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Structural basis for transcription factor ZBTB7A recognition of DNA and effects of ZBTB7A somatic mutations that occur in human acute myeloid leukemia

2023; Elsevier BV; Volume: 299; Issue: 2 Linguagem: Inglês

10.1016/j.jbc.2023.102885

ISSN

1083-351X

Autores

Ren Ren, J.R. Horton, Qin Chen, Jie Yang, Bin Liu, Yun Huang, Robert Blumenthal, Xing Zhang, Xiaodong Cheng,

Tópico(s)

Retinoids in leukemia and cellular processes

Resumo

ZBTB7A belongs to a small family of transcription factors having three members in humans (7A, 7B, and 7C). They share a BTB/POZ protein interaction domain at the amino end and a zinc-finger DNA-binding domain at the carboxyl end. They control the transcription of a wide range of genes, having varied functions in hematopoiesis, oncogenesis, and metabolism (in particular glycolysis). ZBTB7A-binding profiles at gene promoters contain a consensus G(a/c)CCC motif, followed by a CCCC sequence in some instances. Structural and mutational investigations suggest that DNA-specific contacts with the four-finger tandem array of ZBTB7A are formed sequentially, initiated from ZF1–ZF2 binding to G(a/c)CCC before spreading to ZF3–ZF4, which bind the DNA backbone and the 3′ CCCC sequence, respectively. Here, we studied some mutations found in t(8;21)-positive acute myeloid leukemia patients that occur within the ZBTB7A DNA-binding domain. We determined that these mutations generally impair ZBTB7A DNA binding, with the most severe disruptions resulting from mutations in ZF1 and ZF2, and the least from a frameshift mutation in ZF3 that results in partial mislocalization. Information provided here on ZBTB7A–DNA interactions is likely applicable to ZBTB7B/C, which have overlapping functions with ZBTB7A in controlling primary metabolism. ZBTB7A belongs to a small family of transcription factors having three members in humans (7A, 7B, and 7C). They share a BTB/POZ protein interaction domain at the amino end and a zinc-finger DNA-binding domain at the carboxyl end. They control the transcription of a wide range of genes, having varied functions in hematopoiesis, oncogenesis, and metabolism (in particular glycolysis). ZBTB7A-binding profiles at gene promoters contain a consensus G(a/c)CCC motif, followed by a CCCC sequence in some instances. Structural and mutational investigations suggest that DNA-specific contacts with the four-finger tandem array of ZBTB7A are formed sequentially, initiated from ZF1–ZF2 binding to G(a/c)CCC before spreading to ZF3–ZF4, which bind the DNA backbone and the 3′ CCCC sequence, respectively. Here, we studied some mutations found in t(8;21)-positive acute myeloid leukemia patients that occur within the ZBTB7A DNA-binding domain. We determined that these mutations generally impair ZBTB7A DNA binding, with the most severe disruptions resulting from mutations in ZF1 and ZF2, and the least from a frameshift mutation in ZF3 that results in partial mislocalization. Information provided here on ZBTB7A–DNA interactions is likely applicable to ZBTB7B/C, which have overlapping functions with ZBTB7A in controlling primary metabolism. ZBTB7A is a member of BTB/POZ protein interaction domain–containing C2H2 zinc-finger (ZF) proteins (Fig. 1A). This protein is also known as FBI (for factor that binds to the inducer of short transcripts (1Pessler F. Pendergrast P.S. Hernandez N. Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.Mol. Cell. Biol. 1997; 17: 3786-3798Crossref PubMed Scopus (71) Google Scholar)), LRF (for leukemia/lymphoma-related factor (2Davies J.M. Hawe N. Kabarowski J. Huang Q.H. Zhu J. Brand N.J. et al.Novel BTB/POZ domain zinc-finger protein, LRF, is a potential target of the LAZ-3/BCL-6 oncogene.Oncogene. 