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The sfa determinant codes for S fimbrial adhesins which constitute adherence factors of pathogenic Escherichia coli strains. Wehave recently shown that the sfa determinant is transcribed from three prömoters, pA, pB, and pC. In comparison with the promoters pB and pC, promoter pA, which is located in front of the structural gene sfaA, showed very weak activity. Herewe have determined the exact positions ofthe mRNA start points by primer extension studies. We have also shown that mRNAs of 500, 700 and 1400 bases can be detected using oligonucleotide probes specific for the genes sfaB, sfaC and sfaA. SfaB and SfaC arepositive regulators infiuencing fimbriation and the production of the S-specific adhesin which is encoded by the gene sfaS Iocated in the distal half of the determinant. In addition, it is demonstrated that SfaB and SfaC interfere with the regulatory effect of the histone-like protein H-NS, encoded by a locus termed drdX or osmZ. In a drdx+ strain the regulators are necessary for transcription of the sfa determinant. In contrast, sfa expression is activator-independent in a drdx- strain. In this latter genetic background, a substantial fraction of the sfa transcripts is initiated from promoter pA. On the basis of these data we discuss a model for the regulation of this adhesin-specific determinant.
Background:
The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila melanogaster is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA.
Results:
We have investigated whether the function of Brm in pre-mRNA processing in Drosophila melanogaster is mediated by Brm alone or by the SWI/SNF complex. We have analyzed the effects of depleting individual SWI/SNF subunits on pre-mRNA processing throughout the genome, and we have identified a subset of transcripts that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. We have also depleted Brm in larvae and we have shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo.
Conclusions:
We have shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. Our results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes.
TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms
(2011)
The huge majority of myeloma cell lines express TNFR2 while a substantial subset of them failed to show TNFR1 expression. Stimulation of TNFR1 in the TNFR1-expressing subset of MM cell lines had no or only a very mild effect on cellular viability. Surprisingly, however, TNF stimulation enhanced cell death induction by CD95L and attenuated the apoptotic effect of TRAIL. The contrasting regulation of TRAIL- and CD95L-induced cell death by TNF could be traced back to the concomitant NFjBmediated upregulation of CD95 and the antiapoptotic FLIP protein. It appeared that CD95 induction, due to its strength, overcompensated a rather moderate upregulation of FLIP so that the net effect of TNF-induced NFjB activation in the context of CD95 signaling is pro-apoptotic. TRAIL-induced cell death, however, was antagonized in response to TNF because in this context only the induction of FLIP is relevant. Stimulation of TNFR2 in myeloma cells leads to TRAF2 depletion. In line with this, we observed cell death induction in TNFR1-TNFR2-costimulated JJN3 cells. Our studies revealed that the TNF-TNF receptor system adjusts the responsiveness of the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms that generate a highly context-dependent net effect on myeloma cell survival
The correct regulation of cell growth and proliferation is essential during normal animal development. Myc proteins function as transcription factors, being involved in the con-trol of many growth- and proliferation-associated genes and deregulation of Myc is one of the main driving factors of human malignancies.
The first part of this thesis focuses on the identification of directly regulated Myc target genes in Drosophila melanogaster, by combining ChIPseq and RNAseq approaches. The analysis results in a core set of Myc target genes of less than 300 genes which are mainly involved in ribosome biogenesis. Among these genes we identify a novel class of Myc targets, the non-coding small nucleolar RNAs (snoRNAs). In vivo studies show that loss of snoRNAs not only impairs growth during normal development, but that overexpression of several snoRNAs can also enhance tumor development in a neu-ronal tumor model. Together the data show that Myc acts as a master regulator of ribo-some biogenesis and that Myc’s transforming effects in tumor development are at least partially mediated by the snoRNAs.
In the second part of the thesis, the interaction of Myc and the Zf-protein Chinmo is described. Co-immunoprecipitations of the two proteins performed under endogenous and exogenous conditions show that they interact physically and that neither the two Zf-domains nor the BTB/POZ-domain of Chinmo are important for this interaction. Fur-thermore ChIP experiments and Myc dependent luciferase assays show that Chinmo and Myc share common target genes, and that Chinmo is presumably also involved in their regulation. While the exact way of how Myc and Chinmo genetically interact with each other still has to be investigated, we show that their interaction is important in a tumor model. Overexpression of the tumor-suppressors Ras and Chinmo leads to tu-mor formation in Drosophila larvae, which is drastically impaired upon loss of Myc.
