Body-Wide Inactivation of the Myc-Like Mlx Transcription Factor Network Accelerates Aging and Increases the Lifetime Cancer Incidence.

The "Mlx" and "Myc" transcription factor networks cross-communicate and share many common gene targets. Myc's activity depends upon its heterodimerization with Max, whereas the Mlx Network requires that the Max-like factor Mlx associate with the Myc-like factors MondoA or ChREBP. The current work demonstrates that body-wide Mlx inactivation, like that of Myc, accelerates numerous aging-related phenotypes pertaining to body habitus and metabolism. The deregulation of numerous aging-related Myc target gene sets is also accelerated. Among other functions, these gene sets often regulate ribosomal and mitochondrial structure and function, genomic stability, and aging. Whereas "MycKO" mice have an extended lifespan because of a lower cancer incidence, "MlxKO" mice have normal lifespans and a higher cancer incidence. Like Myc, the expression of Mlx, MondoA, and ChREBP and their control over their target genes deteriorate with age in both mice and humans. Collectively, these findings underscore the importance of lifelong and balanced cross-talk between the two networks to maintain proper function and regulation of the many factors that can affect normal aging.

MYC Binding Near Transcriptional End Sites Regulates Basal Gene Expression, Read-Through Transcription and Intragenic Contacts.

The MYC oncoprotein regulates numerous genes involved in cellular processes such as cell cycle and mitochondrial and ribosomal structure and function. This requires heterodimerization with its partner, MAX, and binding to specific promoter and enhancer elements. Here, we show that MYC and MAX also bind near transcriptional end sites (TESs) of over one-sixth of all annotated genes. These interactions are dose-dependent, evolutionarily conserved, stabilize the normally short-lived MYC protein and regulate expression both in concert with and independent of MYC's binding elsewhere. MYC's TES binding occurs in association with other transcription factors, alters the chromatin landscape, increases nuclease susceptibility and can alter transcriptional read-through, particularly in response to certain stresses. MYC-bound TESs can directly contact promoters and may fine-tune gene expression in response to both physiologic and pathologic stimuli. Collectively, these findings support a previously unrecognized role for MYC in regulating transcription and its read-through via direct intragenic contacts between TESs and promoters.

A nanobody-based strategy for rapid and scalable purification of human protein complexes.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENPEuB. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.

The Myc-Like Mlx Network Impacts Aging and Metabolism.

The "Mlx" and "Myc" Networks share many common gene targets. Just as Myc's activity depends upon its heterodimerization with Max, the Mlx Network requires that the Max-like factor Mlx associate with the Myc-like factors MondoA or ChREBP. We show here that body-wide Mlx inactivation, like that of Myc, accelerates numerous aging-related phenotypes pertaining to body habitus and metabolism. The deregulation of numerous aging-related Myc target gene sets is also accelerated. Among other functions, these gene sets often regulate ribosomal and mitochondrial structure and function, genomic stability and aging. Whereas "MycKO" mice have an extended lifespan because of a lower cancer incidence, "MlxKO" mice have normal lifespans and a somewhat higher cancer incidence. Like Myc, Mlx, MondoA and ChREBP expression and that of their target genes, deteriorate with age in both mice and humans, underscoring the importance of life-long and balanced cross-talk between the two Networks to maintain normal aging.

Premature aging and reduced cancer incidence associated with near-complete body-wide Myc inactivation.

MYC proto-oncogene dysregulation alters metabolism, translation, and other functions in ways that support tumor induction and maintenance. Although Myc+/- mice are healthier and longer-lived than control mice, the long-term ramifications of more complete Myc loss remain unknown. We now describe the chronic consequences of body-wide Myc inactivation initiated postnatally. "MycKO" mice acquire numerous features of premature aging, including altered body composition and habitus, metabolic dysfunction, hepatic steatosis, and dysregulation of gene sets involved in functions that normally deteriorate with aging. Yet, MycKO mice have extended lifespans that correlate with a 3- to 4-fold lower lifetime cancer incidence. Aging tissues from normal mice and humans also downregulate Myc and gradually alter many of the same Myc target gene sets seen in MycKO mice. Normal aging and its associated cancer predisposition are thus highly linked via Myc.

Disruption of Multiple Overlapping Functions Following Stepwise Inactivation of the Extended Myc Network.

Myc, a member of the "Myc Network" of bHLH-ZIP transcription factors, supervises proliferation, metabolism, and translation. It also engages in crosstalk with the related "Mlx Network" to co-regulate overlapping genes and functions. We investigated the consequences of stepwise conditional inactivation of Myc and Mlx in primary and SV40 T-antigen-immortalized murine embryonic fibroblasts (MEFs). Myc-knockout (MycKO) and Myc × Mlx "double KO" (DKO)-but not MlxKO-primary MEFs showed rapid growth arrest and displayed features of accelerated aging and senescence. However, DKO MEFs soon resumed proliferating, indicating that durable growth arrest requires an intact Mlx network. All three KO MEF groups deregulated multiple genes and functions pertaining to aging, senescence, and DNA damage recognition/repair. Immortalized KO MEFs proliferated in Myc's absence while demonstrating variable degrees of widespread genomic instability and sensitivity to genotoxic agents. Finally, compared to primary MycKO MEFs, DKO MEFs selectively downregulated numerous gene sets associated with the p53 and retinoblastoma (Rb) pathways and G2/M arrest. Thus, the reversal of primary MycKO MEF growth arrest by either Mlx loss or SV40 T-antigen immortalization appears to involve inactivation of the p53 and/or Rb pathways.

MTCH2 is a mitochondrial outer membrane protein insertase.

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane ß-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.