Targeting of proteins to chromatin in Drosophila melanogaster

Albig, Christian (2020): Targeting of proteins to chromatin in Drosophila melanogaster. Dissertation, LMU München: Medizinische Fakultät

Abstract

Dosage compensation of sex chromosomes in Drosophila melanogaster is an excellent model system to study various aspects of targeting of protein factors to chromatin. Dosage compensation prevents male lethality by up regulating transcription from the single male X chromosome in the ~2 fold range to match the two active X chromosomes in females [reviewed in e.g. (Ferrari et al., 2014; Kuroda et al., 2016; Samata and Akhtar, 2018)]. This up regulation is facilitated by the male specific lethal (MSL) dosage compensation complex (DCC). The DCC binds selectively to ~300 high affinity sites (HAS) on the X chromosome, containing a low complexity GAGA rich sequence motif, the MSL recognition element (MRE) (Alekseyenko et al., 2008; Straub et al., 2008). However, the DCC neglects thousands of other similar sequences in the genome outside of HAS. The DNA binding subunit MSL2 alone can enrich X chromosomal MREs in vitro, although MSL2 misses most MREs within HAS (Villa et al., 2016). The Chromatin Linked Adaptor for MSL Proteins (CLAMP) binds thousands of MREs genome wide and contributes to DCC targeting to HAS (Kaye et al., 2018; Soruco et al., 2013). The role of CLAMP in facilitating MSL2 targeting to HAS was investigated by several approaches. Monitoring MSL2 chromatin binding in vivo by chromatin immunoprecipitation with high throughput sequencing (ChIP seq) showed the requirement of CLAMP for HAS targeting. Next, the interplay between CLAMP and MSL2 in genome wide in vitro DNA binding was studied by DNA immunoprecipitation with high throughput sequencing (DIP seq) (Gossett and Lieb, 2008; Liu et al., 2005; Villa et al., 2016). The data revealed mutual recruitment of both factors to each other’s binding sites and cooperative binding to novel sites. This DNA binding cooperativity extended each other’s binding repertoire to facilitate robust binding of MREs located within HAS, although increased binding to other non functional sites was observed. Both factors interacted directly with each other in co IP experiments, providing an explanation for cooperative DNA binding. Whether CLAMP and MSL2 are required for keeping HAS nucleosome free was studied by assay for transposase accessibly chromatin with high throughput sequencing (ATAC seq) (Buenrostro et al., 2013; Buenrostro et al., 2015). Both factors cooperate to stabilize each other’s binding and to compete with nucleosome positioning at HAS. After successful binding of the DCC to HAS, it interacts with neighboring target genes, which are marked by trimethylation of histone H3K36 (H3K36me3). There, the DCC catalyzes acetylation of H4K16 (H4K16ac) to boost transcription (Akhtar and Becker, 2000; Gelbart et al., 2009; Larschan et al., 2007; Prestel et al., 2010). The DCC employs the chromosome 3D organization, which seems to be invariant between males and females, to transfer from HAS to active genes (Ramirez et al., 2015; Ulianov et al., 2016). The contribution of HAS to the chromosome interaction network was studied by using different chromosome conformation capture techniques. Hi C analysis on sex sorted embryos showed that, H4K16ac and H3K36me3 correlate well with the active compartments (Sexton et al., 2012). Interestingly, compartment switching on the X chromosome between males and females was correlated with H4K16ac and therefore attributed to dosage compensation. The involvement of the Pioneering sites on the X (PionX), a special sub-class of HAS, in chromosome architecture was studied by high resolution 4C seq in male and female cells. Chromosomal segments containing PionX made frequent contact with many loci within the active compartment and even looped over large domains of the inactive compartment (Ghavi-Helm et al., 2014). These long range interactions between PionX with other PionX/HAS were more robust in males compared to females, indicating that the dosage compensation machinery reinforced them. Moreover, de novo induction of DCC assembly in female cells showed that the DCC uses long range interaction within the active compartment to transfer from PionX to target genes marked by H3K36me3 for up regulation of transcription. The chromosomal kinase JIL 1, which catalyzes phosphorylation of histone H3S10, localizes also to actively transcribed genes marked by H3K36me3 and is two fold enriched on the male X chromosome (Jin et al., 2000; Regnard et al., 2011; Wang et al., 2001). JIL 1 is implicated in maintaining overall chromosome organization and preventing the spreading of heterochromatin into the euchromatic part of the X chromosome in both sexes (Cai et al., 2014; Ebert et al., 2004; Jin et al., 1999). Furthermore, JIL 1 localizes to the non LTR retrotransposon arrays of the telomeres to positively regulate their expression (Andreyeva et al., 2005; Silva-Sousa and Casacuberta, 2013; Silva-Sousa et al., 2012). The role of JIL 1 in regulating gene expression was studied using various methods. JIL 1 formed a stable complex with the novel PWWP domain containing protein, JIL 1 Anchoring and Stabilizing Protein (JASPer). The JIL 1 JASPer (JJ) complex specifically enriched H3K36me3 modified nucleosomes in vitro via JASPer’s PWWP domain from a nucleosome library containing 115 different nucleosome types. Consistently, ChIP seq experiments showed that the JJ complex localizes to H3K36me3 chromatin at active gene bodies and at telomeric transposons in vivo. As previously described, the JJ complex is also enriched on the male X chromosome relative to autosomes. Loss of JIL 1 resulted in loss of JASPer enrichment, a small increase in H3K9me2 and a decrease in H4K16ac on the X chromosome shown by spike in ChIP seq. Gene expression analysis by RNA seq showed that the JJ complex positively regulates expression of genes, in particular of genes from the male X chromosome, and of telomeric transposons. Furthermore, the JJ complex associated with the Set1/COMPASS complex and with other remodelling complexes as shown by co IP coupled to mass spectrometry analysis.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 21. Januar 2020
• 1. Bericht­erstatter:in: Becker, Peter