Cryptic Promoter Research Articles

Genomic Basis of Aromatase Excess Syndrome: Recombination- and Replication-Mediated Rearrangements Leading to CYP19A1 Overexpression

Genomic rearrangements at 15q21 have been shown to cause overexpression of CYP19A1 and resultant aromatase excess syndrome (AEXS). However, mutation spectrum, clinical consequences, and underlying mechanisms of these rearrangements remain to be elucidated. The aim of the study was to clarify such unsolved matters. We characterized six new rearrangements and investigated clinical outcome and local genomic environments of these rearrangements and of three previously reported duplications/deletions. Novel rearrangements included simple duplication involving exons 1-10 of CYP19A1 and simple and complex rearrangements that presumably generated chimeric genes consisting of the coding region of CYP19A1 and promoter-associated exons of neighboring genes. Clinical severities were primarily determined by the copy number of CYP19A1 and the property of the fused promoters. Sequences at the fusion junctions suggested nonallelic homologous recombination, nonhomologous end-joining, and replication-based errors as the underlying mechanisms. The breakpoint-flanking regions were not enriched with GC content, palindromes, noncanonical DNA structures, or known rearrangement-associated motifs. The rearrangements resided in early-replicating segments. These results indicate that AEXS is caused by duplications involving CYP19A1 and simple and complex rearrangements that presumably lead to the usage of cryptic promoters of several neighboring genes. Our data support the notion that phenotypes depend on the dosage of CYP19A1 and the characteristics of the fused promoters. Furthermore, we show that the rearrangements in AEXS are generated by both recombination- and replication-mediated mechanisms, independent of the known rearrangement-inducing DNA features or late-replication timing. Thus, AEXS represents a unique model for human genomic disorders.

Mutational activation of the RocR activator and of a cryptic rocDEF promoter bypass loss of the initial steps of proline biosynthesis in Bacillus subtilis

The gamma-glutamyl-phosphate reductase (ProA) interlinks both the anabolic and osmostress adaptive proline biosynthetic routes of Bacillus subtilis. Because no paralogous protein to ProA exists in this microorganism, proA mutants should exhibit a tight proline auxotrophic growth phenotype. Contrary to expectations, proA mutants formed microcolonies on agar plates lacking proline and faster growing Pro(+) suppressor mutants arose. These mutants carried alterations in the rocR-rocDEF region encoding enzymes of the arginine degradation pathway and its transcriptional activator RocR. They were of two types: (i) mutants carrying single amino acid substitutions in RocR resulting in partial inducer-independent variants and (ii) mutants carrying single base-pair changes in the vicinity of the SigL/Sig-54-dependent -12/-24 class rocDEF promoter that activate a cryptic SigA-type promoter. Consequently, enhanced rocDEF transcription should lead to increased cellular amounts of the RocD ornithine aminotransferase, an enzyme that synthesizes the same reaction product as ProA, gamma-glutamic-semialdehyde/delta-1-pyrroline-5-carboxylate. This compound can be enzymatically converted into proline. The Pro(+) suppressors also exhibited a new regulatory pattern by allowing enhanced rocDEF transcription in response to proline availability when ammonium is present. Our work provides an example how flexibly bacteria can genetically develop routes to bypass constraints imposed on their biosynthetic networks and evolve new regulatory mechanisms.

Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene

Trans-splicing, a process involving the cleavage and joining of two separate transcripts, can expand the transcriptome and proteome in eukaryotes. Chimeric RNAs generated by trans-splicing are increasingly described in literatures. The widespread presence of antibiotic resistance genes in natural environments and human intestines is becoming an important challenge for public health. Certain antibiotic resistance genes, such as ampicillin resistance gene (Ampr), are frequently used in recombinant plasmids. Until now, trans-splicing involving recombinant plasmid-derived exogenous transcripts and endogenous cellular RNAs has not been reported. Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a key enzyme involved in cellular cholesterol homeostasis. The 4.3-kb human ACAT1 chimeric mRNA can produce 50-kDa and 56-kDa isoforms with different enzymatic activities. Here, we show that human ACAT1 56-kDa isoform is produced from an mRNA species generated through the trans-splicing of an exogenous transcript encoded by the antisense strand of Ampr (asAmp) present in common Ampr-plasmids and the 4.3-kb endogenous ACAT1 chimeric mRNA, which is presumably processed through a prior event of interchromosomal trans-splicing. Strikingly, DNA fragments containing the asAmp with an upstream recombined cryptic promoter and the corresponding exogenous asAmp transcripts have been detected in human cells. Our findings shed lights on the mechanism of human ACAT1 56-kDa isoform production, reveal an exogenous-endogenous trans-splicing system, in which recombinant plasmid-derived exogenous transcripts are linked with endogenous cellular RNAs in human cells, and suggest that exogenous DNA might affect human gene expression at both DNA and RNA levels.

