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The aim of the research group is to elucidate the molecular mechanisms by which the cell integrates multiple signals to achieve a binary cell fate decision – whether or not to differentiate. Unfolding these mechanisms is critical for the understanding of how cell specialization leads to multicellularity during development, and how impaired signaling can cause abnormal development and diseases such as cancer. The budding yeast S. cerevisiae is an ideal model system to study this problem. In response to a combination of extracellular and intracellular cues budding yeast undergoes a highly conserved cell differentiation program called gametogenesis. Since entry into gametogenesis is controlled by only two master regulators in this model organism, there is unique opportunity to study the molecular and quantitative aspects of this cell fate

Keywords: long noncoding RNAs, transcription, chromatin, cell fate, cell cycle, meiosis, gametogenesis, and yeast

Theme 1: How do long noncoding RNAs control gene expression and cell fate?

Transcription of two long noncoding RNAs controls the cell fate decision leading to gametogenesis.

The master regulatory genes, IME1 and IME4, drive the cell fate decision leading to gametogenesis in budding yeast. This cell fate is also controlled by the mating-type locus. In order to initiate gametogenesis, diploid yeast cells need to express both mating type genes (MATa and MATα). The combined gene product of MATα and MATa, the a1-α2 heterodimer, inhibits RME1 transcription in diploid cells (Fig.1A), but how in haploid cells Rme1 represses IME1 transcription was never understood. We discovered that in cells with the haploid mating-type, expression of IME1 is inhibited by a long noncoding RNA (lncRNA) called IRT1 that is located in the IME1 promoter and induced by the Rme1 transcription factor (Fig.1B). Transcription of this lncRNA recruits the Set2 histone methyltransferase and Set3 histone deacetylase complex to establish repressive chromatin in the IME1 promoter (Fig.1B).  

Figure 1. Schematic overview of how the mating type signal controls entry into gametogenesis.

Figure 1. Schematic overview of how the mating type signal controls entry into gametogenesis.

The chromatin remodeling enzymes Set2 and Set3 are highly conserved from yeast to human, and lncRNAs are also abundant in higher eukaryotes. This raises the interesting possibility that long noncoding RNAs represses gene expression by a conserved mechanism. In addition to IME1, expression of the master regulator IME4 is antagonized by an antisense transcript in haploid cells (Fig. 1B). This antisense transcript in turn is repressed by a1-α2 allowing expression of IME4 in diploid cells (Fig. 1B). The mating-type dependence of IME4 expression led to a second key discovery from this study. When the expression of both the lncRNA in the IME1 promoter and the IME4 antisense transcript is inhibited, cells expressing the haploid mating-type enter gametogenesis with kinetics that are indistinguishable from cells expressing both mating-types (Fig. 1). Thus, transcription of two lncRNAs governs mating-type control of gametogenesis in yeast.

Current Research directions.
The promoter of the master transcription factor for entry into gametogenesis, IME1, integrates nutrient and mating-type signals to make the binary cell fate decision (Fig. 2A). Whole genome transcriptome analysis identified two long ncRNA in the IME1 promoter (Fig. 2B). In cells of the haploid mating-type IME1 is repressed by the lncRNA IRT1 (Fig. 2C). Induction of IRT1 by the transcription factor Rme1 recruits the methyltransferases Set1 and Set2 to methylate histone H3 at lysine 4 and 36. These marks are recognized by the histone deacetylase complexes Set3C and Rpd3(S) and establish a repressive chromatin state in the IME1 promoter (Fig. 2C). The mechanism of cell fate control described here provides a starting point for further investigations.

The goals are: (1) Screen for novel factors required for gene repression by lncRNAs; (2) Investigate how widespread gene repression by lncRNAs is across the genome.

Figure 2. (A) Overview of the signals controlling the IME1 promoter. (B) Annotation of two ncRNAs in the IME1 promoter. (C) Model describing the mechanism by which the lncRNA IRT1 represses IME1.
Figure 2. (A) Overview of the signals controlling the IME1 promoter. (B) Annotation of two ncRNAs in the IME1 promoter. (C) Model describing the mechanism by which the lncRNA IRT1 represses IME1.

Theme 2: How do master regulatory genes integrate multiple signals?

Multiple signals are required to initiate gametogenesis in budding yeast: nutrients such as nitrogen compounds and glucose need to be absent from the medium, cells need to respire (use a non-fermentable carbon source) and must be diploid. These signals drive entry into gametogenesis and all converge on the IME1 promoter (Fig. 1A). This promoter is one of the largest and highly regulated promoters in budding yeast. To understand how these signals are integrated to make a binary cell fate decision, it is essential to identify all the molecular players and pathways involved in acting at the IME1 promoter. The lab will use proteomic and genetic approaches to identify these factors.

The aims outlined here form the beginning of a systematic investigation on how multiple signals are integrated to drive cell fate decisions. It is often not well understood how in higher eukaryotes master regulatory genes make binary decisions that are important for development of an organism. Aberrant expression of these master genes due to impaired signaling can cause abnormal the development and diseases such as cancer. The IME1 promoter serves as a basic model system for studying signal integration at master regulatory genes. The findings from these studies could shed light on how complex promoters are regulated in higher eukaryotes.
© Van Werven Lab 2014 Last updated November, 2016