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One of the most distinctive features of eukaryotic chromosomes is the bundling of DNA together with functionally associated RNA and proteins in chromatin. This allows huge amounts of DNA to be packed inside the very tiny space of the nucleus, and alterations in the structure of chromatin enable access to the DNA for transcription (“reading” genes by production of RNA copies). Much of the current knowledge of chromatin structure and regulation comes from studies of Drosophila melanogaster. When the chromatin structure is open the transcription of a gene can start after recruitment of the necessary factors. The main enzyme for gene transcription is Polymerase II (Pol II). For successful gene transcription, Pol II must not only be recruited to the gene’s promoter, but also escape from a pausing state which occurs soon after transcription initiation. CBP/P300 is one of the co-activators involved in transcriptional activation. In the studies this thesis is based upon, my colleagues and I (hereafter we) discovered a new function for CBP in transcription activation. Using high throughput sequencing techniques, we found that CBP directly stimulates recruitment of Pol II to promoters, and facilitates its release from the paused state, enabling progression to the elongation stage of transcription.

For cells to remember their identity following division during development, the transcriptional state of genes must be transmitted. Intensively studied players involved in this memory are the Polycomb group (PcG) proteins, responsible for maintaining the repressed state of important developmental genes. The core members are Polycomb repressive complex 1 and 2 (PRC1 and PRC2), which are recruited in flies through poorly known mechanisms to target genes by so-called Polycomb response elements (PREs). Using Drosophila mutant cell lines, we showed that (in contrast to previous models) some PREs can recruit PRC1 even when PRC2 is absent. We also observed that at many PREs, PRC1 is needed for recruitment of PRC2 and concluded that targeting PRC complexes to PREs is a much more flexible and variable process than previously thought.

Some phenotypic effects of environmental changes can be transferred to subsequent generations. Previous efforts to identify the mechanisms involved have focused on material (mainly, but not only, DNA) transferred through germ cells. However, organisms’ microbiomes are also transferred to the next generation. Thus, to investigate possible contributions of microbiomes to such transfer, we used fruit flies as the microbiomes they inherit can be easily controlled. We altered some parents’ environmental conditions by lowering the temperature, then grew offspring that received microbiomes from cold-treated and control parents in control conditions and compared their transcriptional patterns. Our results suggest that most of the crosstalk between the microbiome and the fly happens in the gut, and that further investigation of this previously unsuspected mode of inheritance is warranted.

Antagonistic interactions between Polycomb Group (PcG) and Trithorax Group (TrxG) proteins orchestrate the expression of key developmental genes. Distinct maternally loaded repressors establish the silenced state of these genes in cells where they should not be expressed and later PcG proteins sense whether a target gene is inactive and maintain the repression throughout multiple cell divisions. PcG proteins are targeted to genes by DNA elements called Polycomb Response Elements (PREs). The proteins form two major classes of complexes, namely Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2). Mechanistic details of Polycomb repression are not fully understood, however, tri-methylation of Lysine 27 of histone H3 (H3K27me3) is essential for this process. Using Drosophila cell lines deficient for either PRC1 or PRC2, I investigated the role of H3K27 methylation and the interdependence of PRC1 complexes for their recruitment to PREs. My results indicate that recruitment of PcG complexes to PREs proceed via multiple pathways and that H3K27 methylation is not needed for their targeting. However, the methylation is required to stabilize interactions of PRE-anchored PcG complexes with surrounding chromatin.

TrxG proteins prevent erroneous repression of Polycomb target genes where these genes need to be expressed. Ash1 is a TrxG protein which binds Polycomb target genes when they are transcriptionally active. It contains a SET domain which methylates Lysine 36 of histone H3 (H3K36). In vitro, histone H3 methylated at K36 is a poor substrate for H3K27 methylation by PRC2. This prompted a model where Ash1 counteracts Polycomb repression through H3K36 methylation. However, this model was never tested in vivo and does not consider several experimental observations. First, in the ash1 mutant flies the bulk H3K36me2/H3K36me3 levels remain unchanged. Second, in Drosophila, there are two other H3K36-specific histone methyltransferases, NSD and Set2, which should be capable to inhibit PRC2. Third, Ash1 contains multiple evolutionary conserved domains whose roles have not been investigated. Therefore, I asked whether H3K36 methylation is critical for Ash1 to counteract Polycomb repression in vivo and whether NSD and Set2 proteins contribute to this process. I used flies lacking endogenous histone genes and complemented them with transgenic histone genes where Lysine 36 is replaced by Arginine. In these animals, I assayed erroneous repression of HOX genes as a readout for erroneous Polycomb repression. I used the same readout in the NSD or Set2 mutant flies. I also asked if other conserved domains of Ash1 are essential for its function. In addition to SET and domain, Ash1 contains three AT hook motifs as well as BAH and PHD domains. I genetically complemented ash1 loss of function animals with transgenic Ash1 variants, in each, one domain of Ash1 is deleted. I found that Ash1 is the only H3K36-specific histone methyltransferase which counteracts Polycomb repression in Drosophila. My findings suggest that the model, where Ash1 counteracts PcG repression by inhibiting PRC2 via methylation of H3K36, has to be revised. I also showed that, in vivo, Ash1 acts as a multimer and requires SET, BAH and PHD domains to counteract Polycomb repression.

This work led to two main conclusions. First, trimethylation of H3K27 is not essential for targeting PcG proteins to PREs but acts afterwards to stabilize their interaction with the chromatin of the neighboring genes. Second, while SET domain is essential for Ash1 to oppose Polycomb repression, methylation of H3K36 does not play a central role in the process.