Logic of a mammalian metabolic cycle: an oscillated NAD+/NADH redox signaling regulates coordinated histone expression and S-phase progression

Abstract

Many biological activities naturally oscillate. Here, we show that the NAD(+)/NADH ratios (redox status) fluctuate during mammalian cell cycle, with the S-phase redox status being the least oxidative. The S-phase NAD(+)/NADH redox status gates histone expression and S-phase progression, and may provide a genome protection mechanism during S-phase DNA replication as implicated in yeast. Accordingly, perturbing the cellular redox inhibits histone expression and leads to S-phase arrest. We propose that the S-phase NAD(+)/NADH redox status constitutes a redox signaling, which along with the cyclin E/cdk2 signaling regulates histone expression and S-phase progression.

Figure 1

Free cellular NAD⁺/NADH ratios fluctuate during cell cycle. (A) Random HeLa cells (R), or cells separated at the G₁, S- or G₂-phases were subject to FACS analyses (top); H2B mRNA levels (middle) and (free) cellular NAD⁺/NADH ratios (bottom) were measured. (B) The arrow underneath indicates that the re-cultured G₁-phase cells progressed into the G₁/S transition and S-phase (FACS analyses; top), and free cellular NAD⁺/NADH ratios (bottom) and H2B mRNA levels (middle) were measured.

Figure 2

Effects of exogenous NAD(H) on H2B expression in vivo. (A–C) H2B mRNA levels in NAD(H)- treated (4 hrs) cells (A, RT-PCR [NADH]; B, RT-PCR [NAD⁺]; C, real-time PCR [NADH and NAD⁺]). (D) Free intracellular NAD⁺/NADH ratios effected by NAD(H) (0.5 mM, 4 hrs). (E) Cell cycle profiles in control or NAD(H)-treated cells (0.5 mM). Top, BrdU-FACS; bottom, propidium iodide (PI) staining. (F) An H2B-promoter-luciferase reporter assay in control or NAD(H)-treated cells. (G) A chromatin immunoprecipitation (ChIP) assay on control or NAD(H)-treated (0.5 mM, 4 hrs) cells to evaluate the H2B promoter occupancy by p38/GAPDH. Control IgG or anti-Oct-1, anti-TFIIB or anti-p38/GAPDH IgGs were used. The input was either 2% (NAD⁺) or 1% (NADH) of the starting chromatin.

Figure 3

An S-phase NAD⁺/NADH redox window. (A) H2B mRNA levels (RT-PCR) in S-phase (profile shown in Fig. 1B; top) HeLa cells untreated or treated (0.25 mM, 4 hrs) with NADH (top) or NAD⁺ (bottom). (B) An S-phase redox window, see text. (C) Top, free intracellular NAD⁺/NADH ratios in the 30–40% or 80–90% confluence HeLa cells untreated or treated with NAD(H) (4 hrs, 0.5 mM); bottom, cell cycle profiles (BrdU-FACS analyses). (D) H2B mRNA levels (real-time PCR, top; RT-PCR, bottom) in the two cell populations untreated or treated with NAD(H) (4 hrs, 0.5 mM).

Figure 4

A redox window in vitro or in oocytes. (A) The window (in green) is pre-established by pre-existing redox components, the removal of which allows for the biphasic response of H2B transcription to exogenously-enforced NAD⁺ or NAD⁺/NADH ratio titrations. (B) In a crude B cell nuclear extract, high exogenous NAD(H) levels disturb the pre-established redox window (see A) to inhibit the H2B transcription. The IgH template, the transcription of which is not redox-modulated,^, is an internal control. (C) The H4 gene promoter is not redox-sensitive in the crude B cell nuclear extract, neither is the control (immunoglobulin kappa gene promoter).

Figure 5

Coordinated histone expression. (A and B) H2B and other core histone mRNA levels in control or NaN₃-treated (1 mM, 2 hrs) HeLa cells as measured by RT-PCR (A) or quantified by real-time PCR (B). (C and D) correspond to (A and B) but show histone mRNA levels in control or NAD(H)-treated HeLa cells (0.5 mM, 4 hrs).

Figure 6

Redox status and S-phase progression. (A) Cell cycle profiles of HeLa cells treated by NAD(H) (0.5 mM, 24 hrs), analyzed by BrdU-FACS assays (top panel) and propidium iodide (PI) staining (bottom panel). (B) The p21^cip1 expression levels in the redox-perturbed HeLa cells treated with NAD(H) (0.5 mM) for indicated hrs; p21^cip1 levels were examined by immunoblots with p38/GAPDH as a loading control. (C) More complete H2B expression repression in the NAD(H)-treated (0.5 mM, 24 hrs) HeLa cells (bottom, RT-PCR; top, real-time PCR quantification). (D) A model emphasizing that an MMC-based redox signaling along with cyclin E/cdk2 signaling regulates histone expression and S-phase progression.