Histone Deacetylase 9 Activates γ-Globin Gene Expression in Primary Erythroid Cells

Shalini A Muralidhar,Valya Ramakrishnan,Inderdeep S Kalra,Wei Li,Betty S Pace

doi:10.1074/jbc.m110.115725

Abstract

Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of β-hemoglobinopathies probably involve protein modifications by histone deacetylases (HDACs) that mediate γ-globin gene regulation. However, the role of individual HDACs in globin gene expression is not very well understood; thus, the focus of our study was to identify HDACs involved in γ-globin activation. K562 erythroleukemia cells treated with the HbF inducers hemin, trichostatin A, and sodium butyrate had significantly reduced mRNA levels of HDAC9 and its splice variant histone deacetylase-related protein. Subsequently, HDAC9 gene knockdown produced dose-dependent γ-globin gene silencing over an 80-320 nm range. Enforced expression with the pTarget-HDAC9 vector produced a dose-dependent 2.5-fold increase in γ-globin mRNA (p < 0.05). Furthermore, ChIP assays showed HDAC9 binding in vivo in the upstream Gγ-globin gene promoter region. To determine the physiological relevance of these findings, human primary erythroid progenitors were treated with HDAC9 siRNA; we observed 40 and 60% γ-globin gene silencing in day 11 (early) and day 28 (late) progenitors. Moreover, enforced HDAC9 expression increased γ-globin mRNA levels by 2.5-fold with a simultaneous 7-fold increase in HbF. Collectively, these data support a positive role for HDAC9 in γ-globin gene regulation.

Highlights

Hemoglobin switching from fetal ␥-globin to adult ␤-globin gene expression occurs shortly before birth and is usually completed by the first 6 –12 months of life
Changes in histone deacetylases (HDACs) expression levels were quantified in K562 cells after treatment with the HbF inducers hemin (50 ␮M), trichostatin A (TSA) (0.5 ␮M), and NaB (2 mM) [26] for 48 h; ␥-globin mRNA levels were monitored to ensure that adequate induction was achieved (Fig. 1A)
In erythroid cells, the magnitude of HDAC inhibition does not directly correlate with the magnitude of ␥-globin induction by these compounds [13, 34], suggesting that HDAC subtypes or site-specific acetylation may be an important mechanism involved in ␥-globin gene activation

Summary

Introduction

Hemoglobin switching from fetal ␥-globin to adult ␤-globin gene expression occurs shortly before birth and is usually completed by the first 6 –12 months of life. Epigenetic modifications during erythroid maturation that allows interactions between the ␤-globin locus control region and the ␥-globin gene promoters [4, 5] are involved as well Various pharmacological agents, such as butyrate, decitabine, and hydroxyurea, have been shown to induce HbF synthesis in vitro and in vivo (6 – 8); hydroxyurea is the only drug approved for clinical use in sickle cell patients [9]. Limited investigations have been performed to determine the role of Class II HDAC genes (HDAC4, -5, -6, -7, -9, and -10) in ␥-globin gene regulation. These proteins display tissue-specific expression and have the ability to shuttle between the nucleus and cytoplasm of cells. These observations support the dynamic role of HDACs as co-repressors and co-activators in their overall control of gene expression [23]

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Results

Conclusion