MafB (a bZIP transcription factor), ß-catenin (the ultimate target of the Wnt signal transduction pathway that acts as a transcriptional co-activator of LEF/TCF proteins), and WDR77 (a ...transcriptional co-activator of multiple hormone receptors) are important for breast cellular transformation. Unexpectedly, these proteins interact directly with each other, and they have similar genomic binding profiles. Furthermore, while some of these common target sites coincide with those bound by LEF/TCF, the majority are located just downstream of transcription initiation sites at a position near paused RNA polymerase (Pol II) and the +1 nucleosome. Occupancy levels of these factors at these promoter-proximal sites are strongly correlated with the level of paused Pol II and transcriptional activity.
The term “intrinsically disordered region” (IDR) in proteins has been used in numerous publications. However, most proteins contain IDRs, the term refers to very different types of structures and ...functions, and many IDRs become structured upon interaction with other biomolecules. Thus, IDR is an unnecessary, vague, and ultimately confusing concept.
The term “intrinsically disordered region” (IDR) in proteins has been used in numerous publications. However, most proteins contain IDRs, the term refers to very different types of structures and functions, and many IDRs become structured upon interaction with other biomolecules. Thus, IDR is an unnecessary, vague, and ultimately confusing concept.
Enhancers generate nucleosome-depleted regions to regulate DNA-based processes that, unlike RNA polymerase II (Pol II) transcription, do not have dedicated regulatory proteins.Increased accessibility ...of DNA by enhancers facilitates RNA polymerase III (Pol III) transcription, binding of the origin replication complex (ORC) to DNA replication origins, and targeting of the recombination-activating gene (RAG) recombinase for V(D)J recombination.Enhancer-dependent transcription further regulates V(D)J recombination by generating localized regions of trimethylated histone H3-K4 that are recognized by the RAG2 PHD domain.
Enhancers are the key regulators of other DNA-based processes by virtue of their unique ability to generate nucleosome-depleted regions in a highly regulated manner. Enhancers regulate cell-type-specific transcription of tRNA genes by RNA polymerase III (Pol III). They are also responsible for the binding of the origin replication complex (ORC) to DNA replication origins, thereby regulating origin utilization, replication timing, and replication-dependent chromosome breaks. Additionally, enhancers regulate V(D)J recombination by increasing access of the recombination-activating gene (RAG) recombinase to target sites and by generating non-coding enhancer RNAs and localized regions of trimethylated histone H3-K4 recognized by the RAG2 PHD domain. Thus, enhancers represent the first step in decoding the genome, and hence they regulate biological processes that, unlike RNA polymerase II (Pol II) transcription, do not have dedicated regulatory proteins.
Enhancers are the key regulators of other DNA-based processes by virtue of their unique ability to generate nucleosome-depleted regions in a highly regulated manner. Enhancers regulate cell-type-specific transcription of tRNA genes by RNA polymerase III (Pol III). They are also responsible for the binding of the origin replication complex (ORC) to DNA replication origins, thereby regulating origin utilization, replication timing, and replication-dependent chromosome breaks. Additionally, enhancers regulate V(D)J recombination by increasing access of the recombination-activating gene (RAG) recombinase to target sites and by generating non-coding enhancer RNAs and localized regions of trimethylated histone H3-K4 recognized by the RAG2 PHD domain. Thus, enhancers represent the first step in decoding the genome, and hence they regulate biological processes that, unlike RNA polymerase II (Pol II) transcription, do not have dedicated regulatory proteins.
Significance The paper describes an assay for cellular transformation that involves growth in low attachment (GILA). This assay is comparable to the gold-standard soft-agar assay, but it is much ...easier to perform and is suitable for high-throughput drug and genetic screens. We describe such screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.
Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.
Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and ...physiological functions have been unclear. Here we show that SIRT7 is an NAD(+)-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo.
In response to environmental stresses, cells activate stress-response genes and inhibit DNA replication. HBO1 histone acetylase is a coactivator both for AP-1 transcription factors responding to ...stress-activated JNK kinases and also for the Cdt1 licensing factor that ensures that DNA is replicated exactly once per cell cycle. In response to nongenotoxic stress, JNK phosphorylates Jun, an AP-1 transcription factor, leading to increased recruitment of HBO1 and increased transcription of target genes. In addition, JNK phosphorylates Cdt1 on threonine 29, leading to rapid dissociation of HBO1 from replication origins, thereby blocking initiation of DNA replication. Upon relief of stress, HBO1 reassociates with replication origins. Thus, regulated and reciprocal recruitment of the HBO1 coactivator to target genes and replication origins via JNK-mediated phosphorylation of the recruiting transcription and replication licensing factors coordinates the transcriptional and DNA replication response to cellular stress.
► Upon nongenotoxic stress, JNK1 phosphorylates Cdt1 on threonine 29 ► Cdt1-T29 phosphorylation causes rapid dissociation of HBO1 from origins ► Regulated and reciprocal recruitment of HBO1 to enhancers and origins ► JNK1, via HBO1, coordinates the transcriptional and replication response to stress
Nucleosome positioning is critical for gene expression and most DNA-related processes. Here we review the dominant patterns of nucleosome positioning that have been observed and summarize the current ...understanding of their underlying determinants. The genome-wide pattern of nucleosome positioning is determined by the combination of DNA sequence, ATP-dependent nucleosome remodeling enzymes and transcription factors that include activators, components of the preinitiation complex and elongating RNA polymerase II. These determinants influence each other such that the resulting nucleosome positioning patterns are likely to differ among genes and among cells in a population, with consequent effects on gene expression.
Yeast cells undergoing the diauxic response show a striking upstream shift in poly(A) site utilization, with increased use of ORF-proximal poly(A) sites resulting in shorter 3' mRNA isoforms for most ...genes. This altered poly(A) pattern is extremely similar to that observed in cells containing Pol II derivatives with slow elongation rates. Conversely, cells containing derivatives with fast elongation rates show a subtle downstream shift in poly(A) sites. Polyadenylation patterns of many genes are sensitive to both fast and slow elongation rates, and a global shift of poly(A) utilization is strongly linked to increased purine content of sequences flanking poly(A) sites. Pol II processivity is impaired in diauxic cells, but strains with reduced processivity and normal Pol II elongation rates have normal polyadenylation profiles. Thus, Pol II elongation speed is important for poly(A) site selection and for regulating poly(A) patterns in response to environmental conditions.
Histones are rapidly evicted and deposited during transcription by RNA polymerase (Pol) II, but a factor that mediates histone eviction in vivo has not yet been identified. Here, we show that the ...histone chaperone Asf1 associates with promoters and coding regions of transcriptionally active genes. Asf1 mediates histone H3, but not H2B, eviction and deposition during Pol II elongation, suggesting that nucleosome assembly and disassembly occur in a stepwise fashion. Lastly, Asf1 inhibits internal initiation from cryptic promoters within coding regions. These results strongly suggest that Asf1 functions as an elongation factor to disassemble and reassemble histones during Pol II elongation.
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