The H1 Phosphorylation State Regulates Expression of CDC2 and Other Genes in Response to Starvation in Tetrahymena thermophila

Abstract

In Tetrahymena thermophila, highly phosphorylated histone H1 of growing cells becomes partially dephosphorylated when cells are starved in preparation for conjugation. To determine the effects of H1 phosphorylation on gene expression, PCR-based subtractive hybridization was used to clone cDNAs that were differentially expressed during starvation in two otherwise-isogenic strains differing only in their H1s. H1 in A5 mutant cells lacked phosphorylation, and H1 in E5 cells mimicked constitutive H1 phosphorylation. Sequences enriched in A5 cells included genes encoding proteases. Sequences enriched in E5 cells included genes encoding cdc2 kinase and a Ser/Thr kinase. These results indicate that H1 phosphorylation plays an important role in regulating the pattern of gene expression during the starvation response and that its role in transcription regulation can be either positive or negative. Treatment of starved cells with a phosphatase inhibitor caused CDC2 gene overexpression. Expression of the E5 version of H1 in starved cells containing endogenous, wild-type H1 caused the wild-type H1 to remain highly phosphorylated. These results argue that Cdc2p is the kinase that phosphorylates Tetrahymena H1, establish a positive feedback mechanism between H1 phosphorylation and CDC2 expression, and indicate that CDC2 gene expression is regulated by an H1 phosphatase.The organization of nuclear DNA into nucleosomes by histones and the folding of nucleosomes into higher-order chromatin structures are generally believed to compact DNA and make it inaccessible to factors required for transcription (see references 30 and 34). There is now compelling evidence that chromatin structural modifications are actively involved in transcription regulation, and extensive physical and functional interactions among transcription factors, chromatin modifying activities, and nucleosomes are well documented (67). Linker histones (H1 and related proteins) have been strongly implicated in modulating chromatin structure and function at multiple levels (66). They are key components of the nucleosome, the most basic level of the hierarchy of chromatin structure in eukaryotes, where they provide extra protection to DNA wrapped around the core histone octamer, presumably by interacting with the DNA strand where it enters and exits the nucleosome core. Linker histones can affect nucleosome mobility (49) and spacing (6). They also stabilize the folding of nucleosome arrays and the association of folded arrays in vitro (10), probably by interacting with the core histone tails (11). However, in spite of intensive study, there is little agreement regarding the precise location of linker histones in nucleosomes (64) and little insight into the mechanisms by which they influence higher orders of chromatin structure.Linker histones have been implicated in the regulation of transcription and were long thought to serve as global repressors (70). However, a number of studies in a variety of organisms have found that linker histones have highly specific effects...

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“…The steady-state H1 phosphorylation levels in Tetrahymena result from the balance of opposing H1 kinase and phosphatase activities (13,15). One known Tetrahymena H1 kinase, …”

Different Effects ofTetrahymena IFT172Domains on Anterograde and Retrograde Intraflagellar Transport

“…Subtraction was based on a suppression PCR strategy (Gurskaya et al, 1996) using the Clontech PCR-Select cDNA subtraction kit (Clontech, Palo Alto, CA) mainly following the manufacturer's protocol as previously described (Dou et al, 2005). The 0-h mRNA was used as the "driver" and 1-h cilia regeneration mRNA as the "tester"; the ratio of driver to tester was 5:1 with additional SerJ-like cDNA added into the driver pool.…”

“…The hybridization was performed at 42°C overnight in hybridization buffers containing 50% formamide as previously described (Dou et al, 2005) with the same DNA probe used for Southern analysis.…”

“…A neo2 selectable marker (Gaertig et al, 1994) was inserted into the AccI site of the 5Ј MTT1 flanking region as previously described (Dou et al, 2005).…”

Tetrahymena IFT122Ais not essential for cilia assembly but plays a role in returning IFT proteins from the ciliary tip to the cell body

“…20 g total RNA was resolved on 2.2 M formaldehyde-1% agarose gels and blotted to MagnaGraph membranes (Osmonics). Hybridization was performed at 42°C overnight in hybridization buffers containing 50% formamide as previously described (Dou et al, 2005). The probe was labeled by random priming with ␣-[ 32 P]dATP using IFT122A cDNA as the template.…”