Identification of a new set of cell cycle-regulatory genes that regulate S-phase transcription of histone genes in Saccharomyces cerevisiae

Histone mRNA synthesis is tightly regulated to S phase of the yeast Saccharomyces cerevisiae cell cycle as a result of transcriptional and posttranscriptional controls. Moreover, histone gene transcription decreases rapidly if DNA replication is inhibited by hydroxyurea or if cells are arrested in G1 by the mating pheromone alpha-factor. To identify the transcriptional controls responsible for cycle-specific histone mRNA synthesis, we have developed a selection for mutations which disrupt this process. Using this approach, we have isolated five mutants (hpc1, hpc2, hpc3, hpc4, and hpc5) in which cell cycle regulation of histone gene transcription is altered. All of these mutations are recessive and belong to separate complementation groups. Of these, only one (hpc1) falls in one of the three complementation groups identified previously by other means (M. A. Osley and D. Lycan, Mol. Cell. Biol. 7:4204-4210, 1987), indicating that at least seven different genes are involved in the cell cycle-specific regulation of histone gene transcription. hpc4 is unique in that derepression occurs only in the presence of hydroxyurea but not alpha-factor, suggesting that at least one of the regulatory factors is specific to histone gene transcription after DNA replication is blocked. One of the hpc mutations (hpc2) suppresses delta insertion mutations in the HIS4 and LYS2 loci. This effect allowed the cloning and sequence analysis of HPC2, which encodes a 67.5-kDa, highly charged basic protein.

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ANKHD1 Is Essential for Repair of DNA Double-Strand Breaks in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.1762.1762 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1762-1762

Author(s):

Anamika Dhyani ◽

Patricia Favaro ◽

Sara T. Olalla Saad

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Dna Damage ◽

Dna Replication ◽

S Phase ◽

Histone Gene ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna Replication And Repair ◽

Histone Gene Transcription

Abstract ANKHD1, Ankyrin repeat and KH domain-containing protein is highly expressed and plays an important role in the proliferation and cell cycle progression of multiple myeloma (MM) cells. Inhibition of ANKHD1 expression upregulates p21 (CDKN1A, Cyclin Dependent Kinase Inhibitor), a potent cell cycle regulator, and its expression represses p21 promoter. Upregulation of p21 was found to be irrespective of the TP53 mutational status of MM cell lines. A study by our group has shown that ANKHD1 is highly expressed in S phase and that the inhibition of ANKHD1 expression downregulates replication dependent histones suggesting that it might be required for histone transcription (1). Assuming that ANKHD1 might be involved in the transcripitional activation of histones, we studied the effect of ANKHD1 silencing on nuclear protein of the ataxia telangiectasia mutated locus (NPAT), a component of the cell-cycle-dependent histone gene transcription machinery. NPAT associates with histone gene promoters in S phase and suppression of NPAT expression impedes expression of all histone subtypes. In present study, there was a decreased expression of NPAT in ANKHD1 silenced MM cells. Despite the fact that both ANKHD1 and NPAT were localized in the nucleus of MM cells, they did not appear to associate, as observed by confocal microscopy, suggesting at present that ANKHD1 does not modulate histones via NPAT. Since DNA replication is coupled with histone synthesis and downregulation of histones is associated with replication stress and DNA damage, we checked for expression of PCNA (Proliferating Cell Nuclear Antigen), protein involved in DNA replication and repair. PCNA expression was found to be significantly decreased in ANKHD1 inhibited MM cells, suggesting its role in PCNA mediated DNA replication and repair (Fig. 1). To confirm this, we studied the effect of ANKHD1 silencing on some of the components of DNA damage repair (DDR) pathway. We observed increased expression of gamma- H2AX (γ-H2AX i.e Phosphorylated Histone H2AX), marker for DNA double-strand breaks (DSBs) and an early sign of DNA damage induced by replication stress (Fig. 1). We also observed a decrease in phosphorylated CHK2 (Check Point Kinase 2), an essential serine threonine kinase involved in DDR. Accumulation of γ-H2AX on ANKHD1 silencing confirms DNA damage and suggests the possible mechanism of ANKHD1 mediated histones downregulation. In summary, ANKHD1 silencing in MM cells leads to DNA damage (DSBs), suggesting that ANKHD1 is essential for DNA replication and repair. Furthermore, as ANKHD1 negatively regulates p21, and p21 controls DNA replication and repair by interacting with PCNA, we hypothesize that ANKHD1 might be playing role in DNA repair via modulation of the p21-PCNA pathway. Results of the role of ANKHD1 in DNA repair are however preliminary and need to be explored. References: 1) ANKHD1 Is Required for S Phase Progression and Histone Gene Transcription in Multiple Myeloma. Dhyani et al. ASH Abstract; Blood 2014. Figure 1. Western blot analysis of proteins: a) PCNA and b) γ-H2AX, in control and ANKHD1 silenced U266 MM cell line. Tubulin and GAPDH were used as endogenous controls. Figure 1. Western blot analysis of proteins: a) PCNA and b) γ-H2AX, in control and ANKHD1 silenced U266 MM cell line. Tubulin and GAPDH were used as endogenous controls. Disclosures No relevant conflicts of interest to declare.