1999; 18: 365-375Crossref PubMed Scopus (127) Google Scholar, 3Liu C.J. Prazak L. Fajardo M. Yu S. Tyagi N. Di Cesare P.E. Leukemia/lymphoma-related factor, a POZ domain-containing transcriptional repressor, interacts with histone deacetylase-1 and inhibits cartilage oligomeric matrix protein gene expression and chondrogenesis.J. Biol. Chem. 2004; 279: 47081-47091Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar)), OCZF (for osteoclast-derived zinc finger (4Kukita A. Kukita T. Ouchida M. Maeda H. Yatsuki H. Kohashi O. Osteoclast-derived zinc finger (OCZF) protein with POZ domain, a possible transcriptional repressor, is involved in osteoclastogenesis.Blood. 1999; 94: 1987-1997Crossref PubMed Google Scholar)), and POKEMON (for POK erythroid myeloid ontogenic factor (5Maeda T. Hobbs R.M. Merghoub T. Guernah I. Zelent A. Cordon-Cardo C. et al.Role of the proto-oncogene Pokemon in cellular transformation and ARF repression.Nature. 2005; 433: 278-285Crossref PubMed Scopus (294) Google Scholar)), reflecting its multifaceted functions. It is part of a family of three proteins (in humans ZBTB7A, ZBTB7B, and ZBTB7C) that are very similar at the amino end (BTB/POZ) and the ZF-containing carboxyl end, though differing substantially in between (Fig. S1A). ZBTB7A represses the transcription of a wide range of genes (6Constantinou C. Spella M. Chondrou V. Patrinos G.P. Papachatzopoulou A. Sgourou A. The multi-faceted functioning portrait of LRF/ZBTB7A.Hum. Genomics. 2019; 13: 66Crossref PubMed Scopus (21) Google Scholar, 7Gupta S. Singh A.K. Prajapati K.S. Kushwaha P.P. Shuaib M. Kumar S. Emerging role of ZBTB7A as an oncogenic driver and transcriptional repressor.Cancer Lett. 2020; 483: 22-34Crossref PubMed Scopus (21) Google Scholar), including the repression of HIV-1 short transcripts (8Morrison D.J. Pendergrast P.S. Stavropoulos P. Colmenares S.U. Kobayashi R. Hernandez N. FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.Nucleic Acids Res. 1999; 27: 1251-1262Crossref PubMed Scopus (47) Google Scholar); silencing of the tumor suppressor ARF (5Maeda T. Hobbs R.M. Merghoub T. Guernah I. Zelent A. Cordon-Cardo C. et al.Role of the proto-oncogene Pokemon in cellular transformation and ARF repression.Nature. 2005; 433: 278-285Crossref PubMed Scopus (294) Google Scholar), which is expressed from an alternative reading frame of the CDKN2A gene; and repression of glycolytic genes (SLC2A3 [solute carrier family 2, member 3], PFKP, and PKM) (9Liu X.S. Haines J.E. Mehanna E.K. Genet M.D. Ben-Sahra I. Asara J.M. et al.ZBTB7A acts as a tumor suppressor through the transcriptional repression of glycolysis.Genes Dev. 2014; 28: 1917-1928Crossref PubMed Scopus (76) Google Scholar) and fetal hemoglobin genes (10Masuda T. Wang X. Maeda M. Canver M.C. Sher F. Funnell A.P. et al.Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin.Science. 2016; 351: 285-289Crossref PubMed Scopus (228) Google Scholar, 11Martyn G.E. Wienert B. Yang L. Shah M. Norton L.J. Burdach J. et al.Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding.Nat. Genet. 2018; 50: 498-503Crossref PubMed Scopus (182) Google Scholar, 12Yang Y. Ren R. Ly L.C. Horton J.R. Li F. Quinlan K.G.R. et al.Structural basis for human ZBTB7A action at the fetal globin promoter.Cell Rep. 2021; 36: 109759Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Very recently, increased levels of ZBTB7A were shown to modulate host genes related to oxidative responses, leading to coronavirus persistence rather than cell death (13Zhu X. Trimarco J.D. Williams C.A. Barrera A. Reddy T.E. Heaton N.S. ZBTB7A promotes virus-host homeostasis during human coronavirus 229E infection.Cell Rep. 2022; 41: 111540Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). Somatic mutations of ZBTB7A have been identified routinely in acute myeloid leukemia (AML) patients, associated with the t(8;21) translocation (14Lavallee V.P. Lemieux S. Boucher G. Gendron P. Boivin I. Armstrong R.N. et al.RNA-sequencing analysis of core binding factor AML identifies recurrent ZBTB7A mutations and defines RUNX1-CBFA2T3 fusion signature.Blood. 