Internal ribosome entry site-mediated translational regulation of ATF4 splice variant in mammalian unfolded protein response

Activating transcription factor 4 (ATF4) is a master regulator of genes involved in unfolded protein response (UPR) and its translation is regulated through reinitiation at upstream open reading frames. Here, we demonstrate internal ribosome entry site (IRES)-mediated translation of an alternatively spliced variant of human ATF4. This variant that contains four upstream open reading frames in the 5′ leader region was expressed in leukocytes and other tissues. mRNA and protein expression of this variant was activated in the UPR. Its translation was neither inhibited by steric hindrance nor affected by eIF4G1 inactivation, indicating a cap-independent and IRES-dependent mechanism not mediated by ribosome scanning-reinitiation. The IRES activity mapped to a highly structured region that partially overlaps with the third and fourth open reading frames was unlikely attributed to cryptic promoter or splicing, but was activated by PERK-induced eIF2α phosphorylation. Taken together, our findings reveal a new mechanism for translational regulation of ATF4 in mammalian UPR.

Identification of the Control Region of Pancreatic Expression of Bmp4 In Vitro and In Vivo

Bone morphogenetic protein 4 (Bmp4) was recently shown to be related to glucose homeostasis in mouse adult pancreas through the regulation of insulin production. We previously revealed the predominant expression of Bmp4 in adult pancreas by in vivo imaging of transgenic mice. However, the control regions for predominant Bmp4 expression in the adult pancreas are unclear. In this study, we established transgenic (Tg) mice that allow real time in vivo bioluminescence imaging of the enhancer/promoter activity of the Bmp4 gene. Tg mice expressing firefly luciferase with a 7 kb upstream region and 5′-non-coding sequence (three exons and two introns) of the Bmp4 gene showed pancreatic expression of bioluminescence, while the Tg mice bearing luciferase with the 7 kb upstream region alone did not show pancreatic expression of the reporter gene. Interestingly, pancreatic expression of bioluminescence was also present in Tg mice harboring the truncated promoter without exon IA and IB, indicating the presence of a cryptic promoter in front of exon II. Furthermore, the bioluminescence signal was not detected in embryonic pancreas, but increasing signals were observed in neonatal and infantile Tg mice depending on the genotypes observed. These results suggested that a novel mechanism of transcription is involved in pancreatic expression of the Bmp4 gene.

Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter

Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond −461 nucleotides were not detected in the wild type suggesting that this 461bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed.

A Promoter in the Coding Region of the Calcium Channel Gene CACNA1C Generates the Transcription Factor CCAT

The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3′ end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.

Enhanced transgene expression in rice following selection controlled by weak promoters

BackgroundTechniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent.ResultsIn this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos).ConclusionsThe use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.

