Understanding how transcriptional enhancers control over 20,000 protein-coding genes to maintain cell-type-specific gene expression programs in all human cells is a fundamental challenge in ...regulatory biology. Recent studies suggest that gene regulatory elements and their target genes generally occur within insulated neighborhoods, which are chromosomal loop structures formed by the interaction of two DNA sites bound by the CTCF protein and occupied by the cohesin complex. Here, we review evidence that insulated neighborhoods provide for specific enhancer-gene interactions, are essential for both normal gene activation and repression, form a chromosome scaffold that is largely preserved throughout development, and are perturbed by genetic and epigenetic factors in disease. Insulated neighborhoods are a powerful paradigm for gene control that provides new insights into development and disease.
DNA loops, formed by the interaction of two CTCF molecules bound to different sites, are structural and functional units of gene control.
Oncogenes are activated through well-known chromosomal alterations such as gene fusion, translocation, and focal amplification. In light of recent evidence that the control of key genes depends on ...chromosome structures called insulated neighborhoods, we investigated whether proto-oncogenes occur within these structures and whether oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T cell acute lymphoblastic leukemia (T-ALL) and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in nonmalignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells.
There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between ...gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.
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•YY1 generally occupies active enhancers and promoters across cell types•YY1 can form dimers and promote DNA interactions•Perturbation of YY1 binding disrupts enhancer-promoter looping and gene expression•YY1’s structural role accounts for diverse functions reported previously
YY1 is a structural regulator of enhancer-promoter interactions and facilitates gene expression.
Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. ...Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes.
Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in ...the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.
The CCCTC-binding factor (CTCF), which anchors DNA loops that organize the genome into structural domains, has a central role in gene control by facilitating or constraining interactions between ...genes and their regulatory elements
. In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation
or DNA hypermethylation
results in the loss of loop anchors and consequent activation of oncogenes. By contrast, the germ-cell-specific paralogue of CTCF, BORIS (brother of the regulator of imprinted sites, also known as CTCFL)
, is overexpressed in several cancers
, but its contributions to the malignant phenotype remain unclear. Here we show that aberrant upregulation of BORIS promotes chromatin interactions in ALK-mutated, MYCN-amplified neuroblastoma
cells that develop resistance to ALK inhibition. These cells are reprogrammed to a distinct phenotypic state during the acquisition of resistance, a process defined by the initial loss of MYCN expression followed by subsequent overexpression of BORIS and a concomitant switch in cellular dependence from MYCN to BORIS. The resultant BORIS-regulated alterations in chromatin looping lead to the formation of super-enhancers that drive the ectopic expression of a subset of proneural transcription factors that ultimately define the resistance phenotype. These results identify a previously unrecognized role of BORIS-to promote regulatory chromatin interactions that support specific cancer phenotypes.
Transcriptional profiling is a useful strategy to study development and disease. Approaches to isolate RNA from specific cell types, or from specific cellular compartments, would extend the power of ...this strategy. Previous work has shown that isolation of genetically tagged ribosomes (translating ribosome affinity purification; TRAP) is an effective means to isolate ribosome-bound RNA selectively from transgene-expressing cells. However, widespread application of this technology has been limited by available transgenic mouse lines. Here we characterize a TRAP allele (Rosa26 ᶠˢᵀᴿᴬᴾ) that makes this approach more widely accessible. We show that endothelium-specific activation of Rosa26 ᶠˢᵀᴿᴬᴾ identifies endothelial cell-enriched transcripts, and that cardiomyocyte-restricted TRAP is a useful means to identify genes that are differentially expressed in cardiomyocytes in a disease model. Furthermore, we show that TRAP is an effective means for studying translational regulation, and that several nuclear-encoded mitochondrial genes are under strong translational control. Our analysis of ribosome-bound transcripts also shows that a subset of long intergenic noncoding RNAs are weakly ribosome-bound, but that the majority of noncoding RNAs, including most long intergenic noncoding RNAs, are ribosome-bound to the same extent as coding transcripts. Together, these data show that the TRAP strategy and the Rosa26 ᶠˢᵀᴿᴬᴾ allele will be useful tools to probe cell type-specific transcriptomes, study translational regulation, and probe ribosome binding of noncoding RNAs.
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•Poly(isoprene-b-methyl methacrylate) polymers self-assemble in hydrocarbon solvents.•Spheres, worms or vesicles controlled by molar mass, composition and ...concentration.•Post-polymerisation dispersion successful at up to 30% solids content.•Self-assembled nano-objects show thermal responsivity.
The self-assembly of a series of polyisoprene-block-poly(methyl methacrylate) (PI-b-PMMA) block copolymers, in aliphatic hydrocarbon solvents is reported for the first time. The block copolymers were prepared by a change of mechanism polymerisation (CHOMP) whereby living anionic polymerisation was used to prepare polyisoprene macroinitiators for ATRP (PI-Br), which was in turn used to polymerize MMA. The use of anionic polymerisation ensured optimal control over the molar mass and dispersity of the polyisoprene block, in a synthesis that is readily scaled-up, to enable the production of homologous series of block copolymers by varying only the molar mass of the PMMA block. The block copolymers were subsequently dispersed in n-decane or n-hexane, a selective solvent for the PI block, at high solids content of up to 30 wt%. Analysis of the resulting self-assembled nanostructures using DLS and TEM revealed that the block copolymers self-assemble into varying morphologies (spherical micelles, wormlike micelles or vesicles), dependent upon the molar mass and composition of the block copolymer, enabling the construction of a series of “phase-diagrams”. The resultant self-assembled structures were also found to be thermally responsive and, in some cases, underwent a change in morphology upon heating.
Green chemistry places an emphasis on safer chemicals, waste reduction, and efficiency. Processes should be optimized with green chemistry at the forefront of decision making, embedded into research ...at the earliest stage. To assist in this endeavor, we present a spreadsheet that can be used to interpret reaction kinetics via Variable Time Normalization Analysis (VTNA), understand solvent effects with linear solvation energy relationships (LSER), and calculate solvent greenness. With this information, new reaction conditions can be explored in silico, calculating product conversions and green chemistry metrics prior to experiments. The application of this tool was validated with literature case studies. Reaction performance was predicted and then confirmed experimentally for examples of aza-Michael addition, Michael addition, and an amidation. The combined analytical package presented herein permits a thorough examination of chemical reactions, so that the variables that control reaction chemistry can be understood, optimized, and made greener for research and education purposes.
Transferrin‐conjugated gold nanorods are used for efficient targeting, two‐photon imaging, and photothermal therapy of cancer cells under both linearly and circularly polarized femtosecond ...illuminations. The results show that the effective laser energy threshold for therapy can be reduced to around 10 mJ cm−2, which is one order of magnitude lower than the medical safety standard level.
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