Although several studies have applied single-cell approaches to explore gene expression changes in aged brains, they were limited by the relatively shallow sampling of brain cell populations, and thus may have failed to capture aspects of the molecular signatures and dynamics of rare cell types associated with aging and diseases. Here, we set out to investigate the age-dependent dynamics of transcription and chromatin accessibility across diverse brain cell types. With EasySci, an extensively improved single-cell combinatorial indexing strategy, we profiled ~1.5 million single-cell transcriptomes and ~400,000 single-cell chromatin accessibility profiles across mouse brains spanning different ages, genotypes, and both sexes. With a novel computational framework designed for characterizing cellular subtypes based on the expression of both genes and exons, we identified > 300 cell subtypes and deciphered the underlying molecular programs and spatial locations of rare cell types (e.g., pinealocytes, tanycytes) and subtypes. Leveraging these data, we generate a global readout of age-dependent cell population dynamics with high cellular subtype resolution, providing insights into cell types that expand (e.g., rare astrocytes and vascular leptomeningeal cells in the olfactory bulb, reactive microglia and oligodendrocytes) or are depleted (e.g., neuronal progenitors, neuroblasts, committed oligodendrocyte precursors) as age progresses. Furthermore, we explored cell-type-specific responses to genetic perturbations associated with Alzheimer's disease (AD) and identify rare cell types depleted (e.g., mt-Cytb+, mt-Rnr2+ choroid plexus epithelial cells) or enriched (e.g., Col25a1+, Ndrg1+ interbrain and midbrain neurons) in both AD models. Key findings are consistent between males and females, validated across the transcriptome, chromatin accessibility, and spatial analyses. Finally, we profiled a total of 118,240 single-nuclei transcriptomes from twenty-four human brain samples derived from control and AD patients, revealing highly cell-type-specific and region-specific gene expression changes associated with AD pathogenesis. Critical AD-associated gene signatures were validated in both human and mice. In summary, these data comprise a rich resource for exploring cell-type-specific dynamics and the underlying molecular mechanisms in both normal and pathological mammalian aging.

Temporal changes in transcription programs are coupled to control of cell growth and division. We here report that Mediator, a conserved coregulator of eukaryotic transcription, is part of a regulatory pathway that controls mitotic entry in fission yeast. The Mediator subunit cyclin-dependent kinase 8 (Cdk8) phosphorylates the forkhead 2 (Fkh2) protein in a periodic manner that coincides with gene activation during mitosis. Phosphorylation prevents degradation of the Fkh2 transcription factor by the proteasome, thus ensuring cell cycle-dependent variations in Fkh2 levels. Interestingly, Cdk8-dependent phosphorylation of Fkh2 controls mitotic entry, and mitotic entry is delayed by inactivation of the Cdk8 kinase activity or mutations replacing the phosphorylated serine residues of Fkh2. In addition, mutations in Fkh2, which mimic protein phosphorylation, lead to premature mitotic entry. Therefore, Fkh2 regulates not only the onset of mitotic transcription but also the correct timing of mitotic entry via effects on the Wee1 kinase. Our findings thus establish a new pathway linking the Mediator complex to control of mitotic transcription and regulation of mitotic entry in fission yeast. Signaling pathways can control the activation of gene expression programs and thereby regulate cell fate determination. In embryonic stem cells, certain gene expression programs allow the cells to self-renew whereas other programs trigger differentiation into specific cell types as a response to developmental signaling (58). Elucidation of how temporal changes in transcription programs are coupled to control of cell growth and division is therefore of fundamental importance for our understanding of developmental processes.Global gene transcription analysis in yeasts and higher eukaryotes has revealed that a significant proportion of the genome is transcribed in a periodic manner during cell cycle progression (5,15,34,49,55). Correct periodic regulation is believed to play a critical role in normal cell proliferation, and the genes are often deregulated in different forms of cancer (6). Depending on the organism, the number of periodically expressed genes ranges from ϳ400 to more than 1,000 (5, 6, 56). These include genes with well-established roles in cell cycle progression, such as those encoding cyclins, transcription factors and protein kinases.A cluster named CLB2 in budding yeast (35 genes) or cluster 1 in fission yeast (87 genes) is periodically expressed and activated at mitosis and repressed in G 1 of the next cell cycle (4,5,34,56). In budding yeast, transcription of the CLB2 cluster is controlled by the forkhead proteins Fkh1 and Fkh2, which cooperate with Mcm1 (a MADS box protein) and the Ndd1 coactivator (27, 28). In fission yeast, forkhead proteins Sep1 and Fkh2 and the MADS box protein Mbx1 regulate mitotic transcription (12,13,49,53). Deletion of the sep1 gene results in reduced transcription, whereas overexpression of sep1 induces expression of the same genes. In contrast, deletion of fkh2 causes elevated levels of g...

Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression.

Highlights d DNA polymerase z promotes RNA-templated DNA repair and modification d cDNA-, unlike RNA-templated DNA repair, requires endclipping function d RNA-mediated DNA modification proceeds in the absence of recombination genes d Mismatch repair facilitates accurate repair with template RNA

Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events.