Histone H2A.X Phosphorylation and Caspase-Initiated Chromatin
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Jefery et al. Epigenetics & Chromatin (2021) 14:37 https://doi.org/10.1186/s13072-021-00408-5 Epigenetics & Chromatin RESEARCH Open Access Histone H2A.X phosphorylation and Caspase-Initiated Chromatin Condensation in late-stage erythropoiesis Nazish N. Jefery1, Christina Davidson2, Scott A. Peslak3,4, Paul D. Kingsley1, Yukio Nakamura5, James Palis1 and Michael Bulger1* Abstract Background: Condensation of chromatin prior to enucleation is an essential component of terminal erythroid matu- ration, and defects in this process are associated with inefcient erythropoiesis and anemia. However, the mecha- nisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.X in erythropoiesis. Results: We fnd in multiple model systems that this histone is essential for normal maturation, and that the loss of H2A.X in erythroid cells results in dysregulation in expression of erythroid-specifc genes as well as a nuclear conden- sation defect. In addition, we demonstrate that erythroid maturation is characterized by phosphorylation at both S139 and Y142 on the C-terminal tail of H2A.X during late-stage erythropoiesis. Knockout of the kinase BAZ1B/WSTF results in loss of Y142 phosphorylation and a defect in nuclear condensation, but does not replicate extensive transcriptional changes to erythroid-specifc genes observed in the absence of H2A.X. Conclusions: We relate these fndings to Caspase-Initiated Chromatin Condensation (CICC) in terminal erythroid maturation, where aspects of the apoptotic pathway are invoked while apoptosis is specifcally suppressed. Keywords: H2A.X, g-H2A.X, BAZ1B, WSTF, Chromatin condensation, Erythropoiesis, Terminal erythroid maturation, Caspase, Phosphorylation, Apoptosis Introduction nuclear condensation is associated with myelodysplastic Erythropoiesis is a process of enormous magnitude, with syndromes and congenital anemias [3, 4]. Te factors and the adult producing over 2 million red blood cells each pathways that regulate the compaction of the erythroid second [1]. Te generation of a functional erythrocyte genome, however, are poorly understood. from a committed progenitor cell requires signifcant In addition to nuclear condensation and enucleation, changes in gene expression during a time of hemoglobin terminal erythroid maturation is characterized by loss accumulation, rapid cell division and nuclear condensa- of organelles. Tese features are similar to events that tion. In the span of 3–4 cell divisions, erythroid cells con- occur during apoptosis subsequent to caspase activa- dense their nuclei to approximately one-tenth of their tion. Caspase-3 in particular has been implicated in original volume prior to enucleation [2]. Disruption of terminal erythroid maturation, and has been shown to cleave Acinus, which functions in apoptotic chromatin *Correspondence: [email protected] condensation, and lamin B [5–9]. However, radiation 1 Center for Pediatric Biomedical Research, Department of Pediatrics, studies performed in the murine hematopoietic sys- University of Rochester, Rochester, NY, USA tem have revealed that erythroid maturation is asso- Full list of author information is available at the end of the article ciated with a dramatic change in the ability of cells to © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Jefery et al. Epigenetics & Chromatin (2021) 14:37 Page 2 of 15 apoptose. Late erythroid maturation is conventionally Results divided into distinct stages that are separated by cycles Loss of H2A.X results in changes in chromatin of cell division and distinguished by morphology [10, condensation during maturation 11]. Tus, earlier erythroid progenitors (BFU-E and To better understand the role of the variant histone CFU-E) and the most immature erythroid precursor, H2A.X in erythropoiesis, we examined the erythroid proerythroblast (ProE), are readily induced to apoptose compartment in bone marrow of H2A.X KO mice [24]. following clastogenic injury. However, the subsequent Tese mice exhibit macrocytic anemia with decreased stages of maturation, basophilic, polychromatic and numbers of reticulocytes, indicative of reduced produc- orthochromatic erythroblasts (BasoE, PolyE, OrthoE), tion