MYC is a transcription factor, whose expression is elevated or deregulated in many human cancers (up to 70%) and is often associated with aggressive and poorly differentiated tumors. Although MYC is extensively studied, discrepancies have emerged about how this transcription factor works. In primary lymphocytes, MYC promotes transcriptional amplification of virtually all genes with an open promoter, whereas in tumor cells MYC regulates specific sets of genes that have significant prognostic value. Furthermore, the set of target genes that distinguish MYC’s physiological function from the pathological/oncogenic one, whether it exists or not, has not been fully understood yet.
In this study, it could be shown that MYC protein levels within a cell and promoter affinity (determined by E-box presence or interaction with other proteins) of target genes toward MYC are important factors that influence MYC activity. At low levels, MYC can amplify a certain transcriptional program, which includes high affinity binding sites, whereas at high levels MYC leads to the specific up- and down regulation of genes with low affinity. Moreover, the promoter affinity characterizes different sets of target genes which can be distinguished in the physiological or oncogenic MYC signatures.
MYC-mediated repression requires higher MYC levels than activation and formation of a complex with MIZ1 is necessary for inhibiting expression of a subset of MYC target genes.
Normal human brain development is dependent on highly dynamic epigenetic processes for spatial and temporal gene regulation. Recent work identified wide-spread changes in DNA methylation during fetal brain development. We profiled CpG methylation in frontal cortex of 27 fetuses from gestational weeks 12-42, using Illumina 450K methylation arrays. Sites showing genome-wide significant correlation with gestational age were compared to a publicly available data set from gestational weeks 3-26. Altogether, we identified 2016 matching developmentally regulated differentially methylated positions (m-dDMPs): 1767 m-dDMPs were hypermethylated and 1149 hypomethylated during fetal development. M-dDMPs are underrepresented in CpG islands and gene promoters, and enriched in gene bodies. They appear to cluster in certain chromosome regions. M-dDMPs are significantly enriched in autism-associated genes and CpGs. Our results promote the idea that reduced methylation dynamics during fetal brain development may predispose to autism. In addition, m-dDMPs are enriched in genes with human-specific brain expression patterns and/or histone modifications. Collectively, we defined a subset of dDMPs exhibiting constant methylation changes from early to late pregnancy. The same epigenetic mechanisms involving methylation changes in cis-regulatory regions may have been adopted for human brain evolution and ontogeny.
Background
Herpesviruses can infect a wide range of animal species. Herpes simplex virus 1 (HSV-1) is one of the eight herpesviruses that can infect humans and is prevalent worldwide. Herpesviruses have evolved multiple ways to adapt the infected cells to their needs, but knowledge about these transcriptional and post-transcriptional modifications is sparse.
Results
Here, we show that HSV-1 induces the expression of about 1000 antisense transcripts from the human host cell genome. A subset of these is also activated by the closely related varicella zoster virus. Antisense transcripts originate either at gene promoters or within the gene body, and they show different susceptibility to the inhibition of early and immediate early viral gene expression. Overexpression of the major viral transcription factor ICP4 is sufficient to turn on a subset of antisense transcripts. Histone marks around transcription start sites of HSV-1-induced and constitutively transcribed antisense transcripts are highly similar, indicating that the genetic loci are already poised to transcribe these novel RNAs. Furthermore, an antisense transcript overlapping with the BBC3 gene (also known as PUMA) transcriptionally silences this potent inducer of apoptosis in cis.
Conclusions
We show for the first time that a virus induces widespread antisense transcription of the host cell genome. We provide evidence that HSV-1 uses this to downregulate a strong inducer of apoptosis. Our findings open new perspectives on global and specific alterations of host cell transcription by viruses.
Allopolyploid plants are long known to be subject to a homoeolog expression bias of varying degree. The same phenomenon was only much later suspected to occur also in animals based on studies of single selected genes in an allopolyploid vertebrate, the Iberian fish Squalius alburnoides. Consequently, this species became a good model for understanding the evolution of gene expression regulation in polyploid vertebrates. Here, we analyzed for the first time genome-wide allele-specific expression data from diploid and triploid hybrids of S. alburnoides and compared homoeolog expression profiles of adult livers and of juveniles. Co-expression of alleles from both parental genomic types was observed for the majority of genes, but with marked homoeolog expression bias, suggesting homoeolog specific reshaping of expression level patterns in hybrids. Complete silencing of one allele was also observed irrespective of ploidy level, but not transcriptome wide as previously speculated. Instead, it was found only in a restricted number of genes, particularly ones with functions related to mitochondria and ribosomes. This leads us to hypothesize that allelic silencing may be a way to overcome intergenomic gene expression interaction conflicts, and that homoeolog expression bias may be an important mechanism in the achievement of sustainable genomic interactions, mandatory to the success of allopolyploid systems, as in S. alburnoides.
Expression of the MYC oncoprotein, which binds the DNA at promoters of most transcribed genes, is controlled by growth factors in non-tumor cells, thus stimulating cell growth and proliferation.
