomePlex Kit (Sigma) ChIP was performed as previously described NCAPD2 was used as a negative control gene.Verification of target enrichment was performed on a selection of targets using genomic quantitative PCR. DNA fragments were amplified using Fast SYBR® Green Master Mix (Applied Biosystems) according to manufacturers instructions on a 7500 Fast Real-Time PCR System (Applied Biosystems). Promoter specific primers  were desAgilent Technologies mouse promoter 244K (G4490A) 60-mer oligonucleotide arrays (“chips”) covering 17,000 mouse genes and extending 5.5 kb upstream and 2.5 kb downstream of transcriptional start sites were hybridized with 5 µg amplified chromatin per sample. Arrays were annotated to NBI35.1 of the mouse genome. Immunoprecipitated (or isotype control) and total input samples were labelled with Cy5 or Cy3 respectively. Hybridizations were performed and analysed in triplicate using independently differentiated cultures. The isotype control experiment was performed once to confirm no significant enrichment over input chromatin (data not shown).x) <0.01 and P(x) defines the value for each probe, and P.Microarrays were scanned using an Agilent scanner to a resolution of 5 µm. Data were extracted using Agilent G2567AA Feature Extraction Software (v.9.1). The significance of binding events was determined using Agilent Chip Analytics 1.3 software. Initial analysis was done using Chip Analytics defaults settings and then further filtered using the parameters P(http://genome.ucsc.edu/). Negative control genes from the list not called as bound were included. Verification of enrichment was performed on a selection of targets using promoter specific genomic quantitative PCR. Promoter specific primers  were deshttp://burgundy.cmmt.ubc.ca/GOToolBox/) in vitro T-box binding motif TCACACCT http://www.sanger.ac.uk/Software/analysis/nmica/index.shtmlhttp://rsat.ulb.ac.be/rsat/http://weblogo.berkeley.edu/logo.cgi) The GOToolBox , leaving only a glycine and a proline residue attached to the protein. This was concentrated using Amicon Ultra 4 columns (Millipore), and the identity of the resulting protein was confirmed by SDS PAGE and mass spectrometry.The T domain of mouse 32P] dCTP using Klenow fragment. Unincorporated nucleotides were removed using Sephadex G-50 columns . Binding reactions were incubated on ice for 40 minutes in 1× EMSA binding buffer , 0.5% milk powder and 50 ng/µl dI/dC using 30,000 cpm/µl and 8–10 fmol labelled oligomer. Competition reactions using 4 pmol cold oligomers were pre-incubated for 10 min on ice. In supershift experiments goat polyclonal anti-Brachyury N19 antibody  was added after binding and then incubated a further 20 min on ice.Double stranded oligomers containing (i) the core Brachyury consensus binding sequence TCACACCT, (ii) a simple AC repeat, or (iii) the core Brachyury sequence together with the AC repeat, and mutated versions of each, had identical BglII/BamH1 5′ overhangs . These wFigure S1Validation of targets. Box plot showing genomic quantitative PCR of bound promoter regions for targets Axin2, Foxe1, Mapre2, Nkx2.6, Pax3, Rttn, Van Gogh and the published target Nanog, and unbound or negative promoter regions Nanog 3′, 1700010C24Rik and beta actin. Boxes represent the interquartile range, the upper edge being the 75th percentile and lower edge the 25th percentile. The whiskers show the minimum and maximum values. Values above the line are enriched in chromatin immunoprecipitations. Data were obtained from five independent chromatin immunoprecipitations. Probes recognising Nanog were not present on Agilent 244K promoter arrays.(TIF)Click here for additional data file.Figure S2Functional analysis of target genes. Bar charts show Gene Ontology (GO) annotations for (A) biological process; (B) cellular component; and (C) molecular function using the GOToolBox. Horizontal bars represent enrichment ratio (observed frequency/expected frequency) and vertical axis gives the GO term followed by the GO identification number in brackets and hierarchy level. Colour bars indicate statistical significance. GO terms related to the function of Brachyury are highlighted in red boxes.