Micronucleus-specific histone H1 is required for micronuclear chromosome integrity in Tetrahymena thermophila

Abstract

Histone H1 molecules play a key role in establishing and maintaining higher order chromatin structures. They can bind to linker DNA entering and exiting the nucleosome and regulate transcriptional activity. Tetrahymena thermophila has two histone H1, namely, macronuclear histone H1 and micronuclear histone H1 (Mlh1). Mlh1 is specifically localized at micronuclei during growth and starvation stages. Moreover, Mlh1 is localized around micronuclei and forms a specific structure during the conjugation stage. It co-localizes partially with spindle apparatus during micronuclear meiosis. Analysis of MLH1 knock-out revealed that Mlh1 was required for the micronuclear integrity and development during conjugation stage. Overexpression of Mlh1 led to abnormal conjugation progression. RT-PCR analysis indicated that the expression level of HMGB3 increased in ΔMLH1 strains, while the expression level of MLH1 increased in ΔHMGB3 cells during conjugation. These results indicate that micronuclear integrity and sexual development require normal expression level of Mlh1 and that HmgB3 and Mlh1 may functionally compensate each other in regulating micronuclear structure in T. thermophila.

Nuclear localization signal targeting to macronucleus and micronucleus in binucleated ciliate Tetrahymena thermophila

“…Fluorescence microscopic observation showed that the GFP signal (corresponding to GFP-δ) and the mCherry signal (corresponding to γ-mCherry) were both inside the MIC (Figure 3c), suggesting that α protein was first transported into the MIC and cleaved to generate GFP-δ and γ-mCherry proteins. This result is different from the previous finding that HA-tagged α protein does not localize to the MIC (Qiao et al, 2017), probably reflecting the different experimental conditions between our and their experiments (GFP-GST-tag vs. HA-tag and live vs. fixed cells). We used western blotting to confirm whether GFP-α-mCherry protein was indeed cleaved.…”

“…We found that fluorescence signals of GFP-Mlh1 were mostly lost when the cells were fixed with 100% ice-cold methanol, probably because these GFP-tagged proteins were not functional in the MIC and thus not integrated into chromosomes. This idea is supported by the finding that none of HA-tagged-α, HAtagged-β, HA-tagged-γ and HA-tagged-δ fragments were localized in the MAC after fixation with Schaudinn's fixative containing 100% ethanol in a study by Qiao et al (2017). Together, these results indicate that δ and β fragments, but not γ fragment, can be actively transported to the MIC, suggesting the presence of MIC-specific NLS sequences in the δ and β fragments.…”

“…Because the exclusion pore size of the MAC NPC is approximately 50 kDa (Iwamoto et al, 2017), the GFPtagged mature proteins (49-53 kDa) may be mislocalized to the MAC by passive diffusion through the MAC NPCs. This idea is supported by the finding that none of HA-tagged-α, HAtagged-β, HA-tagged-γ and HA-tagged-δ fragments were localized in the MAC after fixation with Schaudinn's fixative containing 100% ethanol in a study by Qiao et al (2017). (a) Diagram represents an Mlh1 precursor protein and its three processed mature forms.…”

“…In T. thermophila, there is another H1-like proteinmicronuclear linker histone-like protein (Mlh1) (gene ID: TTHERM_00471820)-that is specifically localized in the MIC (Supporting Information Figure S1b) and whose amino acid sequence has no homology to MAC-specific H1 and other canonical H1 orthologs (Wu et al, 1994). The gene product of TTHERM_00471820 is expressed as a large precursor protein with a molecular weight (MW) of 70.6 kDa, which is processed into three smaller mature proteins with MWs of 21.7, 23.4 and 25.5 kDa, similar to those of other histone proteins (Allis, Allen, Wiggins, Chicoine, & Richman, 1984;Allis, Glover, & Gorovsky, 1979;Qiao, Xu, Bo, & Wang, 2017). The mechanism targeting these three mature Mlh1 proteins to only the MIC remains unknown.…”

The histone chaperone Nrp1 is required for chromatin stability and nuclear division in Tetrahymena thermophila

“…The MAC and the MIC have different histones, histone modification, and chromatin structure. Micronucleus-specific histone H1 (Mlh1) is different from macronuclear H1 (Hho1) and H1 from other organisms [ 33 , 34 ]. The transcriptionally active MAC contains a histone hvl (H2A variant), and the hvl protein is absent from the MIC, except in the early stages of conjugation [ 35 ].…”