The picky mTORC1 in metabolic enzyme degradation.

In this issue of Molecular Cell, Yi et al.1 demonstrate that reduced mTORC1 activity induces the CTLH E3 ligase-dependent degradation of HMGCS1, an enzyme in the mevalonate pathway, thus revealing a unique connection between mTORC1 signaling and the degradation of a specific metabolic enzyme via the ubiquitin-proteasome system.

FAM120A couples SREBP-dependent transcription and splicing of lipogenesis enzymes downstream of mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that stimulates macromolecule synthesis through transcription, RNA processing, and post-translational modification of metabolic enzymes. However, the mechanisms of how mTORC1 orchestrates multiple steps of gene expression programs remain unclear. Here, we identify family with sequence similarity 120A (FAM120A) as a transcription co-activator that couples transcription and splicing of de novo lipid synthesis enzymes downstream of mTORC1-serine/arginine-rich protein kinase 2 (SRPK2) signaling. The mTORC1-activated SRPK2 phosphorylates splicing factor serine/arginine-rich splicing factor 1 (SRSF1), enhancing its binding to FAM120A. FAM120A directly interacts with a lipogenic transcription factor SREBP1 at active promoters, thereby bridging the newly transcribed lipogenic genes from RNA polymerase II to the SRSF1 and U1-70K-containing RNA-splicing machinery. This mTORC1-regulated, multi-protein complex promotes efficient splicing and stability of lipogenic transcripts, resulting in fatty acid synthesis and cancer cell proliferation. These results elucidate FAM120A as a critical transcription co-factor that connects mTORC1-dependent gene regulation programs for anabolic cell growth.

In vitro analysis of RNA polymerase II elongation complex dynamics.

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.

Cell-Cycle Modulation of Transcription Termination Factor Sen1.

Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.

Determinants of Histone H3K4 Methylation Patterns.

Various factors differentially recognize trimethylated histone H3 lysine 4 (H3K4me3) near promoters, H3K4me2 just downstream, and promoter-distal H3K4me1 to modulate gene expression. This methylation "gradient" is thought to result from preferential binding of the H3K4 methyltransferase Set1/complex associated with Set1 (COMPASS) to promoter-proximal RNA polymerase II. However, other studies have suggested that location-specific cues allosterically activate Set1. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments show that H3K4 methylation patterns on active genes are not universal or fixed and change in response to both transcription elongation rate and frequency as well as reduced COMPASS activity. Fusing Set1 to RNA polymerase II results in H3K4me2 throughout transcribed regions and similarly extended H3K4me3 on highly transcribed genes. Tethered Set1 still requires histone H2B ubiquitylation for activity. These results show that higher-level methylations reflect not only Set1/COMPASS recruitment but also multiple rounds of transcription. This model provides a simple explanation for non-canonical methylation patterns at some loci or in certain COMPASS mutants.

Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation.

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-binding protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-associated factor (TAF) subunits recognize downstream promoter elements, act as coactivators, and interact with nucleosomes. In yeast nuclear extracts, transcription induces stable TAF binding to downstream promoter DNA, promoting subsequent activator-independent transcription reinitiation. In vivo, promoter responses to TAF mutations correlate with the level of downstream, rather than overall, Taf1 cross-linking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.

MAPK13 stabilization via m6A mRNA modification limits anticancer efficacy of rapamycin.

N6-adenosine methylation (m6A) is the most abundant mRNA modification that controls gene expression through diverse mechanisms. Accordingly, m6A-dependent regulation of oncogenes and tumor suppressors contributes to tumor development. However, the role of m6A-mediated gene regulation upon drug treatment or resistance is poorly understood. Here, we report that m6A modification of mitogen-activated protein kinase 13 (MAPK13) mRNA determines the sensitivity of cancer cells to the mechanistic target of rapamycin complex 1 (mTORC1)-targeting agent rapamycin. mTORC1 induces m6A modification of MAPK13 mRNA at its 3' untranslated region through the methyltransferase-like 3 (METTL3)-METTL14-Wilms' tumor 1-associating protein(WTAP) methyltransferase complex, facilitating its mRNA degradation via an m6A reader protein YTH domain family protein 2. Rapamycin blunts this process and stabilizes MAPK13. On the other hand, genetic or pharmacological inhibition of MAPK13 enhances rapamycin's anticancer effects, which suggests that MAPK13 confers a progrowth signal upon rapamycin treatment, thereby limiting rapamycin efficacy. Together, our data indicate that rapamycin-mediated MAPK13 mRNA stabilization underlies drug resistance, and it should be considered as a promising therapeutic target to sensitize cancer cells to rapamycin.

Direct Analysis of Phosphorylation Sites on the Rpb1 C-Terminal Domain of RNA Polymerase II.

