Circadian control of histone turnover during cardiac development and growth - PubMed (original) (raw)

Circadian control of histone turnover during cardiac development and growth

Adrian Arrieta et al. J Biol Chem. 2024 Jul.

Abstract

During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting a role for the circadian clock in temporal control of histone turnover and coordinated cardiomyocyte gene expression. We sought to elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Bmal1 knockdown in neonatal rat ventricular myocytes decreased myocyte size, total cellular protein synthesis, and transcription of the fetal hypertrophic gene Nppb after treatment with serum or the α-adrenergic agonist phenylephrine. Depletion of Bmal1 decreased the expression of clock-controlled genes Per2 and Tcap, as well as Sik1, a Bmal1 target upregulated in adult versus embryonic hearts. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by micrococcal nuclease-quantitative PCR and impaired histone turnover as measured by metabolic labeling of acid-soluble chromatin fractions. Sik1 knockdown in turn decreased myocyte size, while simultaneously inhibiting natriuretic peptide B transcription and activating Per2 transcription. Linking these changes to chromatin remodeling, depletion of the replication-independent histone variant H3.3a inhibited myocyte hypertrophy and prevented phenylephrine-induced changes in clock-controlled gene transcription. Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription. Replication-independent histone turnover is required for transcriptional remodeling of clock-controlled genes in cardiac myocytes in response to growth stimuli.

Keywords: chromatin; histone; myocyte; nucleosome; proteostasis.

Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1

Figure 1

Bmal1 depletion impairs expression of clock-controlled and hypertrophic genes.A, experimental timeline of cultured NRVMs transfected with scrambled or Bmal1-targeted siRNA and treated with increasing serum concentrations. B and C, Bmal1 and GAPDH immunoblots and RT-qPCR demonstrating Bmal1 knockdown. D and E, NRVM lysate protein concentration and cell size measurements. F_–_H, NppbIntron, Per2, and Tcap RT-qPCR. ∗ indicates significant difference between pairwise comparisons, p < 0.05 by two-way ANOVA with Tukey’s post hoc analysis; mean ± SEM. Nppb, natriuretic peptide B; NRVM, neonatal rat ventricular myocyte; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction.

Figure 2

Figure 2

Replication-independent histone turnover is required for myocyte growth and hypertrophic gene expression. NRVMs were transfected with siRNA against H3.3a, and knockdown was confirmed by immunoblot (A and B). H3.3a knockdown results in decreased H3.3a but not H3.3b transcript levels (C), and a decrease in NppbIntron and cell size (D). p Values from unpaired t tests; mean ± SEM. Nppb, natriuretic peptide B; NRVM, neonatal rat ventricular myocyte.

Figure 3

Figure 3

Bmal1 depletion disrupts histone stoichiometry and impairs histone turnover.A, experimental workflow of NRVM transfection and AHA treatment. B, protein lysate concentration and cell size measurement of control or Bmal1 depleted cells. C, immunoblotting of protein lysates for core histones and GAPDH (quantified in D; p values from unpaired t tests). E, diagram of nuclear and chromatin isolation as validated by histone H3 and GAPDH immunoblotting. F, oriole fluorescence stain of total protein and histone H3, H4, H2A, and H2B immunoblots of acid-soluble chromatin fractions. Immunoblot signal was normalized to total protein oriole fluorescence signal from control or Bmal1-depleted NRVM (quantitation shown in G). H, oriole fluorescence staining and streptavidin-HRP blot of acid-soluble chromatin fractions following click-chemistry with biotin-alkyne. I, quantitation of total AHA-labeled acid-soluble chromatin fractions, normalized to oriole fluorescence (_p_-values from unpaired t tests; mean ± SEM). AHA, L-azidohomoalanine; HRP, horseradish peroxidase; NRVM, neonatal rat ventricular myocyte.

