Transcription factor EKLF (KLF1) recruitment of the chaperone HIRA is essential for β-globin 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 -associated and incorporation of and die early in embryogenesis, suggesting that it is essential for histone variants correlates with alterations in . 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 , 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 (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 (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 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. Flag antibody also coprecipitate HIRA. To confirm this inter- Although broad distribution and binding correlations have been action, the immunoprecipitation was also performed with anti- established for HIRA and H3.3 genome-wide (5, 12), a major HIRA. HA-tagged HIRA protein is precipitated efficiently by unresolved question in the field is the mechanism by which HIRA anti-HIRA antibody (Fig. S2, Right). However, EKLF can be and H3.3 are enriched at specific, developmentally critical sites, precipitated by anti-HIRA antibody only in the presence of ectopic not just in the erythroid program but for any transcriptional HIRA protein. These results indicate that EKLF readily interacts output (5, 40). Studies showing selective H3.3 enrichment at the with HIRA after exogenous cotransfection in vivo. β-globin locus, the critical importance of EKLF for its optimal The interaction between endogenous EKLF and HIRA was chromatin and transcriptional configuration, and its direct in- examined in two ways. First, we used murine erythroleukemia teraction with histone H3, all converge on the likelihood that (MEL) cells that express EKLF and HIRA (25) (Fig. S3). MEL these observations are operationally linked. With this in mind, we whole-cell extracts were immunoprecipitated with an anti-EKLF have identified a novel interaction between HIRA and erythroid- antibody (or the IgG antibody) and then blotted and probed with specific transcription factor EKLF by in vitro and in vivo ap- anti-EKLF and anti-HIRA antibodies. A human nonerythroid cell proaches. Importantly, we also find that depletion of HIRA line that does not express EKLF but does express HIRA (293T)

Fig. 1. Endogenous and deletion analysis of EKLF and HIRA interaction. (A) Whole-cell extracts from erythroid MEL and (untransfected) 293T cells (Left) or E13.5 murine fetal livers (Right) were prepared and subjected to immunoprecipitation with an anti- EKLF antibody or an IgG antibody, and immuno- blots were probed with an anti-HIRA antibody. Ten percent of total protein extract was loaded to serve as “input.” For cotransfection studies in 293T cells, schematic diagrams of full-length EKLF or HIRA and the deletion constructs used are shown at the top of B, C, and D. Cells were transfected with HA-tagged HIRA, Flag-tagged full-length EKLF, its deleted derivatives, or vector control, as indicated. Samples were then immunoprecipitated with anti-Flag (B)or anti-HA (C and D) antibodies and probed with anti- EKLF, anti-HA, or anti-Flag (indicated on right). Ex- pression of EKLFΔpro was separately analyzed on a higher percentage acrylamide gel (Fig. S4A). Ten percent of total protein extract was loaded to serve as “input.” ns, a nonspecific band observed after probing with anti-HA (D).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1405422111 Soni et al. Downloaded by guest on September 27, 2021 EKLF Interacts with the HIRA Carboxyl Terminus. Complementation analysis of chicken HIRA-null cells revealed that the amino- and carboxyl-terminal halves of HIRA have distinct roles resulting from region-specific interactions with chromatin and transcrip- tional modulators (44). To locate the binding region(s) between HIRA and EKLF, we constructed two truncation variants of HA-tagged HIRA containing either the amino-terminal half (amino acids 1–443, which overlaps the seven HIRA WD40 repeats) or the carboxyl-terminal half (444–1017). These constructs were trans- fected into 293T cells either alone or together with wild-type full- length EKLF. Although HIRA/1–443 exhibited no interaction, HIRA/444–1017 exhibited similar binding activity to that of the wild-type full-length HIRA protein (Fig. 1D). These data show that EKLF interacts with the region of HIRA that is also known to interact with CABIN1 and the bulk of the ASF1a binding B domain, but not with the WD40 region known to interact with UBN1.

