Numerous studies over the past decade have elucidated a large set of long intergenic noncoding RNAs (lincRNAs) in the human genome. Research since has shown that lincRNAs constitute an important layer of genome regulation across a wide spectrum of species. However, the factors governing their evolution and origins remain relatively unexplored. One possible factor driving lincRNA evolution and biological function is transposable element (TE) insertions. Here, we comprehensively characterize the TE content of lincRNAs relative to genomic averages and protein coding transcripts.Our analysis of the TE composition of 9,241 human lincRNAs revealed that, in sharp contrast to protein coding genes, 83% of lincRNAs contain a TE, and TEs comprise 42% of lincRNA sequence. lincRNA TE composition varies significantly from genomic averages - L1 and Alu elements are depleted and broad classes of endogenous retroviruses are enriched. TEs occur in biased positions and orientations within lincRNAs, particularly at their transcription start sites, suggesting a role in lincRNA transcriptional regulation. Accordingly, we observed a dramatic example of HERVH transcriptional regulatory signals correlating strongly with stem cell-specific expression of lincRNAs. Conversely, lincRNAs devoid of TEs are expressed at greater levels than lincRNAs with TEs in all tissues and cell lines, particularly in the testis.TEs pervade lincRNAs, dividing them into classes, and may have shaped lincRNA evolution and function by conferring tissue-specific expression from extant transcriptional regulatory signals. Recent comprehensive transcriptome sequencing studies uncovered a large class of previously unannotated long noncoding RNA (lncRNA) genes in various species with similar splicing and polyadenylation properties to mRNAs -8. GenomDrosophila [Initial progress has been made to understand the evolution and origins of lncRNAs . The nucosophila , and nemosophila , distantosophila -4,21,22.One important method by which the genome, including lncRNA sequence, evolves is transposable element (TE) insertions. TEs are nucleic acid sequences capable of inserting into genomic DNA that are typically considered 'selfish' genomic parasites and have conquered 45 to 65% of the human genome ,24. DespIn a few known cases, TE proteins required for transposition have seeded novel genes in the host genome -31. Morelinc-ROR), have HERVH elements near the 5' transcript end, suggesting HERVH elements may shape lncRNA regulation in the pluripotent state.Whether TEs have similarly influenced lncRNA sequence and regulation is largely unexplored, but numerous recent studies point to interesting TE-associated lncRNA functions. For example, Alu elements in lncRNAs play a significant role in STAU1-mediated mRNA decay by duplexing with complementary Alu elements in the 3' UTRs of mRNAs . A mutatHere we comprehensively characterize the TE composition of long intergenic noncoding RNAs (lincRNAs) and their functional relationships in the human genome. We find that lincRNAs contain TEs at a far greater rate than protein coding genes and are highly enriched for ERVs and depleted of LINEs and SINEs. TEs have position and orientation preferences in lincRNAs, including a frequent association of LTRs with lincRNA transcription start sites (TSSs) that suggests a role in the genes' origins. In a number of intriguing cases, TE content correlated with lincRNA expression properties. Strikingly, lincRNAs containing HERVH elements exhibit a stem cell-specific expression pattern. These results demonstrate that lincRNAs have nonrandom composition of TEs that strongly correlates with their functional and regulatory properties, suggesting a mechanism for malleable evolution of lincRNAs.To investigate the relationship between lincRNAs and TEs, we first established a reference catalog of TEs in the human genome from RepeatMasker annotations of Hg19. Removing non-TE repeats left 4.5 million TEs, covering 49.9% of the genome. Next, we assembled a catalog of human lincRNAs from RNA sequencing (RNA-Seq) of 28 different tissues and cell lines using methods from our previous human lincRNA annotation effort [Intersection of the lincRNA and TE catalogs revealed that the vast majority 7,710, 83.4%) of lincRNAs overlap at least one TE. In fact, nearly half (41.9%) of lincRNA transcript sequence is TE-derived  and Alu by 1.4-fold (P 1e-29). Other common LINE and SINE families, L2 and MIR, as well as DNA transposons hAT-Charlie and TcMar-Tigger, are also significantly depleted.lincRNAs exhibit many biases in their TE composition relative to genomic averages Figure . The LINP 2e-140) and makes up the most lincRNA sequence of these families.Conversely, retroviral elements ERV1, ERVL-MaLR, ERVL, and ERVK are enriched in lincRNAs Figure . ERVs arTUG1 interacts with methylated Polycomb 2 protein to modulate its recognition of histone modifications [HOTAIR, located in the HOXC cluster, a genomic region known to be nearly devoid of TEs [Linc-ROR, which modulates reprogramming of fibroblasts to a pluripotent state, is almost entirely composed of TE-derived sequence from seven different TE families and has an ERV1 LTR at its TSS  and 2.35 versus 2.09 isoforms/gene (P 1e-49). The correlation between transcript length and these splicing complexity statistics is weak and insufficient to explain the difference.lincRNAs with TEs are larger than those without  than mRNA splice junctions . Previously observed for proteins, MIRs are enriched at splice sites relative to the transcript sequence of both lincRNAs  and proteins  . Taken tNext, we examined the expression patterns of lincRNAs with respect to their TE composition. To this end, we analyzed lincRNA abundance estimates across an RNA-Seq database of the 28 tissues and cell lines used for assembly along with additional iPSC RNA-Seq , refining our observation above that Alu-lincRNAs are more expressed in all tissues but testis. Collectively, these results suggest an intriguing relationship between lincRNA TE composition and expression patterns.Previously, lincRNAs were observed to be far more tissue-specific than protein coding genes . As in Cet al. , we defiThough less conserved than protein coding genes, lincRNAs are more conserved than neutrally evolving sequence by traditional substitution-based statistics ,59-61. FP ~ 0; Figure S8b in Additional file Consistent with observations in mouse , conservTo explore the degree to which the lower conservation of TE-lincRNAs is driven by the TE sequence itself, we compared the conservation scores of TE and non-TE sequence in these genes. The non-TE sequence has slightly greater conservation, with mean PhastCons probability 8.5% versus 7.6% for TE sequence, but still less than that of dTE-lincRNAs. Statistical significance of this comparison is challenging due to the widely different distribution shapes, which is viewed most clearly in the substantially decreased variance in PhyloP scores assigned to TE sequence - most are near zero . AluY drives the 3' peak and is sense oriented in 71% (P 4.7e-4) of the 108 lincRNA 3' ends that it marks. This observation is consistent with the known role of Alu elements in contributing polyadenylation signals to the 3' ends of many protein coding genes [P 6e-47), albeit without a coverage peak . This suggests a minor role for the L1 antisense promoter in initiating lincRNA transcription, which has been documented for protein coding genes [P 2.6e-6).At lincRNA TSSs, both LINE families L1 and L2 tend to be oriented antisense (ng genes . The varP 4e-12) and have a large coverage peak directly at the TSS  and H7-hESCs (P ~ 0). Enrichment of reads could be found at nearly all HERVH elements in lincRNAs - peak calls overlapped 92%  plays a major role in activating transcription in ESCs . We founP ~ 0) and 93% overlap an SP1 peak call (P 4e-67). In contrast, SP1 ChIP-Seq reads are depleted at HERVH-lincRNAs in GM12878 (P 6e-69). Accordingly, SP1 coverage peaks at the TSS of HERVH-lincRNAs in H1-hESC, but not GM12878  and overlap with peak calls , suggesting that many of these lincRNAs have been fully incorporated into pluripotency regulatory networks . Second, in lincRNAs, the flanking LTR7 appears to be evolving slower than HERVH-int. lincRNA LTR7 annotations are significantly more similar to RepeatMasker's LTR7 consensus than are LTR7 annotations outside of lincRNAs , unlike HERVH-int annotations, which are slightly less similar to the consensus in lincRNAs (83.5% versus 84.2%). In every primate genome, LTR7 is present at a greater proportion than HERVH-int in the mapped lincRNA HERVH elements  has a RAltogether, these observations suggest that HERVH insertions may have originated or altered 127 lincRNAs to have stem cell-specific expression by imparting transcriptional regulatory signals. Accordingly, the signal-heavy LTR is more robust to mutation than the HERVH interior.To assess whether the properties of TEs in lincRNAs observed in human carry over to other mammalian genomes, we performed the same analyses on a previously published catalog of mouse lincRNAs built from RNA-Seq of ESCs, lung fibroblasts, and neural precursor cells  and filtP 3e-25) and ERV1 is enriched  - though the specific ERV1 families differ from human. In contrast to human, Alu elements are enriched 1.5-fold (P 1.9e-6), and other SINEs, MIR, B2, and B4, differ insignificantly from the genome. An unknown repeat family named MurSatRep1 is enormously enriched 108-fold (P ~ 0) and overlaps 81 lincRNAs. The ERV1 family MMERGLN-int, recently discovered to undergo significant reduction of DNA methylation between mouse sperm to zygote [P 1.2e-130) and overlaps 10 lincRNAs.Mouse lincRNAs also comprise a nonrandom distribution of TEs. Similar to the human genome, L1 is depleted in lincRNAs . MurSatRep1 appears in the sense orientation in a striking 96% of its 81 lincRNAs (P 4e-32).TEs in mouse lincRNAs also display biased position and orientation. For example, ERVK associates with TSSs, visible as peaked coverage  and neural precursor cells (P 3e-17) but increased expression in ESCs (P 2e-10) relative to dTE-lincRNAs  at the 127 HERVH-lincRNAs. Collectively, these data suggest that HERVH retrotransposition may have shaped pluripotency networks via lincRNA regulation.Exemplifying this phenomenon, we discovered that 127 HERVH-lincRNAs are strikingly enriched for HERVH LTR7 in the sense orientation at their TSSs and exhibit dramatic stem cell-specific expression, observed in both H1-hESCs and iPSCs. Consistent with the notion of TEs contributing promoter regulatory signals, the HERVH elements in these lincRNAs are highly enriched for transcription activation signals in ESCs, but not other cell types. Presence of the activating histone modification H3K4me3 and SP1, a transcription factor previously found to be critical for transcription of HERVH , suggestto iPSCs . SimilarWe found that TEs partition lincRNAs into two classes with divergent properties; 1,531 lincRNAs are devoid of TEs (dTE-lincRNAs), unlike the majority of 7,710 lincRNAs that contain TEs (TE-lincRNAs). This classification of lincRNAs uncovered another example of TE content influencing lincRNA expression as TE-lincRNAs are less expressed in every tissue and cell line, particularly testis. The relative decrease in expression of TE-lincRNAs may be due to lasting effects of well-established TE silencing mechanisms in germline cells , which hTUG1 [linc-ROR [PCAT-1 [SLC7A2-IT1A [BANCR [dTE-lincRNAs also have greater evidence of conservation by substitution-based statistics than TE-lincRNAs . The lower