MDM2-Driven Ubiquitination Rapidly Removes p53 from Its Cognate Promoters

MDM2 is the principal antagonist of the tumor suppressor p53. p53 binds to its cognate DNA element within promoters and activates the transcription of adjacent genes. These target genes include MDM2. Upon induction by p53, the MDM2 protein binds and ubiquitinates p53, triggering its proteasomal degradation and providing negative feedback. This raises the question whether MDM2 can also remove p53 from its target promoters, and whether this also involves ubiquitination. In the present paper, we employ the MDM2-targeted small molecule Nutlin-3a (Nutlin) to disrupt the interaction of MDM2 and p53 in three different cancer cell lines: SJSA-1 (osteosarcoma), 93T449 (liposarcoma; both carrying amplified MDM2), and MCF7 (breast adenocarcinoma). Remarkably, removing Nutlin from the culture medium for less than five minutes not only triggered p53 ubiquitination, but also dissociated most p53 from its chromatin binding sites, as revealed by chromatin immunoprecipitation. This also resulted in reduced p53-responsive transcription, and it occurred much earlier than the degradation of p53 by the proteasome, arguing that MDM2 removes p53 from promoters prior to and thus independent of degradation. Accordingly, the short-term pharmacological inhibition of the proteasome did not alter the removal of p53 from promoters by Nutlin washout. However, when the proteasome inhibitor was applied for several hours, depleting non-conjugated ubiquitin prior to eliminating Nutlin, this compromised the removal of DNA-bound p53, as did an E1 ubiquitin ligase inhibitor. This suggests that the ubiquitination of p53 by MDM2 is necessary for its clearance from promoters. Depleting the MDM2 cofactor MDM4 in SJSA cells did not alter the velocity by that p53 was removed from promoters upon Nutlin washout. We conclude that MDM2 antagonizes p53 not only by covering its transactivation domain and by destabilization, but also by the rapid, ubiquitin-dependent termination of p53–chromatin interactions.

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations

Cell fate is maintained over long timescales, yet molecular fluctuations can lead to spontaneous loss of this differentiated state. Our simulations identified a possible mechanism that explains life-long maintenance of ASE neuron fate in C. elegans by the terminal selector transcription factor CHE-1. Here, fluctuations in CHE-1 level are buffered by the reservoir of CHE-1 bound at its target promoters, which ensures continued che-1 expression by preferentially binding the che-1 promoter. We provide experimental evidence for this mechanism by showing that che-1 expression was resilient to induced transient CHE-1 depletion, while both expression of CHE-1 targets and ASE function were lost. We identified a 130 bp che-1 promoter fragment responsible for this resilience, with deletion of a homeodomain binding site in this fragment causing stochastic loss of ASE identity long after its determination. Because network architectures that support this mechanism are highly conserved in cell differentiation, it may explain stable cell fate maintenance in many systems.

p53-Dependent Repression: DREAM or Reality?

p53 is a major tumor suppressor that integrates diverse types of signaling in mammalian cells. In response to a broad range of intra- or extra-cellular stimuli, p53 controls the expression of multiple target genes and elicits a vast repertoire of biological responses. The exact code by which p53 integrates the various stresses and translates them into an appropriate transcriptional response is still obscure. p53 is tightly regulated at multiple levels, leading to a wide diversity in p53 complexes on its target promoters and providing adaptability to its transcriptional program. As p53-targeted therapies are making their way into clinics, we need to understand how to direct p53 towards the desired outcome (i.e., cell death, senescence or other) selectively in cancer cells without affecting normal tissues or the immune system. While the core p53 transcriptional program has been proposed, the mechanisms conferring a cell type- and stimuli-dependent transcriptional outcome by p53 require further investigations. The mechanism by which p53 localizes to repressed promoters and manages its co-repressor interactions is controversial and remains an important gap in our understanding of the p53 cistrome. We hope that our review of the recent literature will help to stimulate the appreciation and investigation of largely unexplored p53-mediated repression.

Mechanism of HS5-1 eRNA Regulation of Protocadherin α Cluster via R-loop Formation

Enhancers generate bidirectional noncoding enhancer RNAs that may regulate gene expression. At present, mechanisms of eRNA functions are not fully understood. Here, we report an antisense eRNA PEARL that is transcribed from the protocadherin α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA-fragment editing, CRISPRi, and CRISPRa strategies, in conjunction with ChIRP, MeDIP, and DRIP experiments, we find that PEARL regulates Pcdhα expression by forming local R-loop in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancer and target promoters. These findings have important implications regarding mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis

Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development.