Core promoter elements are not essential for transcription in mammals

Core promoter elements are thought to be the docking site for transcription factors binding, and thereby essential for transcription initiation. The role of core promoter elements has been studied for decades in reporter systems. the central model claim that each cis sequence has an epigenetic role in navigating transcription initiation. We use here the MHC class I gene as model to study transcription in a transgenic mouse. MHC class I genes are ubiquitously expressed and subject to both tissue-specific and hormonal regulation. The core promoter contains four conventional elements: CCAAT, a TATAA-like element, an Sp1 binding site and a canonical Inr. The in vivo function of these elements was determined by mutating each individually within the context of the native gene. Surprisingly, none of the elements was essential for transcription since no single mutation eliminated transcription. Indeed, mutation of any one of the elements resulted in increased promoter activity, indicating that these elements function as transcriptional modulators. Further, each of the elements was found to have a distinct function, contributing uniquely to tissue-specificity, hormonal responses or both. The core promoter elements do not affect start site selection, demonstrating that they do not invoke a cryptic promoter. However, they do modulate relative start site usage. The patterns of chromatin modification reflect the expression status of the different promoters. In tissues where the different promoters constructs support active transcription, histone H3K4 trimethylation is high and H3K9 trimethylation is low. Conversely, H3K9 trimethylation is high and H3K4 trimethylation low across the gene in tissues where the promoter constructs are less active. The wild type promoter is activated by interferon, while the Inr and Sp1 mutants repress transcription in response to interferon treatment. Finally, the CAAT element was found to have dual function, both as a transcriptional regulator and as a barrier element. The barrier function correlated with the binding of C/EBP and CTCF to the wild type CAAT element and variegated expression across generations when the CAAT element was mutated. Remarkably, these results demonstrate that none of the elements homologous to canonical core promoter elements are necessary for promoter activity. However, they do contribute to the fine tuning of the tissue specific patterns of expression, extracellular signaling, overall promoter activity and chromatin modifications.

Co-transcriptional histone methylations

Genes transcribed by RNA polymerase II show a stereotypical pattern of histone H3 methylations. H3K4 trimethylation peaks near the transcription start site, with dimethylation peaking further downstream. H3K36 methylation levels increase with distance from the promoter. It is not yet completely understood how this pattern is generated, but the mechanism involves direct interactions between the transcribing polymerase and the methyltransferases Set1/COMPASS (for H3K4) and Set2 (for H3K36). We are working towards a better understanding of how this coupling works. We are also very interested in the function of these co-transcriptional histone modifications. Previous work has shown that methylated H3K36 recruits the histone deacetylase complex Rpd3S and that H3K4me2 recruits the Set3C histone deacetylase complex. Therefore, a major function of co-transcriptional histone methylation is to reset transcribed chromatin to a state less conducive to nucleosome turnover. This pathway represses cryptic promoters inside the transcribed gene, but also slows transcription elongation through this region. We and others have found that these histone methylation-deacetylation pathways can be targeted via overlapping non-coding transcription to regulate nearby promoters. In contrast to H3K4me2 and H3K36me2/3, H3K4me3 correlates with high turnover nucleosomes near transcription start sites. Available data suggests that H3K4me3 is recognized by three complexes containing ING/Yng proteins via their PHD finger domains. The yeast proteins Yng1 and Yng2 (as well as their metazoan counterparts) are subunits of histone acetyltransferase complexes, suggesting that H3K4me3 can target histone acetylation. Since acetylation is correlated with nucleosome remodeling, this putative pathway could be important for gene activation. Surprisingly, the third Yng protein (Pho23, as well as its metazoan counterparts) is a subunit of the Rpd3L histone deacetylase complex, hinting at a more complex series or cycle of acetylation and deacetylation. We are continuing to probe the functions of these pathways and some of our latest work will be presented.

RNAP‐II transcribes two small RNAs at the promoter and terminator regions of the RNAP‐I gene in Saccharomyces cerevisiae

Three RNA polymerases coexist in the ribosomal DNA of Saccharomyces cerevisiae. RNAP-I transcribes the 35S rRNA, RNAP-III transcribes the 5S rRNA and RNAP-II is found in both intergenic non-coding regions. Previously, we demonstrated that RNAP-II molecules bound to the intergenic non-coding regions (IGS) of the ribosomal locus are mainly found in a stalled conformation, and the stalled polymerase mediates chromatin interactions, which isolate RNAP-I from the RNAP-III transcriptional domain. Besides, RNAP-II transcribes both IGS regions at low levels, using different cryptic promoters. This report demonstrates that RNAP-II also transcribes two sequences located in the 5'- and 3'-ends of the 35S rRNA gene that overlap with the sequences of the 35S rRNA precursor transcribed by RNAP-I. The sequence located at the promoter region of RNAP-I, called the p-RNA transcript, binds to the transcription termination-related protein, Reb1p, while the T-RNA sequence, located in the termination sites of RNAP-I gene, contains the stem-loop recognized by Rtn1p, which is necessary for proper termination of RNAP-I. Because of their location, these small RNAs may play a key role in the initiation and termination of RNAP-I transcription. To correctly synthesize proteins, eukaryotic cells may retain a mechanism that connects the three main polymerases. This report suggests that cryptic transcription by RNAP-II may be required for normal transcription by RNAP-I in the ribosomal locus of S. cerevisiae.