conservation levels of TE-lincRNAs is apparently permissive of function as there are many examples of important TE-lincRNAs, including TUG1 , linc-ROlinc-ROR , PCAT-1  [PCAT-1 , SLC7A2-7A2-IT1A , BANCR [A [BANCR , and morThe first scenario is highly plausible. Furthermore, we found evidence that TE insertions may have even played a role in creating those new lincRNAs. Retrotransposons contain promoters to transcribe the element, but, as selfish genomic parasites, typically mutate freely after insertion. This arrangement provides an opportunity for a new lincRNA to arise in the region downstream of an intergenic retrotransposon insertion where the sequence would usually have been evolving neutrally. In our data, we found many examples of TEs associating with lincRNA TSSs; a number of families, particularly ERV LTRs, peak in coverage at the TSS with biased orientation matching the known promoter direction. Whether these TE insertions truly spawned novel lincRNAs or simply donated an alternative TSS to an existing lincRNA will be the focus of future comparative transcriptome analyses. Nonetheless, it appears that TEs may lend regulatory signals to these lincRNAs, exemplified by the stem cell-specific expression of HERVH-lincRNAs.The second scenario - a TE insertion altering a previously existing lincRNA - may also often occur. One hypothesis regarding lincRNA function and evolution proposes a language of independent, small sequence-structure domains . Thus, lMore definitive answers to the evolutionary and regulatory questions raised by this study will require additional computational and experimental analyses. Specifically, this will require deep coverage RNA-Seq datasets to annotate lincRNA loci across primates and eutherian mammals. Such data would generalize trends in the TE composition of lincRNAs and reveal how lineage-specific TEs such as ERVs have shaped transcriptional regulation at lincRNA loci. Future experimental work will focus on exploring the functional role of TE sequence in lincRNAs through detailed mapping of lncRNA molecular interactions. In the meantime, it is now clear that TEs have significantly shaped the noncoding RNA landscape.We built the lincRNA catalog used in this analysis using RNA-Seq experiments from 28 different tissues and cell lines  and recomputed the statistic of interest 100 times. We fit a normal distribution to these null samples and computed For all comparisons of two sets of values where we were interested in whether one set was greater than another, we used a Mann-Whitney rank sum test . This inTo test for enrichment of ChIP-Seq reads in TEs, we used a binomial test modeling each read as a sample from a Bernoulli distribution where the success probability is proportional to the size of the TE family over the size of the alignable genome.P-values in all experiments were corrected for multiple hypothesis testing using Benjamini and Hochberg's false discovery rate procedure [rocedure .We downloaded fastq files of ChIP-Seq reads generated by the ENCODE consortium from UCSC (Additional file ChIPm: chromatin immunoprecipitation; ERV: endogenous retrovirus; ESC: embryonic stem cell; FPKM: fragments per kilobase per million; H3K4me3: trimethylation of lysine 4 on histone 3; iPSC: induced pluripotent stem cell; lincRNA: long intergenic noncoding RNA; LINE: long interspersed nuclear element; lncRNA: long noncoding RNA; LTR: long terminal repeat; SINE: short interspersed nuclear element; TE: transposable element; TSS: transcription start site; UTR: untranslated region.The authors declare that they have no competing interests.DK and JR conceived the study and wrote the manuscript. DK carried out the analyses. All authors have read and approved the manuscript for publication.Supplementary results and figures. [figures. Click here for fileTE content of lincRNAs. Excel table describing TE content of lincRNAs and statistical analysis.Click here for fileTE content of protein coding genes. Excel table describing the TE content of protein coding genes and statistical analysis.Click here for fileRNA-Seq data. Excel table describing RNA-Seq datasets used to build lincRNAs and estimate abundances.Click here for fileTEs in lincRNAs orientation statistics. Excel table describing orientation statistics of TEs in lincRNAs.Click here for fileChIP-Seq data. Excel table describing ChIP-Seq datasets used to study HERHV-lincRNAs.Click here for fileGTF file describing our full lincRNA catalog.Click here for fileGTF file describing only the most expressed isoforms of our lincRNA catalog.Click here for fileCufflinks output file describing abundance estimates for our full lincRNA catalog.Click here for file


In specific regions of the adult mammalian brain, neural stem cells (NSCs) generate new neurons throughout life. Emerging evidence indicate that chromatin-based transcriptional regulation is a key epigenetic mechanism for the life-long function of adult NSCs. In the adult mouse brain, NSCs in the subventricular zone (SVZ) retain the ability to produce both neurons and glia for the life of the animal. In this review, we discuss the origin and function of SVZ NSCs as they relate to key epigenetic concepts of development and potential underlying mechanism of chromatin-based transcriptional regulation. A central point of discussion is how SVZ NSCs – which possess many characteristics of mature, non-neurogenic astrocytes – maintain a “youthful” ability to produce both neuronal and glial lineages. In addition to reviewing data regarding the function of chromatin-modifying factors in SVZ neurogenesis, we incorporate our growing understanding that long non-coding RNAs serve as an important element to chromatin-based transcriptional regulation, including that of SVZ NSCs. Discoveries regarding the epigenetic mechanisms of adult SVZ NSCs may provide key insights into fundamental principles of adult stem cell biology as well as the more complex and dynamic developmental environment of the embryonic brain. While the vast majority of neurons in the adult mammalian brain arise during embryonic development, in specific brain regions, new neurons are born continuously throughout life. Under normal physiological conditions, adult neurogenesis persists in two distinct brain regions – the subgranular zone of the hippocampal dentate gyrus, and the subventricular zone (SVZ) of the lateral ventricles . These aIntriguingly, the adult NSCs that sustain life-long neurogenesis have many characteristics of mature astrocytes, a glial cell population known mostly for their supportive functions, which include the production of trophic factors, modulation of neuronal synapses, and maintenance of the blood–brain barrier . AstrocyEpigenetics is the study of biological mechanisms that orchestrate a specific and heritable pattern of genome function without alterations to the DNA sequence. As with other multi-potent adult stem cell populations, SVZ NSCs must durably maintain an epigenetic state that enables both self-renewal and the production of multiple, differentiated cell types. This stem cell epigenetic state is likely to involve (1) the active expression of specific genes, (2) the repression of certain other loci, and (3) the maintenance of genes required for lineage specification . How is in vitro  and generate OB interneurons when transplanted back to the SVZ in vivo . These NSCs reside directly adjacent to the ventricle in a region that has been loosely termed the SVZ. Throughout life, these SVZ NSCs produce neuroblasts that migrate anteriorly to the OB where they differentiate into neurons and integrate into local circuits . Several distinct interneuron subtypes are generated by the SVZ Type B1 cells give rise to transit amplifying precursors (type C cells), which generate neuroblasts (type A cells) that migrate to the OB . Based oo the OB . Type A o the OB . This cho the OB . The RMSen face, the thin apical endings of type B1 cells cluster together at the center of a rosette of ependymal in a repeated “pinwheel” pattern , and – like SVZ type B1 cells – maintain apical contact with the adjacent brain ventricle. Unlike adult SVZ NPCs, however, radial glia extend a thin radial process from their soma to the pial surface of the developing brain. Throughout development, radial glia maintain this apical–basal contact: as the brain grows with the progressive addition of newly born cells, radial glial cells elongate their basal process, and both apical and basal contacts are preserved .In the mouse, neuroepithelial cells begin transforming into radial glia at around embryonic day 9 (E9). Shortly after this time, these cells begin expressing glial markers including GLAST, BLBP, and, in some species, GFAP . FurtherMultiple cell-fate analyses demonstrate that radial glia – when considered as a population – give rise to neurons and glia in essentially all parts of the brain. However, this is not to say that each individual radial glial cell is cell-intrinsically capable of giving rise to the entire diversity of cell types found in the adult brain. For instance, radial glia in the dorsal regions generate different types of neurons than those in the ventral brain. Furthermore, the developmental potential of embryonic neural precursors also changes over time. That is, early embryonic radial glia generate different neural cell types than those later in development. Interestingly, as we next discuss, it appears that this “progressive” change in developmental potential is in part cell-intrinsic and epigenetically stable.The mammalian cortex has a laminar structure, and the development of the six layers of major projection neurons proceeds in an “inside out” manner: neurons of the deepest layer are born first, and the more superficial neurons are born later in a sequential fashion, with the outer-most neurons born last . Interesin vivo cell fate analysis support this lineage relationship  cortical astrocytes into functional inhibitory interneurons . Histone variants also exist and likely play a role in the epigenetics of transcriptional regulation, but we do not include these in our discussion, as we are not aware of data regarding their function in adult SVZ neurogenesis.There are two basic mechanisms for modifying chromatin states: (1) non-covalent modifications of protein–protein and protein–DNA interactions including ATP-dependent chromatin remodeling, and (2) covalent changes to chromatin such as DNA methylation and histone amino acid modifications. Core histones are subject to over 100 different post-translational modifications, and while most are still poorly understood, it is clear that some of these covalent changes strongly correlate with gene expression. For instance, histone acetylation is generally associated with local transcription. Histone methylation can be associated with either transcriptional activation or repression, depending in part on which particular residue is modified. In a combinatorial fashion, it is likely that such covalent modifications comprise a “histone code” that determines local chromatin structure and thus epigenetically imposes specific transcriptional programs .As with our neural developmental discourse above, a central concept to our mechanistic discussion below relates to the observation that while most radial glia progressively lose their neurogenic potential, SVZ type B1 cells retain the ability to produce neurons throughout life. For this review, we next discuss select chromatin-based mechanisms that have been shown to regulate adult SVZ neurogenesis.Histone acetylation – the first described histone modification – is associated