(TIF)Click here for additional data file.Figure S3Interaction of the mouse Brachyury T domain with DNA. (A) Sequences surrounding bound probes are enriched for an (AC)n repeat relative to their genomic neighbours. The motif was generated using the NestedMICA position weight matrix. This may represent a secondary Brachyury recognition motif. (B) Electrophoretic mobility shift assays. Panel a: Binding reactions using 32P-labelled TCACACCT. Lane 1, no protein; lane 2, control protein derived from empty vector; lanes 3–6, mouse T domain protein: lane 4 includes excess unlabelled probe; lane 5 includes excess unlabelled mutated probe; lane 6 is a ‘supershift’ using anti-T N-19 . Notice that the Brachyury T domain binds the T site oligonucleotide and that binding is competed by cold wild-type oligonucleotide but not by a mutated oligonucleotide. Panel b: Lanes 7–9 include 32P-labelled TCACACCT; lane 10 uses the indicated mutated version of this oligonucleotide. Note that the Brachyury T domain does not bind the mutated oligonucleotide. Panel c: Binding reaction using a 32P-labelled AC repeat oligonucleotide. Lanes 11–13 as panel a; lane 14 includes excess unlabelled probe; lane 15 includes excess of an unlabelled mutated probe; lane 16 is a ‘supershift’. Notice that the Brachyury T domain binds the AC repeat oligonucleotide weakly but that binding does not seem to be competed by cold wild-type oligonucleotide. The complex however is ‘supershifted’ using the Brachyury antibody. Panel d: Binding reactions using a 32P labelled motif that includes both the T site TCACACCT and an AC repeat. Lanes 17–20 show that Brachyury binds this oligonucleotide, and that binding is competed by cold wild-type oligonucleotide. Lanes 21 and 22 show that binding is not competed significantly by unlabelled oligonucleotides in which either motif is mutated. Lane 23 shows a ‘supershift’. Experiments in (a–d) were performed under identical conditions and exposed for the same times.(TIF)Click here for additional data file.Figure S4The expression domains of Brachyury, Wnt3a and Axin2 overlap in E7.75 mouse embryos. (A) Expression of Axin2 analysed using a fluorescein labelled antisense probe detected with NBT/BCIP (purple). (B) The embryo in (A) analysed using a digoxigenin labelled antisense Brachyury probe detected with INT/Mg phosphate (brown). (C) Expression of Wnt3a analysed using a fluorescein labelled antisense probe detected with NBT/BCIP (purple). (D) The embryo in (C) analysed using a digoxigenin labelled antisense Brachyury probe detected with INT/Mg phosphate (brown). All embryos orientated as in (A). Scale bars are 200 µm.(TIF)Click here for additional data file.Figure S5The expression domains of Brachyury and Fgf8 overlap in the primitive streak of E7.75 mouse embryos. (A). Expression of Fgf8 analysed using a fluorescein labelled antisense probe detected with NBT/BCIP (purple). (B). The embryo in (A) analysed using a digoxigenin labelled antisense Brachyury probe detected with INT/Mg phosphate (brown). Black bars are 200 µm.(TIF)Click here for additional data file.Figure S6Verification of anti Brachyury antibody. (A) Immunohistochemistry of E9.5 embryo using Santa Cruz anti-human T C19 with nickel enhanced DAB substrate. Staining is present in the notochord (arrowhead), pre-somitic mesoderm (arrow) and tailbud. Staining was absent in controls in which primary or secondary antibodies were omitted. (B) Expression of Brachyury RNA in an E9.5 embryo studied by in situ hybridisation. Note similarity to (A). Bars in (A) and (B) represent 250 µm. (C) Western blot testing antibody specificity. Size markers are shown to the left. Lane 1: Mouse Brachyury reticulocyte lysate translation product; lane 2: unprogrammed reticulocyte lysate translation product; lane 3: Immunoprecipitated material derived from Brachyury reticulocyte lysate translation product; lane 4: Supernatant of immunoprecipitated material in lane 3; lane 5: Immunoprecipitated material derived from day 4 embryoid bodies; lane 6: Supernatant of immunoprecipitated material in lane 5; lane 7: Immunoprecipitated material derived from day 4 embryoid bodies, having omitted first antibody; lane 8: Supernatant of immunoprecipitated material in lane 7. All immunoprecipitations used Santa Cruz anti-T C19. Western blots used R&D Systems anti-T as a primary antibody and SantaCruz