Core promoter mutation contributes to abnormal gene expression in bladder cancer

Background Bladder cancer is one of the most mortal cancers. Bladder cancer has distinct gene expression signature, highlighting altered gene expression plays important roles in bladder cancer etiology. However, the mechanism for how the regulatory disorder causes the altered expression in bladder cancer remains elusive. Core promoter controls transcriptional initiation. We hypothesized that mutation in core promoter abnormality could cause abnormal transcriptional initiation thereby the altered gene expression in bladder cancer. Methods In this study, we performed a genome-wide characterization of core promoter mutation in 77 Spanish bladder cancer cases. Results We identified 69 recurrent somatic mutations in 61 core promoters of 62 genes and 28 recurrent germline mutations in 20 core promoters of 21 genes, including TERT, the only gene known with core promoter mutation in bladder cancer, and many oncogenes and tumor suppressors. From the RNA-seq data from bladder cancer, we observed altered expression of the core promoter-mutated genes. We further validated the effects of core promoter mutation on gene expression by using luciferase reporter gene assay. We also identified potential drugs targeting the core promoter-mutated genes. Conclusions Data from our study highlights that core promoter mutation contributes to bladder cancer development through altering gene expression.

Mandatory coupling of zygotic transcription to DNA replication in early Drosophila embryos

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesized that collision-avoidance mechanisms safeguard the onset of zygotic transcription in these cycles. To explore this hypothesis, we used real-time imaging of transcriptional events at the onset of each interphase. Endogenously tagged RNA polymerase II (RNAPII) abruptly formed clusters before nascent transcripts accumulated, indicating recruitment prior to transcriptional engagement. Injection of inhibitors of DNA replication prevented RNAPII clustering, blocked formation of foci of the pioneer factor Zelda, and largely prevented expression of transcription reporters. Knockdown of Zelda or the histone acetyltransferase CBP prevented RNAPII cluster formation except at the replication-dependent (RD) histone gene locus. We suggest a model in which the passage of replication forks allows Zelda and a distinct pathway at the RD histone locus to reconfigure chromatin to nucleate RNAPII clustering and promote transcriptional initiation. The replication dependency of these events defers initiation of transcription and ensures that RNA polymerases transcribe behind advancing replication forks. The resulting coordination of transcription and replication explains how early embryos circumvent collisions and promote genome stability.

Recognition of acetylated histone by Yaf9 regulates metabolic cycling of transcription initiation and chromatin regulatory factors

How transcription programs rapidly adjust to changing metabolic and cellular cues remains poorly defined. Here, we reveal a function for the Yaf9 component of the SWR1-C and NuA4 chromatin regulatory complexes in maintaining timely transcription of metabolic genes across the yeast metabolic cycle (YMC). By reading histone acetylation during the oxidative and respiratory phase of the YMC, Yaf9 recruits SWR1-C and NuA4 complexes to deposit H2A.Z and acetylate H4, respectively. Increased H2A.Z and H4 acetylation during the oxidative phase promotes transcriptional initiation and chromatin machinery occupancy and is associated with reduced RNA polymerase II levels at genes—a pattern reversed during transition from oxidative to reductive metabolism. Prevention of Yaf9-H3 acetyl reading disrupted this pattern of transcriptional and chromatin regulator recruitment and impaired the timely transcription of metabolic genes. Together, these findings reveal that Yaf9 contributes to a dynamic chromatin and transcription initiation factor signature that is necessary for the proper regulation of metabolic gene transcription during the YMC. They also suggest that unique regulatory mechanisms of transcription exist at distinct metabolic states.

Identification of Enhancers and Promoters in the Genome by Multidimensional Scaling

The positions of enhancers and promoters on genomic DNA remain poorly understood. Chromosomes cannot be observed during the cell division cycle because the genome forms a chromatin structure and spreads within the nucleus. However, high-throughput chromosome conformation capture (Hi-C) measures the physical interactions of genomes. In previous studies, DNA extrusion loops were directly derived from Hi-C heat maps. Multidimensional Scaling (MDS) is used in this assessment to more precisely locate enhancers and promoters. MDS is a multivariate analysis method that reproduces the original coordinates from the distance matrix between elements. We used Hi-C data of cultured osteosarcoma cells and applied MDS as the distance matrix of the genome. In addition, we selected columns 2 and 3 of the orthogonal matrix U as the desired structure. Overall, the DNA loops from the reconstructed genome structure contained bioprocesses involved in transcription, such as the pre-transcriptional initiation complex and RNA polymerase II initiation complex, and transcription factors involved in cancer, such as Foxm1 and CREB3. Therefore, our results are consistent with the biological findings. Our method is suitable for identifying enhancers and promoters in the genome.