with active transcription . Many lyHistone deacetylation is associated with transcriptional silencing, and this covalent change is catalyzed by histone deacetylases (HDACs), of which there are four classes: class I , class II , class III (Sirt1–7), and class IV (HDAC11).Subventricular zone neurosphere cultures treated with class I and II HDAC inhibitors exhibit increased neuronal production and decreased oligodendrocyte differentiation, suggesting a role for this chromatin modification in neural fate determination . Class IIn the postnatal SVZ, HDAC1 is expressed in GFAP-positive cells – presumably including type B1 cells – and HDAC2 is expressed in the migratory type A cells . It appePten tumor suppressor, leading to their transcriptional repression. These data support a model in which TLX regulates SVZ NSC function in part through chromatin-based mechanisms. More generally, the above studies indicate that histone acetylation is a critical component of chromatin-based transcriptional regulation required for SVZ neurogenesis.Histone deacetylases are known to interact with transcription factors and can be targeted to specific loci. Tailless (TLX) is a transcription factor expressed in SVZ type B1 cells and is required for NSC self-renewal and adult neurogenesis . TLX intDNA methyltransferases (DNMTs) mediate the covalent addition of methyl groups to the C5 position of cytosine residues. In mammalian cells, this DNA methylation typically occurs within CpG dinucleotides. In promoter regions, DNA methylation generally correlates with transcriptional repression. DNMT1 is constitutively expressed in dividing cells, and during DNA replication, this enzyme symmetrically propagates methylation to the nascent daughter strand. While DNMT1 appears to serve primarily to maintain DNA methyl marks, DNMT3A and DNMT3B are de novo methyltransferases, capable to catalyzing cytosine methylation at new genomic locations  transcription factor; during the neurogenic phase of embryonic development, CpG sites in these response elements are methylated, which blocks STAT binding  proteins are a family of enzymes that can convert 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which may be an important intermediate to DNA demethylation via base-excision repair. Recently, Dnmt3a have severely diminished SVZ neurogenesis, resulting in smaller OBs. Genome-wide analysis of DNA methylation and other histone modifications suggested a novel mechanism of activation, that Dnmt3a methylates DNA proximal to the promoters of neurogenic genes like Dlx2, which antagonizes the placement of repressive histone modifications. These data suggest that SVZ type B1 cells require Dnmt3a to help maintain epigenetic activation of specific loci. Interestingly, Dnmt3a is not expressed in non-neurogenic brain regions, suggesting that this de novo DNA methyltransferase is a key component to the maintenance of adult neurogenesis.While DNA methylation is well known for its repressive role at gene promoters, DNA methylation is also abundant within many gene bodies. While the role of non-promoter DNA methylation still remains unclear, in the adult SVZ, DNMT3A-dependent non-promoter DNA methylation appears to facilitate the transcription of key neurogenic genes . Mice nuDrosophila, repress or activate transcription, respectively, to control the specification and maintenance of cell identity. By assembling into large multi-protein complexes that modify chromatin structure, they organize the genome regionally into transcriptionally silent or active states.The polycomb group (PcG) and trithorax group (trxG) gene products, originally described in Polycomb group complexes silence gene expression by a number of different mechanisms including the covalent modification of histones by ubiquitination and methylation. Of all of the histone modifications mediated by PcG complexes, histone 3 lysine 27 trimethylation (H3K27me3) is the best understood. Polycomb repressive complex 2 (PRC2) contains EZH2, a catalytically active core component that trimethylates H3K27. Bmi1 and Ring1b are components of the polycomb repressive complex 1 (PRC1) that recognizes H3K27me3 and participates in transcriptional silencing through multiple mechanisms, including chromatin compaction. In embryonic brain development, members of both PRC1 and PRC2 are necessary for regulating the transition from neurogenesis to gliogenesis, suggesting that chromatin-based mechanisms play a key role in establishing the developmental potential of neural precursor cells .Bmi1-deficient mouse cells generate fewer SVZ neurospheres as compared to wildtype control  is the prototypic trxG homolog of the mixed lineage leukemia (MLL) family. There are several MLL family members, and these appear to have independent roles in development. In addition to non-covalent chromatin remodeling processes, MLL proteins can catalyze histone 3 lysine 4 trimethylation (H3K4me3) – a mark that is permissive for transcription – and recruit H3K27me3-specific demethylases  in Mll1-deleted cells, suggesting that other trxG family members can compensate for loss of MLL1. However, in the absence of MLL1, the Dlx2 locus is enriched for repressive H3K27me3, correlating with the failure of proper Dlx2 upregulation. MLL family members can physically interact with and recruit H3K27-specific demethylases UTX and JMDJ3 to specific loci. Thus, taken together, the data support a model in which MLL1 is required for the recruitment of H3K27-specific demethylase activity to specific neurogenic loci for neuronal differentiation.ogenesis . In SVZ Drosophila genome harbors polycomb and trithorax response elements – specific sequences to which specific subunits of PcG and trxG bind – however, it does not appear that mammalian cells contain such genomic elements. A number of different mechanisms for PcG and trxG complex targeting in mammalian cells have been described including recruitment via transcription factor interactions and the localization to specific CpG islands. More recently, studies have emerged demonstrating the critical role of lncRNAs in the targeting of chromatin-modifying activities to specific loci.While it is clear that PcG and trxG factors interact at specific genetic loci, how such targeting is achieved in mammalian cells is not well understood. The in vivo and in vitro studies established its critical role in X chromosome inactivation via polycomb-dependent mechanisms . Are lncRNA involved in the targeting of MLL1 to specific genomic regions  in adult SVZ cells? At present, our understanding of lncRNA function in brain development is very limited: mice null for lncRNA Evf2 have abnormal interneuron development and function  for lncRNA expression analysis. Integration of these diverse experimental approaches stringently identified ~100 lncRNAs with potential roles in SVZ neurogenesis, including Dlx1as, a lncRNA encoded from an ultraconserved region of the Dlx1/2 bigene cluster.To identify specific lncRNAs with potential roles in adult neurogenesis, we recently performed a comprehensive analysis of lncRNA expression in the SVZ NSC lineage . First, Dlx1/2 bigene cluster encodes two homeodomain-containing transcription factors, DLX1 and DLX2, which are critical neurogenic regulators in both embryonic brain development and adult SVZ neurogenesis . Mice null for Dlx1/2 fail to develop GABAergic cortical and OB interneurons and do not survive; Dlx1-null mice survive to adulthood but suffer from epilepsy.The Dlx1as is transcribed from a TSS adjacent to the I12a enhancer, through the I12b enhancer, and the second exon overlaps Dlx1 on the opposite strand . Interestingly, in autistic probands, single nucleotide polymorphisms (SNPs) have been found at the DLX1/2 locus, including the ultraconserved regions that correspond to Dlx1as , and that Dlx and Hox genes appear to have co-evolved, perhaps the lncRNA-based mechanisms that regulate the function of polycomb and trithorax chromatin-modifying factors are also conserved among these evolutionarily conserved gene clusters .shRNA-mediated ogenesis . The mecMll1-deficient SVZ cells, the Dlx2 promoter region is enriched for both H3K4me3 and H3K27me3 , H3K27me3, and binding of the p300 co-transcriptional activator is a signature of poised transcriptional enhancers. Replacement of H3K27me3 with H3K27 acetylation (H3K27ac) correlates with enhancer element activation. Thus, activation of poised enhancers may require the removal of H3K27me3 and the enzymatic addition of H3K27ac . These rDlx1/2 locus is required for adult SVZ neurogenesis from type B1 cells, which arise from ventral brain radial glia in early postnatal development. Expression of Dlx1 and Dlx2 is also required for the generation of specific interneuron populations from ventral brain radial glial cells during embryonic development. Thus, the Dlx1/2 bigene cluster likely exists in a transcriptionally poised state in certain embryonic radial glia as well as adult type B1 cells. Although the nature of this epigenetic state is still under investigation, based on our discussions above, we may speculate that a specific chromatin signatures at the Dlx1/2 locus in part underlies the neurogenic potential of glial cells in both the embryo and adult SVZ. Consistent with this notion, we may further hypothesize that neurogenic loci – including their regulatory regions – become progressively repressed at the level of chromatin in non-neurogenic, non-SVZ astrocytes. Is the poised state of Dlx1/2 in SVZ type B1 cells related to chromatin modifications at the intragenic enhancers, promoter regions, or both? Which chromatin-modifying factors are required to establish and maintain this ability to rapidly activate the potent Dlx1 and Dlx2 neurogenic transcription factors? Are lncRNAs required for the local recruitment of PcG and trxG factors? By focusing in on the complex orchestration of chromatin-based modifications that occur at Dlx1/2 in neurogenic glial cells through development and into adulthood, we may discover fundamental principles of the epigenetics that underlie both regional and temporal differences in developmental potential of neural precursors.Activation of the Dlx1/2 in a transcriptionally poised state may involve polycomb factors at both enhancer and promoter regions. Dlx1/2 expression requires trxG MLL1 protein and possibly the action of H3K27me3-specific demethylases, which have been implicated in enhancer and promoter activation. Additionally, the activity of HATs and/or inhibition of HDAC activity are also likely required at the Dlx1/2 bigene cluster for efficient neurogenesis. The binding of PRC2 complexes to Dlx2 non-promoter regions appears to be antagonized by DNMT3A-dependent de novo DNA methylation, indicating an interplay between histone and DNA modifications. Furthermore, polycomb and trithorax proteins must somehow become localized to Dlx1/2 regulatory elements, and emerging data implicate lncRNAs in the targeting of chromatin-modifying complexes, including MLL1 and EZH2. Clearly, as we continue to explore the epigenetic landscape neural development, we will need to investigate in parallel the multitude of chromatin modifications. By experimentally testing models of the functional interactions between chromatin modifications and their effectors at critical genomic regions such as Dlx1/2, we may discover mechanistic themes relevant to the epigenetics of embryonic development as well as adult stem cell populations.From our discussion above, it is apparent that the transcriptional regulation of key developmental regulators involves the complex interplay of multiple