D anti-goat IgG HRP linked secondary antibody. (D) Strategy to create ES cell clones lacking Brachyury. Clones were created using 65 bp ShRNA duplexes targeting the first exon of Brachyury (T). Sequences were inserted into the XhoI/HindIII site of the pSingle ShRNA vector (Clontech) which includes a tetracyclin-controlled transcriptional repressor that in turn regulates the expression of the ShRNA sequence. Selection of stable lines is achieved by culture in G418 and induction of ShRNA expression occurs through addition of 1 µg/ml doxycycline. (E) Western blot analysis of day 5 embryoid body extracts from clones containing ShRNA constructs targeted to Brachyury exon 1 (T1) or a scrambled version of this sequence (Ts), either treated with doxycycline (+) or left untreated (−). Samples were immunoprecipitated as in (C). Note loss of Brachyury band in lane 3.(TIF)Click here for additional data file.Table S1Full gene list.(DOC)Click here for additional data file.Table S2Targets involved in key signalling pathways.(DOC)Click here for additional data file.Table S3Genes associated with germ cell development.(DOC)Click here for additional data file.Table S4Targets identified as transcription factors.(DOC)Click here for additional data file.Table S5Conservation of AC repeats.(DOC)Click here for additional data file.Table S6Quantitative PCR primers.(DOC)Click here for additional data file.Table S7In situ hybridisation, T box, and electrophoretic mobility shift assay primers.(DOC)Click here for additional data file.Table S8Genomic quantitative PCR primers.(DOC)Click here for additional data file.


To study transcriptional regulation by distant enhancers, we devised a system of easilymodified reporter plasmids for integration into single-copy targeting cassettes in clones of HuH7, a human hepatocellular carcinoma. The plasmid constructs tested transcriptional function of a 35-kb region that contained the rat albumin gene and its upstream flanking region. Expression of integrants was analyzed in two orientations, and compared to transient expression of non-integrated plasmids. Enhancers were studied in their natural positions relative to the promoter and localizedby deletion. All constructs were also analyzed by transient transfection assays. In addition to the known albumin gene enhancer (E1 at −10 kb), we demonstrated two new enhancers, E2 at −13, and E4 at +1.2 kb. All three enhancers functioned in both transient assays and integrated constructs. However, chromosomal integration demonstrated several differences from transient expression. For example, analysis of E2 showed that enhancer function within the chromosome required a larger gene region than in transient assays. Another conserved region, E3 at −0.7 kb, functioned as an enhancer in transient assays but inhibited the function of E1 and E2 when chromosomally integrated. The enhancers did not show additive or synergistic behavior,an effect consistent with competition for the promoter or inhibitory interactions among enhancers. Growth arrest by serum starvation strongly stimulated the function of some integrated enhancers, consistent with the expected disruption of enhancer-promoter looping during the cell cycle. In addition to the core promoter and its proximal regulatory element, many genes have enhancers, which vary in size, location, strength, and mode of action. Ranging from 50–1500 bp, enhancers can activate their target promoters from great distances (up to 1 mb) and may reside within introns or even on different chromosomes enhancer trappingIndividual enhancers may vary in promoter specificity and strength, and combinations may additively, synergistically, or competitively stimulate transcription 5–106 enhancers in the genome, i.e., an enhancer every 3–30 kb There has been great progress in finding enhancers. Conserved or ultraconserved regions in non-coding DNA frequently identify enhancers, but do not always correlate with transcriptional function locus control region (LCR), a complex of enhancers and other functional elements that render position-independence to transgenes Albumin gene.Since the discovery of enhancers, transient assay in reporter plasmids has provided a standard test of function independent of chromosomal context. The limitations of