2016; 127: 2498-2501Crossref PubMed Scopus (50) Google Scholar, 15Hartmann L. Dutta S. Opatz S. Vosberg S. Reiter K. Leubolt G. et al.ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation.Nat. Commun. 2016; 7: 11733Crossref PubMed Scopus (45) Google Scholar, 16Faber Z.J. Chen X. Gedman A.L. Boggs K. Cheng J. Ma J. et al.The genomic landscape of core-binding factor acute myeloid leukemias.Nat. Genet. 2016; 48: 1551-1556Crossref PubMed Scopus (177) Google Scholar, 17Kawashima N. Akashi A. Nagata Y. Kihara R. Ishikawa Y. Asou N. et al.Clinical significance of ASXL2 and ZBTB7A mutations and C-terminally truncated RUNX1-RUNX1T1 expression in AML patients with t(8;21) enrolled in the JALSG AML201 study.Ann. Hematol. 2019; 98: 83-91Crossref PubMed Scopus (19) Google Scholar, 18Christen F. Hoyer K. Yoshida K. Hou H.A. Waldhueter N. Heuser M. et al.Genomic landscape and clonal evolution of acute myeloid leukemia with t(8;21): an international study on 331 patients.Blood. 2019; 133: 1140-1151Crossref PubMed Scopus (74) Google Scholar, 19Opatz S. Bamopoulos S.A. Metzeler K.H. Herold T. Ksienzyk B. Braundl K. et al.The clinical mutatome of core binding factor leukemia.Leukemia. 2020; 34: 1553-1562Crossref PubMed Scopus (49) Google Scholar). This t(8;21) (q22;q22) translocation results in a RUNX1–RUNX1T1 fusion gene (20Rowley J.D. Identificaton of a translocation with quinacrine fluorescence in a patient with acute leukemia.Ann. Genet. 1973; 16: 109-112PubMed Google Scholar, 21Erickson P. Gao J. Chang K.S. Look T. Whisenant E. Raimondi S. et al.Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt.Blood. 1992; 80: 1825-1831Crossref PubMed Google Scholar), which is one of the most frequent chromosomal aberrations in AML. Despite the causative role of the RUNX1–RUNX1T1 fusion in leukemia initiation, this fused gene alone is insufficient to induce leukemia (22Rhoades K.L. Hetherington C.J. Harakawa N. Yergeau D.A. Zhou L. Liu L.Q. et al.Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model.Blood. 2000; 96: 2108-2115Crossref PubMed Google Scholar, 23Yuan Y. Zhou L. Miyamoto T. Iwasaki H. Harakawa N. Hetherington C.J. et al.AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10398-10403Crossref PubMed Scopus (368) Google Scholar, 24Higuchi M. O'Brien D. Kumaravelu P. Lenny N. Yeoh E.J. Downing J.R. Expression of a conditional AML1-ETO oncogene bypasses embryonic lethality and establishes a murine model of human t(8;21) acute myeloid leukemia.Cancer Cell. 2002; 1: 63-74Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 25Schwieger M. Lohler J. Friel J. Scheller M. Horak I. Stocking C. AML1-ETO inhibits maturation of multiple lymphohematopoietic lineages and induces myeloblast transformation in synergy with ICSBP deficiency.J. Exp. Med. 2002; 196: 1227-1240Crossref PubMed Scopus (126) Google Scholar, 26Schessl C. Rawat V.P. Cusan M. Deshpande A. Kohl T.M. Rosten P.M. et al.The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice.J. Clin. Invest. 2005; 115: 2159-2168Crossref PubMed Scopus (186) Google Scholar). In fact, ectopic expression of wildtype ZBTB7A prevents RUNX1–RUNX1T1-mediated clonal expansion of human CD34+ cells (27Redondo Monte E. Wilding A. Leubolt G. Kerbs P. Bagnoli J.W. Hartmann L. et al.ZBTB7A prevents RUNX1-RUNX1T1-dependent clonal expansion of human hematopoietic stem and progenitor cells.Oncogene. 