Eukaryotic Gene Transcription Research Articles

Functional Operons in Secondary Metabolic Gene Clusters in Glarea lozoyensis (Fungi, Ascomycota, Leotiomycetes).

ABSTRACTOperons are multigene transcriptional units which occur mostly in prokaryotes but rarely in eukaryotes. Protein-coding operons have not been reported in the Fungi even though they represent a very diverse kingdom of organisms. Here, we report a functional operon involved in the secondary metabolism of the fungus Glarea lozoyensis belonging to Leotiomycetes (Ascomycota). Two contiguous genes, glpks3 and glnrps7, encoding polyketide synthase and nonribosomal peptide synthetase, respectively, are cotranscribed into one dicistronic mRNA under the control of the same promoter, and the mRNA is then translated into two individual proteins, GLPKS3 and GLNRPS7. Heterologous expression in Aspergillus nidulans shows that the GLPKS3-GLNRPS7 enzyme complex catalyzes the biosynthesis of a novel pyrrolidinedione-containing compound, xenolozoyenone (compound 1), which indicates the operon is functional. Although it is structurally similar to prokaryotic operons, the glpks3-glnrps7 operon locus has a monophylogenic origin from fungi rather than having been horizontally transferred from prokaryotes. Moreover, two additional operons, glpks28-glnrps8 and glpks29-glnrps9, were verified at the transcriptional level in the same fungus. This is the first report of protein-coding operons in a member of the Fungi.

Unraveling the association between mRNA expressions and mutant phenotypes in a genome-wide assessment of mice

High-throughput gene expression profiling has revealed substantial leaky and extraneous transcription of eukaryotic genes, challenging the perceptions that transcription is strictly regulated and that changes in transcription have phenotypic consequences. To assess the functional implications of mRNA transcription directly, we analyzed mRNA expression data derived from microarrays, RNA-sequencing, and in situ hybridization, together with phenotype data of mouse mutants as a proxy of gene function at the tissue level. The results indicated that despite the presence of widespread ectopic transcription, mRNA expression and mutant phenotypes of mammalian genes or tissues remain associated. The expression-phenotype association at the gene level was particularly strong for tissue-specific genes, and the association could be underestimated due to data insufficiency and incomprehensive phenotyping of mouse mutants; the strength of expression-phenotype association at the tissue level depended on tissue functions. Mutations on genes expressed at higher levels or expressed at earlier embryonic stages more often result in abnormal phenotypes in the tissues where they are expressed. The mRNA expression profiles that have stronger associations with their phenotype profiles tend to be more evolutionarily conserved, indicating that the evolution of transcriptome and the evolution of phenome are coupled. Therefore, mutations resulting in phenotypic aberrations in expressed tissues are more likely to occur in highly transcribed genes, tissue-specific genes, genes expressed during early embryonic stages, or genes with evolutionarily conserved mRNA expression profiles.

Abundance and distribution of RNA polymerase II in Arabidopsis interphase nuclei.

RNA polymerase II (RNAPII) is responsible for the transcription of most eukaryotic protein-coding genes. Analysing the topological distribution and quantification of RNAPII can contribute to understanding its function in interphase nuclei. Previously it was shown that RNAPII molecules in plant nuclei form reticulate structures within euchromatin of differentiated Arabidopsis thaliana nuclei rather than being organized in distinct 'transcription factories' as observed in mammalian nuclei. Immunosignal intensity measurements based on specific antibody labelling in maximum intensity projections of image stacks acquired by structured illumination microscopy (SIM) suggested a relative proportional increase of RNAPII in endopolyploid plant nuclei. Here, photoactivated localization microscopy (PALM) was applied to determine the absolute number and distribution of active and inactive RNAPII molecules in differentiated A. thaliana nuclei. The proportional increase of RNAPII during endopolyploidization is confirmed, but it is also shown that PALM measurements are more reliable than those based on SIM in terms of quantification. The single molecule localization results show that, although RNAPII molecules are globally dispersed within plant euchromatin, they also aggregate within smaller distances as described for mammalian transcription factories.

Structural basis of transcription initiation by RNA polymerase II.

Transcription of eukaryotic protein-coding genes commences with the assembly of a conserved initiation complex, which consists of RNA polymerase II (Pol II) and the general transcription factors, at promoter DNA. After two decades of research, the structural basis of transcription initiation is emerging. Crystal structures of many components of the initiation complex have been resolved, and structural information on Pol II complexes with general transcription factors has recently been obtained. Although mechanistic details await elucidation, available data outline how Pol II cooperates with the general transcription factors to bind to and open promoter DNA, and how Pol II directs RNA synthesis and escapes from the promoter.