this approach are exemplified by the β-globin Alb—a classical marker of the liver phenotype—established paradigms for gene expression controlled by distant enhancers. The 16.5-kb rat Alb gene has 15 exons and a single liver-specific enhancer (“E1”) at −10 kb, a remarkable distance at the time of its discovery Alb expression. Our analysis characterizeda 35-kb region containing the full Alb gene and 18-kb upstream segment. The region includes E1 and several other conserved non-coding regions, potentially novel regulatory elements.Prior studies of recombinase mediated cassette exchange (RMCE) that uses inverted LoxP sites to prevent re-excision of the integrated transgene and allows insertion of DNA segments that do not contain a selection marker Alb-gene reporters validated the experimental system by demonstrating that transcription of Alb is regulated by a much more complicated system of distant enhancers than revealed by previous studies.We adapted an efficient system of Alb transcription controls because they express high levels of Alb mRNA like fetal and adult hepatocytes Alb combined with a GFP reporter (We used human HuH7 cells to study reporter .Origin of Replication Complex Subunit 5 (ORC5L) and 78 kb from the 5′-end of Reelin (RELN)  . Both gePrior to analysis of gene expression, gancyclovir-resistant isolates were first screened by PCR to demonstrate integration and determine orientation of gene constructs . SoutherTo work outanalysis of enhancer function, we studied the albumin promoter alone (plasmid P) or combined with the intact 17.4 kb upstream region (plasmid 1+2+P) that contains well-characterized enhancer E1 . Early sTranscription oftransiently transfected plasmids is controlled only by regulatory elements within the plasmid construct. In contrast, chromosomal integration allows interaction with transcription control elements outside of the construct. To control for such positional effects, constructs were compared at different integration sites—HuH7-9 or Huh7-10—and in two orientations, designated O1 and O2 . By flowAlb enhancer, so subsequent analysis was limited to HuH7-9, comparing O1 and O2 to discriminate positional effects.The promoter and enhancer regions functioned in all integrants. However, expression of the promoter and relative stimulation by enhancers showed significant positional differences. In HuH7-9, the promoter hadsimilar strength in both orientations, while the enhancer region gave 10-fold stimulation in O1 and4-fold in O2. In HuH7-10 cells, the promoter gave 6-fold higher expression than in HuH7-9, with only 2 to 3-fold additional stimulation added by enhancer region. Nevertheless, the combined enhancer and promoter gave gene expression comparable to O1 in HuH7-9 cells. The results in HuH7-10 suggested promoter stimulation by a non-Prior studies localizedE1 from −10.1 to −9.2 kb Transient transfection demonstrated that Regions 1, 2, and ALB each significantly enhanced gene expression, 10-, 4-, and 12-fold, respectively . The conWhen integrated, Region 1 and ALB each caused significant enhancement in either orientation . Gene exThe largest plasmid construct, 1+2+P+ALB, combined all of the regulatory regions.Its large size led to low efficiency of integration, and we obtained only a single integrant, in O1. The combined stimulation by Regions 1, 2 and ALB in this clone was not greater than individual component segments, an effect also noted in transient expression.Thus, transient transfection and stable integration in either orientation demonstrated enhancer function. However, genomic integration revealed complex relationships among regulatory elements within the constructs, andpositional effects suggested interaction with additional elements outside of the constructs.The regional analysis and pattern of sequence conservation both suggested enhancers throughout the 35-kb region. We first focused on Region 1. Deletion mapping—with constructs analyzed by both transient transfection and stable integration—identified a new enhancer, E2 . E1 and Alb gene itself caused strong transcriptional enhancement, and contains conserved noncoding regions within introns 2 and 4 .The two arresting conditions had significantly different effects on gene expression . ConflueAlb, a distinctive