2020; 39: 3195-3205Crossref PubMed Scopus (12) Google Scholar), and retroviral expression of wildtype ZBTB7A inhibits cell growth in the t(8;21) translocation–positive AML cell line Kasumi-1 (15Hartmann L. Dutta S. Opatz S. Vosberg S. Reiter K. Leubolt G. et al.ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation.Nat. Commun. 2016; 7: 11733Crossref PubMed Scopus (45) Google Scholar) (possibly by inhibiting glycolysis, see Discussion section). Recent studies of a population mutational spectrum have suggested that additional genetic lesions, including ZBTB7A mutations, are required for development of RUNX1–RUNX1T1-associated diseases (16Faber Z.J. Chen X. Gedman A.L. Boggs K. Cheng J. Ma J. et al.The genomic landscape of core-binding factor acute myeloid leukemias.Nat. Genet. 2016; 48: 1551-1556Crossref PubMed Scopus (177) Google Scholar, 19Opatz S. Bamopoulos S.A. Metzeler K.H. Herold T. Ksienzyk B. Braundl K. et al.The clinical mutatome of core binding factor leukemia.Leukemia. 2020; 34: 1553-1562Crossref PubMed Scopus (49) Google Scholar) as well as in a variety of solid tumors (28Liu X.S. Liu Z. Gerarduzzi C. Choi D.E. Ganapathy S. Pandolfi P.P. et al.Somatic human ZBTB7A zinc finger mutations promote cancer progression.Oncogene. 2016; 35: 3071-3078Crossref PubMed Scopus (30) Google Scholar). Given the importance of understanding the role of naturally occurring ZBTB7A mutations in disease development, we studied selected ZBTB7A mutations within the C-terminal ZF DNA-binding domain and their effects on binding to sequence elements from the ZBTB7A-responsive promoters. We used biophysical methods to investigate the detailed interactions of the ZBTB7A DNA-binding domain with relevant promoter elements and determined precisely how known human ZBTB7A mutations interfere with DNA binding and cellular localization. In sum, our study provides significant insights into DNA sequence recognition by both native and disease-associated mutants of the human transcription factor ZBTB7A. Previous ZBTB7A chromatin immunoprecipitation sequencing experiments were carried out in human K562 (myelogenous leukemia), HUDEP-2 (umbilical erythroid progenitor), and hematopoietic stem and progenitor–derived cells (10Masuda T. Wang X. Maeda M. Canver M.C. Sher F. Funnell A.P. et al.Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin.Science. 2016; 351: 285-289Crossref PubMed Scopus (228) Google Scholar, 11Martyn G.E. Wienert B. Yang L. Shah M. Norton L.J. Burdach J. et al.Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding.Nat. Genet. 2018; 50: 498-503Crossref PubMed Scopus (182) Google Scholar). These studies identified ZBTB7A-binding profiles containing a consensus G(a/c)CCC motif (Fig. 1B) and detected a strong peak of ZBTB7A occupancy in the promoter of the CDKN2A gene (p14ARF) in HUDEP-2 cells but not in K562 cells (Fig. 1C; this difference is discussed later). The human p14ARF promoter region contains multiple putative ZBTB7A-binding sites (5Maeda T. Hobbs R.M. Merghoub T. Guernah I. Zelent A. Cordon-Cardo C. et al.Role of the proto-oncogene Pokemon in cellular transformation and ARF repression.Nature. 2005; 433: 278-285Crossref PubMed Scopus (294) Google Scholar), so we chose the three at −970, −100, and +20 that include the GaCCC motif (oligos #1–3 in Fig. 1D; position numbers are relative to the first transcribed base). We chose three additional oligonucleotides for comparison. One is the −370 p14ARF site, which includes a GcCCC motif (oligo #4), which resembles the −200 element of the fetal globin promoter (oligo #5) in that both contain an 11 bp segment with two stretches of C:G base pairs separated by two random base pairs. Finally, we included a ZBTB7A consensus binding sequence (oligo #6), which had been identified by cyclic amplification and selection of targets (CAST) (5Maeda T. Hobbs R.M. Merghoub T. Guernah I. Zelent A. Cordon-Cardo C. et al.Role of the proto-oncogene Pokemon in cellular transformation and ARF repression.Nature. 