Dynamic interactions between RNA polymerase II and various mRNA-processing and chromatin-modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code."

Identification of a potent and selective covalent Pin1 inhibitor.

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.

Sex-dependent metabolic remodeling of kidneys revealed by arteriovenous metabolomics.

Sex is a fundamental biological variable important in biomedical research, drug development, clinical trials, and prevention approaches. Among many organs, kidneys are known to exhibit remarkable structural, histological, and pathological differences between sexes. However, whether and how kidneys display distinct metabolic activities between sexes is poorly understood. By developing kidney-specific arteriovenous (AV) metabolomics combined with transcriptomics, we report striking sex differences in both basal metabolic activities and adaptive metabolic remodeling of kidneys after a fat-enriched ketogenic diet (KD), a regimen known to mitigate kidney diseases and improve immunotherapy for renal cancer. At the basal state, female kidneys show highly accumulated aldosterone and various acylcarnitines. In response to the KD, aldosterone levels remain high selectively in females but the sex difference in acylcarnitines disappears. AV data revealed that, under KD, female kidneys avidly take up circulating fatty acids and release 3-hydroxybutyrate (3-HB) whereas male kidneys barely absorb fatty acids but consistently take up 3-HB. Although both male and female kidneys take up gluconeogenic substrates such as glycerol, glutamine and lactate, only female kidneys exhibit net glucose release. Kidney transcriptomics data incompletely predict these sex differences, suggesting post-transcriptional/translational regulation mechanisms. This study provides foundational insights into the sex-dependent and diet-elicited metabolic flexibility of the kidneys in vivo, serving as a unique resource for understanding variable disease prevalence and drug responses between male and female kidneys.

Circulating biomarkers of kidney angiomyolipoma and cysts in tuberous sclerosis complex patients.

Patients with tuberous sclerosis complex (TSC) develop multi-organ disease manifestations, with kidney angiomyolipomas (AML) and cysts being one of the most common and deadly. Early and regular AML/cyst detection and monitoring are vital to lower TSC patient morbidity and mortality. However, the current standard of care involves imaging-based methods that are not designed for rapid screening, posing challenges for early detection. To identify potential diagnostic screening biomarkers of AML/cysts, we performed global untargeted metabolomics in blood samples from 283 kidney AML/cyst-positive or -negative TSC patients using mass spectrometry. We identified 7 highly sensitive chemical features, including octanoic acid, that predict kidney AML/cysts in TSC patients. Patients with elevated octanoic acid have lower levels of very long-chain fatty acids (VLCFAs), suggesting that dysregulated peroxisome activity leads to overproduction of octanoic acid via VLCFA oxidation. These data highlight AML/cysts blood biomarkers for TSC patients and offers valuable metabolic insights into the disease.

A set of Saccharomyces cerevisiae integration vectors for fluorescent dye labeling of proteins.

Protein fusions are frequently used for fluorescence imaging of individual molecules, both in vivo and in vitro. The SNAP, CLIP, HALO (aka HaloTag7), and DHFR protein tags can be linked to small molecule dyes that provide brightness and photo-stability superior to fluorescent proteins. To facilitate fluorescent dye tagging of proteins in the yeast Saccharomyces cerevisiae, we constructed a modular set of vectors with various combinations of labeling protein tags and selectable markers. These vectors can be used in combination to create strains where multiple proteins labeled with different colored dyes can be simultaneously observed.

Comparative characterization of 3D chromatin organization in triple-negative breast cancers.

Triple-negative breast cancer (TNBC) is a malignant cancer subtype with a high risk of recurrence and an aggressive phenotype compared to other breast cancer subtypes. Although many breast cancer studies conducted to date have investigated genetic variations and differential target gene expression, how 3D chromatin architectures are reorganized in TNBC has been poorly elucidated. Here, using in situ Hi-C technology, we characterized the 3D chromatin organization in cells representing five distinct subtypes of breast cancer (including TNBC) compared to that in normal cells. We found that the global and local 3D architectures were severely disrupted in breast cancer. TNBC cell lines (especially BT549 cells) showed the most dramatic changes relative to normal cells. Importantly, we detected CTCF-dependent TNBC-susceptible losses/gains of 3D chromatin organization and found that these changes were strongly associated with perturbed chromatin accessibility and transcriptional dysregulation. In TNBC tissue, 3D chromatin disorganization was also observed relative to the 3D chromatin organization in normal tissues. We observed that the perturbed local 3D architectures found in TNBC cells were partially conserved in TNBC tissues. Finally, we discovered distinct tissue-specific chromatin loops by comparing normal and TNBC tissues. In this study, we elucidated the characteristics of the 3D chromatin organization in breast cancer relative to normal cells/tissues at multiple scales and identified associations between disrupted structures and various epigenetic features and transcriptomes. Collectively, our findings reveal important 3D chromatin structural features for future diagnostic and therapeutic studies of TNBC.