high-level phenotypic marker of hepatocytes. Our analysis exploited the hepatocyte-like phenotype of Alb-expressing HuH7 cells, where function of Alb transcriptional regulatory elements is similar—though perhaps not identical—to normal hepatocytes.We set out to create an experimental system of gene integration into a controlled chromosomal location with the eventual goal of resolving specific molecular interactions within a complex locus. The model of gene expression was Alb enhancers, E2 and E4. The weak activity of the previously characterized E1 has seemed inadequate to explain the very strong expression of Alb in hepatocyte-derived cells, so the new observations eliminate a significant discrepancy. Comparative sequence analysis showed that E2 and E4 have features typical of hepatocyticenhancers, with putative sites for their characteristic binding factors, HNF4, FOXA/HNF3, and C/EBP (http://genome.ucsc.edu/cgi-bin/hgTracks). Phylogenetic comparisons demonstrated that enhancers have conserved regions flanked by specific binding sites that may be poorly conserved The analysis validated the gene targeting system by demonstratingtwo novel nd C/EBP . MoreoveAlb gene expression require further investigation.Prior RMCE studies have analyzed orientation-dependent effects of flanking sequences on transgenes, position-dependent silencing of the β-globin LCR, and transcriptional interference at a complex locus Another difference between transient and integrated expression was revealed by deletion mapping of E2, which was localized to an 0.8 kb genomic interval that contains two conserved regions.The E2Δ3 construct eliminated one conserved region and still functioned as an enhancer in transient assays. In contrast, enhancer function after integration required both conserved regions, revealing an essential activity that is dispensable for transient expression from a plasmid.Transfection of the largest plasmid, 1+2+P+ALB, was challenging due to its size and low copy number, but even this large plasmid functioned in transient assays at a level indicating enhancer-stimulated gene expression. Both transient assay and chromosomal integration produced intermediatereporter stimulation, indicating that the enhancer regions were not additive or synergistic. The level was much less than the maximum capacity of the promoter since the 1+P construct was about 3-fold stronger. Because each can separately stimulatetranscription, the lack of additive stimulation suggestedenhancer competition. This could result from competition for binding to the same element within the promoter or competition for a limiting trans factor. Alternatively, the effect could represent binding of the enhancers to each other in competition with their binding of the promoter, or an indirect boundary function where one enhancer establishes a local chromatin structure that obstructs access of the other to the promoter. Any of these functions might modulate during development, so they are all possible mechanisms for enhancer switching.Alb locus does not show complete position independence. Perhaps additional elements outside the 35 kb region contribute to higher-order Alb regulation Transgenic studies of the β-globin gene cluster and its LCR led to the concept that position independence is an ideal criterion for demonstrating that a regulatory region is intact Alb region. Natural Alb expression is consistent with these observations, because proliferating hepatocytes or cell lines have lowerexpression than the quiescent hepatocytes of normal liver Alb gene constructs will provide an ideal platform for this research.It is well established that promoters and enhancers loop together, but looping represents different kinds of interactions. The distant LCR dynamically oscillates between β- and γ-globin genes by the distant LCR, transitory looping within a more stable chromatin hub Alb-Afp locus, and for Cre-mediated recombination into mammalian cells (NdeI and HindIII sites of pUC19 (pLL1) and also transferred to pBR322 (pLL2). The low copy number of the latter facilitates cloning of large genomic DNA segments. The plasmids incorporate the following features. Two loxP sites in opposite orientation allow Cre-mediated insertional recombination with the targeting