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Recruitment of the Histone Chaperone HIRA Is Essential for Β-Globin Gene Expression

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Recruitment of the Histone Chaperone HIRA Is Essential for Β-Globin Gene Expression Transcription factor EKLF (KLF1) recruitment of the histone chaperone HIRA is essential for β-globin gene expression Shefali Sonia, Nikolay Pchelintsevb, Peter D. Adamsb,c, and James J. Biekera,d,e,1 aDepartment of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029; bInstitute of Cancer Sciences, University of Glasgow, Glasgow G61 1BD, Scotland; cBeatson Institute for Cancer Research, Glasgow G61 1BD, Scotland; and dBlack Family Stem Cell Institute, eTisch Cancer Institute, Mount Sinai School of Medicine, New York, NY 10029 Edited by Gary Felsenfeld, National Institutes of Health, Bethesda, MD, and approved August 12, 2014 (received for review March 25, 2014) The binding of chromatin-associated proteins and incorporation of and die early in embryogenesis, suggesting that it is essential for histone variants correlates with alterations in gene expression. proper development and survival (19, 20). The precise nature of These changes have been particularly well analyzed at the mamma- HIRA’s absolute requirement for vertebrate development, however, lian β-globin locus, where transcription factors such as erythroid remains to be elucidated. HIRA family member proteins are char- Krüppel-like factor (EKLF), which is also known as Krüppel-like factor acterized by seven tryptophan-aspartic acid (WD) repeats con- 1 (KLF1), play a coordinating role in establishing the proper chroma- served at the amino terminus, predicted to form a β-propeller tin structure and inducing high-level expression of adult β-globin. structure, and the presence of nuclear localization signals. The We had previously shown that EKLF preferentially interacts with carboxyl-terminal region of HIRA is responsible for its inter- histone H3 and that the H3.3 variant is differentially recruited to action with proteins Pax-3 (21), HIRIP, and core histones (22). the β-globin promoter. We now find that a novel interaction be- Although mutational studies have identified key residues that play tween EKLF and the histone cell cycle regulation defective homolog an important role in specifying H3.3 deposition (3, 5), and more A (HIRA) histone chaperone accounts for these effects. HIRA is not recently two key H3.3 residues were shown to be important for its only critical for β-globin expression but is also required for activation recognition by DAXX protein (23, 24), it is still unclear how H3.3 is of the erythropoietic regulators EKLF and GATA binding protein 1 targeted to transcriptionally active regions. BIOCHEMISTRY (GATA1). Our results provide a mechanism by which transcription Erythroid Krüppel-like factor (EKLF), which is also known as factor-directed recruitment of a generally expressed histone chap- Krüppel-like factor 1 (KLF1), an erythroid cell-specific zinc finger β erone can lead to tissue-restricted changes in chromatin compo- protein, is a key player in activating mammalian -globin gene transcription by virtue of its binding ability to its cognate CACCC nents, structure, and transcription at specific genomic sites during β differentiation. sequence element at the -globin promoter (25). Genetic ablation of EKLF leads to a loss of specific DNaseI hypersensitive site in the proximal β-globin promoter and a lack of DNase hypersensi- fficient packaging of DNA in a highly organized chromatin tivity at hypersensitive site 3 at the distal locus control region (26), Estructure inside the cell is a remarkable characteristic of all indicating that EKLF is required for the chromatin reorganization eukaryotic organisms. Chromatin assembly is a stepwise process at the β-globin promoter. EKLF-null embryos die of anemia at that requires histone chaperones to deposit histones in forming embryonic day (E)14.5, because definitive erythroid cells fail to nucleosomes. These chaperones are essential to facilitate ordered produce β-globin transcripts in vivo, leading to a profound β-thal- assembly of nucleosomes for both replication-dependent and assemia (27, 28). It is now recognized that EKLF is a global -independent events (1). The incorporation of histone variants also modulates chromatin Significance dynamics. Histone variant H3.3 differs from its canonical coun- terpart H3.1 in only five amino acid residues but has a very distinct β function. H3.3 is incorporated into nucleosomes independent of The -like globin locus has provided a long-standing model DNA replication and also serves as an epigenetic mark of active for the study of cell-specific and developmental control of chromatin (2, 3). H3.3 incorporation into nucleosomes contributes transcription and chromatin structure. We have previously to their destabilization, thus facilitating transcription (4). Hence, shown that the replication-independent histone H3.3 variant is H3.3 is highly enriched at active promoters and gene bodies of enriched at the active adult β-globin promoter. Although the actively transcribed genes and also at regulatory sites of both erythroid