Modulation of the transcription regulatory program in yeast cells committed to sporulation

BACKGROUND: Meiosis in budding yeast is coupled to the process of sporulation, where the four haploid nuclei are packaged into a gamete. This differentiation process is characterized by a point of transition, termed commitment, when it becomes independent of the environment. Not much is known about the mechanisms underlying commitment, but it is often assumed that positive feedback loops stabilize the underlying gene-expression cascade. RESULTS: We describe the gene-expression program of committed cells. Sporulating cells were transferred back to growth medium at different stages of the process, and their transcription response was characterized. Most sporulation-induced genes were immediately downregulated upon transfer, even in committed cells that continued to sporulate. Focusing on the metabolic-related transcription response, we observed that pre-committed cells, as well as mature spores, responded to the transfer to growth medium in essentially the same way that vegetative cells responded to glucose. In contrast, committed cells elicited a dramatically different response. CONCLUSION: Our results suggest that cells ensure commitment to sporulation not by stabilizing the process, but by modulating their gene-expression program in an active manner. This unique transcriptional program may optimize sporulation in an environment-specific manner

A Dynamic View of Domain-Motif Interactions

Many protein-protein interactions are mediated by domain-motif interaction, where a domain in one protein binds a short linear motif in its interacting partner. Such interactions are often involved in key cellular processes, necessitating their tight regulation. A common strategy of the cell to control protein function and interaction is by post-translational modifications of specific residues, especially phosphorylation. Indeed, there are motifs, such as SH2-binding motifs, in which motif phosphorylation is required for the domain-motif interaction. On the contrary, there are other examples where motif phosphorylation prevents the domain-motif interaction. Here we present a large-scale integrative analysis of experimental human data of domain-motif interactions and phosphorylation events, demonstrating an intriguing coupling between the two. We report such coupling for SH3, PDZ, SH2 and WW domains, where residue phosphorylation within or next to the motif is implied to be associated with switching on or off domain binding. For domains that require motif phosphorylation for binding, such as SH2 domains, we found coupled phosphorylation events other than the ones required for domain binding. Furthermore, we show that phosphorylation might function as a double switch, concurrently enabling interaction of the motif with one domain and disabling interaction with another domain. Evolutionary analysis shows that co-evolution of the motif and the proximal residues capable of phosphorylation predominates over other evolutionary scenarios, in which the motif appeared before the potentially phosphorylated residue, or vice versa. Our findings provide strengthening evidence for coupled interaction-regulation units, defined by a domain-binding motif and a phosphorylated residue

Macrophage-Secreted CSF1 Transmits a Calorie Restriction-Induced Self-Renewal Signal to Mammary Epithelial Stem Cells

Calorie restriction enhances stem cell self-renewal in various tissues, including the mammary gland. We hypothesized that similar to their intestinal counterparts, mammary epithelial stem cells are insulated from sensing changes in energy supply, depending instead on niche signaling. The latter was investigated by subjecting cultures of mammary epithelial stem cells for 8 days to in vivo paracrine calorie-restriction signals collected from a 4-day-conditioned medium of individual mammary cell populations. Conditioned medium from calorie-restricted non-epithelial cells induced latent cell propagation and mammosphere formation—established markers of stem cell self-renewal. Combined RNA-Seq, immunohistochemistry and immunofluorescence analyses of the non-epithelial population identified macrophages and secreted CSF1 as the energy sensor and paracrine signal, respectively. Calorie restriction-induced pStat6 expression in macrophages suggested that skewing to the M2 phenotype contributes to the sensing mechanism. Enhancing CSF1 signaling with recombinant protein and interrupting the interaction with its highly expressed receptor in the epithelial stem cells by neutralizing antibodies were both affected stem cell self-renewal. In conclusion, combined in vivo, in vitro and in silico studies identified macrophages and secreted CSF1 as the energy sensor and paracrine transmitter, respectively, of the calorie restriction-induced effect on mammary stem cell self-renewal

Experimental evidence of phosphorylation-mediated modulation of domain-motif interactions.

<p>*Numbers in parentheses indicate the distance between the motif and the proximal phosphorylation site/s.</p

Phosphorylation events as double switches.

<p>(<b>A</b>) A protein (black horizontal line) includes a segment that matches two sequence patterns: the first is typical for SH3 domain binding (green), and the second typifies SH2 domain binding (red). The non-phosphorylated form binds SH3 and not SH2 (upper), while phosphorylation inverts the binding preferences (lower). (<b>B</b>) Specificity switches within the PDZ domain family. A protein (black horizontal line) includes a segment that may bind distinct PDZ domains (upper). The non-phosphorylated form binds PDZ^a and not PDZ^b, while phosphorylation inverts these binding preferences (lower).</p

Frequency of various phylogenetic traces of motif-phosphorylation coupling.

<p>The stacked-bar graph details the relative frequency of the three possible phylogenetic traces of the interaction-regulation units (for either intra-motif phosphorylation or near-motif phosphorylation sites): (i) co-appearance of the motif and the potentially phosphorylated residue in the same organism (grey), (ii) the motif appeared before the potentially phosphorylated residue (cyan) (iii) the potentially phosphorylated residue appeared before the motif (red). For each domain we tested if the distribution of the various scenarios deviates from random by a χ² test. Asterisks denote statistically significant results (based on <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341.s008" target="_blank">Table S6</a>).</p

Dual sequence patterns used for the identification of potential double switches in human proteins.

<p>Column titles include sequence patterns for motifs that bind SH3 or class I WW domains (in red), and row titles include sequence patterns for motifs that bind different types of SH2 domains, upon motif phosphorylation (in blue). Each table cell includes a merged sequence pattern that hints at a dual binding potential of the motif to both SH2 and SH3 (or WW) domains. The columns under class I WW and SH3-1 titles represent the strict analysis scheme. Sequence patterns were extracted from the ELM database <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002341#pcbi.1002341-Gould1" target="_blank">[14]</a>. (i) An example for a dual motif. The PP.Y.N. sequence pattern is composed of the SH2^Grb2 Y.N. and the class I WW PP.Y patterns. (ii) Note that this sequence pattern encompasses seven positions.</p