Title

Identification and characterization of Hhp1p, a heterochromatin-associated protein in Tetrahymena thermophila

Date of Award

1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

C. David Allis

Keywords

Hhp1p, heterochromatin-associated protein in Tetrahymena

Subject Categories

Cell Biology | Molecular Biology

Abstract

The identification and characterization of heterochromatin-associated proteins has greatly improved our understanding of heterochromatin assembly and dynamics in eukaryotes. In this dissertation, I identified and cloned a 28 KDa polypeptide, Hhp1p, from Tetrahymena macronuclei that possesses many features of the heterochromatin-associated protein HP1 from Drosophila. Like other HP1-like proteins, Hhp1p contains both a chromo domain and a chromo shadow domain. However, a short domain between the chromo domain and its shadow domain in Hhp1p displays some features of linker histone H1. Thus, this protein is being referred to as Hhp1p (for H1/HP1-like protein). In Tetrahymena, Hhp1p specifically localizes in transcriptionally active macronuclei where Hhp1p displays a punctate staining pattern.

To further investigate Hhp1p function, I have disrupted all of the expressed copies of the HHP1 gene in somatic macronuclei. HHP1 knock-out ($\Delta$HHP1) cells grow at normal rates, demonstrating that Hhp1p is not essential in Tetrahymena. However, the survival rate of $\Delta$HHP1 cells is markedly reduced compared to that of wildtype cells during prolonged starvation. Upon starvation, $\Delta$HHP1 cells display a roughly 1.5-fold increase in total macronuclear size along with a reduction in the size of the electron-dense chromatin bodies. In contrast, in wildtype cells, Hhp1p becomes hyperphosphorylated during prolonged starvation concomitant with an increase in chromatin body size. In addition, the activation of two starvation-induced genes is reduced in $\Delta$HHP1 cells, while transcription rates of growth-related genes are comparable to wildtype cells. These results suggest that Hhp1p functions in heterochromatin assembly and/or maintenance, and it is required for the appropriate expression of certain genes in physiological states such as starvation.

To investigate the potential relationship between Hhp1p and linker histone H1, a double knockout strain was created and studied. It appears that knocking out H1 in $\Delta$HHP1 cells partially rescues some wildtype phenotypes, suggesting that H1 may function as a suppressor of HHP1 in certain physiological states. These results, together with preliminary biochemical evidence that Hhp1:H1 products are detected in cross-linked macronuclei, suggest that linker histone H1 may interact with Hhp1p in vivo.

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