Illegitimate V(D)J Recombination Involving Notch1 and Bcl11b in Precursor T-Cell Lymphoblastic Leukemia/Lymphoma

Abstract 1323V(D)J recombination of antigen receptor loci confers diversity to the mammalian immune system. However, off-target, illegitimate V(D)J recombination between non-antigen receptor loci, leading to site-specific interstitial deletion has been reported in humans and mice with lymphoid malignancies. These recurrent, site-specific interstitial deletions are thought to be oncogenic, leading to activation of proto-oncogenes or deletion of tumor suppressor genes. Notch1 and Bcl11b are known targets for illegitimate V(D)J recombination in murine precursor T-cell leukemia/lymphoma (pre-T-LBL). The Notch1 5'-deletions remove exon1 and 2; as a consequence Notch1 transcription initiates from a cryptic promoter immediately 5' of exon 25. These alternative transcripts are translated from exon 28, and lack the N-term ligand binding domain, resulting in a Notch1 protein that is independent of ligand binding. The known Bcl11b deletions excise exon2 and 3, and result in loss of function of the Bcl11b tumor suppressor gene. The frequency with which Notch1 and Bcl11b deletions occur in concert is unknown. Here we examined both Notch1 and Bcl11b deletion mutations, as well as Notch1 mutations involving the PEST, HD, and TM domains in primary murine pre-T LBL samples (N=44), and murine pre-T LBL cell lines (N=21), that arose in specific genetically engineered mouse strains (Scl-Lmo1, NUP98-PHF23, HoxA9, Lin28B, Survivin-TCR). Analysis of 44 primary tumor samples identified 17 Notch1 5'-interstitial deletions (38.6%) and 8 Bcl11b deletions (18.1%), while analysis of 21 cell lines revealed 10 Notch1 5' deletions (47.6%) and 5 Bcl11b deletions (24%). The percent of Notch1 PEST, HD, and TM mutations were 84.1%, 13.6%, and 4.5%, respectively, in the primary tumors and 95%, 5%, and 0% in the cell lines.Two primary tumor samples showed several different 5' deletion mutations, representing multiple independent leukemic clones. Of the 13 primary tumor/cell line pairs we analyzed, 5 pairs showed identical Notch1 and Bcl11b mutations. However, for 7 pairs, the primary tumor was oligoclonal with respect to Notch1 and Bcl11b mutations, with one clone being biologically selected for growth in vitro. In 3 cases, Bcl11b deletion was not detected in primary tumor but was detected in the cell line, suggesting that the Bcl11b deletion was a secondary event in these cell lines. Notch1 PEST mutations were present in almost all cell lines, and were often accompanied by an additional Notch1 mutation (5'deletion, HD, or TM). Conversely, 5' deletion, HD, and TM mutations were mutually exclusive. These findings support the hypothesis that the 5'deletion, HD, and TM mutations lead to ligand independent activation, while the PEST domain mutations prevent proteasomal degradation leading to persistence of the transcriptionally active intracellular Notch1. In several cases, samples had an identical PEST mutation, but distinct 5' deletion or HD mutations, indicating the PEST mutation preceded the 5' deletion or HD mutation. Three of 21 cell lines had both a 5' deletion and Bcl11b deletion, however there was no statistically significant correlation between these events.One 5'-recombination recognition sequence (RSS) and 2 3'-RSSs have been reported for Notch1 5' deletions. H3K4me3 ChIP-sequence data clearly demonstrated alternative transcript initiation from exon 25 in cell lines with a 5' Notch1 deletion. Curiously, one cell line (106A) which was negative for the 5' deletion showed a marked increase of H3K4Me3 at Notch1 exon25, suggesting transcription initiation at this alternative site, and leading to the hypothesis that this cell line may have a variant form of Notch1 5' deletion which was undetectable by conventional assays. We were able to identify an alternative RSS for this cell line, leading to a novel 5' Notch1 deletion. We then reanalyzed our samples and found an additional example of this alternate 5' deletion, and a third sample which displayed yet another form of Notch1 5' deletion. Of note, this deletion did not have a cryptic RSS, and had developed in a RAG1 deficient mouse. In summary, we have identified three novel forms of Notch1 5' deletions, shown that Notch1 mutations are very common in murine pre-T LBL, shown that PEST mutations generally precede 5'deletion mutations, and shown that 5' Notch1 deletions are not consistently associated with other illegitimate VDJ recombinase-mediated events (such as Bcl11b deletions). Disclosures:No relevant conflicts of interest to declare.