Comparison of transcriptional initiation by RNA polymerase II across eukaryotic species

The preinitiation complex (PIC) for transcriptional initiation by RNA polymerase (Pol) II is composed of general transcription factors that are highly conserved. However, analysis of ChIP-seq datasets reveals kinetic and compositional differences in the transcriptional initiation process among eukaryotic species. In yeast, Mediator associates strongly with activator proteins bound to enhancers, but it transiently associates with promoters in a form that lacks the kinase module. In contrast, in human, mouse, and fly cells, Mediator with its kinase module stably associates with promoters, but not with activator-binding sites. This suggests that yeast and metazoans differ in the nature of the dynamic bridge of Mediator between activators and Pol II and the composition of a stable inactive PIC-like entity. As in yeast, occupancies of TATA-binding protein (TBP) and TBP-associated factors (Tafs) at mammalian promoters are not strictly correlated. This suggests that within PICs, TFIID is not a monolithic entity, and multiple forms of TBP affect initiation at different classes of genes. TFIID in flies, but not yeast and mammals, interacts strongly at regions downstream of the initiation site, consistent with the importance of downstream promoter elements in that species. Lastly, Taf7 and the mammalian-specific Med26 subunit of Mediator also interact near the Pol II pause region downstream of the PIC, but only in subsets of genes and often not together. Species-specific differences in PIC structure and function are likely to affect how activators and repressors affect transcriptional activity.

Re-evaluating the role of nucleosomal bivalency in early development

Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome1; posttranslational modifications of these histone proteins influence the activity of the associated genomic regions to regulate cell identity2–4. Traditionally, trimethylation of histone 3-lysine 4 (H3K4me3) is associated with transcriptional initiation5–10, whereas trimethylation of H3K27 (H3K27me3) is considered transcriptionally repressive11–15. The apparent juxtaposition of these opposing marks, termed “bivalent domains”16–18, was proposed to specifically demarcate of small set transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status19–22. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure H3K4me3/H3K27me3 bivalency genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model16,18: from naïve mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread, does not resolve with differentiation, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against interpreting bivalent domains as specific markers of developmentally poised genes.

DOT1L complex regulates transcriptional initiation in human erythroleukemic cells

DOT1L, the only H3K79 methyltransferase in human cells and a homolog of the yeast Dot1, normally forms a complex with AF10, AF17, and ENL or AF9, is dysregulated in most cases of mixed-lineage leukemia (MLLr), and has been believed to regulate transcriptional elongation on the basis of its colocalization with RNA polymerase II (Pol II), the sharing of subunits (AF9 and ENL) between the DOT1L and super elongation complexes, and the distribution of H3K79 methylation on both promoters and transcribed regions of active genes. Here we show that DOT1L depletion in erythroleukemic cells reduces its global occupancy without affecting the traveling ratio or the elongation rate (assessed by 4sUDRB-seq) of Pol II, suggesting that DOT1L does not play a major role in elongation in these cells. In contrast, analyses of transcription initiation factor binding reveal that DOT1L and ENL depletions each result in reduced TATA binding protein (TBP) occupancies on thousands of genes. More importantly, DOT1L and ENL depletions concomitantly reduce TBP and Pol II occupancies on a significant fraction of direct (DOT1L-bound) target genes, indicating a role for the DOT1L complex in transcription initiation. Mechanistically, proteomic and biochemical studies suggest that the DOT1L complex may regulate transcriptional initiation by facilitating the recruitment or stabilization of transcription factor IID, likely in a monoubiquitinated H2B (H2Bub1)-enhanced manner. Additional studies show that DOT1L enhances H2Bub1 levels by limiting recruitment of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex. These results advance our understanding of roles of the DOT1L complex in transcriptional regulation and have important implications for MLLr leukemias.

Sequence determinants, function, and evolution of CpG islands

In vertebrates, cytosine-guanine (CpG) dinucleotides are predominantly methylated, with ∼80% of all CpG sites containing 5-methylcytosine (5mC), a repressive mark associated with long-term gene silencing. The exceptions to such a globally hypermethylated state are CpG-rich DNA sequences called CpG islands (CGIs), which are mostly hypomethylated relative to the bulk genome. CGIs overlap promoters from the earliest vertebrates to humans, indicating a concerted evolutionary drive compatible with CGI retention. CGIs are characterised by DNA sequence features that include DNA hypomethylation, elevated CpG and GC content and the presence of transcription factor binding sites. These sequence characteristics are congruous with the recruitment of transcription factors and chromatin modifying enzymes, and transcriptional activation in general. CGIs colocalize with sites of transcriptional initiation in hypermethylated vertebrate genomes, however, a growing body of evidence indicates that CGIs might exert their gene regulatory function in other genomic contexts. In this review, we discuss the diverse regulatory features of CGIs, their functional readout, and the evolutionary implications associated with CGI retention in vertebrates and possibly in invertebrates.