2005; 433: 278-285Crossref PubMed Scopus (294) Google Scholar), which combines the two features together: a GaCCC motif as well as a segment of four C:G base pairs. Previously, we used a ZBTB7A recombinant fragment including residues 370 to 500, which encompassed the four-finger DNA-binding domain, in a study of binding to the fetal globin promoter (12Yang Y. Ren R. Ly L.C. Horton J.R. Li F. Quinlan K.G.R. et al.Structural basis for human ZBTB7A action at the fetal globin promoter.Cell Rep. 2021; 36: 109759Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). During the course of that study, AlphaFold (DeepMind) (29Jumper J. Evans R. Pritzel A. Green T. Figurnov M. Ronneberger O. et al.Highly accurate protein structure prediction with AlphaFold.Nature. 2021; 596: 583-589Crossref PubMed Scopus (9075) Google Scholar) released protein structure predictions for the human proteome including ZBTB7A, which predicted two additional folded helices preceding the ZF domain (Fig. S1, B and C). We thus generated a longer fragment, including residues 341 to 505, that covers the N-terminal putative helices plus five additional amino acids (including two basic residues) at the C terminus (Fig. 1A). However, using fluorescence polarization (FP), we found that both fragments bind the 16-bp CAST sequence (oligo #6) equally well (Fig. 1E). The more N- and C-proximal additions to the ZF region contributed little to the binding affinities. Specifically, the dissociation constant KD values (∼80 nM) were approximately the same for the two constructs. Next, we compared the binding affinities for the four p14ARF promoter sequences (oligos #1–4) (Fig. 1F). Oligos #1 to 3, each containing a single GaCCC element, exhibited 2 to 3 times reduced binding affinity relative to that of oligo #4. Oligo #4, taken from the ARF promoter −370 site, has the same affinity (KD ∼ 80 nM) as that of the CAST oligo and the −200 site from the fetal globin promoter (Fig. 1G). All three higher-affinity oligos (#4–6) share the same features of a 5′ G(a/c)CCC motif and a 3′ C:G-rich segment. This enhanced affinity is understandable given that the predicted recognition sequence for a four-ZF array is approximately 12 bp, using the rule of one-finger recognizing three adjacent DNA base pairs (30Choo Y. Klug A. Physical basis of a protein-DNA recognition code.Curr. Opin. Struct. Biol. 1997; 7: 117-125Crossref PubMed Scopus (212) Google Scholar, 31Wolfe S.A. Nekludova L. Pabo C.O. DNA recognition by Cys2His2 zinc finger proteins.Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 183-212Crossref PubMed Scopus (804) Google Scholar). A 5-bp GaCCC motif should be bound by just two fingers, which would presumably result in reduced binding affinity relative to sequences bound by all four fingers. In addition to p14ARF promoter, others showed that ZBTB7A binds to the promoters of glycolytic genes (SLC2A3, PFKP, and PKM) (9Liu X.S. Haines J.E. Mehanna E.K. Genet M.D. Ben-Sahra I. Asara J.M. et al.ZBTB7A acts as a tumor suppressor through the transcriptional repression of glycolysis.Genes Dev. 2014; 28: 1917-1928Crossref PubMed Scopus (76) Google Scholar), which contain the G(a/c)CCC motif (Fig. 1D). SLC2A3 encodes the glucose transmembrane transporter GLUT3 (32Kayano T. Fukumoto H. Eddy R.L. Fan Y.S. Byers M.G. Shows T.B. et al.Evidence for a family