chromatin modifications and their effectors. For instance, based on our current mechanistic models, the maintenance of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The drought tolerance (DT) of plants is a complex quantitative trait. Under natural and artificial selection, drought tolerance represents the crop survival ability and production capacity under drought conditions . To understand the regulation mechanism of varied drought tolerance among rice genotypes, 95 diverse rice landraces or varieties were evaluated within a field screen facility based on the ‘line–source soil moisture gradient’, and their resistance varied from extremely resistant to sensitive. The method of Ecotype Targeting Induced Local Lesions in Genomes (Ecotilling) was used to analyze the diversity in the promoters of 24 transcription factor families. The bands separated by electrophoresis using Ecotilling were converted into molecular markers. STRUCTURE analysis revealed a value of K = 2, namely, the population with two subgroups , which coincided very well with the UPGMA clusters (NTSYS-pc software) using distance-based analysis and InDel markers. Then the association analysis between the promoter diversity of these transcription factors and the DT index/level of each variety was performed. The results showed that three genes were associated with the DT index and that five genes were associated with the DT level. The sequences of these associated genes are complex and variable, especially at approximately 1000 bp upstream of the transcription initiation sites. The study illuminated that association analysis aimed at Ecotilling diversity of natural groups could facilitate the isolation of rice genes related to complex quantitative traits. DST  could be located to the mutation site by the aid of leaf width because the gene controlled simultaneously both the drought-tolerant and leaf width traits lcyE and crtRB1 alleles responsible for beta-carotene levels in maize In recent years, various meteorological disasters, especially droughts, have seriously and frequently affected agricultural production and threatened food security in China. Although stress-resistant varieties have been developed by the use of conventional breeding methods, these methods require a long period of testing and evaluation and lead to be too costly. Currently, it is urgent for modern biotechnology to resolve stress-resistant breeding problems. Molecular breeding should be based on the collection of drought-tolerant (DT) cultivars all over the world and the comprehension of the drought-resistant mechanisms International Rice Research Institute, this approach has been used to screen genes of interest in the rice germplasm pool and in mutants Cucumis spp. was used for the implementation of Ecotilling to identify new allelic variants of eIF4E that control virus susceptibility TILLING  provides a reverse genetics strategy that is cost-effective and high-throughput AtHDG11, an HD-START protein, is an important regulatory element in flower development, but its over-expression in a T-DNA mutant clearly enhances the drought tolerance of Arabidopsis DST, which controls leaf morphology; it can enhance rice drought tolerance after suppression Under drought stress, there are a large number of proteins expressed in plants, including transcription factors, which are a very important class of regulatory factors cis-element, a promoter is an important component of a gene, and it has an important role in gene function research. The research about promoter research has currently become a hot topic. An important reason for this is that gene functional changes are caused by changes in the promoter. In rice and Arabidopsis, there are direct evidences to indicate that the gene spatiotemporal expression profile will also be changed, and unexpected changes in its function occur with changes in the sequence of the promoter. The aforementioned AtHDG11 has been validated as a flower development gene, and it enhanced the drought tolerance of Arabidopsis when it was inserted in a super-expression promoter HKT1;1 has been inserted into a new position using an enhancer trap expression system and was only expressed specifically in the mature root stele. This led to a reduction in the root-to-shoot transfer of Na+; plants with reduced shoot Na+ also have increased salinity tolerance, but plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulate high shoot Na+ and grow poorly Xa13 is a key gene in pollen development. Promoter mutations in Xa13 cause downregulation of expression during the host–pathogen interaction, resulting in the fully recessive xa13, which grants race-specific resistance against bacterial blight Arabidopsis thaliana in low-phosphate medium, the pLPR1Bay0 allele became a null allele because a 16-bp deletion was located upstream of the LPR1 transcription start site and caused a loss of function of LPR1 As a Drought is one of the major agricultural disasters in China. To reduce losses in agricultural production caused by drought, we have long been engaged in breeding and theoretical research on drought resistance in rice at the Shanghai Agrobiological Gene Center. A set of rice DT germplasm has been collected and evaluated under both normal and drought stress conditions. In this study, the promoter polymorphism of twenty-four rice transcription factor families was analyzed using Ecotilling technology based on these materials. Drought-related transcription factors were identified by association analysis using the software TASSEL.Using the methods and facilities of DT evaluation based on the ‘line-source soil moisture gradient’ CEL1 digestion between Nipponbare and testing materials were detected. A band phenotype appeared for the first time was marked as 1, and the band phenotypes emerged later were marked in turn. There were a maximum of 11 alleles in one primer pair that could be detected. Each primer pair detected an average of 3.36 alleles in these genes. There were 69 genes with 2 alleles, 52 genes with 3 alleles, 46 gene markers with 4 alleles and 23 gene markers with 5 alleles, which accounted for 34.2%, 25.7%, 22.8%, and 11.4% of the total number of polymorphic markers, respectively.The genome data of 24 transcription factor families were downloaded from the GRAMENE database. Based on 1000–1200 bp upstream sequences of genes, 505 pairs of primers were designed for Ecotilling detection . PCR proP(D)) sharply increased from k = 1 to k = 2 but slowly after k = 2  shared by the marker locus variation is shown in the Electronic supplementary material, The association between the DT traits and TILLING markers was calculated using a MLM function based on a simple model using TASSEL2.0 OsAE128 marker, with two alleles, was significantly associated with the DT level trait. Accessions with allele 1 (A1) had the lower DT level and 44 accessions with A1 belong to the upper subpopulation in OsZIM14 marker with two alleles, all accessions with A1 belong to the lower subpopulation except for the variety of DJAUB. For other gene markers, alleles distributed two subpopulations. But it is also interesting that 11 accessions with A2 in the gene OsGRF5 were originated from Philippines and Brazil varieties.The least significant differences (LSDs) were calculated to determine the differences among the accession groups defined by alleles or their haplotypes of associated markers . The genOsALF11, OsAE128 and OsZIM14  revealed that the sequence polymorphism altered the plant cis-acting regulatory DNA elements. Compared to Shenshuidao1, a cauliflower mosaic virus 35S site (TCTCTCTCT) was appended in the insertion fragment of the promoter of the OsALF11 gene in Huhan3. Then the site was moved to the −77 position in front of the transcription initiation site, while the site in other varieties was proximal to the transcription initiation site . The family is not only involved in rice growth and development but also plays an important role in stress responses involving the dehydration-responsive element-binding protein and the ethylene-responsive element-binding factor OsZIM14 gene can control the development of plant inflorescences Transcription factors play important roles in plant growth, development and environmental response. In this study, only three genes were found to be associated with DT index and five genes with DT level in the 24 transcription factor families which they were from five gene families . Of themOsGRF5 and OsAE25 were also found at the QTL regions in a mapping population derived from a cross upland rice To adapt to the environmental changes during plant long-term evolution, the promoter region has participated in a series of changes, and some of them have caused changes in gene expression patterns. As an evolutionary effect, plants were enhanced or weakened in their ability to respond to changes in the environment OsAE128 marker belong to the upper and lower subpopulation except for several accessions. In addition, A1 from OsZIM14 marker belong to the lower subpopulation with higher DT level except for one variety. It suggested that there were not only subspecies nature but also DT nature. A2 from OsGRF5 marker were originated from specific region. This kind of information is useful in the DT breeding program based on germplasm with distant genetic relations, by which the genetic diversity of modern varieties can be broadened.The estimation of allelic effects showed diverse patterns for associated markers in our present study . Allele Ninety-five domestic and foreign rice varieties were collected . Three lhttp://ricetfdb.bio.uni-potsdam.de/v3.0/) http://www.gramene.org/). The primers were designed using Primer Premier Software.The data of the rice transcription factors were downloaded from PlnTFDB (3.0) . The Ecotilling electrophoresis bands were converted into molecular markers. Then the genetic structure of all 95 samples was investigated with the model-based method implemented in STRUCTURE2 (http://pritch.bsd.uchicago.edu/structure.html). The association between DT index, DT level and TILLING markers was detected based on a simple model in TASSEL2.0 (http://www.maizegenetics.net). The detailed methods were available in our previous publication The polymorphisms of the genetic population were analyzed using 30 InDels that were widely distributed across the whole genome . The sequence complexity was shown using Vector NTI 8 software. The plant cis-acting regulatory DNA elements were located using PLACE (http://www.dna.affrc.go.jp/PLACE).Based on the results of an associated analysis, the polymorphism markers associated with DT traits were selected for further analysis. According to band phenotype and DT level, more than 5 varieties were selected to isolate the promoter fragments that were inserted into the pGEM-T easy Vector  for sequencing with a repeat. If the sequence from the repeat was not coincident, the fragment was submitted to resequence. Multiple alignments of the promoter sequences were performed using Clustal X software Click here for additional data file.Table S2Distribution of InDel markers on the rice chromosomes.(DOCX)Click here for additional data file.Table S3The association analysis of drought tolerant traits with the diversity of 204 transcription factor promoters. R2 indicates the proportion of phenotypic variation against 204 transcription factors. P indicates the association analysis of drought tolerant traits with the diversity of 204 transcription factor promoters. The red font indicates the significantly associated genes.(XLSX)Click here for additional data file.