HIRA Alters EKLF Transcriptional Activity at Erythroid Promoters. To elucidate the functional consequence of the interaction between EKLF and HIRA, we analyzed its effect on EKLF transcriptional activity by cotransfection with a human β-globin gene promoter/ luciferase reporter into K562 erythroleukemic cells (45). As Fig. 2. HIRA alters EKLF-mediated transcriptional activity of its target pro- expected, EKLF activates the reporter; although transfection of moters. K562 cells were transfected with luciferase reporters containing the HIRA alone had no effect, it enhanced EKLF activity (Fig. 2) in adult β-globin promoter together with expression plasmids for EKLF and HIRA. a manner similar to that seen with CBP/P300 (36). Luciferase activity was normalized against Renilla activity from a cotransfected Recent studies of EKLF transactivation mechanisms have control vector. The relative luciferase activity reflects the values obtained from demonstrated that not all targets are equivalently affected (29). triplicate experiments. We find this to also be true for HIRA’s effect on EKLF. For ex-

ample, EKLF activation of the BKLF promoter is enhanced in the BIOCHEMISTRY presence of HIRA but is not at the AHSP or p21 promoters (Fig. was used as a negative control. We find that endogenous EKLF S5). The β-globin and BKLF promoters share some similarities and HIRA interact (Fig. 1A, Left). Second, cell extracts prepared in their promoter architecture (e.g., INI and DPE elements), from embryonic mouse fetal liver cells isolated at a stage when they unlike the AHSP promoter (46). In addition, EKLF/TAF9 inter- are primarily erythroid (E13.5) were immunoprecipitated using actions are critical for β-globin but not AHSP promoter activity. anti-EKLF antibody or control IgG antibody. Our results show We conclude that HIRA–EKLF interactions play an important that EKLF interacts with HIRA in these primary erythroid cells role at selected erythroid promoters by modulating EKLF activity. (Fig. 1A, Right). Together these data support the cotransfection results and further demonstrate that endogenous EKLF and HIRA Is Required for Erythropoiesis in ES Cells. To directly ascertain HIRA proteins interact in vivo. the role of HIRA in erythroid genetic regulation, we first compared normal ES cells and genetically modified ES cells lacking HIRA The EKLF Zinc Finger Domain Interacts with HIRA. To map the sub- (47). We conducted a 6-d time course for the differentiation of ES domain of EKLF that is responsible for interacting with HIRA, cells into embryoid bodies (EBs) as a cell culture model of hema- topoietic development (48) and evaluated the expression of β-globin two EKLF deletion constructs were used. The first construct, Δ and the transcriptional regulators GATA-2, GATA-1, and EKLF. EKLF Pro (Fig. 1B), contains the zinc finger domain of EKLF GATA-2 plays a critical role early in hematopoietic development (amino acids 287–376) along with a Flag tag at the amino terminus. Δ (49), whereas EKLF and GATA-1 are downstream targets that are The second construct, EKLF ZnF (Fig. 1C), contains the proline- essential for establishment of erythroid differentiation. EBs from – rich domain of EKLF (amino acids 19 305; no tag). We performed HIRA-null cells are smaller in comparison with WT (Fig. S6A), but two immunoprecipitations, one using the Flag antibody coupled to most importantly we find that the onset of β-globin mRNA is greatly − − M2 agarose beads to pull down EKLFΔPro and another using reduced in differentiating HIRA / ES cells (Fig. 3A). However, a HA-specific antibody to pull down HIRA. The results show that there is also a dramatic reduction in the levels of the erythroid HIRA can be immunoprecipitated with EKLF proteins that con- regulators EKLF and GATA-1 (Fig. 3B) and in expression of the tain the zinc finger domain (full-length and EKLFΔPro) (Fig. 1B Ter119 terminal differentiation marker (Fig. S6B). Because there and Fig. S4A), but not with EKLF containing only its proline-rich is no change in levels of the hematopoietic regulator GATA-2 in −/− domain protein (EKLFΔZnF) (Fig. 1C). These data suggest that the HIRA ES cells compared with WT (Fig. 3B), we conclude the zinc finger domain of EKLF is primarily responsible for its that depletion of HIRA exerts an erythroid, but not hemato- interaction with HIRA. poietic, cell-specific effect. Because the zinc finger domain is also the DNA binding do- β main of EKLF, we determined whether the ability of EKLF to HIRA Is Necessary for Activation of -Globin During Cell Differentiation. Because the absence of HIRA led to a down-regulation of not only bind to DNA and interact with HIRA could be uncoupled. For β this we used a mutant EKLF that is compromised in its ability to -globin expression but also of the upstream transcription factors EKLF and GATA-1, we determined whether MEL cells could be bind to DNA (43). Wild-type EKLF or EKLF ZnF(m123) (a full- alternatively used to address whether HIRA is required for length variant containing mutations that alter critical histidines β-globin expression. Treatment of MEL cells with hexamethylene known to be important for coordinating zinc within each finger) bis-acetamide (HMBA) or DMSO results in high-level induction were cotransfected with HA-tagged HIRA, immunoprecipitated of erythroid genes, including β-globin, and eventual terminal dif- using anti-EKLF, and analyzed using anti-HIRA. We find that the ferentiation (50, 51). The expression of EKLF, GATA-1, and wild type and the zinc finger mutant of EKLF attain a slightly re- importantly, HIRA proteins, are stable during the differentiation duced level of interaction with HIRA after normalization with the of MEL cells (Fig. S3). MEL cells were individually or coinfected input (Fig. S4B). We conclude that the DNA-binding and with short hairpin (sh)RNA-expressing lentiviruses that target HIRA-binding activities of EKLF are separable. different regions of HIRA (52) (scheme outlined in Fig. S7A). The