3D imaging of Sox2 enhancer clusters in embryonic stem cells.

Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.

Clinical implications of MTA proteins in human cancer.

Metastasis-associated gene or metastasis tumor antigen 1 (MTA1) is a new member of cancer progression-related gene family. It was first identified in rat mammary adenocarcinoma and later recognized as an important constituent of nucleosomal remodeling complex (NuRD), displaying dual regulatory functions as a co-repressor and co-activator for a large number of genes. Chromatin remodelers are ATP-dependent multi-protein chromatin modifying machines. These complexes alter the nucleosome positioning regulating the accessibility of genomic DNA to various transcription factors and thus modulate eukaryotic gene transcription. Since its identification two decades ago, MTA1 has been reported to be overexpressed in many cancers. Moreover, its overexpression has also been correlated with transformation and tumor progression. Furthermore, MTA1 has been shown to modulate the response of several tumor suppressor genes like p53 and oncogenes like c-myc. Taken together, current literature suggests that MTA proteins, especially MTA1, act as a master co-regulatory molecule involved in the carcinogenesis and progression of various malignant tumors. The primary focus of this review is to provide an overview of the MTA proteins with special emphasis on its role in cancer and use as a marker for cancer progression and potential target for therapy.

Binding of the termination factor Nsi1 to its cognate DNA site is sufficient to terminate RNA polymerase I transcription in vitro and to induce termination in vivo.

Different models have been proposed explaining how eukaryotic gene transcription is terminated. Recently, Nsi1, a factor involved in silencing of ribosomal DNA (rDNA), was shown to be required for efficient termination of rDNA transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Nsi1 contains Myb-like DNA binding domains and associates in vivo near the 3' end of rRNA genes to rDNA, but information about which and how DNA sequences might influence Nsi1-dependent termination is lacking. Here, we show that binding of Nsi1 to a stretch of 11 nucleotides in the correct orientation was sufficient to pause elongating Pol I shortly upstream of the Nsi1 binding site and to release the transcripts in vitro. The same minimal DNA element triggered Nsi1-dependent termination of pre-rRNA synthesis using an in vivo reporter assay. Termination efficiency in the in vivo system could be enhanced by inclusion of specific DNA sequences downstream of the Nsi1 binding site. These data and the finding that Nsi1 blocks efficiently only Pol I-dependent RNA synthesis in an in vitro transcription system improve our understanding of a unique mechanism of transcription termination.

Abstract 4600: P-TEFb is a therapeutic target in human bortezomib-resistant multiple myeloma cells

Abstract Despite proteasome inhibitor (e.g., bortezomib/btz and carfilzomib/cfz) success in multiple myeloma (MM), drug resistance remains a key problem. MM cells commonly over-express Mcl-1, a protein implicated in survival and drug-resistance. Mcl-1 exhibits rapid protein turnover (half-life < 1h), suggesting that efficient transcriptional machinery for de novo synthesis may be required for its constitutive expression in MM. Eukaryotic protein-coding gene transcription is controlled by RNA polymerase II (RNAPII), which is regulated by negative (N-TEF) and positive elongation factors (P-TEF). P-TEFb, a holoenzyme Cdk9/cyclin T complex, activates RNAPII by phosphorylating the S2 site within the carboxy-terminal domain (CTD). Here, the role of P-TEFb in Mcl-1 maintenance was investigated in MM cells. Anti-apoptotic Bcl-2 family members profiling revealed high Mcl-1 protein levels in virtually all MM lines tested and up-regulation in drug-resistant lines (e.g., btz-resistant PS-R cells). Similar patterns occurred in human MM xenograft mouse models and primary CD138+ MM cells. Ectopic Mcl-1 over-expression strikingly diminished btz lethality, arguing that Mcl-1 is a critical determinant of btz-resistance. All tested MM cell lines, primary MM specimens, and murine xenograft samples displayed constitutive P-TEFb activation i.e., high CTD (S2) phosphorylation, which correlated with a) up-regulation of P-TEFb subunits i.e., Cdk9 (42 and 55kD isoforms) and its partner cyclin T1 (but not T2a/b or K); b) marked 42kD T186 phosphorylation, while drug-resistant cells also displayed sharply increased 55kD T186 phosphorylation; and c) constitutive P-TEFb complex assembly and RNAPII interactions, but little inhibitory association with HEXIM which binds to P-TEFb via 7SK snRNP. The function of P-TEFb in Mcl-1 regulation was then validated by genetic and pharmacological strategies. First, Cdk9 or cyclin T1 shRNA knock-down dramatically inhibited CTD S2 phosphorylation and down-regulated Mcl-1. Second, pan-Cdk inhibitors e.g., dinaciclib rapidly inhibited P-TEFb and down-regulated Mcl-1, leading to apoptosis prior to cell cycle perturbations. These events primarily reflected P-TEFb complex disassembly, but not levels of P-TEFb subunits or T186 phosphorylation. Third, like Mcl-1 knock-down, Cdk9 shRNA or a specific Cdk9 inhibitor significantly restored susceptibility of btz-resistant cells to btz or cfz, and also sensitized them to the BH3-mimetic ABT-737 which does not target Mcl-1. Finally, pan-Cdk inhibitors reduced tumor burden and eliminated human CD138+ cells in the bone marrow of murine models bearing drug-naïve or btz-resistant MM cells. Collectively, these findings implicate constitutive activation of P-TEFb in maintenance of high Mcl-1 levels in MM, and argue that P-TEFb may serve as a rational target to overcome btz-resistance. This study was supported by the MD Anderson SPORE in Myeloma. Citation Format: Yun Dai, Shuang Chen, Liang Zhou, Yu Zhang, Yun Leng, Xin-Yan Pei, Hui Lin, Robert Z. Orlowski, Steven Grant. P-TEFb is a therapeutic target in human bortezomib-resistant multiple myeloma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4600. doi:10.1158/1538-7445.AM2014-4600

