Most of the knowledge about H2Bub1 function comes from yeast studies utilizing mutant strains with mutation of K123 in H2B (Sun, 2002). Unfortunately, this approach cannot be utilized in mammal systems due to the high number of H2B genes (Marzluff, 2002). In the present study it was decided to use a “knockdown-overexpression” system to overcome this problem. In this system endogenous H2B is depleted with siRNAs and at the same time synthetic H2B, introduced via stable plasmid transfection, is overexpressed. By changing the codons of the H2B sequence it is possible to make the overexpressed protein resistant to siRNAs that target H2B. Exchange of amino acids that undergo post-translational modifications, e.g. lysine 120 that is subjected to monoubiquitination, allows to investigate their functions in more detail.
4.1.1. Overexpression of Flag-H2B
The H2B overexpression construct was based on pcDNA5/TO plasmid (Fig.5A). To differentiate from endogenous H2B (endo-H2B) a Flag-tag was added to N-terminus of the overexpressed protein. Two constructs were created: H2B with wild-type protein sequence (Flag-H2B) and mutant H2B that cannot undergo ubiquitination (K120R). To test the constructs H1299 cells were transiently transfected with the corresponding plasmids and Flag-H2B expression was examined by Western blot (Fig. 5B). Both constructs were properly expressed, however the amount of H2B expressed from the transgene was far lower than the amount of endogenous protein. Examination of H2Bub1 levels showed that Flag-H2B was successfully ubiquitinated, although in a far lower amount than endogenous H2B, whereas the K120R mutant was not ubiquitinated (Fig. 5C). Confocal immunofluorescence staining of
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overexpressed proteins demonstrated that both of them correctly localize to the nucleus (Fig.
5D), suggesting that Flag-H2B and K120R proteins are incorporated into chromatin.
Fig. 5. H2B constructs are correctly expressed in H1299 cells.
(A) Scheme of Flag-H2B and K120R overexpressing constructs.
(B) H1299 cells were transiently transfected with Flag-H2B or K120R expressing constructs for 24h followed by H2B depletion for 48h. Protein lysates were collected and analyzed by Western blot using H2B antibodies. Different exposures of the same film are shown.
(C) Transient transfection of H1299 cells with H2B constructs was performed for 24h. Samples were analysed by Western blot with antibodies to H2Bub1 and H2B as a loading control.
(D) H1299 were transfected as in (C) and visualized by immunofluorescence using antibodies to H2B or Flag-tag. Scale bar corresponds to 20 µm.
46 4.1.2 H2B depletion leads to cell cycle arrest
The next step in creating a “knockdown-overexpression” for H2B was establishing an H2B knockdown in human cells. Due to nucleotide sequence dissimilarity between H2B genes it was not possible to target all of them with a low number of siRNAs, thus we focused on targeting individual H2B transcripts with siRNA. To establish efficient H2B knockdown, expression of different histone genes was examined in H1299 cells (Fig. 6A). It was observed that the amount of mRNA transcripts can vary up to s thousand fold between different H2B genes. The highest levels of transcription were observed for HIST1H2BK, HIST1H2BC, HIST1H2BD, HIST1H2BJ, HIST1H2BN, HIST1H2BE, HIST1H2BG and HIST1H2BO.
Unfortunately, the commercially available siRNAs did not show the efficient targeting for most of the transcripts, so taking into account the expression levels and feasibility of targeting, the following genes were chosen for knockdown: HIST1H2BC, HIST1H2BG and HIST1H2BO. The sequences of chosen siRNAs also allowed targeting of other H2B genes.
Transfection of H1299 cells with individual siRNA demonstrated a significant decrease in the expression of corresponding H2B genes for each of siRNAs (Fig. 6B) as well as for siRNA mix (Fig. 6C).