Here in this thesis, it is shown that MYC interacts with SPT5, a subunit of the RNA polymerase II (Pol II) elongation factor DSIF. MYC recruits SPT5 to promoters of genes and is required for its association with Pol II. The transfer of SPT5 is mediated by CDK7 activity on TFIIE, which evicts it from Pol II and allows SPT5 to bind Pol II.
MYC is required for fast and processive transcription elongation, consistent with known functions of SPT5 in yeast. In addition, MYC increases the directionality of promoters by stimulating sense transcription and by suppressing the synthesis of antisense transcripts.
The results presented in this thesis suggest that MYC globally controls the productive assembly of Pol II with general elongation factors to form processive elongation complexes in response to growth-factor stimulation of non-tumour cells. However, MYC is found to be overexpressed in many tumours, and is required for their development and progression.
In this thesis it was found that, unexpectedly, such overexpression of MYC does not further enhance transcription but rather brings about squelching of SPT5. This reduces the processivity of Pol II on selected set of genes that are known to be repressed by MYC, leading to a decrease in growth-suppressive gene transcription and uncontrolled tumour growth
In eukaryotes, the enormously long DNA molecules need to be packaged together with histone proteins into nucleosomes and further into compact chromatin structures to fit it into the nucleus. This nuclear organisation interferes with all phases of transcription that require the polymerase to bind to DNA. During transcription – the process in which the hereditary information stored in DNA is transferred to many transportable RNA molecules - nucleosomes form a physical obstacle for polymerase progression. Thus, transcription is usually accompanied by processes mediating nucleosome destabilisation, including post-translational histone modifications (PTMs) or exchange of canonical histones by their variant forms. To the best of our knowledge, acetylation of histones has the highest capability to induce chromatin opening. The lysine modification can destabilise histone-DNA interactions within a nucleosome and can serve as a binding site for various chromatin remodelers that can modify the nucleosome composition. For example, H4 acetylation can impede chromatin folding and can stimulate the exchange of canonical H2A histone by its variant form H2A.Z at transcription start sites (TSSs) in many eukaryotes, including humans. As histone H4, H2A.Z can be post-translationally acetylated and as acetylated H4, acetylated H2A.Z is enriched at TSSs suggested to be critical for transcription. However, thus far, it has been difficult to study the cause and consequence of H2A.Z acetylation.
Even though, genome-wide chromatin profiling studies such as ChIP-seq have already revealed the genomic localisation of many histone PTMs and variant proteins, they can only be used to study individual chromatin marks and not to identify all factors important for establishing a distinct chromatin structure. This would require a comprehensive understanding of all marks associated to a specific genomic locus. However, thus far, such analyses of locus-specific chromatin have only been successful for repetitive regions, such as telomeres.
In my doctoral thesis, I used the unicellular parasite Trypanosoma brucei as a model system for chromatin biology and took advantage of its chromatin landscape with TSSs comprising already 7% of the total T. brucei genome (humans: 0.00000156%). Atypical for a eukaryote, the protein-coding genes are arranged in long polycistronic transcription units (PTUs). Each PTU is controlled by its own ~10 kb-wide TSS, that lies upstream of the PTU. As observed in other eukaryotes, TSSs are enriched with nucleosomes containing acetylated histones and the histone variant H2A.Z. This is why I used T. brucei to particularly investigate the TSS-specific chromatin structures and to identify factors involved in H2A.Z deposition and transcription regulation in eukaryotes. To this end, I established an approach for locus-specific chromatin isolation that would allow me to identify the TSSs- and non-TSS-specific chromatin marks. Later, combining the approach with a method for quantifying lysine-specific histone acetylation levels, I found H2A.Z and H4 acetylation enriched in TSSs-nucleosomes and mediated by the histone acetyltransferases HAT1 and HAT2. Depletion of HAT2 reduced the levels of TSS-specific H4 acetylation, affected targeted H2A.Z deposition and shifted the sites of transcription initiation. Whereas HAT1 depletion had only a minor effect on H2A.Z deposition, it had a strong effect on H2A.Z acetylation and transcription levels. My findings demonstrate a clear link between histone acetylation, H2A.Z deposition and transcription initiation in the early diverged unicellular parasite T. brucei, which was thus far not possible to determine in other eukaryotes. Overall, my study highlights the usefulness of T. brucei as a model system for studying chromatin biology. My findings allow the conclusion that H2A.Z regardless of its modification state defines sites of transcription initiation, whereas H2A.Z acetylation is essential co-factor for transcription initiation. Altogether, my data suggest that TSS-specific chromatin establishment is one of the earliest developed mechanisms to control transcription initiation in eukaryotes.