Uncovering new functions for histone variants: a role for H2A.Z in silencing retrotransposons
Creators: name: Shen, Qian
Publisher: name: The Australian National University
Year: 2016
Subjects: LINE-1s
H2A.Z
RNAPII
Trophoblast stem cells
CD69

description: Eukaryotic genomes must be, on the one hand, highly compacted by wrapping their DNA around histones to form nucleosomes while on the other, still remain accessible to the transcriptional machinery in a developmental and cell-type specific manner. Various epigenetic-based mechanisms exist that can regulate DNA accessibility ensuring the proper regulation of gene transcription. One epigenetic modification in particular that impacts all aspects of genome function and organization is the replacement of canonical histones with their variant forms. Among the histone variants that are most extensively studied is the essential and evolutionary conserved variant, H2A.Z. Recent studies have revealed many layers of regulation and complex functions of H2A.Z in modulating gene expression. Notably, H2A.Z has the ability to both activate and repress transcription but it remains unresolved how H2A.Z affects gene expression in these opposing ways. At a genome-wide level, H2A.Z is found at active transcription start sites, inactive promoters and of relevance to this thesis, it is also found at satellite repeats including those present at constitutive heterochromatin and the centromere. To gain new insights into the function of H2A.Z and how it can perform these opposing roles, this thesis has examined the transcriptional role of H2A.Z in regulating two different types of RNAPII transcribed elements: the retrotransposon, LINE-1 and a coding gene important for T-cell development, Cd69. Long Interspersed Nucleotide Element-1 (LINE-1) is one of most impactful and still active transposable elements (TEs) that occupy 18%-20% of mammalian genomes. Given that H2A.Z is targeted to repetitive mouse DNA sequences found at heterochromatin and the centromere, which is necessary for chromatin compaction, I wondered whether this role might extend to other repetitive DNA elements. Specifically, I explored the role of H2A.Z in LINE-1 transcription in four different cell types that were undifferentiated (mouse trophoblast stem cells (TSCs)), differentiated (trophoblast giant cells (TGCs) and mouse L929 cells) and committed (mouse embryonic fibroblasts (MEFs)). The results showed significant variability in H2A.Z occupancy relative to H2A at LINE-1s in these different cell types. Moreover, the enrichment of H2A.Z was inversely correlated with LINE-1 RNA expression. Specifically, relative high levels of LINE-1 transcripts were observed in TSCs compared to MEFs and L929 cells and this was correlated with a lower level of H2A.Z in the promoter and coding region of the LINE-1 element. Furthermore, when TSCs were differentiated into TGCs in vitro, there was a dramatic reduction in LINE-1 expression and this repression was positively correlated with a gain of H2A.Z on LINE-1s. These observations suggested that H2A.Z was a repressor of LINE-1 transcription. To directly test this hypothesis, a H2A.Z knockdown system was successfully established in both L929 and TSCs. Indeed, the loss of H2A.Z activated LINE-1 transcription in both L929 cells and TGCs following its differentiation from TSCs. These data shows unequivocally that H2A.Z is a repressor of LINE-1 expression. Unexpectedly though, the inhibition of H2A.Z expression in TSCs decreased LINE-1 expression rather than increasing it. Further investigations revealed that the level of acetylated H2A.Z (acH2A.Z) at the promoter region of LINE-1s differed between TSCs and TGCs. Significantly, there was a higher level of acH2A.Z at LINE-1s in TSCs compared to TGCs consistent with acH2A.Z being a mark of active transcription and the higher level of transcription seen in TSCs. These results demonstrate for the first time that H2A.Z is a regulator of LINE-1 transcription and whether it activates or represses these repetitive elements is dependent upon the abundance and the acetylation state of H2A.Z. To further explore the role of H2A.Z in the regulation of gene transcription, I chose to study the activation of the Cd69 promoter during T-cell development. The advantage of studying T-cell development is that it is possible to obtain a pure population of T-cells at different stages of development. The Cd69 gene was of interest because it is a surface marker that accompanies induction or repression through different stages of thymocyte development. This enabled an investigation of when during development H2A.Z is recruited to the Cd69 promoter, and the relationship between this recruitment with gene activation and the engagement of RNA polymerase II (RNAPII). Surprisingly, the data demonstrated that H2A.Z was present on the Cd69 promoter early in development when it is inactive and prior to the recruitment of RNAPII. Furthermore, H2A.Z was subsequently removed at a developmental stage prior to the binding of RNAPII suggesting again that H2A.Z may be a repressor of gene transcription during development. In conclusion, this thesis has revealed new unexpected functions for H2A.Z in the regulation of LINE-1 and Cd69 transcription.