New insights into promoter–enhancer communication mechanisms revealed by dynamic single-molecule imaging

Establishing cell-type-specific gene expression programs relies on the action of distal enhancers, cis-regulatory elements that can activate target genes over large genomic distances — up to Mega-bases away. How distal enhancers physically relay regulatory information to target promoters has remained a mystery. Here, we review the latest developments and insights into promoter–enhancer communication mechanisms revealed by live-cell, real-time single-molecule imaging approaches.

The Seed Development Factors TT2 and MYB5 Regulate Heat Stress Response in Arabidopsis

HEAT SHOCK FACTOR A2 (HSFA2) is a regulator of multiple environmental stress responses required for stress acclimation. We analyzed HSFA2 co-regulated genes and identified 43 genes strongly co-regulated with HSFA2 during multiple stresses. Motif enrichment analysis revealed an over-representation of the site II element (SIIE) in the promoters of these genes. In a yeast 1-hybrid screen with the SIIE, we identified the closely related R2R3-MYB transcription factors TT2 and MYB5. We found overexpression of MYB5 or TT2 rendered plants heat stress tolerant. In contrast, tt2, myb5, and tt2/myb5 loss of function mutants showed heat stress hypersensitivity. Transient expression assays confirmed that MYB5 and TT2 can regulate the HSFA2 promoter together with the other members of the MBW complex, TT8 and TRANSPARENT TESTA GLABRA 1 (TTG1) and that the SIIE was involved in this regulation. Transcriptomic analysis revealed that TT2/MYB5 target promoters were enriched in SIIE. Overall, we report a new function of TT2 and MYB5 in stress resistance and a role in SIIE-mediated HSFA2 regulation.

Nonlinear control of transcription through enhancer-promoter interactions

Chromosome structure in mammals is thought to regulate transcription by modulating the three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated interactions and topologically associating domains (TADs)1–4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. To address this question we use a novel assay to position an enhancer at a large number of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. Quantitative analysis of hundreds of cell lines reveal that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a non-linear relationship. Mathematical modeling and validation against experimental data further provide evidence that nonlinearity arises from transient enhancer-promoter interactions being memorized into longer-lived promoter states in individual cells, thus uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism for how enhancers control transcription across large genomic distances despite rarely meeting their target promoters, and for how TAD boundaries can block distal enhancers. We finally show that enhancer strength additionally determines not only absolute transcription levels, but also the sensitivity of a promoter to CTCF-mediated functional insulation. Our unbiased, systematic and quantitative measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.

Mechanisms of enhancer action: the known and the unknown

Differential gene expression mechanisms ensure cellular differentiation and plasticity to shape ontogenetic and phylogenetic diversity of cell types. A key regulator of differential gene expression programs are the enhancers, the gene-distal cis-regulatory sequences that govern spatiotemporal and quantitative expression dynamics of target genes. Enhancers are widely believed to physically contact the target promoters to effect transcriptional activation. However, our understanding of the full complement of regulatory proteins and the definitive mechanics of enhancer action is incomplete. Here, we review recent findings to present some emerging concepts on enhancer action and also outline a set of outstanding questions.

Characterization of the binding motif for the T3SS master regulator LcrF in Yersinia pseudotuberculosis

LcrF is the master regulator that positively regulates the Ysc type III secretion system (T3SS) in Yersinia and shares high similarity to the DNA binding domain of the T3SS master regulator ExsA in Pseudomonas aeruginosa. Based on these features, bioinformatics analysis has predicted putative LcrF binding site in its target promoters. Here we experimentally characterized its binding motif. An A-rich LcrF binding region in the lcrG promoter sequence, a typical regulatory target of LcrF, was first confirmed. To obtain detailed information, this binding region was cloned into a synthetized promoter and mutations in this region were further constructed. We demonstrated that the 5’-AAAAA-n5-GnCT-3’ sequence is required for LcrF regulation and this motif is strictly located 4-bp upstream of a noncanonical promoter, in which the -35 and -10 elements are separated by a 21-bp spacer. Consistently, the putative binding motif was found in promoters of nine T3SS related operons or genes positively regulated by LcrF. Transcriptome analysis further confirmed that LcrF specifically activates T3SS genes in Yersinia. Collectively, our data suggest that LcrF is evolved to be a specific T3SS activator with stringent sequence requirement for transcriptional regulation.