Soni et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 Discussion We have shown that ablation of HIRA leads to a deficiency in erythropoiesis due to lower levels of the critical EKLF and GATA-1 transcription factors. In addition, depletion of HIRA disrupts expression of an important erythroid target, β-globin, even in the presence of normal levels of EKLF and GATA-1. As a result, HIRA plays critical roles in both the onset and main- tenance of erythropoiesis. Its control of specific targets during terminal erythroid differentiation is brought about, at least in part, by its interaction with and recruitment by EKLF to these restricted sites. Knowledge of the regulation and mode of action of these molecules raises a number of interesting biochemical Fig. 3. Depletion of HIRA effects hematopoietic differentiation during EB and developmental issues. differentiation. mRNA expression levels of adult β-globin (A), EKLF, GATA-1, and GATA-2 (B) at indicated days (d) after EB differentiation of wild-type and Developmental Convergence of EKLF and HIRA Function. EKLF is − − HIRA / ES cells were monitored by quantitative RT-PCR (normalized to highly restricted in its developmental expression pattern, because it GAPDH levels). Values are presented relative to expression level at day 0, is first expressed in the mesodermal blood islands of the yolk sac at which was set to 1. Results are the average of triplicates from a single ex- E7.5, then solely in the fetal liver as the site of erythropoiesis periment that is representative of two experiments. The error bars reflect SD. is changed during early development (55). Although ablation of EKLF yields visibly “normal” primitive yolk sac cells with slight effects on embryonic β-like globin expression, morphologically ab- expression of HIRA was reduced in the shHIRA #2 and in + errant definitive erythroid cells that are profoundly deficient in the shHIRA #1 2 MEL cells at both the protein and mRNA adult β-globin expression appear after the switch to fetal liver, levels but not by shHIRA #1 alone (Fig. S7B). Importantly, leading to embryonic lethality by E14.5 (27, 28). HIRA also plays depletion of HIRA had no effect on the expression of EKLF and a critical role in early developmental processes: null embryos exhibit GATA-1 (Fig. S7B). lethalitybyE10–11 due to defects that begin during gastrulation at We used these and two additional stably infected cell lines to β E6 and alter subsequent formation of the mesoderm and primitive study the effect of HIRA depletion on the expression of -globin streak (19). This can alter expression of important tissue-restricted after their treatment with DMSO to induce differentiation and β regulators in development, such as MyoD during myogenic differ- expression of adult -globin. As monitored after 4 d of differ- entiation (56) and VEGFR1 in vascular development and angio- entiation, the shHIRA#2-, #3-, and #4-expressing MEL cells β genesis (52). Analogous to our observations showing that erythroid exhibit a dramatic drop in -globin activation in proportion to lineage differentiation (EKLF, GATA-1, and β-globin) but not the level of RNA and protein knockdown (Fig. 4). No effect on β early hematopoiesis (GATA-2) is affected, HIRA-null ES cells -globin induction was observed in cells infected with the empty are able to generate neuronal precursor cells but not mature vector, a scrambled shRNA, or shHIRA #1 alone (Fig. 4). These neurons (47). Although we have focused on specific targets, results are also supported by a detailed time course of differ- absence of HIRA likely has additional effects deleterious to entiation and monitoring RNA isolated every 24 h after differ- erythroid maturation. entiation induction (Fig. S8). We conclude that HIRA reduction β It is intriguing that BMP4 expression is significantly down- leads to a significant drop in the activation of -globin, inde- regulated in HIRA-null embryos (19), because this signaling pendent of any effect on EKLF or GATA-1 expression. Coupled pathway plays a critical role in directing the onset of EKLF and with the data in Fig. 3, we can also conclude that HIRA is thus GATA1 expression (57, 58). As a result, HIRA is appropriately not only required for erythroid onset but also continuously re- placed to exert a significant biological effect in tissues within quired for maintenance of proper expression even after erythro- which EKLF expression and erythropoietic differentiation begins. poiesis has been underway.