of human glucose transporter-like proteins. Sequence and gene localization of a protein expressed in fetal skeletal muscle and other tissues.J. Biol. Chem. 1988; 263: 15245-15248Abstract Full Text PDF PubMed Google Scholar). Once inside the cell, glucose is converted to pyruvate via a cascade of ten chemical reactions (33Bian X. Jiang H. Meng Y. Li Y.P. Fang J. Lu Z. Regulation of gene expression by glycolytic and gluconeogenic enzymes.Trends Cell Biol. 2022; 32: 786-799Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) (glycolysis; Fig. S2). In addition to the three characterized glycolytic genes, we found that ZBTB7A occupies the promoters of all ten glycolytic genes in K562 or HUDEP-2 cells or both (Fig. S2). This control of glycolytic genes is independent from that of globin genes, as mutant HUDEP-2 cells harboring the -195C-to-G substitution at the γ-globin gene promoter showed no effect on the binding activity of ZBTB7A at the promoters of the glycolytic genes (Fig. S2). Furthermore, an RNA-Seq analysis of ZBTB7A knockout K562 cells revealed increased expression of the genes for glucose transporters (SLC2A1 and SLC2A3), phosphoglycerate mutases (PGM2 and PGM3), and enolase (ENO2). However, there was no change in expression of the kinase genes involved in glycolysis—hexokinase (HK1), pyruvate kinase (PKM), and phosphofrutokinase (PFKP and PFKM) (27Redondo Monte E. Wilding A. Leubolt G. Kerbs P. Bagnoli J.W. Hartmann L. et al.ZBTB7A prevents RUNX1-RUNX1T1-dependent clonal expansion of human hematopoietic stem and progenitor cells.Oncogene. 2020; 39: 3195-3205Crossref PubMed Scopus (12) Google Scholar). To gain insight into the structural basis for ZBTB7A recognition of the GaCCC motif, we cocrystallized the four-finger DNA-binding domain (residues 370–500) with three 15-bp DNA oligos, each containing a single GACCC with 5′ overhangs or blunt ends (Fig. 2). These complexes crystallized in three different space groups, and the structures were determined to resolutions of 2.25, 2.85, and 3.09 Å, respectively (Table S1). In space groups C2221 (Protein Data Bank [PDB] ID: 7N5W) and P21212 (PDB ID: 7N5U), we observed the three fingers ZF1–ZF3, whereas ZF4 was disordered (Fig. 2, A and B). In both space groups, ZF1 and ZF2 lie in the DNA major groove, whereas ZF3 points away from DNA in varied directions. In space group P21, we observed two protein–DNA complexes (Fig. 2C). Complex A contains all four fingers, with ZF1–ZF2 binding in the DNA major groove and ZF3 and ZF4 pointing away from DNA (Fig. 2C). The ZF3–ZF4 pair is stabilized via protein–protein interactions with complex B, which harbors only the first two fingers (Fig. 2C). In sum, we observed four ZBTB7A–DNA complexes that included a GaCCC element (Fig. 2D). In all four complexes, ZF1–ZF2 were very similar, having an rmsd of <1 Å. The main differences between these four complexes involved crystal packing interactions with neighboring molecules, resulting in different conformations of ZF3–ZF4, some of which were disordered and lacked electron density. In the absence of specific contacts, ZF3–ZF4 adopted various conformations (Fig. 2E), and this was achieved via a series of rotations of main-chain torsion angles involving the glycine residue in the linker between the two fingers (i.e., Gly434 between ZF2 and ZF3). This glycine—the most flexible of amino acids—is conserved in the corresponding linkers between pairs of fingers (bright red in Fig. 3A). In fact, these glycines are very highly conserved among vertebrate orthologs of ZBTB7A as phylogenetically distant from humans as whale sharks (Fig. S1D). An equivalent conformational switch point within the linker was previously identified in the 