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Distinct replication-independent and -dependent phases of histone gene expression during the Physarum cell cycle.

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1933 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1933-1937 ◽

Cited By ~ 5

Author(s):

J J Carrino ◽

V Kueng ◽

R Braun ◽

T G Laffler

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Dna Replication ◽

Dna Synthesis ◽

S Phase ◽

Histone Gene ◽

Histone Mrna ◽

Histone Gene Expression ◽

Tightly Coupled ◽

G2 Phase

During the S phase of the cell cycle, histone gene expression and DNA replication are tightly coupled. In mitotically synchronous plasmodia of the myxomycete Physarum polycephalum, which has no G1 phase, histone mRNA synthesis begins in mid-G2 phase. Although histone gene transcription is activated in the absence of significant DNA synthesis, our data demonstrate that histone gene expression became tightly coupled to DNA replication once the S phase began. There was a transition from the replication-independent phase to the replication-dependent phase of histone gene expression. During the first phase, histone mRNA synthesis appears to be under direct cell cycle control; it was not coupled to DNA replication. This allowed a pool of histone mRNA to accumulate in late G2 phase, in anticipation of future demand. The second phase began at the end of mitosis, when the S phase began, and expression became homeostatically coupled to DNA replication. This homeostatic control required continuing protein synthesis, since cycloheximide uncoupled transcription from DNA synthesis. Nuclear run-on assays suggest that in P. polycephalum this coupling occurs at the level of transcription. While histone gene transcription appears to be directly switched on in mid-G2 phase and off at the end of the S phase by cell cycle regulators, only during the S phase was the level of transcription balanced with the rate of DNA synthesis.

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Distinct replication-independent and -dependent phases of histone gene expression during the Physarum cell cycle

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1933-1937.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1933-1937

Author(s):

J J Carrino ◽

V Kueng ◽

R Braun ◽

T G Laffler

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Dna Replication ◽

Dna Synthesis ◽

S Phase ◽

Histone Gene ◽

Histone Mrna ◽

Histone Gene Expression ◽

Tightly Coupled ◽

G2 Phase

During the S phase of the cell cycle, histone gene expression and DNA replication are tightly coupled. In mitotically synchronous plasmodia of the myxomycete Physarum polycephalum, which has no G1 phase, histone mRNA synthesis begins in mid-G2 phase. Although histone gene transcription is activated in the absence of significant DNA synthesis, our data demonstrate that histone gene expression became tightly coupled to DNA replication once the S phase began. There was a transition from the replication-independent phase to the replication-dependent phase of histone gene expression. During the first phase, histone mRNA synthesis appears to be under direct cell cycle control; it was not coupled to DNA replication. This allowed a pool of histone mRNA to accumulate in late G2 phase, in anticipation of future demand. The second phase began at the end of mitosis, when the S phase began, and expression became homeostatically coupled to DNA replication. This homeostatic control required continuing protein synthesis, since cycloheximide uncoupled transcription from DNA synthesis. Nuclear run-on assays suggest that in P. polycephalum this coupling occurs at the level of transcription. While histone gene transcription appears to be directly switched on in mid-G2 phase and off at the end of the S phase by cell cycle regulators, only during the S phase was the level of transcription balanced with the rate of DNA synthesis.

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Transcription of alpha-tubulin and histone H4 genes begins at the same point in the Physarum cell cycle.

The Journal of Cell Biology ◽

10.1083/jcb.102.5.1666 ◽

1986 ◽

Vol 102 (5) ◽

pp. 1666-1670 ◽

Cited By ~ 14

Author(s):

J J Carrino ◽

T G Laffler

Keyword(s):

Cell Cycle ◽

Gene Transcription ◽

Blot Hybridization ◽

S Phase ◽

Histone Gene ◽

Histone H4 ◽

Alpha Tubulin ◽

Periodic Cell ◽

G2 Phase ◽

Histone Gene Transcription

In naturally synchronous plasmodia of Physarum polycephalum, both tubulin and histone gene transcription define periodic cell cycle-regulated events. Using a slot-blot hybridization assay and Northern blot analysis, we have demonstrated that a major peak of accumulation of both alpha-tubulin and histone H4 transcripts occurs in late G2 phase. Nuclear transcription assays indicate that both genes are transcriptionally activated at the same point in the cell cycle: mid G2 phase. While the rate of tubulin gene transcription drops sharply at the M/S-phase boundary, the rate of histone gene transcription remains high through most of S phase. We conclude that the cell cycle regulation of tubulin expression occurs primarily at the level of transcription, while histone regulation involves both transcriptional and posttranscriptional controls. It is possible that the periodic expression of both histone and tubulin genes is triggered by a common cell cycle regulatory mechanism.

