Scaling of cellular proteome with ploidy

Ploidy changes are frequent in nature and contribute to evolution, functional specialization and tumorigenesis. Analysis of model organisms of different ploidies revealed that increased ploidy leads to an increase in cell and nuclear volume, reduced proliferation, metabolic changes, lower fitness, and increased genomic instability, but the underlying mechanisms remain poorly understood. To investigate how the gene expression changes with cellular ploidy, we analyzed isogenic series of budding yeasts from 1N to 4N. We show that mRNA and protein abundance scales allometrically with ploidy, with tetraploid cells showing only threefold increase in proteins compared to haploids. This ploidy-specific scaling occurs via decreased rRNA and ribosomal protein abundance and reduced translation. We demonstrate that the Tor1 activity is reduced with increasing ploidy, which leads to rRNA gene repression via a novel Tor1-Sch9-Tup1 signaling pathway. mTORC1 and S6K activity are also reduced in human tetraploid cells and the concomitant increase of the Tup1 homolog Tle1 downregulates the rDNA transcription. Our results revealed a novel conserved mTORC1-S6K-Tup1/Tle1 pathway that ensures proteome remodeling in response to increased ploidy.

Comprehensive Profiling of Mammalian Tribbles Interactomes Implicates TRIB3 in Gene Repression

The three human Tribbles (TRIB) pseudokinases have been implicated in a plethora of signaling and metabolic processes linked to cancer initiation and progression and can potentially be used as biomarkers of disease and prognosis. While their modes of action reported so far center around protein–protein interactions, the comprehensive profiling of TRIB interactomes has not been reported yet. Here, we have developed a robust mass spectrometry (MS)-based proteomics approach to characterize Tribbles’ interactomes and report a comprehensive assessment and comparison of the TRIB1, -2 and -3 interactomes, as well as domain-specific interactions for TRIB3. Interestingly, TRIB3, which is predominantly localized in the nucleus, interacts with multiple transcriptional regulators, including proteins involved in gene repression. Indeed, we found that TRIB3 repressed gene transcription when tethered to DNA in breast cancer cells. Taken together, our comprehensive proteomic assessment reveals previously unknown interacting partners and functions of Tribbles proteins that expand our understanding of this family of proteins. In addition, our findings show that MS-based proteomics provides a powerful tool to unravel novel pseudokinase biology.

Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor

The RNA-guided CRISPR-associated Cas9 proteins have been widely applied in programmable genome recombination, base editing or gene regulation in both prokaryotes and eukaryotes. SpCas9 from Streptococcus pyogenes is the most extensively engineered Cas9 with robust and manifold functionalities. However, one inherent limitation of SpCas9 is its stringent 5′-NGG-3′ PAM requirement that significantly restricts its DNA target range. Here, to repurpose SpCas9 as a universal gene repressor, we generate and screen variants of the deactivated SpCas9 (SpdCas9) with relaxed 5′-CAT-3′ PAM compatibility that can bind to the start codon ATG of almost any gene. Stepwise structure-guided mutations of the PAM-interacting residues and auxiliary PAM-proximal residues of the SpdNG (5′-NG-3′ PAM) create a PAM-flexible variant SpdNG-LWQT that preferentially accommodates 5′-NRN-3′ PAMs. SpdNG-LWQT is demonstrated to be effective in gene repression with the advantage of customizable sgRNA design in both Escherichia coli and Saccharomyces cerevisiae. This work validates the feasibility of purposeful PAM expansion of Cas9 towards signature PAMs and establishes a universal SpdCas9-based gene repressor.

Transcriptional repressor Gal80 recruits corepressor complex Cyc8–Tup1 to structural genes of the Saccharomyces cerevisiae GAL regulon

Under non-inducing conditions (absence of galactose), yeast structural genes of the GAL regulon are repressed by Gal80, preventing interaction of Gal4 bound to UASGAL promoter motifs with general factors of the transcriptional machinery. In this work, we show that Gal80 is also able to interact with histone deacetylase-recruiting corepressor proteins Cyc8 and Tup1, indicating an additional mechanism of gene repression. This is supported by our demonstration that a lexA–Gal80 fusion efficiently mediates repression of a reporter gene with an upstream lexA operator sequence. Corepressor interaction and in vivo gene repression could be mapped to a Gal80 minimal domain of 65 amino acids (aa 81-145). Site-directed mutagenesis of selected residues within this domain showed that a cluster of aromatic-hydrophobic amino acids (YLFV, aa 118-121) is important, although not solely responsible, for gene repression. Using chromatin immunoprecipitation, Cyc8 and Tup1 were shown to be present at the GAL1 promoter in a wild-type strain but not in a gal80 mutant strain under non-inducing (derepressing) growth conditions. Expression of a GAL1–lacZ fusion was elevated in a tup1 mutant (but not in a cyc8 mutant) grown in derepressing medium, indicating that Tup1 may be mainly responsible for this second mechanism of Gal80-dependent gene repression.

