Molecular mechanism of dBigH1 action

Autor/a

Tatarski, Miloš

Director/a

Azorín, F.

Tutor/a

Agell i Jané, Neus

Data de defensa

2018-09-21

Pàgines

155 p.



Departament/Institut

Universitat de Barcelona. Facultat de Farmàcia i Ciències de l'Alimentació

Resum

INTRODUCTION: For decades it was known that many species contain embryo specific linker histone H1 variants that replace the somatic H1 during early embryogenesis. This is especially important because the early embryo shows typically zero to very little activity of transcription, and the first cleavages of the embryo depend exclusively on maternally deposited factors that are important for transcription and chromatin assembly. The first species shown to contain an embryo specific histone H1 was the sea urchin. Other species like the mouse, Xenopus or the zebrafish followed. Even in humans there are embryo specific H1 variants. Drosophila seemed to be an exception to this, until in 2013 the first linker histone H1 variant was discovered that was called dBigH1. Like other embryo H1 variants, dBigH1 is expressed in the early embryo and disappears when cellularization starts and it gets replaced by the somatic H1. Likewise, to its counterparts in other species, dBigH1 is responsible for the inhibition of transcription during the early stage of fly development. OBJECTIVES: In this thesis, we addressed the questions about the mechanism of inhibition of dBigH1 as well as the factors that are responsible for its deposition into chromatin. RESULTS: To answer the first question, we used an in vitro system for chromatin reconstitution based on an extract from early Drosophila embryos (DREX) that contains dBigH1 and all other factors needed for proper chromatin assembly. We then used the reconstituted chromatin in transcription experiments using HeLa nuclear extract that contains all factors needed for transcription. We saw that transcription for chromatin reconstituted in DREX could be reduced when the extract was previously depleted from dBigH1 using specific antibodies against it. By adding back recombinant dBigH1 to the depleted extract we were able to restore the initial lever of transcription. This showed us that dBigH1 was the repressive factor, as it was already confirmed in vivo. We then used a truncated construct of dBigH1 where we depleted the N-terminal domain of the protein. This was of particular interest as the N-terminal region of dBigH1 is the one that differs most form the somatic H1. It is much longer and more importantly very enriched with acidic residues, something that is very unique amongst all embryo specific H1 variants. We saw that when using the truncated construct, transcription was inhibited to a much lesser extent than with the full length dBigH1, proposing that the N-terminal domain is indeed responsible for the inhibition of transcription. To answer the second question about the factors needed for dBigH1 deposition, we used Drosophila testis to study dBigH1 in vivo. dBigH1 shows a very similar expression pattern in testis as the chromatin remodeler ACF1. This is why we decided to investigate a possible interaction between those two proteins. Additionally, we knew that ACF1 uses NAP1 as a histone chaperone for H1 in some species, so we also asked if NAP1 could play a role in dBigH1 deposition as well. Indeed, we saw that when using flies deficient for ACF1 we see much less dBigH1 in the testis tip where the germal stem cells (GSC) reside, suggesting that ACF1 plays an important role in dBigH1 deposition. In accordance, we see more dBigH1 in the GSCs when using flies overexpressing ACF1. At the same time, we can see that when depleting NAP1 from DREX, we see more dBigH1.

Paraules clau

Biologia molecular; Biología molecular; Molecular biology; Ciències de la salut; Ciencias biomédicas; Medical sciences; Cromatina; Chromatin; Epigenètica; Epigenética; Epigenetics

Matèries

577 - Bioquímica. Biologia molecular. Biofísica

Àrea de coneixement

Ciències de la Salut

Documents

MILOS TATARSKI_PhD_THESIS.pdf

3.079Mb

 

Drets

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