cassette. Two I-SceI sites flank the cloning linker to allow incision of integrated gene segments. Unique restriction sites that do not cut the Alb-Afp locus , or cut the entire locus only once , were arranged so that individual regions could be joined or excised through simple forced directional cloning strategies. Other restriction enzyme sites were arranged to facilitate locus assembly or to provide general cloning sites.Cloning vectors Locus Linker 1 and 2  were constructed for assembly of restriction enzyme fragments excised from genomic clones of the rat an cells . A 290 ban cells  and S1 wGFP was amplified by PCR from pEGFP-N1  with primers that added terminal SgfI and SmaI sites , detected with a Dark Reader Transilluminator , and purified using a QIAEX II gel extraction kit . Ligations, performed with standard procedures, were electroporated into DH10B (Invitrogen) or SURE . Deletion mapping of enhancer-containing regions was carried out with  primers .PvuII fragment containing the cassette pL1-HY-TK-1L R colonies were maintained under continuous antibiotic selection. Southern blot analysis (not illustrated) demonstrated fourclones that contained single cassette copies. These were screened by transfection of pL1-CMV-Neo-1L alone or in cotransfection with pCMV-Cre/pBS185 6 cells. HuH7-9 had the greatest efficiency of targeted integration and was used as the standard system for analysis of albumin gene constructs after an initial comparison with HuH7-10 (see A 2.5 kb 7-10 see  below.6 cells were transfected with 2.5 µg of DNA in Lipofectamine (Invitrogen) according to the manufacturer's protocol. Cells were fed with fresh medium at 24 hr, then trypsinized, washed, and analyzed by flow cytometry at 48 hr. For targeted integration, cells were reselected with 350 µg/ml hygromycin B solution for one or two passages before stable transfection. Then 1×106 cells were transfected with 4 µg of gene constructs in pLL1 or pLL2 and 1 µg pCMV-Cre (Clontech), using Lipofectamine LTX with PLUS reagent (Invitrogen), on 60 mm plates. At 24 hr, cells were split 1∶2 to 100 mm plates. 1 µM gancyclovir was added at 48 hr HuH7-9 and HuH7-10 cells were maintained in Williams E medium supplemented with 1% L-Glutamine, 1% Pen/Strep, and 5% fetal bovine serum at 37°C in 5% CO2. For transient assays, 0.5×10To study the effects of growth arrest, control and GFP-expressing cell clones were seeded at low density (∼25% confluence) and fed with Williams E medium containing 5% or 0.1% serum 24 hr after plating. Clones were analyzed by flow cytometry 48 and 96 hr following the change of medium.AvrII or PvuII and self-ligated at 1–2 ng/µl. Inverse PCR of the cassette-genomic junctions used primers Target F and Target R. PCR products (1–4 kb) were sequenced using the same primers.Genomic DNA was digested with 6cells were trypsinized, washed, resuspended in cold PBS containing 1% FBS, and analyzed with a FACSCalibur flow cytometer . Forward and side scatter measurements were used to differentiate live and dead cells. Measurements for 20,000 cell events in stable clones, or 60,000 cell events in transient transfection assays, were further analyzed with FCS Express software . For transient assays, average fluorescence intensity was determined by histogram subtraction of the distribution determined for untransfected cells from distribution of transfected cells. Since all transfections utilized the same DNA concentration of transfected plasmid, the mean fluorescence was then corrected for relative plasmid copy number in each transfection. For stable clones, values determined were mean fluorescence intensity, and position of the peak of highest gene expression. In most cases, at least two separate clones were analyzed in each orientation.1–2×10Figure S1Linker for assembly of the Alb-AFP gene region. The linker was cloned into NdeI and HindIII sites of pUC19. Restriction enzyme sites in large bold type were used for assembly of rat Alb and AFP gene segments. L1 and 1L are loxP sites in opposite orientations.(DOCX)Click here for additional data file.Table S1Plasmids.(DOCX)Click here for additional data file.Table S2PCR primers.(DOCX)Click here for additional data file.Table S3Bands detected by Southern blot.(DOCX)Click here for additional data file.