Molecular and Cellular Biology最新文献

Glioma, originating from neuroglial progenitor cells, is a type of intrinsic brain tumor with poor prognosis. temozolomide (TMZ) is the first-line chemotherapeutic agent for glioma. Exploring the mechanisms of circTTLL13 underlying TMZ resistance in glioma is of great significance to improve glioma treatment. Bioinformatics was adopted to identify target genes. The circular structure of circTTLL13 and its high expression in glioma cells were disclosed by quantitative real time-PCR (qRT-PCR) and PCR-agarose gel electrophoresis. Functional experiments proved that oxidized LDL receptor 1 (OLR1) promotes TMZ resistance of glioma cells. CircTTLL13 enhances TMZ resistance of glioma cells via modulating OLR1. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pulldown, mRNA stability, N6-methyladenosine (m⁶A) dot blot and RNA total m⁶A quantification assays were implemented, indicating that circTTLL13 stabilizes OLR1 mRNA via recruiting YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and promotes m⁶A methylation of OLR1 pre-mRNA through recruiting methyltransferase-like 3 (METTL3). TOP/FOP-flash reporter assay and western blot verified that circTTLL13 activates Wnt/β-catenin signaling pathway by regulating OLR1. CircTTLL13 promotes TMZ resistance in glioma through regulating OLR1-mediated Wnt/β-catenin pathway activation. This study offers an insight into the efficacy improvement of TMZ for glioma treatment.

The highly conserved retromer complex controls the fate of hundreds of receptors that pass through the endolysosomal system and is a central regulatory node for diverse metabolic programs. More than 20 years ago, retromer was discovered as an essential regulator of endosome-to-Golgi transport in yeast; since then, significant progress has been made to characterize how metazoan retromer components assemble to enable its engagement with endosomal membranes, where it sorts cargo receptors from endosomes to the trans-Golgi network or plasma membrane through recognition of sorting motifs in their cytoplasmic tails. In this review, we examine retromer regulation by exploring its assembled structure with an emphasis on how a range of adaptor proteins shape the process of receptor trafficking. Specifically, we focus on how retromer is recruited to endosomes, selects cargoes, and generates tubulovesicular carriers that deliver cargoes to target membranes. We also examine how cells adapt to distinct metabolic states by coordinating retromer expression and function. We contrast similarities and differences between retromer and its related complexes: retriever and commander/CCC, as well as their interplay in receptor trafficking. We elucidate how loss of retromer regulation is central to the pathology of various neurogenerative and metabolic diseases, as well as microbial infections, and highlight both opportunities and cautions for therapeutics that target retromer. Finally, with a focus on understanding the mechanisms that govern retromer regulation, we outline new directions for the field moving forward.

The smallest subunit of the human Origin Recognition Complex, hOrc6, is required for DNA replication progression and plays an important role in mismatch repair (MMR) during S-phase. However, the molecular details of how hOrc6 regulates DNA replication and DNA damage response remain to be elucidated. Orc6 levels are elevated upon specific types of genotoxic stress, and it is phosphorylated at Thr229, predominantly during S-phase, in response to oxidative stress. Many repair pathways, including MMR, mediate oxidative DNA damage repair. Defects in MMR are linked to Lynch syndrome, predisposing patients to many cancers, including colorectal cancer. Orc6 levels are known to be elevated in colorectal cancers. Interestingly, tumor cells show reduced hOrc6-Thr229 phosphorylation compared to adjacent normal mucosa. Further, elevated expression of wild-type and the phospho-dead forms of Orc6 results in increased tumorigenicity, implying that in the absence of this "checkpoint" signal, cells proliferate unabated. Based on these results, we propose that DNA-damage-induced hOrc6-pThr229 phosphorylation during S-phase facilitates ATR signaling in the S-phase, halts fork progression, and enables assembly of repair factors to mediate efficient repair to prevent tumorigenesis. Our study provides novel insights into how hOrc6 regulates genome stability.

Nucleosome dynamics in the coding region of a transcriptionally active locus is critical for understanding how RNA polymerase II progresses through the gene body. Histone acetylation and deacetylation critically influence nucleosome accessibility during DNA metabolic processes like transcription. Effect of such histone modifications is context and residue dependent. Rather than effect of individual histone residues, the network of modifications of several histone residues in combination generates a chromatin landscape that is conducive for transcription. Here we show that in Saccharomyces cerevisiae, crosstalk between deacetylation of the H4 N-terminal tail residue H4K16 and acetylation of the H3 core domain residue H3K56, promotes RNA polymerase II progression through the gene body. Results indicate that deacetylation of H4K16 precedes and in turn induces H3K56 acetylation. Effectively, recruitment of Rtt109, the HAT responsible for H3K56 acetylation is essentially dependent on H4K16 deacetylation. In Hos2 deletion strains, where H4K16 deacetylation is abolished, both H3K56 acetylation and RNA polymerase II recruitment gets significantly impaired. Notably, H4K16 deacetylation and H3K56 acetylation are found to be essentially dependent on active transcription. In summary, H4K16 deacetylation promotes H3K56 acetylation and the two modifications together work towards successful functioning of RNA polymerase II during active transcription.

Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops and self-associating domains. Purified cohesin diffuses along DNA in an ATP-independent manner but can be propelled by transcribing RNA polymerase. In conjunction with a cofactor, the complex also extrudes DNA loops in an ATP-dependent manner. In this study we examine transcription-driven translocation of cohesin under various conditions in yeast. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by an inducible gene. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers capture mobilized cohesin demonstrates that transcription-driven complexes translocate processively in vivo. Together, this study reveals unexplored limitations to cohesin movement on chromosomes.

The primary cilium functions as a cellular sensory organelle and signaling antenna that detects and transduces extracellular signals. Mutations in the human gene CILK1 (ciliogenesis associated kinase 1) cause abnormal cilia elongation and faulty Hedgehog signaling, associated with developmental disorders and epilepsy. CILK1 is a protein kinase that requires dual phosphorylation of its TDY motif for activation and its extended C-terminal intrinsically disordered region (IDR) mediates targeting to the basal body and substrate recognition. Proteomics previously identified katanin-interacting protein (KATNIP), also known as KIAA0556, as a CILK1 interacting partner. In this study we discovered that CILK1 colocalizes with KATNIP at the basal body and the CILK1 IDR is sufficient to mediate binding to KATNIP. Deletion analysis of KATNIP shows one of three domains of unknown function (DUF) is required for association with CILK1. KATNIP binding with CILK1 drastically elevated CILK1 protein levels and TDY phosphorylation in cells. This resulted in a profound increase in phosphorylation of known CILK1 substrates and suppression of cilia length. Thus, KATNIP functions as a regulatory subunit of CILK1 that potentiates its actions. This advances our understanding of the molecular basis of control of primary cilia.

CDKN2A/B deletion or silencing is common across human cancer, reinforcing the general importance of bypassing its tumor suppression in cancer formation or progression. In rhabdomyosarcoma (RMS) and neuroblastoma, two common childhood cancers, the three CDKN2A/B transcripts are independently expressed to varying degrees, but one, ARF, is uniformly silenced. Although TGFβ induces certain CDKN2A/B transcripts in HeLa cells, it was unable to do so in five tested RMS lines unless the cells were pretreated with a broadly acting methyltransferase inhibitor, DZNep, or one targeting EZH2. CDKN2A/B induction by TGFβ correlated with de novo appearance of three H3K27Ac peaks within a 20 kb cis element ∼150 kb proximal to CDKN2A/B. Deleting that segment prevented their induction by TGFβ but not a basal increase driven by methyltransferase inhibition alone. Expression of two CDKN2A/B transcripts was enhanced by dCas9/CRISPR activation targeting either the relevant promoter or the 20 kb cis elements, and this "precise" manipulation diminished RMS cell propagation in vitro. Our findings show crosstalk between methyltransferase inhibition and TGFβ-dependent activation of a remote enhancer to reverse CDKN2A/B silencing. Though focused on CDKN2A/B here, such crosstalk may apply to other TGFβ-responsive genes and perhaps govern this signaling protein's complex effects promoting or blocking cancer.

Positive transcription elongation factor b (P-TEFb) regulates expression of diverse sets of genes within mammalian cells that have implications in several human disease pathogeneses. However, mechanisms of functional regulation of P-TEFb complex through regulation of its stability are poorly known. In this study, we show an important role of C-terminus of Hsc70-interacting protein (CHIP aka STUB1) in regulation of overall level of CDK9 and thus P-TEFb complex within mammalian cells. STUB1 acts as a ubiquitin E3 ligase for proteasomal degradation of CDK9 involving N-terminal lysine 3 (K3) residue. Whereas, overexpression of STUB1 enhances, its knockdown reduces overall CDK9 degradation kinetics within mammalian cells. Interestingly, owing to the same region of binding within CDK9, CyclinT1 protects CDK9 from STUB1-mediated degradation. Factors that cooperatively bind with CyclinT1 to form functional complex also protects CDK9 from degradation by STUB1. Knockdown of STUB1 enhances CDK9 expression and thus P-TEFb complex formation that leads to global increase in RNA polymerase II CTD phosphorylation and transcriptional activation of diverse P-TEFb target genes. Thus, we describe an important functional role of STUB1 in regulation of transcription through modulation of overall level of P-TEFb complex formation within mammalian cells.

Target of rapamycin complex 1 (TORC1) is activated in response to nutrient availability and growth factors, promoting cellular anabolism and proliferation. To explore the mechanism of TORC1-mediated proliferation control, we performed a genetic screen in fission yeast and identified Sfp1, a zinc-finger transcription factor, as a multicopy suppressor of temperature-sensitive TORC1 mutants. Our observations suggest that TORC1 phosphorylates Sfp1 and protects Sfp1 from proteasomal degradation. Transcription analysis revealed that Sfp1 positively regulates genes involved in ribosome production together with two additional transcription factors, Ifh1/Crf1 and Fhl1. Ifh1 physically interacts with Fhl1, and the nuclear localization of Ifh1 is regulated in response to nutrient levels in a manner dependent on TORC1 and Sfp1. Taken together, our data suggest that the transcriptional regulation of the genes involved in ribosome biosynthesis by Sfp1, Ifh1, and Fhl1 is one of the key pathways through which nutrient-activated TORC1 promotes cell proliferation.