Figure 4

Figure 4

Sik1 is a novel clock-controlled gene with cardiac-specific chromatin organization that is altered in response to growth stimuli.A, diagram of analyses to identify Bmal1 targets upregulated in the adult as compared to the embryonic heart and to identify cardiac-specific Bmal1 candidates. g:Profiler analyses indicating Bmal1-regulated gene ontologies common to the liver, kidney, and heart (B), and Bmal1-regulated gene ontologies specific to the heart (C). D, overlap of Bmal1 ChIP-seq data (21) from indicated mouse tissues and ATAC-seq and RNA-seq data of Sik1 from hearts of mice subjected to transaortic constriction-mediated cardiac hypertrophy (6). E, overlay of Mef2A, Nkx2.5, Tbx5, SRF, GATA4, RNAPII, and H3K27Ac ChIP-seq data of Sik1 from embryonic and adult mouse hearts (35). F, ATAC-seq data of the Sik1 loci generated from NRVM, demonstrating increased Sik1 chromatin accessibility after treatment with PE (38). ATAC-seq, assay for transposase-accessible chromatin sequencing; ChIP-seq, chromatin immunoprecipitation sequencing; NRVM, neonatal rat ventricular myocytes; PE, phenylephrine; RNAPII, RNA polymerase II; Sik1, salt inducible kinase 1.

Figure 5

Figure 5

Bmal1 is required for Sik1 promoter accessibility and transcriptional induction in response to a growth stimulus.A, diagram of MNase assay, showing accessibility and degradation of open regions of chromatin. B, optimization of MNase for examination of chromatin compaction. In addition, 0.1U for 5 min led to uniform digestions: this global pattern was unaffected by Bmal1 depletion. C, change in promoter DNA (indicative of MNase degradation) at Per2 and two Sik1 loci highlighted in Fig. 4_E_ and effect of Bmal1 depletion (p values from unpaired t tests). D, experimental workflow of PE treatment. Clock-controlled gene transcripts measured by RT-qPCR and effect of PE on hypertrophy and nascent Nppb transcription (NppbIntron). E, effect of PE on nascent Sik1 transcription (Sik1Intron) and the role of Bmal1 on this process (F). For D and E, ∗ indicates p < 0.05 by one-way ANOVA with Tukey’s post hoc analysis for pairwise comparisons in the same treatment condition; for (F), ∗ indicates p < 0.05 by two-way ANOVA with Tukey’s post hoc analysis for pairwise comparisons. G, experimental workflow for PE treatment and Bmal1 depletion. Abundance of Bmal1 and fetal gene transcripts (Nppb, Nppa, and Acta1) were assessed by RT-qPCR, and hypertrophy via cell size measurements. Transcription of additional clock-controlled gene levels measured by RT-qPCR include Per2Intron, TcapIntron, and SikIntron (∗ indicates p < 0.05 by two-way ANOVA with Tukey’s post hoc analysis for pairwise comparisons). H, experimental workflow for Sik1 depletion. Myocyte hypertrophy was assessed by cell size, NppbIntron RT-qPCR, and total cellular protein measurements. Per2Intron RT-qPCR performed to examine clock-controlled gene transcription (p values from unpaired t test; mean ± SEM). PE, phenylephrine; Nppb, natriuretic peptide B; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; Sik1, salt inducible kinase 1.

Figure 6

Figure 6

Replication-independent histone variant H3.3a is required for clock-controlled gene transcription in response to α-adrenergic stimulation.A, experimental workflow of PE treatment and Bmal1 depletion. B, effect of PE on histone H3.3a as measured by RT-qPCR. Hypertrophy was assessed by NppbIntron RT-qPCR and cell size. Clock-controlled gene levels were measured by RT-qPCR, including Bmal1, TcapIntron, and Sik1Intron (∗ indicates p < 0.05 by two-way ANOVA with Tukey’s post hoc analysis for pairwise comparisons; mean ± SEM). C, summary diagram: in response to a hypertrophic stimulus, Bmal1 binds transcriptionally inactive and inaccessible clock-controlled target genes and activates their transcription via coordination of histone turnover. Sik1 then executes prohypertrophic actions, while Per2 serves to negatively regulate Bmal1 transcriptional activity. Bmal1 depletion impairs this chromatin remodeling cascade. PE, phenylephrine; Nppb, natriuretic peptide B; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; Sik1, salt inducible kinase 1.

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