ERK signaling dissolves ERF Repression Condensates in Living Embryos

ABSTRACTPhase separation underlies the organization of the nucleus, including the biogenesis of nucleoli and the packaging of heterochromatin. Here we explore the regulation of transcription factor condensates involved in gene repression by ERK signaling in gastrulating embryos of a simple proto-vertebrate (Ciona). ERK signaling induces nuclear export of the transcriptional repressor ERF, which has been linked to various human developmental disorders. Using high resolution imaging we show that ERF is localized within discrete nuclear condensates that dissolve upon ERK activation. Interestingly, we observe dynamic pulses of assembly and dissociation during the cell cycle, providing the first visualization of a nuclear phase separation process that is regulated by cell signaling. We discuss the implications of these observations for producing sharp on/off switches in gene activity and suppressing noise in cell-cell signaling events.

Genetic screen for suppressors of increased silencing in rpd3 mutants in Saccharomyces cerevisiae identifies a potential role for H3K4 methylation

Several studies have identified the paradoxical phenotype of increased heterochromatic gene silencing at specific loci that results from deletion or mutation of the histone deacetylase (HDAC) gene RPD3. To further understand this phenomenon, we conducted a genetic screen for suppressors of this extended silencing phenotype at the HMR locus in Saccharomyces cerevisiae. Most of the mutations that suppressed extended HMR-silencing in rpd3 mutants without completely abolishing silencing were identified in the histone H3 lysine 4 methylation (H3K4me) pathway, specifically in SET1, BRE1 and BRE2. These second site mutations retained normal HMR silencing, therefore appear to be specific for the rpd3Δ extended silencing phenotype. As an initial assessment of the role of H3K4 methylation in extended silencing, we rule out some of the known mechanisms of Set1p/H3K4me mediated gene repression by HST1, HOS2 and HST3 encoded HDACs. Interestingly, we demonstrate that the RNA Polymerase III complex remains bound and active at the HMR-tDNA in rpd3 mutants despite silencing extending beyond the normal barrier. We discuss these results as they relate to the interplay among different chromatin modifying enzyme functions and the importance of further study of this enigmatic phenomenon.

Variant PCGF1-PRC1 links PRC2 recruitment with differentiation-associated transcriptional inactivation at target genes

Polycomb repressive complexes-1 and -2 (PRC1 and 2) silence developmental genes in a spatiotemporal manner during embryogenesis. How Polycomb group (PcG) proteins orchestrate down-regulation of target genes upon differentiation, however, remains elusive. Here, by differentiating embryonic stem cells into embryoid bodies, we reveal a crucial role for the PCGF1-containing variant PRC1 complex (PCGF1-PRC1) to mediate differentiation-associated down-regulation of a group of genes. Upon differentiation cues, transcription is down-regulated at these genes, in association with PCGF1-PRC1-mediated deposition of histone H2AK119 mono-ubiquitination (H2AK119ub1) and PRC2 recruitment. In the absence of PCGF1-PRC1, both H2AK119ub1 deposition and PRC2 recruitment are disrupted, leading to aberrant expression of target genes. PCGF1-PRC1 is, therefore, required for initiation and consolidation of PcG-mediated gene repression during differentiation.

MAX mutant small-cell lung cancers exhibit impaired activities of MGA-dependent noncanonical polycomb repressive complex

The MYC axis is disrupted in cancer, predominantly through activation of the MYC family oncogenes but also through inactivation of the MYC partner MAX or of the MAX partner MGA. MGA and MAX are also members of the polycomb repressive complex, ncPRC1.6. Here, we use genetically modified MAX-deficient small-cell lung cancer (SCLC) cells and carry out genome-wide and proteomics analyses to study the tumor suppressor function of MAX. We find that MAX mutant SCLCs have ASCL1 or NEUROD1 or combined ASCL1/NEUROD1 characteristics and lack MYC transcriptional activity. MAX restitution triggers prodifferentiation expression profiles that shift when MAX and oncogenic MYC are coexpressed. Although ncPRC1.6 can be formed, the lack of MAX restricts global MGA occupancy, selectively driving its recruitment toward E2F6-binding motifs. Conversely, MAX restitution enhances MGA occupancy to repress genes involved in different functions, including stem cell and DNA repair/replication. Collectively, these findings reveal that MAX mutant SCLCs have either ASCL1 or NEUROD1 or combined characteristics and are MYC independent and exhibit deficient ncPRC1.6-mediated gene repression.

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss of function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.