Extensive molecular mapping of TCRα/δ- and TCRβ-involved chromosomal translocations reveals distinct mechanisms of oncogene activation in T-ALL

AbstractChromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) and are generally believed to result from illegitimate V(D)J recombination events. However, molecular characterization and evaluation of the extent of recombinase involvement at the TCR-oncogene junction has not been fully evaluated. In the present study, screening for TCRβ and TCRα/δ translocations by FISH and ligation-mediated PCR in 280 T-ALLs allowed the identification of 4 previously unreported TCR-translocated oncogene partners: GNAG, LEF1, NKX2-4, and IL2RB. Molecular mapping of genomic junctions from TCR translocations showed that the majority of oncogenic partner breakpoints are not recombinase mediated and that the regulatory elements predominantly used to drive oncogene expression differ markedly in TCRβ (which are exclusively enhancer driven) and TCRα/δ (which use an enhancer-independent cryptic internal promoter) translocations. Our data also imply that oncogene activation takes place at a very immature stage of thymic development, when Dδ2-Dδ3/Dδ3-Jδ1 and Dβ-Jβ rearrangements occur, whereas the bulk leukemic maturation arrest occurs at a much later (cortical) stage. These observations have implications for T-ALL therapy, because the preleukemic early thymic clonogenic population needs to be eradicated and its disappearance monitored.

Chd1 chromatin remodelers maintain nucleosome organization and repress cryptic transcription

Chromatin organization is essential for defining transcription units and maintaining genomic integrity in eukaryotes. In this study, we found that deletion of the Schizosaccharomyces pombe Chd1 chromatin remodelers, hrp1 and hrp3, causes strong, genome-wide accumulation of antisense transcripts. Nucleosome mapping revealed a specific role for Chd1 remodelers in the positioning of nucleosomes in gene coding regions. Other mutations associated with enhanced cryptic transcription activity, such as set2Δ, alp13Δ and FACT complex subunit pob3Δ, did not, or only mildly, affect nucleosome positioning. These data indicate several mechanisms in the repression of cryptic promoter activity in eukaryotic cells.

Set3 HDAC Mediates Effects of Overlapping Noncoding Transcription on Gene Induction Kinetics

The Set3 histone deacetylase complex (Set3C) binds histone H3 dimethylated at lysine 4 (H3K4me2) to mediate deacetylation of histones in 5'-transcribed regions. To discern how Set3C affects gene expression, genome-wide transcription was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3 primarily caused changes during transition periods, as genes were induced or repressed. Surprisingly, a majority of Set3-affected genes are overlapped by noncoding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2 instead of me3 over promoter regions, due to either reduced H3K4me3 or ncRNA transcription from distal or antisense promoters. Set3C also represses internal cryptic promoters, but in different regions of genes than the Set2/Rpd3S pathway. Finally, Set3C stimulates some genes by repressing an overlapping antagonistic antisense transcript. These results show that overlapping noncoding transcription can fine-tune gene expression, not via the ncRNA but by depositing H3K4me2 to recruit the Set3C deacetylase.