Transcription factories are nuclear domains where gene transcription takes placealthough the molecular basis for their formation and maintenance are unknown. In thisstudy, we explored how the properties of chromatin as a polymer may contribute to thestructure of transcription factories. We found that transcriptional active chromatincontains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Singlefibre analysis showed that this modification spans the entire body of the gene.Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active andinactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in thechromatin fibre. The result of this change in flexibility is that chromatin couldbehave like a multi-block copolymer with repetitions of stiff-flexible(active-inactive chromatin) components. Copolymers with such structure self-organizethrough spontaneous phase separation into microdomains. Consistent with such modelH4K16ac chromatin form foci that associates with nascent transcripts. We propose thattranscription factories are the result of the spontaneous concentration of H4K16acchromatin that are in proximity, mainly in cis. Transcription in eukaryotes is organized in transcription factories (TFs), which arenuclear domains where several genes are grouped to be transcribed together ,23-5]. -5. 3-5].Chromatin at the TF is decondensed  and contin vivo or in vitro by using Bromo-Uridine (BrU) or BrUTPrespectively. The area covered by acetylated histones was larger than that stained bythe nascent transcripts  , suggesting that co-linear active genes expressedat the same time, in agreement with the analysis of expression data using FDCP mix cells . The slin system, where the rod is the stiff activeTU. The multi-block copolymers function as amphiphiles whose components segregateinto domains to avoid unfavourable contact with each other. In these systems,complete phase separation is prevented by the covalent linkage between the components [Collinear active TUs are enriched in H4K16Ac which confers stiffness and inhibitsinter-fiber interaction -17. In tmponents . The rodmponents  , which reduces RNA pol II transcription by 98%; and heatshock (1h 45°C), which releases RNA pol II from the DNA   before joining aTF. This is conceptually very different from current transcription factory model,which proposes that genes are targeted to TFs to “enhance production byconcentrating the relevant machines, resources, and expertise in one place” .For in vivo transcription, cells grown on coverslips were incubated in presence of2.5 mM BrU for several min.For in vitro transcription, cells grown on coverslips were treated as described .3 cells in 5 μl) were spotted onto a 22 × 50mm glass slide and 5 μl of lyses buffer were added ). After 10 min at 20°C, the slide was tilted to allow the drop to run down.Samples were air-dried and fixed in 4% Paraformaldehyde for 10 min. Clusters weredefined as two o more hyper acetylated tracks in less than 100 Kb. For quantificationof clusters of hyper acetylated chromatin between 150 and 200 tracks of hyperacetylated chromatin were analysed.Cells  or heat shock(Hs) for 1h at 45°C. Both treatments led to the release of RNA pol II fromthe genes. These foci were also resistant to NaCl extraction (cellspermeabilised with 0.05% Triton X100 for 5 min in PBS at 4°C followed by10 min extraction with 2M NaCl for 10 min). The images were pseudo-coloured fordisplay. The bottom bar shows the scale of pseudo-colours used.Stability of H4 K16Ac foci. Resistance of H4 K16Ac foci to various treatmentsthat disrupted transcription or chromatin structure. The aspect of H4 K16Acfoci did not change after DRB treatment (2h 150 Click here for file(a) TheH4K16Ac foci were disassembled by formamide treatment, as can be seen from thechange in the staining pattern, which is more diffuse and less intense than thecontrol. The images were pseudo-coloured for display. (b) Thedeconstruction of the foci was quantified by the change in the pixel intensityvariation coefficient (SD/mean). This analysis was performed by measuring themean intensity and the standard deviation (SD) of the H4K16Ac signal of thenuclear areas in at least 200 cells for each treatment. The images werepseudo-coloured for display. The bottom bar shows the scale of pseudo-coloursused.H4 K16Ac foci are disassembled by formamide. The resistance of H4 K16Ac foci toformamide treatment. Cells were incubated for 5 min in PBS with differentconcentrations of formamide  then fixed with 4%paraformaldehyde and immunolabelled with H4K16Ac antibodies. Click here for file


Fungal laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet. Fungal laccases are phenol oxidases belonging to the group of multi-copper enzymes that catalyze the oxidation of a great variety of phenolic compounds and aromatic amines using molecular oxygen as electron acceptor . Almost 1.1. Trametes versicolor 290, the highest laccase activity value (2500 U L-1) was achieved in cultures grown in the presence of 400 μM Cu2+, corresponding to a 18-fold higher level than in the absence of copper [Trametes spp. The laccase activity of Trametes versicolor MB 52, Trametes versicolor MB 54, Trametes suaveolens MB 51 was induced by 1 mM Cu2+, reaching values of about 9000, 10000, and 7000 U L-1, respectively, while none of these strains produced laccase activity when grown on culture medium without additional Cu+2 [et al. [Trametes versicolor (CBS100.29). The highest values were obtained by cultures supplemented with 3.5 mM Cu2+, showing an increase of 11-fold and maximum values around 8000 U L-1. More recently, a higher laccase production induced by copper was observed by the Trametes sp. 48424. In this case, the highest laccase activity in the presence of 1 mM Cu2+ was 15273 U L-1 [Regulation of laccase expression by metals is widespread in fungi. In y value 200 U L-1 f copper  have stunal Cu+2 . Lorenzo [et al.  studied 73 U L-1 .Pleurotus spp. the induction of laccase by copper has been widely documented. Giardina et al. [-1, growing Pleurotus ostreatus (ATCC MYA-2306) in nutrient-rich medium, with addition of 150 µM CuSO4, whereas laccase production resulted between 0.5 and 4 U L-1 in the presence of copper traces. More recently, a laccase production of 70000 U L-1 was observed when this fungus was grown in semi-synthetic growth media formulated with agro-industrial by-products and supplemented with 150 µM CuSO4 (unpublished data). Even in a et al.  obtainedT. versicolor [Ceriporiopsis subvermispora [P. ostreatus [P. sajor-caju [Coriolopsis rigida [Trametes pubescens [P. ostreatus (ATCC MYA-2306), Cu2+ has a marked effect on the induction of the examined isoenzymes POXC, POXA3 and POXA1b. The effect of Cu2+on laccase synthesis can be explained not only at transcriptional level. The results of Palmieri et al. [2+ concentration of 1 mM decreases the extracellular proteolytic activity, thus impairing laccase degradation.In all the analysed fungi , regulai et al.  showed t+, and Mn+2 ions have also been reported as modulators of laccase transcription [+2 acts as an inducer of laccase transcription in P. sajor caju [Clitocybula dusenii and Nematoloma frowardii [C. subvermispora [Agcription ,10. It ijor caju , Clitocyrowardii , but it 1.2. 1 [Phenolic and aromatic compounds structurally related to lignin or lignin derivatives are routinely added to fungal cultures to increase laccase production , 24. Lac1 -47. LaccPhlebia spp. followed by P. ostreatus [Pycnoporus cinnabarinus [The effect of veratryl alcohol on laccase production varies also with the composition of basal media used , enhancistreatus . Howeverabarinus . P. pulmonarius, ferulic acid and vanillin produce the highest levels of laccase activity, inducing the expression of lcc3 isoenzyme, not detectable in basal condition [P. ostreatus laccase induction has shown ABTS (0.5-1 mM) as the most significant inducer, enhancing laccase activity production up to ﬁve-fold. 2,5-Xylidine, guaiacol and ferulic acid (1 mM) also increase laccase activity from two to three-fold, whereas veratryl alcohol does not stimulate enzyme production [P. ostreatus (ATCC MYA-2306) also produces a different laccase isoenzyme pattern [Among the 15 different phenolic compounds tested in ondition . Screenioduction . Moreove pattern . Laccase induction has also been demonstrated in the presence of dyes, the induction level being highly sensitive to small differences in their chemical structures . P. pulmonarius and Botryosphaeria rhodina cultures, the induction seems to be more efficient when the inducer was added to the cultures at inoculum time [P. ostreatus, Rigidoporus lignosus and Trametes modesta medium conditioning is usually performed after 2-5 days from inoculation in order to avoid growth inhibition [Besides molecular structures and concentration, the inductive effect of phenolic compounds depends also on the time of their addition to the cultures . In P. plum time , 51, whehibition , 53. HigT. versicolor, both 1-hydroxybenzotriazole and 2,5-xylidine effectively activate lcc transcription, whereas no induction is observed in the presence of either veratric acid or ferulic acid [lcc1, from T. villosa is induced by the addition of 2,5-xylidine, but a second gene is constitutively expressed under the tested conditions [Trametes sp. I62, nine structural closely related aromatic compounds appear to have different effects on laccase gene expression. The three laccase genes of this fungus are differentially expressed in response to these compounds, with specific induction patterns being observed for each molecule tested [The induction of aromatic compounds has been found to occur at transcriptional level, differing not only among the various fungi analyzed, but also among the different isonzymes of the same organism. In nditions . In the e tested . Trametes sp. AH28-2, where three novel laccase genes, lacA, lacB, and lacC, have been recently identified, it has been reported that different aromatic compounds can selectively induce the production of distinct laccase isozymes, with o-toluidine inducing the expression of LacA and 3,5-dihydroxytoluene mainly stimulating the production of LacB. Analysis by competitive Real Time Polymerase Chain Reaction showed that the accumulation of laccase RNA transcripts is accompanied by the increase of corresponding enzyme activity in cultures [In the basidiomycete cultures . Volvariella volvacea, lcc1 transcript titres are differentially tuned by the addition of various aromatic compounds, whereas lcc4 transcription is not affected by these molecules [In olecules . 1.3. Laccase activity has also been shown to be dependent on the concentration and nature of carbon and nitrogen sources as well as on their ratio. Nitrogen source plays a key role in laccase production, with effects depending on its nature and concentration in culture media . Change lcc1 and lcc2 transcript levels increased 100-fold in comparison with limiting nitrogen conditions [lcc1 in Tramtes trogii is preferentially induced by organic nitrogen sources compared with inorganic ones [P. sajor-caju are differentially induced at the transcriptional level by nitrogen sources, with a positive regulation by nitrogen occurring for lac2 and lac4, and no effect being detected for lac1 and lac3 [Laccase induction at transcriptional level has been reported to occur under non-limiting nitrogen levels in the ligninolytic basidiomycete I62 (CECT 20197), where nditions . Expressnic ones . Individand lac3 . P. ostreatus IMI 395545 submerged cultures [-1.As far as carbon sources are concerned, it has been demonstrated that supplementation of substrates, like glucose that are readily utilizable and efficiently metabolized by the microorganism, results in high levels of laccase activity . Studiescultures  have sho-1) inhibit laccase transcription in basidiomycete I62 [T. pubescens [Trametes sp. AH28-2 [In some fungi, laccase expression has been found to be subjected to catabolite repression: this mechanism is thought to be an energy-saving response. High carbon levels  [Cis-acting MREs have been discovered in multiple copies in the Saccharomyces cerevisiae metallothionein promoter where they are essential for efficient metal-inducible transcription [lap2 promoter region suggests the existence of a carbon catabolite repressor similar to the CreA isolated in A. nidulans [lap2 gene expression caused, for example, by high concentrations of copper. Interestingly, neither xenobiotic response elements (XREs) nor antioxidant response elements (ARE) could be detected in the lap upstream region in accordance with the lack of transcriptional induction observed in the presence of aromatic substances [The promoter region of the gene coding for the major laccase isoenzyme LAP2 from . (2002) . This up. (2002)  and a “g. (2002) . Cis-actcription . The prenidulans . HSE andbstances .Gaeumannomyces graminis, two of which are inverted and 51bp apart. In addition, this promoter contains two putative ACE1 (activation of cup1 expression protein) binding sites situated between the TATA box and ATG codon [S. cerevisiae, ACE binding site is the recognition site for the ACE1 copper-responsive transcription factor, which binds its target sequence in a copper-loaded conformation, activating the transcription of several genes [lcs from C. subvermispora [Four putative MRE consensus sequences have been also found in the promoter region (~ 1kb long) of the copper inducible LAC2 laccase from TG codon . First dal genes . An ACE rmispora .lac1, lac2, lac3 and lac4, extending 724bp, 214bp, 840bp and 1740bp upstream of the start codons, respectively) from P. sajorcaju displaying differential regulation in response to different stimuli [consensus sequences putatively involved in nitrogen and carbon regulation such as CreA, Mig and NIT2 [P. sajor-caju laccase genes [lac4 gene, whilst sequences with perfect homology with XRE have been detected in lac1 and lac4 promoters. Interestingly, laccase transcriptional analysis indicated that both lac1 and lac4 are regulated by the addition of 2,5-xylidine and ferulic acid, while lac2 and lac3, both of which appear to lack XRE, are constitutively expressed under these conditions.Soden and Dobson  have per stimuli . A numbeand NIT2  binding lacA, lacB and lacC) from Trametes sp. AH28-2, has been investigated [consensus sequences have been identified in the lacA, lacB and lacC promoter regions . Differences in copy number and distribution of XREs among the three genes (seven XREs in lacA and only two in lacB and lacC) are supposed to be responsible for their different responses to aromatic compounds: lacA is induced by all tested aromatic compounds, lacB is induced mainly by guaiacol and 3,5-dihydroxytoluene, whilst lacC transcript levels are not detectable in the presence of these compounds. The overlapping of one XRE element with the TATA box in the lacC promoter is probably responsible for the absence of induction of this gene by aromatic compounds.The differential expression of three laccase genes , one of which is annotated as a ferroxidase-like. The sequence similarity and the intron–exon structure, along with the degree of identity among the corresponding protein sequences, allowed us to individuate two “laccase subfamilies”, while poxa3 gene is evolutionary distant from both subfamilies [P. ostreatus laccase genes, extending 500bp upstream of the start codon, have been analysed, revealing very little identity degree among them. In silico analysis allowed the identification of several putative response elements, differentially distributed along the promoter sequences . It is worth noting that the promoters of the most abundantly produced proteins (POXC and POXA3) display the highest number of signals belonging to different classes. Furthermore some characteristics are peculiar to each subfamily, i.e. the high number of ARE, and the absence of XRE and C and N nutrient responsive element in the poxa1b sub-family. Twelve putative laccase genes have been identified in the recently sequenced families . The pro3. in silico analysis of many laccase promoters, only a few reports go through the molecular mechanisms underlying laccase regulation by different stimuli.Despite the extensive data available by P. ostreatus (ATCC MYA-2306), the formation of protein complexes on specific MREs identified in the promoter regions of the two copper induced laccase genes poxc and poxa1b [poxc and poxa1b promoters (four MREs in each promoter region) are recognized by fungal proteins, except for one MRE in poxc promoter region, that is located downstream of the transcription-initiation site. Interestingly, the formation of complexes between the identified MREs and fungal proteins was found to occur in the absence of metal ions, suggesting the involvement of a 25 kDa negative-acting regulatory factor able to repress gene expression when bound to laccase promoter. Moreover, the role of specific nucleotides of the identified MREs in complex formation has been analysed, leading to the determination of an affinity scale of the tested MREs and to the definition of the optimal binding sequence. Results of UV-cross-linking analyses indicated that the highest affinity sequence binds also a 30 kDa factor. However, when a GC-rich region is located adjacent to the MRE site, this interaction is inhibited by the binding of a 15 kDa protein. A similar behaviour was also found in some metallothionein promoters where a regulatory role of Sp1, binding to this GC-rich region, in MRE-mediated transcriptional activation has been demonstrated [Some reports have been devoted to the comprehension of molecular aspects leading to laccase induction by copper. In d poxa1b , was verd poxa1b . EMSAs anstrated .P. chrysosporium (Pc-ACE1-like) trough complementation of a S. cerevisiae ace1∆ strain [mco1 gene, inducing gene expression. Using an in vitro transcriptional activation system, it was also demonstrated that Pc-ACE1-like is able to activate the transcription of a promoter-reporter construct in the presence of only one ACE element [P. chrysosporium cultures also caused an increase of mco2 mRNA levels, although mco2 promoter regions lacks ACE elements. This effect may be ascribed to a different mechanism, possibly involving the generation of reactive oxygen species [An ACE1-like factor has been isolated in ∆ strain . This fa element . Intrigu species .C. subvermispora (Cs-ACE1-like). Specific binding of this factor to its cognate ACE sequence in the promoter of the copper induced lcs laccase was demonstrated by EMSA assays. This factor exhibited structural differences along with a lower complementation efficiency of the above mentioned S. cerevisiae strain, in comparison with its ortologue from P. chrysosporium [S. cerevisiae and its ortologues. Although binding capacities of Cs-ACE1 to ACE elements is unaltered, its structural features may be responsible of the rather weak complementation of the S. cerevisiae ace1 ∆ strain.An ACE1-like factor was also isolated from osporium . In partC. neoformans, where LAC1 is a copper regulated laccase acting as a virulence factor [C. neoformans was also found to be induced during glucose starvation, stimulated by copper, iron and calcium and repressed at elevated temperatures [Mechanisms driving copper regulation have been also analysed in the pathogenic fungus e factor . In thise factor , harboureratures . The comeratures . via a conserved signalling cascade whose terminal component is the zinc-finger transcription factor PacC/Rimp1p [pacC mutants mimicking acidity or alkaline phenotypes allowed to infer the involvement of PacC in modulating laccase transcription in the plant pathogenic fungus F. oxysporum. As a fact, expression of lcc3 occurs in acidic conditions and is down regulated by PacC, while expression of lcc5 occurs only at pH 6 and is up regulated by the same factor. The presence of PacC binding consensus sites in lcc3 and lcc5 promoter regions further supports these data.In plant pathogens, ambient pH may affect the expression of putative virulence factors, including laccases. Responses to pH variations occur C/Rimp1p . PacC isMagnaporthe oryzae, the causal agent of rice blast disease. Deletion of Moatf1 gene, an ortologue of Schizosaccharomyces pombe ATF/CREB involved in oxidative stress response, caused retarded vegetative growth of mycelia together with reduced transcriptional level of laccases and peroxidases. As a fact, the basic leucine zipper transcription factor Moatf1 was found to be necessary for full virulence of the fungus by regulating the transcription of laccases and peroxidases to impair reactive oxygen species-mediated plant defence [Oxidative stress response has been investigated in the fungus  defence . cis acting elements is only partially known. Further studies may provide additional clues to unravel the mechanism underlying laccase transcriptional regulation and to verify its general occurrence in different species.In conclusion, the reported analyses, seen as a whole, suggest a complex picture of laccase regulation phenomena whose precise molecular mechanisms through a variety of putative


