The Brar and Ünal Labs

TRANSCRIPTIONAL CONTROL OF CELLULAR REJUVENATION AND DIFFERENTIATION

Leveraging Meiosis-Specific Rejuvenation Pathways to Extend Lifespan

The beneficial effects of gametogenesis on cellular aging and health have been primarily studied in the budding yeast S. cerevisiae. This organism shares striking similarities with metazoans regarding the cellular manifestations of aging including perturbed nuclear and nucleolar integrity, mitochondrial dysfunction, decreased lysosomal acidity and loss of protein homeostasis. Over time, these types of age-associated damage eventually lead to cell death. Interestingly, when yeast cells undergo meiotic differentiation, gametes no longer contain any of the organelle defects that were present in the aged precursor cells. In fact, each of the four gametes is born young, independent of the progenitor’s age (Ünal et al, 2011). These findings demonstrate that meiotic differentiation naturally resets the aging clock by eliminating age-associated damage. The meiotic transcription factor (TF) Ndt80 is the key driver of gamete rejuvenation, however the subset of its gene targets that extend lifespan remains unknown. To identify the complement of genes involved in cellular rejuvenation, we have conducted an unbiased genetic screen using custom-made meiotic cDNA libraries (Sing et al, 2022). This gain-of-function screen led to 80 “rejuvenation candidates“ that we are currently examining.

Repurposing Endogenous Stress Pathway Activity to Drive Cellular Rejuvenation (in collaboration with the Brar Lab)

Ectopic expression of Ndt80 can extend the lifespan of mitotically growing aged cells, demonstrating that aspects of meiotic rejuvenation are portable and can be repurposed to reverse the detrimental effects of age in somatic cells (Ünal et al, 2011).Interestingly, almost every known stress response pathway and its associated TF is expressed during discrete windows of gametogenesis in yeast (Brar et al, 2012). Many stress response TFs are induced, directly or indirectly, by Ndt80, and thus are candidates for driving cellular rejuvenation.

Both beneficial and harmful effects of stress pathways on cellular quality control and longevity have been established; however, the underlying basis for this yin-yang relationship remains enigmatic. We propose that this results from dosage effects—these pathways can be beneficial when transiently activated at low levels (a phenomenon known as hormesis), as seen during gametogenesis but are toxic when hyper-activated, as is often the case in the non-physiological contexts in which they are typically studied. Gametogenesis thus provides an unmined discovery platform in which to uncover the physiological roles of stress pathways in a context without toxic, saturated or persistent inputs.

We propose that gametogenesis is a physiological manifestation of hormesis, whereby moderate and transient stress response pathway activation drives cellular rejuvenation. To test this hypothesis, we are probing the transcriptional subprograms that are driven by key stress response TFs during gametogenesis to identify gene targets with roles in cellular quality control and meiotic rejuvenation. We are also determining the degree of stress pathway activation during gametogenesis with the ultimate goal of engineering mitotic cells to mimic these programs.

Investigating LUTI-based Gene Regulation in Meiosis and Beyond (in collaboration with the Brar Lab)

Only a fraction of the Ndt80-dependent transcripts are canonical; i.e. fully mapping to one of ~6000 annotated open reading frames (ORFs) within the yeast genome. The rest contain alternative transcription start sites within or outside of the known ORFs (Brar et al 2012). Among the newly identified meiotic transcripts, we have dissected the mechanism of action for a distinct class that we termed LUTIs – for Long Undecoded Transcript Isoforms (Chen and Tresenrider 2017; Chia et al 2017; Cheng and Otto et al 2018; Tresenrider et al 2021). LUTIs are expressed from an alternative promoter, which is located upstream of the canonical promoter near the coding region. Activation of the LUTI promoter downregulates protein synthesis by a two-pronged mechanism: First, transcription from the LUTI promoter establishes chromatin that silences the canonical promoter in cis, thereby blocking the expression of the protein-coding transcript isoform. Second, the LUTI mRNA cannot be translated into a functional protein, because the upstream open reading frames (uORFs) in its 5’ leader restrict ribosome access to the coding region. Consequently, a single transcription factor can synchronously activate and repress protein synthesis for distinct sets of genes, depending whether it binds to a canonical or an alternative LUTI promoter, respectively. To identify new regulators of LUTI-based regulation, we have taken an unbiased genetic approach by fusing the 5’ leader of a LUTI-regulated gene in front of a reporter gene, and by selecting mutants that gain the ability to express the reporter despite elevated levels of LUTI expression (LUTI suppressors). This approach has enabled us to uncover new players in LUTI-based regulation. In parallel, we are investigating the functional consequences of LUTI-based regulation in meiosis by examining the genes regulated by this mechanism.

Extending our initial discoveries in budding yeast, we have demonstrated that the human proto-oncogene MDM2 is regulated by a LUTI (Hollerer et al. 2019). As human embryonic stem cells (hESCs) differentiate into endoderm, MDM2 LUTI expression is upregulated while the protein-coding MDM2 transcript shows the opposite trend. Because MDM2 is the principal antagonist of p53, a tumor suppressor that promotes mesendodermal gene expression and inhibits pluripotency factors, we propose that MDM2 LUTI is important for proper endoderm differentiation. To this end, we are testing whether the LUTI-based repression requires the same components used in budding yeast and whether MDM2 LUTI is necessary for endoderm differentiation. We are also developing reporter systems to screen for new players of LUTI-based regulation in human cells.