Degree Type

Dissertation

Date of Award

2011

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Kristen M. Johansen

Second Advisor

Jorgen Johansen

Abstract

Epigenetic regulations play a crucial role in control of gene expression and development. Histone modification is one of the epigenetic mechanisms that contribute to regulation of chromatin structure and gene expression. In Drosophila melanogaster, JIL-1, the predominant interphase histone H3S10 kinase, localizes specifically to euchromatic interband regions of polytene chromosomes, and is upregulated two-fold on the male X chromosome. Genetic interaction assays with JIL-1 hypomorphic and null allelic combinations demonstrated that JIL-1 can counterbalance the gene-silencing effect of the three major heterochromatin components Su(var)3-9, Su(var)3-7, and HP1a on position-effect variegation. Our data suggested that the epigenetic H3S10ph mark functions to counteract heterochromatic spreading and gene silencing in Drosophila .

JIL-1 can be divided into four main domains including a NH2-terminal domain (NTD), the first kinase domain (KDI), the second kinase domain (KDII), and a COOH-terminal domain (CTD). Transgenic analysis demonstrated that the CTD of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes, but that both COOH- and NH2-terminal sequences were necessary for upregulation on the male X chromosome. Another construct ∆CTD that lacks the CTD domain but has histone H3S10 kinase activity can be localized to chromatin by the NTD and was able to rescue autosomes as well as partially rescue male X polytene chromosome morphology.

Furthermore, to study the role of histone H3S10 phosphorylation in transcription we examined the distribution of JIL-1 and histone H3S10 phosphorylation under both heat shock and non-heat shock conditions. There was no redistribution or upregulation of JIL-1 or histone H3S10 phosphorylation found at transcriptionally active puffs after heat shock treatments. Also in JIL-1 null mutant backgrounds, heat shock-induced puffs were strongly labeled by the antibody to the elongating form of RNA polymerase II (Pol IIoser2), indicating that Pol IIoser2 is actively involved in heat shock-induced transcription in the absence of histone H3S10 phosphorylation. These results suggested a model where transcriptional defects in the absence of histone H3S10 phosphorylation are a result of structural alterations of chromatin rather than direct effects on Pol II activation.

To further study the interplay between JIL-1 and transcription regulation, a genome-wide analysis of JIL-1 kinase binding sites by ChIP-seq was conducted and combined with an analysis of whole genome transcription level changes by RNA-seq in the absence of JIL-1. We found that most of the identified JIL-1 binding peaks locate around 200 bp upstream of transcription start sites and that in the absence of H3S10 phosphorylation by JIL-1 a number of normally actively expressed genes were repressed, whereas some inactive genes were activated. Moreover, in the absence of JIL-1 the gene expression level changes of two JIL-1 target genes showed alterations in H3K9 dimethylation levels. Taken together, all these observations suggested a model that H3S10 phosphorylation mainly facilitates gene expression of active genes by maintaining an open chromatin structure at promoter regions by counteracting heterochromatization.

DOI

https://doi.org/10.31274/etd-180810-2773

Copyright Owner

Weili Cai

Language

en

Date Available

2012-04-28

File Format

application/pdf

File Size

145 pages

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