SAGA, TFIID and regulation of transcription through chromatin

Abstract

Chromatin has an important role in eukaryotic transcription. Research into this role is ongoing and genome-wide analysis has correlated various histone modifications to multiple elements in active and silent genes, such as enhancers, promoters and coding regions. Modifications often serve to recruit complexes involved in the process of transcription, such as TFIID to H3K4me3 via the TAF3 subunit. Chromatin modifying complexes include the co-activator complex SAGA, which acetylates lysine-9 and -14 on histone H3. The work described in this thesis was aimed at exploring multiple aspects of chromatin signaling. Both the writing and the reading of several histone modifications were investigated, to better understand the role of chromatin in transcription. In chapter 2 we set out to study crosstalk of H3K4me3 and acetylation of histone H3 via SAGA’s subunit SGF29 in mammalian cells. SGF29 is required to recruit SAGA to H3K4me3. We focused on the endoplasmic reticulum (ER) stress target genes, which were shown to recruit SAGA upon ER stress induction. Strikingly, we discovered that on these genes not only H3K14ac decreases in SGF29 knockdown cells, but also that this knockdown results in a decrease of the H3K4me3 modification. Furthermore, both H3K4me3 and SGF29 were present prior to ER stress, suggesting a ‘poised’ state for the ER stress target genes. We hypothesize that at least on ER stress target genes, SAGA’s role is two-fold. Prior to ER stress, SAGA recruitment is involved in maintenance of the H3K4me3, possibly via the recruitment of MLL-complexes. Upon induction SAGA is required for acetylation of the promoter and transcriptional induction. In an addendum to this chapter the development of a quantitative mass spectrometric-based method to study histone modifications in an unbiased and antibody independent manner is described. The global histone acetylation levels in SGF29 knockdown cells were determined and showed a reduction in H3K9acK14ac. This is in line for what was found in chapter 2 on the ER stress genes, thereby showing that this method is useful to study histone modifications. In chapter 3 work aimed at identifying the HAT responsible for H3K4 acetylation in mammalian cells is presented. A series of knockdown and overexpression experiments revealed a role for HBO1 and the HBO1 complex. Chapter 4 describes an investigation of the multivalent engagement of the general transcription factor TFIID to promoters, both in vitro and in vivo. Synergistic binding of TFIID to both DNA and histone modifications on promoter nucleosomes is revealed. These results accentuate the co-operative role of DNA and histones in the process of transcription initiation. Finally, chapter 5 discusses concepts and implications for future studies emerging from the work presented in this thesis.