Molecular biology is a rapidly evolving field that has led to the development of increasingly sophisticated technologies to improve our capacity to study cellular processes in much finer detail. Transcription is the first step in protein expression and the major point of regulation of the components that determine the characteristics, fate and functions of cells. The study of transcriptional regulation has been greatly facilitated by the development of reporter genes and transcription factor expression vectors, which have become versatile tools for manipulating promoters, as well as transcription factors in order to examine their function. The understanding of promoter complexity and transcription factor structure offers an insight into the mechanisms of transcriptional control and their impact on cell behaviour. This review focuses on some of the many applications of molecular cut-and-paste tools for the manipulation of promoters and transcription factors leading to the understanding of crucial aspects of transcriptional regulation. Proteins are the building blocks of cells, as integral parts of the cell structure and effectors of its functions. Though translation of the mRNA message, as well as modification and correct folding of the polypeptide chains are essential for the protein composition of a cell, expression of all the components begins by transcription from DNA. Thus, synthesis of mRNA is the first step that controls eukaryotic gene expression. Transcription consists of three main phases: initiation, when RNA polymerase II (Pol II) is recruited to the promoter and begins RNA synthesis; elongation, during which the transcripts are completed and termination, when Pol II and the full length mRNA disengage from the DNA template . The proThe mechanisms of transcriptional regulation are tailored to individual genes. Constitutive genes are mainly house-keeping proteins that form each cell and confer its basic functions. Transcription of these genes is constant, at a pace depending on the need and half-life of each protein. Regulated genes are proteins expressed in response to signals from the cell and its environment. These signals are transmitted to the nucleus in the form of active transcription factors that trigger transcription by binding to response elements mainly in the 5′ sequences or promoters of genes, activating the transcriptional machinery. A wide range of genes are expressed on demand, controlled by different mechanisms according to their function. Thus immediate early genes (IEG) are rapidly and transiently induced by modulation of the chromatin structure and PIC pre-assembly, with elongation being the main point of regulation ,5. On thA wide variety of highly sophisticated technologies have been developed to study molecular events in more detail. However, in this review, we will concentrate on those using molecular cut-and-paste as a strategy to understand the mechanisms that drive and regulate transcription.The transcriptional regulation of a particular gene has been traditionally studied by analysing the abundance of the messenger RNA by Northern blot, RNA protection RT-PCR or quantitative RT-PCR . ChangesThe level of detail that could be studied increased considerably with the development of reporter genes. These consist of the 5′-flanking region or promoter of the gene of interest pasted to a gene, which becomes an indicator of the promoter’s activity . The recThe bacterial enzyme, chloramphenicol acetyltransferase (CAT), catalyses the transfer of the acetyl group from acetyl-CoA to the substrate chloramphenicol. CAT has been widely used in recombinant constructs, because it is relatively stable in mammalian cells and has no eukaryotic equivalent that could interfere with the assay, so the activity measured corresponds exclusively to the construct introduced in the cells and reflects the activity of the promoter under study. The neuropeptide Y (NPY) promoter was characterised pasting 700 bp of the 5′-flanking sequence into a CAT reporter. It was shown that vasoactive intestinal peptide (VIP) induces the NPY promoter, and specific inhibitors attributed this activity to the cAMP-dependent protein kinase A (PKA) . On the Plasmodium falciparum. Its AT-rich genome and extensive repeats make the identification of regulatory sequences very difficult. Using a CAT reporter construct for transient expression in the parasite, a 5′ region of a gene called maebl was identified as harbouring the regulatory activity for the expression of this gene [The reporter strategy has also provided the possibility of studying gene expression in complex organisms, such as the malaria parasite, his gene .14-labelled chloramphenicol, analysing the products by thin layer chromatography and quantifying the amount of acetylated chloramphenicol by scintillation counting or by exposure to X-ray films and densitometry. Additionally, the level of chloramphenicol conversion is highly dependent on the strength of the promoter, making analysis of weak promoters difficult [However, measuring CAT activity is a lengthy and laborious procedure that involves incubation of extracts from transfected cells with Cifficult . These letc. In particular β-galactosidase, encoded by the Lac Z gene from E. coli, has been very useful for determining or normalising transfection efficiency. This enzyme catalyses the hydrolysis of β-galactoside sugars, such as lactose, and its activity can be assayed with a variety of substrates detected with a spectrophotometer, fluorometer, luminometer or in situ with histochemical staining. Enzymatic activity can also generate colour compounds that can be directly visualised on the microscope, such as the blue colour generated by the cleavage of X-gal, allowing direct visualisation of transfected cells. However, some mammalian cells have endogenous lysosomal β-galactosidase activity, so using higher pH of 7–8, pre-heating extracts to 50 °C or using negative controls becomes an important precaution [Other commonly-used reporter genes include β-galactosidase, growth hormone (GH), β-glucuronidase (GUS), alkaline phosphatase (AP), green fluorescent protein (GFP), ecaution .Photinus pyralis was cloned [Renilla reniformis) luciferase. Renilla has a different substrate requirement from the firefly luciferase, but its emission can also be detected by a luminometer in a dual assay that provides a reproducible, accurate and sensitive method to measure transcriptional activity in a simple and rapid detection assay.In the 1980s, the luciferase gene from the firefly s cloned  and shows cloned . In the s cloned . Furtheretc. In a breast cancer cell line expressing the progesterone receptor (PgR+), progesterone (Pg) was found to increase cytokine activation of iNOS, while it has not effect in PgR− cells. The additive effect of Pg results in an increase of cell death, which might have implications for breast cancer biology [The luciferase reporter gene has been used in a wide variety of studies examining complex interactions of transcription factors and molecules on promoters and their role in transcriptional regulation, sometimes providing links between systems and processes. For example, while studying the transcriptional regulation of the inducible nitric oxide synthase (iNOS or NOSII), we showed an interaction between the hormonal and immune systems. iNOS is an important component of inflammation and innate immunity, fulfilling a role of non-specific anti-microbial defence by secreting high levels of nitric oxide (NO), which quickly kills invading organisms. The toxicity of high levels of NO implies that the enzyme must be tightly regulated starting by transcription, which is only induced in response to stimulation by immune cytokines, bacterial compounds,  biology .Sometimes, determining regulatory sequences and crucial marks, such as the TSS, are not straight forward, as was the case of the human caspase family of proteins. Caspases are cysteine proteases that play an important role in cell death by apoptosis. Their promoters still remain largely uncharacterised, which was particularly intriguing in the case of caspase-2, because two isoforms with opposing effects on apoptosis had been described. A long isoform (casp-2L) was shown to induce programmed cell death, whereas the short isoform (casp-2S) could suppress it. In order to understand how expression of these isoforms is regulated, the 5′-flanking region of the casp-2 gene was cloned, and 100 bp fragments encompassing potential TSSs were introduced into a luciferase construct. This vector contains a viral SV40 enhancer, but requires insertion of a TSS to drive luciferase expression. It was shown that casp-2L uses a stronger TSS that determines the inclusion of a different exon 1 from casp-2S. In turn, the short isoform has a weaker TSS leading to an exon 1 that is skipped in casp-2L and causes a frame-shift that generates an early stop codon explaining the shorter protein. Putative promoters of the two isoforms were analysed in luciferase reporters, showing that casp-2L has a much stronger promoter than casp-2S, which correlates to the higher levels of the long isoform in cells . The indRecombinant reporter vectors can be manipulated in order to characterise the precise role of un-translated regions (UTR) in the transcription of the gene they regulate. Particularly large promoters, such as the human iNOS, can be studied using this strategy. Up to 16 kb of the iNOS promoter have been analysed, inserting various 5′-flanking sequences of the gene into a luciferase reporter vector ,23. SeveRegulatory sequences are also be found in the 3′UTRs of genes. These are mainly implicated in the control of translation and mRNA stability, but in some cases, have been shown to play a role in transcription as well. 3′UTRs can be examined by insertion downstream of reporter genes, as was done for the mixed lineage leukaemia (MLL) proto-oncogene. Rearrangements of MLL with different partner genes involving the 3′UTR are associated with the development of acute leukaemia. Cloning the MLL 3′UTR downstream of luciferase showed strong repression of the gene, and a deletion analysis localised this effect to a 900 bp region of the proximal 3′UTR. The decrease in luciferase activity was paralleled by a marked reduction of mRNA, yet its stability was not compromised, suggesting a transcriptional mechanism. Insertion of a polyadenylation