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ANKHD1 Is Required for S Phase Progression and Histone Gene Transcription in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.2002.2002 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2002-2002

Author(s):

Anamika Dhyani ◽

Patricia Favro ◽

Sara T O Saad

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Gene Transcription ◽

Cell Cycle Progression ◽

S Phase ◽

Histone Gene ◽

Cycle Progression ◽

Promoter Sequences ◽

Core Histones ◽

Histone Gene Transcription

Abstract Background ANKHD1, Ankyrin repeat and KH domain-containing protein 1 is highly expressed in CD138+ cells of patients with multiple myeloma (MM) as well as in MM cell lines (U266, RPMI 8226 ,MM1S and MM1R. Our microarray studies showed modulation of several histone variants after ANKHD1 silencing (not published). Furthermore ANKHD1 silencing in MM cell lines resulted in S phase arrest (1). As genes involved in histone transcription are upregulated in S phase, and ANKHD1 downregulation inhibits cell cycle progression at S phase, we hypothesized that ANKHD1 might be a protein that gets upregulated in S phase and plays a role in histone gene transcription. Hence, in the present study ANKHD1 expression was sought at different phases of cell cycle and a possible interaction of ANKHD1 with histones was also investigated, in addition to the effect of ANKHD1 downregulation on histone expression in a MM cell line. Methods MM cell line U266 was synchronized at G1 phase by serum starvation (16h), S phase by double thymidine block (2mM) followed by release in 24 μM deoxycytidine (4h) or G2 phase by nocodazole treatment(50ng/ml;16h). Cells were stained with PI and analyzed by flow cytometry for DNA content. Percentage of cells in G1, S, or G2/M was calculated using the ModFit program. Western blot was then carried out for ANKHD1 expression in U266 cells untreated or synchronized at different G1, S and G2 phases of cell cycle. ANKHD1 expression was inhibited by lentiviral mediated ANKHD1shRNA transduction and its effect on expression of histones was studied by qPCR and immunoblot. Further chromatin immunoprecipitation (ChIP) assay was performed to study the interaction between ANKHD1 and histones using EZ-Magna ChIP™ A kit(Millipore) followed by qPCR with primers specific to core histones promoter region. Immunofluorescence was performed to determine the localization of ANKHD1 before and after leptomycin B treatment of U266 cells. Results. In the present study, endogenous ANKHD1 expression showed a clear cell-cycle-dependence, peaking during S phase, when cells were synchronized by double thymidine block followed by deoxycytidine release. Further down-regulation of ANKHD1 expression in U266 cells by lentiviral mediated shRNA against ANKHD1 resulted in a significant reduction of histones (p<0.05) at both mRNA and protein levels. Chromatin immunoprecipitation followed by qPCR with primers specific to core histones promoter region showed that ANKHD1, though not IgG (negative control) coprecipitated with histone gene chromatin, thus confirming the interaction between the core histone promoter regions and ANKHD1. Fold enrichment (mean ± sd) of promoter sequences bound to ANKHD1 were 7.74 ± 0.048, 7.78± 0.129 and 7.06± 0.178 for histones H2B/r, H3/c and H4/e, respectively. Immunofluorescence after leptomycin B treatment (20ng/ml) of U266 wild type cells for 24 hours showed accumulation of ANKHD1 inside nucleus as compared to untreated cells where ANKHD1 was found to be predominantly in cytoplasm. This suggests transport of ANKHD1 between nucleus and cytoplasm. Conclusion ANKHD1 expression peaks during S phase of cell cycle and downregulation of ANKHD1 protein by shRNA results in downregulation of histones. ANKHD1 interacts with histone gene promoter sequences and modulates histones transcription. These results suggest that ANKHD1 might be an important component of the machinery required for histone mRNA expression and cell-cycle progression. Furthermore, ANKHD1 protein which was earlier reported to be localized predominantly in cytoplasm (1) is herein suggested to shuttle between cytoplasm and nucleus thereby playing a role in gene regulation. Extensive studies are required to understand the mechanism underlying the regulation of gene transcription by ANKHD1. References: 1) ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells. Dhyani et al. FEBS letters 2012. Disclosures No relevant conflicts of interest to declare.

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Maximal binding levels of an H1 histone gene-specific factor in S-phase correlate with maximal H1 gene transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4576 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4576-4578 ◽

Cited By ~ 20

Author(s):

S Dalton ◽

J R Wells

Keyword(s):

Cell Cycle ◽

Nucleic Acids ◽

Gene Transcription ◽

S Phase ◽

Histone Gene ◽

Specific Factor ◽

Synchronized Cells ◽

Phase Regulation ◽

G2 Phase ◽

Binding Component

Levels of trans-acting factor (H1-SF) binding to the histone H1 gene-specific motif (5'-AAACACA-3' [L. S. Coles and J. R. E. Wells, Nucleic Acids Res. 13:585-594, 1985]) increase 12-fold from G1 to S-phase in synchronized cells and decrease again in G2 phase of the cell cycle. Since the H1 element is required for S-phase expression of H1 genes (S. Dalton and J. R. E. Wells, EMBO J. 7:49-56, 1988), it is likely that the increased levels of H1-SF binding component play an important role in S-phase regulation of H1 gene transcription.

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