Transcript elongation factors: shaping transcriptomes after transcript initiation.

Elongation is a dynamic and highly regulated step of eukaryotic gene transcription. A variety of transcript elongation factors (TEFs), including modulators of RNA polymerase II (RNAPII) activity, histone chaperones, and histone modifiers, have been characterized from plants. These factors control the efficiency of transcript elongation of subsets of genes in the chromatin context and thus contribute to tuning gene expression programs. We review here how genetic and biochemical analyses, primarily in Arabidopsis thaliana, have advanced our understanding of how TEFs adjust plant gene transcription. These studies have revealed that TEFs regulate plant growth and development by modulating diverse processes including hormone signaling, circadian clock, pathogen defense, responses to light, and developmental transitions.

RNA Polymerase II Forms Transcription Networks in Rye and Arabidopsis Nuclei and Its Amount Increases with Endopolyploidy

RNA polymerase II (RNAPII) is responsible for the transcription of most eukaryotic genes. In mammalian nuclei, RNAPII is mainly localized in relatively few distinct transcription factories. In this study - applying super-resolution microscopy - it is shown that in plants, inactive (non-phosphorylated) and active (phosphorylated) RNAPII modifications compose distinct ‘transcription networks' within the euchromatin. These reticulate structures sometimes attach to each other, but they are absent from heterochromatin and nucleoli. The global RNAPII distribution within nuclei is not influenced by interphase chromatin organization such as Rabl (rye) versus non-Rabl (Arabidopsis thaliana) orientation. Replication of sister chromatids without cell division causes endopolyploidy, a phenomenon widespread in plants and animals. Endopolyploidy raises the number of gene copies per nucleus. Here, it is shown that the amounts of active and inactive RNAPII enzymes in differentiated 2-32C leaf nuclei of A. thaliana proportionally increase with rising endopolyploidy. Thus, increasing the transcriptional activity of cells and tissues seems to be an important function of endopolyploidy.

Unveiling translocation intermediates of RNA polymerase.