signal upstream of the repressive region restored mRNA synthesis, demonstrating that in order to repress, the 3′UTR has to be part of the transcribed unit. It was discovered that the repressive region of the MLL 3′UTR has the capacity to retain Pol II slowing the rate of transcription. The fusion with partner genes abrogates this effect leading to MLL overexpression and leukaemogenesis .in vitro by electrophoretic mobility shift assay (EMSA) that also allows identification of the binding transcription factors with specific antibodies [Identifying regulatory regions in promoters advanced the understanding of transcriptional regulation, but the role of transcription factors and their response to signalling pathways still remained obscure. Once a promoter-region was identified as bearing regulatory activity, its sequence can be examined for consensus transcription factor-binding sites that can be verified tibodies . Once cotibodies , AP-1 [2tibodies , STAT-1 tibodies  and Oct-tibodies ,30. The tibodies . With thtibodies .The function of transcription factors can be studied directly and independently of the genes they regulate with the construction of transcription factor-specific reporter genes. In this approach, several copies of a binding element are placed in tandem upstream of a luciferase gene and its core promoter. Thus, four tandem copies of the κB enhancer that binds NFκB showed that NFκB-driven gene expression is dependent on p38 MAP kinase, which is partly due to activation of the TATA binding protein (TBP) . We usedTranscription factor-specific reporters have also proven invaluable to refine DNA binding sites by determining the base requirements for protein interaction. This approach is particularly useful for the analysis of long binding sites, such as the 21 bases long RE1. The Neurone Restricted Silencing Factor  that binds RE1 is a transcriptional repressor that silences neuronal genes in non-neuronal cells. A search of the data base for genes containing RE1 revealed a certain variability in the base composition of the core consensus sequence, so in order to define the crucial bases for REST binding and function, different RE1 sequences were introduced in a CAT reporter. Repressive activity was assayed and matched with REST binding, identifying eight variable nucleotides that are not critical for REST binding, while the other 13 are more conserved . The rolThe number of consensus elements in promoters and the complexity of transcriptional activation were clear indications that transcription factors generally do not act alone. They cooperate to induce activation, amplify or repress other protein’s signals, recruit other factors or co-factors to the DNA, interact with the basal machinery, and some of them can bend or modify the DNA structure. Some of these interactions occur through direct protein-protein binding, suggesting that the distance between binding sites might be important. In order to study these effects, Bertolino and Singh constructed a reporter vector containing an octamer site (Oct or POU) together with a core promoter and distal enhancer regulating the β-globin gene, whose RNA was measured directly to evaluate transcriptional activation . They weSuch studies began unravelling the complexity of promoter structure, locating regulatory elements as far as 100 kbp from the TSS and leading to the concept of enhancers. These are DNA sequences containing transcription factor binding sites, sometimes in clusters that regulate the activity of core promoters. It is the core promoters surrounding the TSS that serve as the docking site for the basal transcription machinery, ultimately leading to the assembly of the PIC with binding of Pol II ,38. The The difficulty of assessing the contribution of particular transcription factors to transcriptional activation of a gene is complicated even further by the existence of transcription factor families with the same DNA-binding specificity. For example, Oct binding sites are recognised by several members of the POU family, such as Oct-1, Oct-2 and Pit-1; Sp1 binding sites are bound by Sp-1, Sp-2 and Sp-3 and the GATA motif by GATA-1, GATA-2, GATA-3 and GATA-6 . An ingeN- and C- terminal domains that recruit chromatin-modifying enzymes, including Histone de-acetylases (HDAC). So, by binding to its DNA element, REST causes tightening of the DNA nucleosome structure, making it inaccessible to the transcription machinery [N- and C- terminal domains were deleted, while preserving the DNA binding domain. To facilitate detection, a c-myc-epitope tag was incorporated at the C-terminal, as well as GFP as a marker for transfection efficiency. This construct acts in a dominant-negative (DN) capacity to compete-out endogenous REST binding to RE1 [A widely used method to understand the role of a protein is to inhibit either its activity by chemicals or its expression by siRNA and study the effect of this inhibition. It is also possible to use molecular tools to modify transcription factors in such a way that they lose their transcriptional activity. The transcription factor, REST, mentioned earlier, is composed of a zinc finger DNA-binding domain with affinity for the RE1 element and achinery . A modifg to RE1 . Adenovig to RE1  that demg to RE1 ,47.i.e., synaptic vesicles and dense core granules. These proteins are missing at the mRNA level indicating a transcriptional block that is reversed by introduction of the DN-REST [We used this DN-REST construct to demonstrate that inappropriate expression of REST is responsible for an overall repression of the secretory pathway in a neuroendocrine variant cell line. Isolated from the rat pheochromocytoma PC12 line, the variant A35C lacks expression of markers of neurone-specific organelles,  DN-REST . The conMany transcriptional responses are very fast and transient, often as a result of the quick activation and degradation of the transcription factors involved. Some transcription factors are small or processed and, therefore, difficult to follow and visualise by conventional biochemical methods. A powerful approach to overcome these hurdles is the use of chimeric transcription factors, such as GAL4-VP16, that contain the DNA-binding domain of the yeast GAL4 transcription factor coupled to the activating domain of the herpes simplex VP16 protein . The UASlacZ was introduced to serve as an indicator of activity. When GAL4-VP16 was inserted in the intracellular domain (ICD), nuclear localisation in vivo could be detected as activation of the UAS-LacZ transcription, whereas if inserted in the extracellular domain, access to the nucleus could not be shown. These experiments demonstrated that in response to extracellular signalling, Notch is cleaved and the ICD translocated to the nucleus. Though the presence of VP16 in these constructs increased the observed transcriptional activity, if GAL4 was inserted alone in the Notch ICD, transcriptional response was also detected, proving that Notch transduces extracellular signals by regulating expression of specific genes [C-terminal fragment of APP and a luciferase reporter gene with the EGFR promoter, it was confirmed that γ-secretase cleavage of various targets results in the translocation of ICDs to the nucleus to regulate gene expression [The GAL4 system was used to study the nuclear localisation and transcriptional activity of elusive factors, such as Notch, a member of a transmembrane receptor family that transduce intercellular signals controlling cell fate . The funic genes . Using tic genes . Indeed,pression .HIS4 core promoter was coupled with a Gal4-binding site 55 bp upstream of the TATA box, and this template was linked to streptavidin-coated magnetic beads via 5′-biotin. The beads were incubated with nuclear extracts from yeast transfected or not with Gal4-VP16, and the complexes formed were recovered by endonuclease cleavage. The components of the complexes were then analysed by quantitative mass spectrometry and Western blotting [The chimeric constructs combining DNA-binding domains with transcription-activating domains have been used for a wide range of studies, including the most fundamental transcription machinery. Thus the yeast blotting . A compain vivo. Transgenic mice have been generated introducing markers, such as β-gal, GFP or other fluorescent probes into the genome, downstream of the desired gene. Expression of the probe is regulated by the promoter of the studied gene, providing a marker to follow expression of genes throughout development and the different tissues and organs of the body [The advent of reporter genes offered possibilities of studying transcriptional regulation the body ,55.in vivo genetic manipulation uses mobile DNA elements or transposons that “jump” through the genome. Though no function has been found for these elements, it was observed that, on occasion, insertions occur in regulatory regions changing gene expression. Transposons can also integrate in coding regions, therefore altering expression or even silencing the disrupted gene or causing mutations when exiting an integration site that is not repaired properly. These elements and the transposases involved in their movement have been developed into a widely used strategy to study and discover genes implicated in disease [A different strategy of  disease . The eng disease .Plasmodium falciparum, the main parasite causing malaria in humans and responsible for the more serious clinical cases of the disease [Plasmodium falciparum led to the identification of CAF-1 (ccr4-not associated factor 1), a component of the carbon carbolite repressor protein 4 (CCR4) that causes severe attenuation of intraerythrocytic growth in vitro. Functional characterisation of the CAF-1 null mutant revealed that it regulates mRNA decay. Using microarray analysis, we showed that CAF-1 has a critical role in temporal gene regulation, as its disruption delays overall gene expression, eventually inducing premature egress from red blood cells, thus attenuating the growth rate [in vivo, while the transposon technology is a forward genetics strategy that provides means to identify novel genes and understand their functions and associated phenotypes.The transposon technology has been applied to a variety of systems, including the study of genetic expression in  disease . The parwth rate . TransgeThe capability to manipulate regulatory sequences and accurately probe their activity led to the understanding of many regulatory mechanisms of gene expression . Thus, t