A compendium of chromatin contact maps reflecting regulation by chromatin remodelers in budding yeast.

We herein employ in situ Hi-C with an auxin-inducible degron (AID) system to examine the effect of chromatin remodeling on 3D genome organization in yeast. Eight selected ATP-dependent chromatin remodelers representing various subfamilies contribute to 3D genome organization differently. Among the studied remodelers, the temporary depletions of Chd1p, Swr1p, and Sth1p (a catalytic subunit of the Remodeling the Structure of Chromatin [RSC] complex) cause the most significant defects in intra-chromosomal contacts, and the regulatory roles of these three remodelers in 3D genome organization differ depending on the chromosomal context and cell cycle stage. Furthermore, even though Chd1p and Isw1p are known to share functional similarities/redundancies, their depletions lead to distinct effects on 3D structures. The RSC and cohesin complexes also differentially modulate 3D genome organization within chromosome arm regions, whereas RSC appears to support the function of cohesin in centromeric clustering at G2 phase. Our work suggests that the ATP-dependent chromatin remodelers control the 3D genome organization of yeast through their chromatin-remodeling activities.

Identification of Three Sequence Motifs in the Transcription Termination Factor Sen1 that Mediate Direct Interactions with Nrd1.

The Nrd1-Nab3-Sen1 (NNS) complex carries out the transcription termination of non-coding RNAs (ncRNAs) by RNA polymerase II (Pol II) in yeast, although the detailed interactions among its subunits remain obscure. Here we have identified three sequence motifs in Sen1 that mediate direct interactions with the Pol II CTD interaction domain (CID) of Nrd1, determined the crystal structures of these Nrd1 interaction motifs (NIMs) bound to the CID, and characterized the interactions in vitro and in yeast. Removal of all three NIMs abolishes NNS complex formation and gives rise to ncRNA termination defects.

Selective Kinase Inhibition Shows That Bur1 (Cdk9) Phosphorylates the Rpb1 Linker In Vivo.

Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription. Cdk7/Kin28, Cdk9/Bur1, and Cdk12/Ctk1 phosphorylate the polymerase and other factors to drive the dynamic exchange of initiation and elongation complex components over the transcription cycle. We engineered strains of the yeast Saccharomyces cerevisiae for rapid, specific inactivation of individual kinases by addition of a covalent inhibitor. While effective, the sensitized kinases can display some idiosyncrasies, and inhibition can be surprisingly transient. As expected, inhibition of Cdk7/Kin28 blocked phosphorylation of the Rpb1 C-terminal domain heptad repeats at serines 5 and 7, the known target sites. However, serine 2 phosphorylation was also abrogated, supporting an obligatory sequential phosphorylation mechanism. Consistent with our previous results using gene deletions, Cdk12/Ctk1 is the predominant kinase responsible for serine 2 phosphorylation. Phosphorylation of the Rpb1 linker enhances binding of the Spt6 tandem SH2 domain, and here we show that Bur1/Cdk9 is the kinase responsible for these modifications in vivo.

Hantaan virus surveillance in small mammals at firing points 10 and 60, Yeoncheon, Gyeonggi Province, Republic of Korea.

We used epidemiological data and indirect fluorescent antibody tests to determine the Hantaan virus (HTNV) antibody-positive (Ab+) prevalence in small mammals captured at firing point 10 (FP-10) and firing point 60 (FP-60), Gyeonggi Province, near the demilitarized zone, Republic of Korea (ROK), from 2001 to 2005. We used these data, combined with the partial M segment amplified from HTNV recovered from lung tissues of Apodemus agrarius, to clarify the genetic diversity and phylogenetic relationships among HTNV strains in the ROK. Of the eight species of rodents and one insectivore species captured, A. agrarius accounted for 93.4% and 88.5% at FP-10 and FP-60, respectively. Only two species of rodents, A. agrarius and Micromys minutus, were HTNV Ab+. The overall HTNV Ab+ prevalence for A. agrarius captured at FP-10 and FP-60 was 23.3% (121/520) and 14.5% (94/647), respectively. The hantaviral reverse transcription-polymerase chain reaction-positive rate of Ab+ A. agrarius was 74.2% (167/215), and the phylogenetic trees, based on the 269-nucleotide G2-encoding M segment, demonstrated that HTNV strains from FP-10 and FP-60 were distantly segregated from HTNV of other geographic regions in Korea and China. These data are useful in the development of risk reduction strategies for the prevention of hantavirus infections among military personnel, especially during training or the event of hostilities, and civilian populations.