Translocation of RNA polymerase (RNAP) is a robust target for regulation of gene expression in prokaryotes and eukaryotes (1⇓–3). During elongation, RNAP frequently encounters a broad range of DNA/RNA conformations (RNA hairpins, curved and cruciform DNA), DNA-binding proteins, DNA lesions, and misincorporation events at the 3′ ends of the RNA. These encounters impede forward translocation, leading to RNAP pausing (3). There are many protein factors that strengthen or weaken pausing by targeting translocation, such as archaeal/eukaryotic Spt5 and bacterial NusG/RfaH (4) or N/Nun proteins of lambdoid phages (3). In metazoa, RNAP II pausing in promoter-proximal regions plays a role in having polymerases in place for a rapid transcription response to environmental stimuli, such as heat-shock or cell differentiation, and maintaining a basal level of gene expression (5, 6). Translocation blocks also initiate RNAP backtracking (7). Backtracking events disengage the 3′ RNA end from the RNAP catalytic site, which stabilizes pausing; this type of event broadly controls gene transcription in eukaryotes (8). Forward translocation of RNAP along DNA has long been regarded as the movement of the RNA–DNA hybrid through the catalytic cleft, which vacates the active center, termed i+1 site, for an NTP binding. In this sense, the mechanism of translocation has been primarily focused on the hybrid movement, with only limited emphasis on the DNA sequences surrounding the hybrid (1, 2, 9). However, several reports indicate that the DNA sequences immediately upstream and downstream from the hybrid regulate translocation (7, 10⇓–12). In PNAS, Silva et al. (13) identify an important component of the translocation mechanism using millisecond molecular dynamics (MD) simulation of translocation of yeast RNAP II: Translocation of the hybrid occurs before entry of the template DNA base to the i+1 site, where it can pair …

Uncovering transcription factor binding pockets in the tail subdomain of the human mediator complex (943.2)

The Mediator Complex plays a major role in regulating gene transcription in eukaryotes. One of its fundamental roles is to integrate inputs from a variety of transcription factors to control the gene’s transcriptional state. The majority of these transcription factors are thought to bind to the Mediator’s Tail subdomain. In humans, the Tail is composed of eight subunit proteins, whose structure and arrangement remain unknown, largely due to difficulties in expressing and purifying them. Moreover, only a few interactions between transcription factors and some of the Tail subunits have been found.To overcome difficulties in protein expression and interaction analysis, we used the Leishmania cell‐free protein expression system and pair‐wise measurements of protein interactions via the AlphaScreen assay. These methods allowed us to build a model of the interactions between protein subunits in the Mediator Tail. As validation, we pulled down all the component proteins in the Tail subdomain from a single cell‐free co‐expression. The same techniques were used to test the binding of various transcription factors to the Tail subunits. Our results show that different families of transcription factors bind to distinct groups of subunits in the Tail. Combined with our model of the Tail, this data reveals binding pockets on the Tail’s surface.Grant Funding Source: Supported by Australian Research Council

Identification and characterization of the SET domain gene family in maize

Histone lysine methylation plays a pivotal role in a variety of developmental and physiological processes through modifying chromatin structure and thereby regulating eukaryotic gene transcription. The SET domain proteins represent putative candidates for lysine methyltransferases containing the evolutionarily-conserved SET domain, and important epigenetic regulators present in eukaryotes. In recent years, increasing evidence reveals that SET domain proteins are encoded by a large multigene family in plants and investigation of the SET domain gene family will serve to elucidate the epigenetic mechanism diversity in plants. Although the SET domain gene family has been thoroughly characterized in multiple plant species including two model plant systems, Arabidopsis and rice, through their sequenced genomes, analysis of the entire SET domain gene family in maize was not completed following maize (B73) genome sequencing project. Here, we performed a genome-wide structural and evolutionary analysis of maize SET domain genes from the latest version of the maize (B73) genome. A complete set of 43 SET domain genes (Zmset1-43) were identified in the maize genome using Blast search tools and categorized into seven classes (Class I-VII) based on phylogeny. Chromosomal location of these genes revealed that they are unevenly distributed on all ten chromosomes with seven segmental duplication events, suggesting that segmental duplication played a key role in expansion of the maize SET domain gene family. EST expression data mining revealed that these newly identified genes had temporal and spatial expression pattern and suggested that many maize SET domain genes play functional developmental roles in multiple tissues. Furthermore, the transcripts of the 18 genes (the Class V subfamily) were detected in the leaves by two different abiotic stress treatments using semi-quantitative RT-PCR. The data demonstrated that these genes exhibited different expression levels in stress treatments. Overall, our study will serve to better understand the complexity of the maize SET domain gene family and also be beneficial for future experimental research to further unravel the mechanisms of epigenetic regulation in plants.