11-finger protein CTCF (34Hashimoto H. Wang D. Horton J.R. Zhang X. Corces V.G. Cheng X. Structural basis for the versatile and methylation-dependent binding of CTCF to DNA.Mol. Cell. 2017; 66: 711-720.e3Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), suggesting that the flexibility of the linker between pair of fingers plays a critical role, perhaps allowing the multifinger array greater versatility in its sequence recognition, as well as in binding varied lengths of DNA duplex. Next, we cocrystallized the CAST sequence (oligo #6), which contains both a 5′ GaCCC motif and 3′ four C:G bp, in complex with the short (residues 370–500) and the long fragments (residues 341–505) of ZBTB7A. As noted previously, both fragments bind the DNA equally (Fig. 1E), and both fragments crystallized, in the space groups of P6 and P21, respectively (Fig. 3, B and C). The complex structures were determined to resolutions of 2.62 and 2.99 Å, respectively (Table S1). Although crystallized in two different space groups, the two structures are highly similar, with an rmsd of <0.9 Å over 109 pairs of Cα atoms. The additional N-terminal residues included in the longer fragment were disordered, and we did not observe the AlphaFold-predicted helices (Fig. S1C). All four ZF units occupy and wrap along the DNA major groove (Fig. 3D). The first two fingers, ZF1 and ZF2, interact with the 5′ sequence GaCCC, ZF3 provides a spacer (via sequence-independent DNA phosphate binding), and ZF4 interacts with the 3′ sequence (CCCC). As in conventional protein–DNA interactions (35Luscombe N.M. Laskowski R.A. Thornton J.M. Amino acid-base interactions: a three-dimensional analysis of protein-DNA interactions at an atomic level.Nucleic Acids Res. 2001; 29: 2860-2874Crossref PubMed Scopus (785) Google Scholar, 36Patel A. Horton J.R. Wilson G.G. Zhang X. Cheng X. Structural basis for human PRDM9 action at recombination hot spots.Genes Dev. 2016; 30: 257-265Crossref PubMed Scopus (35) Google Scholar), seven C:G base pairs (at bp positions 1, 3, 4, 5, 9, 10, and 11; labeled in Fig. 3B) were recognized by lysine, arginine, and histidine residues via direct hydrogen bonds with the O6 and N7 atoms of guanines: guanine G1 by Lys396 of ZF1 (Fig. 3E), G3 by Arg399 of ZF1 (Fig. 3G), G4 by Arg421 of ZF2 (Fig. 3H), G5 by Lys424 of ZF2 (Fig. 3I), G9 by Arg477 of ZF4 (Fig. 3K), G10 by His480 of ZF4 (Fig. 3L), and G11 by Arg483 of ZF4 (Fig. 3M). Two negatively charged residues, Asp423 of ZF2 and Asp479 of ZF4, provide a hydrogen bond with cytosines C3 and C8, respectively (Fig. 3, G and J). The A:T base pair at position 2 forms van der Waals contacts with the Cα atom of Gly393 and the side chain aliphatic carbon of Lys396 (via the methyl group of T2) (Fig. 3F). All these DNA-contacting residues of the human ortholog are fully conserved among other vertebrate orthologs of ZBTB7A (Fig. S1D), and 9 of 10 are conserved among the human ZBTB7A family members ZBTB7B and 7C (Fig. S1A). Among the 11 bp covered by the four ZF units, two base pairs at positions 6 and 7 are not involved in direct interactions with the DNA bases, and these correspond to the position of ZF3 (which makes base-independent backbone contacts). Among the base-specific interactions, seven of them are in the bottom strand (T2, G3, G4, G5, G9, G10, and G11), whereas the G1, C3, and C8 are in the top strand. In general, more-specific DNA-binding proteins recognize both strands (37Lin M. Guo J.T. New insights into protein-DNA binding specificity from hydrogen bond based comparative study.Nucleic Acids Res. 2019; 47: 11103-11113Crossref PubMed Scopus (28) Google Scholar). The cross-strand interaction of Lys396–G1 has been previously observed in Lys413 