Krüppel-like factor (EKLF), which is also known as active and inactive genes (5–7). The importance of H3.3 is fur- Krüppel-like factor 1 (KLF1), transcription factor interacts with ther highlighted by the recent observation that H3FA3, one of histone H3, it does not distinguish between the H3.1 or H3.3 the two genes encoding H3.3, is mutated in pediatric malignant variants. We now show that EKLF interacts with the H3.3 brain tumors and that these mutations are proposed to drive tu- chaperone named histone cell cycle regulation defective homo- mor formation (8, 9). log A (HIRA) and that this enables its selective recruitment of H3.3 deposition is mediated by distinct factors at specific genomic HIRA to the promoter. To our knowledge, our studies implicate regions, with histone cell cycle regulation defective homolog A HIRA for the first time in establishment of erythropoiesis and (HIRA)beinginvolvedindepositionofH3.3atpromotersandinthe explain how critical protein interactions can lead to directed body of active genes (10–12). Further supporting these observations, changes in histone variants at restricted sites. the biochemical isolation and characterization of protein complexes containing preassembled histone H3.1 and H3.3 from human cells Author contributions: S.S. and J.J.B. designed research; S.S. performed research; N.P. and P.D.A. revealed that H3.1 associates with the chaperone chromatin as- contributed new reagents/analytic tools; S.S. and J.J.B. analyzed data; S.S. and J.J.B. wrote the sembly factor-1, whereas H3.3 is incorporated into chromatin by the paper; and N.P. and P.D.A. supplied reagents, protocols, and constructive commentary. HIRA chaperone complex consisting of HIRA, CABIN1, and The authors declare no conflict of interest. UBN1 together with the histone-binding protein ASF1 (10, 13–17). This article is a PNAS Direct Submission. In mammals, HIRA was originally identified as a gene that is 1To whom correspondence should be addressed. Email: [email protected]. deleted in DiGeorge syndrome (18). HIRA plays an important This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. role at gastrulation, and HIRA-null mice are grossly abnormal 1073/pnas.1405422111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1405422111 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 regulator of erythroid gene expression (29–31) because its activation impairs hematopoietic development in mouse ES cells. Our data target repertoire includes protein-stabilizing, heme biosynthetic show that HIRA is not only required for transcriptional activation pathway, red cell membrane protein, cell cycle, and transcription of globin genes but also for activation of erythropoietic regulators, factor genes in both primitive and definitive cells. such as EKLF and GATA-1, during erythroid differentiation. EKLF can undergo multiple modifications, including phos- phorylation (32), sumoylation (33), ubiquitination (34), and acet- Results ylation (35, 36), and these in turn alter its ability to interact with EKLF and HIRA Interact in Vivo. Because only one of the five amino modifiers (e.g., CBP/p300, Sin3A) and chromatin remodelers acid differences between the H3.1 and H3.3 variants reside in (e.g., SWI/SNF). Lys-288 acetylation is critical for recruitment of its region of interaction with EKLF (37), we tested whether CBP to the β-globin locus, modification of histone H3, occupancy a modified histone H3 might alter the interaction, particularly by EKLF, opening of chromatin structure, and transcription of given that most of its modifications are localized to the amino tail adult β-globin (37). EKLF helps to coordinate this process by the (41) that overlaps the EKLF interaction region (37). However, specific association of its zinc finger domain with the histone H3 using an in vitro array containing all known modifications of H3 amino terminus. These interactions likely play a crucial role in and H4 (Active Motif), we find no discrimination by EKLF under establishing the correct 3D structure at the β-like globin locus (38) conditions whereby the CBX7 chromodomain (42) discriminates and transcription factories in vivo that enable efficient coordinate its modified H3 targets (Fig. S1). As a result, we investigated expression of select EKLF target genes (39). whether EKLF might recruit histone H3.3 to the β-globin pro- Previous work from our laboratory demonstrated that the moter via its chaperone, HIRA. replication-independent H3.3, but not the replication-dependent Coimmunoprecipitation assays were performed after cotrans- H3.1, is enriched on the β-globin promoter after the induction of fection of Flag-tagged EKLF and HA-tagged HIRA (or their differentiation of erythroid MEL cells (37). Because only one of empty vector controls) into 293T cells. EKLF but not HIRA alone the five amino acid differences reside in this region, affinity dif- can be precipitated efficiently by the anti-Flag antibody (Fig. S2, ferences with EKLF might not account for the differential H3.3 Left). However, only in the presence of EKLF protein can the anti- recruitment to the actively transcribing region of the globin gene.
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