HIRA Recruitment to the β-Globin Promoter Requires EKLF. To ad- dress the functional consequence of the interaction of HIRA with EKLF, first we studied HIRA occupancy at the β-globin promoter by chromatin immunoprecipitation (ChIP) of MEL cells by a benzonase-based, non–cross-linked protocol, using primers that overlap known EKLF binding sites at the β-pro- moter and the locus control region (LCR) (53). As shown in Fig. 5A, HIRA is present, and its occupancy increases after induction of differentiation, solely at the adult β-globin promoter but not at the upstream hypersensitive sites 2 and 3 (HS2 and HS3; critical components of the LCR) or the necdin promoter (used as a negative control). This demonstrates that HIRA interacts pre- cisely within the same region as EKLF at the promoter but not at the EKLF-bound upstream enhancer elements. Next we addressed whether the presence of HIRA on the β-globin promoter is EKLF dependent by monitoring its chromatin occupancy after using RNA interference to knock down EKLF β levels. We used two MEL stable cells lines expressing two different Fig. 4. HIRA is required for -globin expression. MEL cells were infected in- doxycycline (Dox)-inducible shEKLF RNAs that have been pre- dividually with lentiviral shRNAs directed against different regions of HIRA (1, 2, 3, 4) empty vector (V), or a scrambled control (Scr). Results from two viously shown to decrease EKLF expression and thus its occupancy separate experiments are shown (Left and Right). Protein and mRNA levels of at and activation of the β-globin promoter (54). We find that HIRA β β HIRA (Insets; -actin is protein loading control) were monitored in the infected occupancy at the -globin promoter is adversely affected in pro- cell lines for this experiment. Stably infected shHIRA MEL cells were subjected portion to the extent of EKLF knockdown in the two MEL lines, to treatment with DMSO for 96 h to induce differentiation, and β-globin even without induction of differentiation (Fig. 5B). These results, mRNA levels were monitored by quantitative RT-PCR in comparison with the together with our interaction assays, support the idea that EKLF is uninduced cells. Results are the average of triplicates from a single experiment required for recruitment of HIRA to the β-globin promoter. that is representative of up to three experiments. The error bars reflect SD.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1405422111 Soni et al. Downloaded by guest on September 27, 2021 have been shown to interact with HIRA (12, 25, 59), their ability to recruit it to a specific site had not been addressed. In this context, it is of interest that the EKLF zinc fingers are primarily responsible for its interaction with HIRA, because they are also critical for EKLF contact with histone H3 (37). Protein binding irrespective of an intact finger structure raises the speculation that EKLF/HIRA interactions may remain relevant at sites that do not contain a cognate EKLF DNA binding element. The EKLF zinc finger region is also its interaction site for protein Brg1 (60, 61). EKLF acetylated at K288 shows a higher affinity for the SWI/SNF chromatin remodeling complex (that minimally includes Brg1 and Baf155) and is a more potent ac- tivator of the β-globin promoter in in vitro reconstituted chro- matin (36, 62). These observations tie together well with HIRA, which also forms a protein complex with Brg1, Baf170, and Baf155, as shown by endogenous coimmunoprecipitation in HeLa cells and by their preferential coenrichment at active promoters and enhancers (12). HIRA is part of stable multiprotein complex that consists of UBN1, CABIN1, and ASF1a proteins (16). UBN1 interacts with the HIRA WD40 region (63) and ASF1a with a central “B” region adjacent to this module (13). On the other hand, CABIN1 interacts with the non-WD40, C-terminal region of HIRA (16), which we have shown also binds EKLF. As a result, it is possible that these interactions are antagonistic, as has been shown for Mef2 in myogenesis (64). The ASF1a component of the complex directly interacts with histone H3.3, as does EKLF. In this case these interactions are nonoverlapping, because the EKLF zinc Fig. 5. EKLF mediates the binding of HIRA to the β-globin promoter. We