Widespread evidence of cooperative DNA binding by transcription factors in Drosophila development

Regulation of eukaryotic gene transcription is often combinatorial in nature, with multiple transcription factors (TFs) regulating common target genes, often through direct or indirect mutual interactions. Many individual examples of cooperative binding by directly interacting TFs have been identified, but it remains unclear how pervasive this mechanism is during animal development. Cooperative TF binding should be manifest in genomic sequences as biased arrangements of TF-binding sites. Here, we explore the extent and diversity of such arrangements related to gene regulation during Drosophila embryogenesis. We used the DNA-binding specificities of 322 TFs along with chromatin accessibility information to identify enriched spacing and orientation patterns of TF-binding site pairs. We developed a new statistical approach for this task, specifically designed to accurately assess inter-site spacing biases while accounting for the phenomenon of homotypic site clustering commonly observed in developmental regulatory regions. We observed a large number of short-range distance preferences between TF-binding site pairs, including examples where the preference depends on the relative orientation of the binding sites. To test whether these binding site patterns reflect physical interactions between the corresponding TFs, we analyzed 27 TF pairs whose binding sites exhibited short distance preferences. In vitro protein–protein binding experiments revealed that >65% of these TF pairs can directly interact with each other. For five pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. This study demonstrates how DNA-binding motifs can be used to produce a comprehensive map of sequence signatures for different mechanisms of combinatorial TF action.

MED12 mutations in human diseases

The Mediator Complex plays key roles in activating gene transcription in eukaryotes. Mediator of RNA polymerase II transcription subunit 12 homolog (MED12) is a subunit of the Mediator Complex and regulates the activity of the complex. MED12 is involved in a variety of cellular activities, and mutations in MED12 gene impair MED12 activities and are associated with several diseases, including Opitz-Kaveggia syndrome, Lujan syndrome, uterine leiomyomas and prostate cancer. This review will discuss the biological function of MED12 and the relationship between MED12 mutations and diseases.

Pollen-Expressed Transcription Factor 2 Encodes a Novel Plant-Specific TFIIB-Related Protein that Is Required for Pollen Germination and Embryogenesis in Arabidopsis

Pollen germination and embryogenesis are important to sexual plant reproduction. The processes require a large number of genes to be expressed. Transcription of eukaryotic nuclear genes is accomplished by three conserved RNA polymerases acting in association with a set of auxiliary general transcription factors (GTFs), including B-type GTFs. The roles of B-type GTFs in plant reproduction remain poorly understood. Here we report functional characterization of a novel plant-specific TFIIB-related gene PTF2 in Arabidopsis. Mutation in PTF2 caused failure of pollen germination. Pollen-rescue revealed that the mutation also disrupted embryogenesis and resulted in seed abortion. PTF2 is expressed prolifically in developing pollen and the other tissues with active cell division and differentiation, including embryo and shoot apical meristem. The PTF2 protein shares a lower amino acid sequence similarity with other known TFIIB and TFIIB-related proteins in Arabidopsis. It can interact with TATA-box binding protein 2 (TBP2) and bind to the double-stranded DNA (dsDNA) as the other known TFIIB and TFIIB-related proteins do. In addition, PTF2 can form a homodimer and interact with the subunits of RNA polymerases (RNAPs), implying that it may be involved in the RNAPs transcription. These results suggest that PTF2 plays crucial roles in pollen germination and embryogenesis in Arabidopsis, possibly by regulating gene expression through interaction with TBP2 and the subunits of RNAPs.

Mediator Recruitment to Heat Shock Genes Requires Dual Hsf1 Activation Domains and Mediator Tail Subunits Med15 and Med16

The evolutionarily conserved Mediator complex is central to the regulation of gene transcription in eukaryotes because it serves as a physical and functional interface between upstream regulators and the Pol II transcriptional machinery. Nonetheless, its role appears to be context-dependent, and the detailed mechanism by which it governs the expression of most genes remains unknown. Here we investigate Mediator involvement in HSP (heat shock protein) gene regulation in the yeast Saccharomyces cerevisiae. We find that in response to thermal upshift, subunits representative of each of the four Mediator modules (Head, Middle, Tail, and Kinase) are rapidly, robustly, and selectively recruited to the promoter regions of HSP genes. Their residence is transient, returning to near-background levels within 90 min. Hsf1 (heat shock factor 1) plays a central role in recruiting Mediator, as indicated by the fact that truncation of either its N- or C-terminal activation domain significantly reduces Mediator occupancy, whereas removal of both activation domains abolishes it. Likewise, ablation of either of two Mediator Tail subunits, Med15 or Med16, reduces Mediator recruitment to HSP promoters, whereas deletion of both abolishes it. Accompanying the loss of Mediator, recruitment of RNA polymerase II is substantially diminished. Interestingly, Mediator antagonizes Hsf1 occupancy of non-induced promoters yet facilitates enhanced Hsf1 association with activated ones. Collectively, our observations indicate that Hsf1, via its dual activation domains, recruits holo-Mediator to HSP promoters in response to acute heat stress through cooperative physical and/or functional interactions with the Tail module.

