Heat shock transcription factor 1 regulates the fetal γ-globin expression in a stress-dependent and independent manner during erythroid differentiation

Abstract

Heat shock transcription factor 1 (HSF1) is a highly versatile transcription factor that, in addition to protecting cells against proteotoxic stress, is also critical during diverse developmental processes. Although the functions of HSF1 have received considerable attention, its potential role in β-globin gene regulation during erythropoiesis has not been fully elucidated. Here, after comparing the transcriptomes of erythrocytes differentiated from cord blood or adult peripheral blood hematopoietic progenitor CD34⁺ cells in vitro, we constructed the molecular regulatory network associated with β-globin genes and identified novel and putative globin gene regulators by combining the weighted gene coexpression network analysis (WGCNA) and context likelihood of relatedness (CLR) algorithms. Further investigation revealed that one of the identified regulators, HSF1, acts as a key activator of the γ-globin gene in human primary erythroid cells in both erythroid developmental stages. While during stress, HSF1 is required for heat-induced globin gene activation, and HSF1 downregulation markedly decreases globin gene induction in K562 cells. Mechanistically, HSF1 occupies DNase I hypersensitive site 3 of the locus control region upstream of β-globin genes via its canonical binding motif. Hence, HSF1 executes stress-dependent and -independent roles in fetal γ-globin regulation during erythroid differentiation.

Introduction

The human β-globin gene locus consists of five functional globin genes (embryonic ε, fetal ^Gγ and ^Aγ, and adult δ and β) within a 70 kb domain, arranged tandemly corresponding to their progressive activation during ontology development. The β-globin genes are primarily regulated by a locus control region (LCR) containing five DNase I hypersensitive sites (HS1 to HS5) located upstream of the embryonic ε-globin gene [1]. The β-globin locus enables to establish a three-dimensional chromosomal interaction conformation by looping of the LCR into the proximity with gene promoters to initiate transcription [2].

During development, human β-globin genes undergo two waves of developmental switch, namely β-globin switching [3]. The first switch (the ε−γ globin switch) takes place during the first six to eight weeks of gestation, accompanying with hematopoietic site shifts from yolk sac to fetal liver, which consequently silences the embryonic ε-globin and activates two fetal γ-globin genes (^Gγ and ^Aγ). The second switch (γ-β globin switch) occurs at birth, when the hematopoietic sites migrate to bone marrow and spleen, leading to the gradual repression of fetal (^Gγ and ^Aγ) globin expression and induction of adult (δ and β) globin expression.

The β-globin locus, as a paradigm of developmentally regulated multigene loci, has been intensively investigated. Two opposing, but complementary, mechanisms have been proposed for the β-globin switches [3]. Gene competition model demonstrates that full activation of certain globin gene(s) at the proper developmental stage is (are) controlled by the LCR, which preferentially enhances the closest gene expression in “open” chromatin; and autonomous gene silencing model holds that embryonic (ε-) and fetal (γ-) globin are appropriately repressed during development.

Since the reactivation of fetal γ-globin gene has important clinical and therapeutic implications [4], multiple factors governing the fetal γ-globin gene repression in adult-stage erythroid cells have been identified and characterized. For example, transcription factors, including B-cell lymphoma/leukemia 11A (BCL11A) [5], testosterone receptor (TR) 2 and TR4[6,7], leukemia/lymphoma-related factor (LRF) [8], sex determining region Y-box (SOX6) [9], MYB [10] and others [11], have been intensively studied. Additionally, epigenetic modifiers are also associated with fetal γ-globin regulation in erythroid cells at adult stage, e.g., DNA methyltransferase 1 (DNMT1) [12], lysine-specific histone demethylase 1 (LSD1) [13] and histone deacetylase 1 and 2[14]. Although these studies significantly increased the understanding of γ-globin gene regulation, it is still under intense investigation to yield new pharmacological agents. Therefore, novel fetal γ-globin reactivators and novel therapeutic agents are still needed.

The mammalian heat shock transcription factor (HSF) family consists of four members (HSF1, HSF2, HSF3 and HSF4), of which HSF1 is the master regulator. HSF1 is responsible for the activation of heat shock proteins (HSPs) under conditions of acute proteotoxic stress [15]. In Hsf1-knockout mice (Hsf1^−/−) and fibroblasts obtained from Hsf1^−/− mice, heat stress did not trigger HSP activation, indicating that HSF1 is essential for the development of thermotolerance to maintain cellular integrity under stress [[16], [17], [18], [19]]. HSF1 is a highly conserved transcription factor and is composed of one DNA-binding domain (DBD), three heptad-repeat domains (HR-A, HR-B and HR-C) and one transcriptional activation domain. Among them, the DBD is the best-conserved region of HSF1 and is considered the signature domain for recognizing heat shock response elements (HSEs) [[20], [21], [22]], which contain at least three inverted repeats of consensus sequence of nGAAn [23].

In addition to protecting cells against proteotoxic stress, HSF1 is also involved in various important physiological processes during development and survival, as indicated by accumulating evidence [[24], [25], [26], [27], [28], [29], [30], [31]]. Hsf1-knockout mice, under stress-free conditions, exhibit multiple defects, such as increased prenatal lethality, growth retardation, enlarged ventricles, neurodegeneration, female infertility and progressive myelin loss [17,32,33]. Moreover, both single gene-based and genome-scale studies have revealed that a large number of genes that are not classified as HSP genes or molecular chaperones are subject to HSF1-dependent control [34].

In this study, by integrating weighted gene co-expression network analysis (WGCNA) and context likelihood of relatedness (CLR) algorithms, we constructed a transcriptional regulatory network associated with globin genes and identified novel putative globin regulators. Among these putative regulators, HSF1 involved in the control of γ-globin gene expression. Further study showed that HSF1 knockdown significantly decreased γ-globin gene expression in primary erythroid cells. Additionally, HSF1 is essential for fetal γ-globin gene induction upon heat stress. Mechanistically, HSF1 bound to the LCR HS3 site via its canonical binding motif to regulate fetal γ-globin and adult β-globin expression during erythropoiesis.