fingers interact with the amino terminal tail of H3 (37), but BIOCHEMISTRY used a benzonase-based ChIP protocol in the absence of crosslinker and ASF1a interacts with the globular region of H3 (65, 66). HIRA sonication with a mixture of four anti-HIRA antibodies that led to a highly interacts with yet a different surface on ASF1a (13). enriched and specific signal. (A) Quantitative ChIP analysis of HIRA occupancy at the β-globin promoter, globin LCR hypersensitive sites HS2 and HS3, or These latter structural considerations enable a relatively necdin was performed with anti-HIRA antibodies or control IgG in MEL cells straightforward visualization of a HIRA/ASF1a/H3.3/EKLF in- without differentiation (day 0) or after DMSO-induced differentiation (72 h), teraction. However, placing this within the larger context of as indicated. (Inset) Agarose gel of the product after end-point PCR. (B)Pro- chromatin remodelers, and given that the multitasking EKLF zinc tein levels of EKLF after Dox-inducible knockdown (2 d) of stable MEL cells fingers also confer DNA recognition specificity at the β-globin that express inducible shEKLFs (46) (Inset; β-actin is the protein loading con- promoter, is clearly more complex. The end result, however, of trol). HIRA occupancy at the β-major promoter was measured by quantitative such recruitment by EKLF of HIRA is incorporation of H3.3 and ChIP using HIRA or IgG antibodies on untreated (−) or Dox-treated (+)control establishment of an open chromatin domain at the β-locus (37, 38, (parental) or shEKLF MEL cell lines 1 or 2. Results are the average of biological 59). Because nucleosomal core particles containing H3.3 are rel- triplicates from a single experiment that is representative of two experiments. atively less stable than those with H3.1 (4), transcriptional acti- vation of the β-globin gene is more readily accomplished (37). Constraints on Protein Interactions/Structural Considerations at the Materials and Methods β -Globin Promoter. Our studies demonstrate that directed re- A non–cross-linked/benzonase protocol was used for ChIP. Lentiviral shRNA- cruitment of the HIRA histone H3.3 chaperone to the develop- β expressing particles were used for RNA interference. Details of these and mentally controlled adult -globin gene is accomplished by its other experimental approaches are provided in SI Materials and Methods. interaction with the EKLF transcription factor. However, EKLF recruitment is discriminatory: HIRA was selectively recruited to ACKNOWLEDGMENTS. We thank Li Xue and Ismael Oumzil for technical the β-promoter and not to the upstream HS2 and HS3 sites within help; Debasree Dutta and Soumen Paul for HIRA shRNAs; Emily Bernstein the LCR. These data also provide an explanation for the selective for the CBX7 chromodomain expression plasmid; Simon Elsässer, Ariane enrichment of histone H3.3 at the promoter during erythroid Chapgier, and David Allis for the HIRA-null ES cell lines; Francois Morle for the differentiation (37), particularly because a large majority of HIRA shEKLF MEL cell lines; Annalisa Mancini, Carol McDonald, and Vikrant Singh for technical advice; and Xiajun Li and members of the J.J.B. laboratory for binding sites are enriched for H3.3 (12). Depletion of HIRA discussion throughout the course of the work. The Quantitative RT-PCR fa- decreases β-globin expression, and reduction of EKLF decreases cility is supported by the Mount Sinai School of Medicine. This work was HIRA recruitment. Although a number of transcription factors supported by National Institutes of Health Grant R01 DK46865 (to J.J.B.).

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