of KLF4 and Lys328 of ZNF410 (38Liu Y. Olanrewaju Y.O. Zheng Y. Hashimoto H. Blumenthal R.M. Zhang X. et al.Structural basis for Klf4 recognition of methylated DNA.Nucleic Acids Res. 2014; 42: 4859-4867Crossref PubMed Scopus (74) Google Scholar, 39Lan X. Ren R. Feng R. Ly L.C. Lan Y. Zhang Z. et al.ZNF410 uniquely activates the NuRD component CHD4 to silence fetal hemoglobin expression.Mol. Cell. 2021; 81: 239-254.e8Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The ability to switch, between Lys396–G1 interaction in GaCCC and Lys396–G2 interaction of the opposite strand of GcCCC (12Yang Y. Ren R. Ly L.C. Horton J.R. Li F. Quinlan K.G.R. et al.Structural basis for human ZBTB7A action at the fetal globin promoter.Cell Rep. 2021; 36: 109759Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) (akin to the Lys424–G5 interaction [Fig. 3I]), allows ZBTB7A to bind sequences that vary at the second position of G(a/c)CCC. In addition, the cross-strand interaction between Asp423–C3 and Asp47–C8 are common among the ZF units containing an Arg–Asp electrostatic stabilization pair, for example, Arg421–Asp423 of ZF2 (Fig. 3H) and Arg477–Asp479 of ZF4 (Fig. 3K). Most importantly, our structural characterization of ZBTB7A in complex with DNA elements illustrated that ZF1–ZF2 binding of G(a/c)CCC is nearly identical in structure with either the full consensus sequence or just the 5′ part. In addition, ZBTB7A displayed the greatest reduction in binding affinity in response to single-base pair substitutions in the DNA sequence recognized by ZF1 and ZF2, followed by a smaller impact on binding in DNA recognized by ZF4, and the least in DNA protected by ZF3 (12Yang Y. Ren R. Ly L.C. Horton J.R. Li F. Quinlan K.G.R. et al.Structural basis for human ZBTB7A action at the fetal globin promoter.Cell Rep. 2021; 36: 109759Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). These observations suggest that the specific contacts with the ZF array (in this case, four fingers) are formed sequentially; putatively starting with ZF1–ZF2 binding to GaCCC before spreading to ZF3–ZF4, which respectively bind to the backbone and 3′ end of CCCC sequence. However, it is also possible that ZF3 binding contributes to nonspecific DNA association during the period of short-range linear diffusion (sliding) while searching for specific binding sites (40Halford S.E. An end to 40 years of mistakes in DNA-protein association kinetics?.Biochem. Soc. Trans. 2009; 37: 343-348Crossref PubMed Scopus (189) Google Scholar, 41Esadze A. Iwahara J. Stopped-flow fluorescence kinetic study of protein sliding and intersegment transfer in the target DNA search process.J. Mol. Biol. 2014; 426: 230-244Crossref PubMed Scopus (47) Google Scholar, 42Yang X.W. Liu J. Observing protein one-dimensional sliding: methodology and biological significance.Biomolecules. 2021; 11: 1618Crossref PubMed Scopus (1) Google Scholar). The involvement of two fingers versus four fingers might reflect the observed differences in DNA-binding affinities (Fig. 2D). Although ZF3 is not involved in DNA base–specific interactions, it did contribute direct contacts with the DNA phosphate groups via five residues (Tyr438, Asn450, Lys454, Asn455, and Arg458; Fig. 3N). All five of these phosphate-binding residues are fully conserved among vertebrate orthologs (Fig. S1D), and in human ZBTB7C, though there are two substitutions in ZBTB7B (N450S and R458H; Fig. S1A). ZF3 also forms interfinger interactions with ZF2 and ZF4. These ZF3-mediated interactions are important for positioning the succeeding ZF4 appropriately into the DNA major groove. We next considered naturally occurring disease-

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