Examining the effect of knockdowns on global H2B level with Western blot is difficult due to huge amounts of produced protein. To overcome this problem the effect on cellular processes upon H2B depletion was examined. H1299 cells were transfected with siRNA mix containing siRNAs to HIST1H2BC, HIST1H2BG and HIST1H2BO for 48h. After that the cell cycle profile was monitored by flow cytometry using propidium iodide (PI) and Bromodeoxyuridine (BrdU) staining (Fig. 6D). Upon H2B knockdown cells synchronized in G1/S phase agreeing with the idea that H2B is synthesized at the end of G1 phase and without it cell is not able to proceed to S-phase. To test whether G1-arrest is a specific response of H1299 cells or it is a more general effect H2B knockdown was also performed in HEK293 cells (Fig. 6E), which demonstrated a similar and even more pronounced increase in the G1/early S fraction. Described observations suggest that knocking down of several H2B genes is sufficient to affect cellular metabolism and cause a G1-arrest or block S-phase entry.
47 Fig. 6. H2B knockdown inhibits cell cycle progression.
(A) Expression of H2B genes in H1299 cells. RNA was isolated from H1299 cells followed by cDNA synthesis. Expression of H2B was analysed by qRT-PCR. Number of copies was calculated based on C(t) values of genomic DNA with known copy number and indicated as “Rel. gDNA units“. Mean
±SD, n = 3.
(B, C) H1299 cells were transfected with control siRNA, individual siRNAs against HIST1H2BC, HIST1H2BG and HIST1H2BO (B) or a mix of them (C) for 48h. After that RNA was isolated followed by cDNA synthesis. Gene expression was examined by qRT-PCR and normalized to RPLP0 expression (indicated as “Rel. mRNA levels”). Mean ±SD, n = 3.
(D) H1299 cells were transfected with siRNA mix as in (C). After 48h of knockdown cells were incubated with BrdU for 1h followed by fixation, incubation with FITC-coupled anti-BrdU antibody and staining with PI. Cell cycle distribution was measured by flow cytometry. Mean ±SD, n = 3.
(E) HEK293 cells were transfected and analysed as in (D). Cell cycle distribution was measured by flow cytometry. Mean ±SD, n = 3.
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4.1.3 “Knockdown - overexpression” approach is not applicable for human cells
After checking H2B overexpression and knockdown their combination was tested as an approach to study H2Bub1 function. H1299 were transiently transfected with Flag-H2B for 24h or K120 followed by H2B depletion for 48h (Fig 5B). Unfortunately, no significant depletion of endogenous H2B was observed. To examine if replacement of endo-H2B with Flag-H2B increases with longer overexpression or knockdowns H1299 were stably transfected with Flag-H2B or K120R (Fig. 7A). Two clones for each construct were analyzed but no significant increase in overexpressed H2B was observed. Depletion of endogenous H2B also did not enhance replacement of endo-H2B with transgene overexpression.
Fig.7. Optimizing Flag-H2B overexpression.
(A) H1299 cells were stably transfected with Flag-H2B or K120R construct. Clones were selected using Hygromycin-containing medium. Cells were transfected with mix of siRNAs against HIST1H2BC, HIST1H2BG and HIST1H2BO or control siRNA for 48h. Protein lysates were analyzed by Western blot with antibodies for H2Bub1 and H2B.
(B) U2OS cells expressing the Tet-R were stably transfected with Flag-H2B or K120R constructs and grown on Hygromycin. Cells were pre-treated with doxycyclin for 24h followed by H2B knock down as in (A) for 48h. Proteins were collected and analysed by Western blot using antibodies to H2Bub and H2B.
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The next step was creation of stable cell lines with inducible Flag-H2B expression were created using U2OS cells expressing Tet-R (Monroe, 2003) since the inducible expression helps to avoid negative selection. To replace endo-H2B with overexpressed Flag-H2B or K120R U2OS Tet-R cells were treated with doxycyclin for 24h to induce transgene expression followed by H2B knockdown for 48h (Fig.7B). Unfortunately, this approach did not lead to increased expression of synthetic proteins and no effect of H2B knockdown was observed.
Analyzing all used approaches it can be concluded that overexpressed synthetic H2B is not able to replace endogenous protein. Increasing the overexpression to the cellular levels was technically possible neither with transient nor with stable construct expression. Knocking down some of H2B genes did not increase ratio of synthetic H2B to total H2B either.
Summing up, H2Bub1 function is difficult to decipher by knockdown-overexpression system in mammalian cells.