Computational Reconstruction of Transcriptional Relationships from ChIP-Chip Data

Eukaryotic gene transcription is a complex process, which requires the orchestrated recruitment of a large number of proteins, such as sequence-specific DNA binding factors, chromatin remodelers and modifiers, and general transcription machinery, to regulatory regions. Previous works have shown that these regulatory proteins favor specific organizational theme along promoters. Details about how they cooperatively regulate transcriptional process, however, remain unclear. We developed a method to reconstruct a Bayesian network (BN) model representing functional relationships among various transcriptional components. The positive/negative influence between these components was measured from protein binding and nucleosome occupancy data and embedded into the model. Application on S.cerevisiae ChIP-Chip data showed that the proposed method can recover confirmed relationships, such as Isw1-Pol II, TFIIH-Pol II, TFIIB-TBP, Pol II-H3K36Me3, H3K4Me3-H3K14Ac, etc. Moreover, it can distinguish colocating components from functionally related ones. Novel relationships, e.g., ones between Mediator and chromatin remodeling complexes (CRCs), and the combinatorial regulation of Pol II recruitment and activity by CRCs and general transcription factors (GTFs), were also suggested. protein binding events during transcription positively influence each other. Among contributing components, GTFs and CRCs play pivotal roles in transcriptional regulation. These findings provide insights into the regulatory mechanism.

ATX1-Generated H3K4me3 Is Required for Efficient Elongation of Transcription, Not Initiation, at ATX1-Regulated Genes

Tri-methylated H3 lysine 4 (H3K4me3) is associated with transcriptionally active genes, but its function in the transcription process is still unclear. Point mutations in the catalytic domain of ATX1 (ARABIDOPSIS TRITHORAX1), a H3K4 methyltransferase, and RNAi knockdowns of subunits of the AtCOMPASS–like (Arabidopsis Complex Proteins Associated with Set) were used to address this question. We demonstrate that both ATX1 and AtCOMPASS–like are required for high level accumulation of TBP (TATA-binding protein) and Pol II at promoters and that this requirement is independent of the catalytic histone modifying activity. However, the catalytic function is critically required for transcription as H3K4me3 levels determine the efficiency of transcription elongation. The roles of H3K4me3, ATX1, and AtCOMPASS–like may be of a general relevance for transcription of Trithorax-activated eukaryotic genes.

Spatial organization of the chicken beta-globin gene domain in erythroid cells of embryonic and adult lineages

BackgroundThe β-globin gene domains of vertebrate animals constitute popular models for studying the regulation of eukaryotic gene transcription. It has previously been shown that in the mouse the developmental switching of globin gene expression correlates with the reconfiguration of an active chromatin hub (ACH), a complex of promoters of transcribed genes with distant regulatory elements. Although it is likely that observations made in the mouse β-globin gene domain are also relevant for this locus in other species, the validity of this supposition still lacks direct experimental evidence. Here, we have studied the spatial organization of the chicken β-globin gene domain. This domain is of particular interest because it represents the perfect example of the so-called ‘strong’ tissue-specific gene domain flanked by insulators, which delimit the area of preferential sensitivity to DNase I in erythroid cells.ResultsUsing chromosome conformation capture (3C), we have compared the spatial configuration of the β-globin gene domain in chicken red blood cells (RBCs) expressing embryonic (3-day-old RBCs) and adult (9-day-old RBCs) β-globin genes. In contrast to observations made in the mouse model, we found that in the chicken, the early embryonic β-globin gene, Ε, did not interact with the locus control region in RBCs of embryonic lineage (3-day RBCs), where this gene is actively transcribed. In contrast to the mouse model, a strong interaction of the promoter of another embryonic β-globin gene, ρ, with the promoter of the adult β-globin gene, βA, was observed in RBCs from both 3-day and 9-day chicken embryos. Finally, we have demonstrated that insulators flanking the chicken β-globin gene domain from the upstream and from the downstream interact with each other, which places the area characterized by lineage-specific sensitivity to DNase I in a separate chromatin loop.ConclusionsTaken together, our results strongly support the ACH model but show that within a domain of tissue-specific genes, the active status of a promoter does not necessarily correlate with the recruitment of this promoter to the ACH.