Andrea Schram

In eukaryotic cells, the process of copying a DNA template into RNA is called gene transcription or expression. This is highly regulated at many different levels by a wide plethora of proteins. Chromatin is one of the important regulators of gene expression. Nucleosomes are the basic building blocks of chromatin and are formed by DNA wrapped around a histone octamer (two copies of histones H2A, H2B, H3 and H4) and the linker histone H1. For a long time the histones were regarded as merely being packaging material to fit the DNA into the cell’s nucleus. However, later it became apparent that the protruding (N-terminal) tails of the histones can be post-translationally modified. They are subjected to chemical modifications like acetyl, methyl, phospho and ubiquitin. These modifications can act alone or context-dependent to activate or repress transcription. Furthermore, one modification can lead to the addition or removal of the next. Genome-wide analyses of different modifications have correlated certain modification states, their genome-wide loci and level of gene expression. For example, nucleosomes surrounding the transcription start site (TSS) of active genes are often hyperacetylated on histones H3 and H4. Also trimethylation of histone H3 of lysine residue 4 (H3K4me3) is associated with the promoter of active genes. Promoters are the minimal DNA sequence at the start of a gene required to start transcription. The three categories of proteins involved in chromatin biology are writers; proteins that add the modification, erasers; proteins that remove modifications and readers; proteins that are recruited to modifications. Two important readers in the process of transcription are TFIID and SAGA, which both contain a subunit that binds to H3K4me3. SAGA also contains a writer subunit to acetylate histones. This thesis describes the role of SAGA and TFIID in the regulation of transcription through chromatin. Furthermore, a writer was identified for a novel acetylation of histone H3 on lysine 4 (H3K4ac).

The role of SAGA in transcription was investigated using genes involved in the cell’s reaction to stress in the endoplasmic reticulum (ER), which is a compartment of the cell specialized in folding of proteins. It is known that SAGA is recruited to these ER stress genes upon ER stress. These genes were used to dissect the exact role of SAGA and its H3K4me3 binding subunit SGF29. Experiments showed that not only acetylation of the promoter of ER stress genes but also presence of H3K4me3 is dependent on SGF29. This is surprising because by itself SAGA cannot establish this modification. Thus most likely SAGA and SGF29 binding to H3K4me3 recruits other protein complexes to maintain presence of H3K4me3 to ensure transcription in case of a stress situation. Lastly, both SAGA and SGF29 are required for survival of the cells after ER stress.

H3K4ac is a relatively new identified modification and is also associated with active transcription. With the help of a screen, the HBO1 protein was identified to be a writer for this modification. This was confirmed by an experiment where HBO1 was over-expressed in cells leading to an increase of H3K4ac.

TFIID is a very important player in the process of transcription initiation. Previously, its subunit TAF3 was identified to bind to H3K4me3. In this thesis we further characterized the binding of TFIID to promoter nucleosomes. We discovered that this binding is multivalent and depends on several different features found in the promoter region. In addition to H3K4me3, acetylation of histone H3 and the presence of the TATA sequence in the DNA increase the binding of TFIID. We have also determined the composition of the TFIID complex in human cells, which turned out to be similar to yeast TFIID. These analyses also suggested that TFIID exists in different compositions, probably dependent on the situation at the specific genes.

Taken together the results described in this thesis have added to our knowledge of SAGA, TFIID and chromatin in the regulation of transcription.