of RBCs; these phenotypic changes are accompanied are resistant to apoptotic cell death [10, 11]. Tis is cor- by increased cell size (mean corpuscular volume, MCV) related with high expression of apoptosis inhibitor Bcl- compared to WT mice (Additional fle 2: Fig. S1a, b). xL at these later stages [12, 13]. Tus, while erythroid Cell numbers for other hematopoietic lineages are not maturation invokes components of a caspase-mediated signifcantly afected (Additional fle 2: Fig. S1a), indicat- apoptotic pathway, apoptosis is specifcally inhibited. ing that H2A.X loss specifcally impacts the erythroid Normal erythroid maturation requires the histone lineage. Next, we quantifed erythroblast intermediates H2A variant, H2A.X [14]. Loss of histone H2A.X in in bone marrow (Additional fle 2: Fig. S1c) [25] and erythroid cells in mice leads to a decrease in enucleated found an increase in total Ter119+ cells compared to WT cells and dyserythropoiesis, along with the appearance (Additional fle 1: Fig. S1d), consistent with an increased of Howell–Jolly bodies, which are nuclear remnants erythropoietic drive in response to anemia. In addition, indicative of inefcient enucleation, in mature red H2A.X KO mice have an increased proportion of more blood cells. Collectively, these features resemble phe- immature (basophilic) erythroblasts in the bone marrow notypes in patients with myelodysplastic syndrome and and a corresponding decrease in the proportion of the thus indicate defects in normal erythroid maturation most mature (orthochromatic) erythroblasts (Additional [14]. While the best studied function of histone H2A.X fle 1: Fig. S1e), indicating potential defects in terminal is its role in amplifying the signal for DNA repair upon erythroid maturation. Taken together, these data demon- phosphorylation of S139 in its C-terminal tail region strate that loss of H2A.X leads to dysregulated terminal (H2A.X pS139 or γ-H2A.X), other functions have been maturation and an inability to maintain steady-state lev- described [15–19]. H2A.X has been implicated in the els of erythrocyte production. maintenance of genomic integrity, in condensation of Terminal maturation of erythroid precursors is char- chromatin during spermatogenesis and in silencing acterized by a substantial decrease in nuclear size prior of embryonic genes in ES cells [15–17, 20, 21]. His- to enucleation, associated with chromatin condensation. tone H2A.X phosphorylation has also been implicated Increased MCV results in defects in nuclear maturation, in apoptotic pathways. DNA damage is thought to as seen in megaloblastic anemias due to folate or B12 induce dephosphorylation of H2A.X C-terminal tyros- defciencies [26]. Tus, the increased MCV of erythro- ine 142 (Y142) subsequent to phosphorylation of S139 blasts in H2A.X KO mice may refect improper nuclear (γ-H2A.X). In the absence of Y142 dephosphorylation, maturation. Consistent with this, we fnd that basophilic or if Y142 is re-phosphorylated, the dual phosphoryla- erythroblasts exhibit a higher nuclear:cytoplasmic (N:C) tion can be recognized by apoptotic signaling factors ratio compared to WT (Additional fle 2: Fig. S1f), sug- [22, 23]. gesting that nuclear maturation in H2A.X KO erythroid In this study, we fnd that loss of histone H2A.X in cells is impaired. Taken together, these data indicate that erythroid cells results in improper terminal matura- histone H2A.X plays a role in normal erythropoiesis, tion characterized by defective nuclear maturation and and may be required for normal nuclear maturation and dysregulation of erythroid gene expression. In addition, chromatin condensation. murine erythroid cells exhibit increased γ-H2A.X foci at the basophilic erythroblast stage while also exhibiting Prevalence of H2A.X and phosphorylated H2A.X Y142 phosphorylation. We generated knockout (KO) cell in terminal erythroid maturation lines and performed transcriptome profling in order to Phosphorylation of histone H2A.X at S139 (γ-H2A.X) is study the efects the loss of H2A.X or the Y142 kinase, an early signal for DNA repair pathways in response to BAZ1B, have on erythroid maturation. Tese data sug- DNA breaks. To determine if γ-H2A.X is present dur- gest that histone H2A.X serves multiple roles in late- ing