Set2 methylation of histone H3 lysine 36 suppresses histone exchange on transcribed genes

Set2-mediated methylation of histone H3 at Lys 36 (H3K36me) is a co-transcriptional event that is necessary for the activation of the Rpd3S histone deacetylase complex, thereby maintaining the coding region of genes in a hypoacetylated state. In the absence of Set2, H3K36 or Rpd3S acetylated histones accumulate on open reading frames (ORFs), leading to transcription initiation from cryptic promoters within ORFs. Although the co-transcriptional deacetylation pathway is well characterized, the factors responsible for acetylation are as yet unknown. Here we show that, in yeast, co-transcriptional acetylation is achieved in part by histone exchange over ORFs. In addition to its function of targeting and activating the Rpd3S complex, H3K36 methylation suppresses the interaction of H3 with histone chaperones, histone exchange over coding regions and the incorporation of new acetylated histones. Thus, Set2 functions both to suppress the incorporation of acetylated histones and to signal for the deacetylation of these histones in transcribed genes. By suppressing spurious cryptic transcripts from initiating within ORFs, this pathway is essential to maintain the accuracy of transcription by RNA polymerase II.

Multivalent di-nucleosome recognition enables the Rpd3S histone deacetylase complex to tolerate decreased H3K36 methylation levels

The Rpd3S histone deacetylase complex represses cryptic transcription initiation within coding regions by maintaining the hypo-acetylated state of transcribed chromatin. Rpd3S recognizes methylation of histone H3 at lysine 36 (H3K36me), which is required for its deacetylation activity. Rpd3S is able to function over a wide range of H3K36me levels, making this a unique system to examine how chromatin regulators tolerate the reduction of their recognition signal. Here, we demonstrated that Rpd3S makes histone modification-independent contacts with nucleosomes, and that Rpd3S prefers di-nucleosome templates since two binding surfaces can be readily accessed simultaneously. Importantly, this multivalent mode of interaction across two linked nucleosomes allows Rpd3S to tolerate a two-fold intramolecular reduction of H3K36me. Our data suggest that chromatin regulators utilize an intrinsic di-nucleosome-recognition mechanism to prevent compromised function when their primary recognition modifications are diluted.

Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function

A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3′ splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3′ ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3′ ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence.

Model for MLL translocations in therapy-related leukemia involving topoisomerase IIβ-mediated DNA strand breaks and gene proximity

Topoisomerase poisons such as the epipodophyllotoxin etoposide are widely used effective cytotoxic anticancer agents. However, they are associated with the development of therapy-related acute myeloid leukemias (t-AMLs), which display characteristic balanced chromosome translocations, most often involving the mixed lineage leukemia (MLL) locus at 11q23. MLL translocation breakpoints in t-AMLs cluster in a DNase I hypersensitive region, which possesses cryptic promoter activity, implicating transcription as well as topoisomerase II activity in the translocation mechanism. We find that 2-3% of MLL alleles undergoing transcription do so in close proximity to one of its recurrent translocation partner genes, AF9 or AF4, consistent with their sharing transcription factories. We show that most etoposide-induced chromosome breaks in the MLL locus and the overall genotoxicity of etoposide are dependent on topoisomerase IIβ, but that topoisomerase IIα and -β occupancy and etoposide-induced DNA cleavage data suggest factors other than local topoisomerase II concentration determine specific clustering of MLL translocation breakpoints in t-AML. We propose a model where DNA double-strand breaks (DSBs) introduced by topoisomerase IIβ into pairs of genes undergoing transcription within a common transcription factory become stabilized by antitopoisomerase II drugs such as etoposide, providing the opportunity for illegitimate end joining and translocation.

Conditional Mutagenesis of a Novel Choline Kinase Demonstrates Plasticity of Phosphatidylcholine Biogenesis and Gene Expression in Toxoplasma gondii

The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length (∼70-kDa) TgCK displayed a low affinity for choline (K(m) ∼0.77 mM) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by ∼60%, which was abolished in the off state of the mutant (Δtgck(i)). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments.