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1.  Crystallization and preliminary X-ray analysis of the NKX2.5 homeodomain in complex with DNA 
As part of an effort to elucidate the molecular basis for the pathogenesis of NKX2.5 mutations in congenital heart disease using X-ray crystallography, the NKX2.5 homeodomain has been crystallized in complex with a specific DNA element, the −242 promoter region of atrial natriuretic factor. Crystals of the homeodomain–DNA complex diffracted X-rays to 1.7 Å resolution and belonged to space group P65, with unit-cell parameters a = b = 71.5, c = 94.3 Å. The asymmetric unit contained two molecules of the NKX2.5 homeodomain and one double-stranded oligonucleotide.
PMCID: PMC2581709  PMID: 18997347
2.  Crystal Structure of the Human NKX2.5 Homeodomain in Complex with DNA Target 
Biochemistry  2012;51(32):6312-6319.
NKX2.5 is a homeodomain containing transcription factor regulating cardiac formation and function, and its mutations are linked to congenital heart disease. Here we provide the first report of the crystal structure of the NKX2.5 homeodomain in complex with double-stranded DNA of its endogenous target, locating within the proximal promoter –242 site of the atrial natriuretic factor gene. The crystal structure, determined at 1.8 Å resolution, demonstrates that NKX2.5 homeodomains occupy both DNA binding sites separated by five nucleotides without physical interaction between themselves. The two homeodomains show identical conformation despite the differences in the DNA sequences they bind, and no significant bending of the DNA was observed. Tyr54, absolutely conserved in NK2 family proteins, mediates sequence-specific interaction with the TAAG motif. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients.
PMCID: PMC3448007  PMID: 22849347
3.  Binding Polymorphism in the DNA Bound State of the Pdx1 Homeodomain 
PLoS Computational Biology  2013;9(8):e1003160.
The subtle effects of DNA-protein recognition are illustrated in the homeodomain fold. This is one of several small DNA binding motifs that, in spite of limited DNA binding specificity, adopts crucial, specific roles when incorporated in a transcription factor. The homeodomain is composed of a 3-helix domain and a mobile N-terminal arm. Helix 3 (the recognition helix) interacts with the DNA bases through the major groove, while the N-terminal arm becomes ordered upon binding a specific sequence through the minor groove. Although many structural studies have characterized the DNA binding properties of homeodomains, the factors behind the binding specificity are still difficult to elucidate. A crystal structure of the Pdx1 homeodomain bound to DNA (PDB 2H1K) obtained previously in our lab shows two complexes with differences in the conformation of the N-terminal arm, major groove contacts, and backbone contacts, raising new questions about the DNA recognition process by homeodomains. Here, we carry out fully atomistic Molecular Dynamics simulations both in crystal and aqueous environments in order to elucidate the nature of the difference in binding contacts. The crystal simulations reproduce the X-ray experimental structures well. In the absence of crystal packing constraints, the differences between the two complexes increase during the solution simulations. Thus, the conformational differences are not an artifact of crystal packing. In solution, the homeodomain with a disordered N-terminal arm repositions to a partially specific orientation. Both the crystal and aqueous simulations support the existence of different stable binding conformers identified in the original crystallographic data with different degrees of specificity. We propose that protein-protein and protein-DNA interactions favor a subset of the possible conformations. This flexibility in DNA binding may facilitate multiple functions for the same transcription factor.
Author Summary
All organisms require the capability to control gene expression. In eukaryotes, transcription factors play an important role in gene regulation by recognizing specific DNA control regions associated with each gene. The DNA binding domains of transcription factors belong to evolutionarily conserved families with different protein folds. An example is the homeodomain family. Although this DNA binding domain has been studied for a long time, the properties that determine DNA binding specificity are still not clear. We previously showed in a crystal structure that the homeodomain of a transcription factor Pdx1 (Pancreatic and duodenal homeobox 1) binds DNA in 2 different conformations. In this paper, we used Molecular Dynamics simulations to show that both of these conformations are stable in solution. This is surprising since it is often assumed that proteins recognize DNA by finding a single lowest energy state. This study shows that transcription factors may bind DNA in an ensemble of conformations. This scenario may facilitate their finding the correct binding site among the 3 billion basepairs of DNA in the human genome. It may also provide flexibility in the DNA sequence that homeodomains can recognize to promote gene transcription.
PMCID: PMC3738460  PMID: 23950697
4.  Foxp2 Inhibits Nkx2.1-Mediated Transcription of SP-C via Interactions with the Nkx2.1 Homeodomain 
The transcription factor (TF) Foxp2 has been shown to partially repress surfactant protein C (SP-C) transcription, presumably through interaction of an independent repressor domain with a conserved Foxp2 consensus site in the SP-C promoter. We explored the role of interactions between Foxp2 and the homeodomain TF Nkx2.1 that may contribute to the marked reduction in SP-C expression accompanying phenotypic transition of alveolar epithelial type II (AT2) to type I (AT1) cells. Foxp2 dose-dependently inhibited Nkx2.1-mediated activation of SP-C in MLE-15 cells. While electrophoretic mobility shift assays and chromatin immunoprecipitations revealed an interaction between Foxp2 and the conserved consensus motif in the SP-C promoter, Nkx2.1-mediated activation of the 318-bp proximal SP-C promoter (which lacks a Foxp2 consensus) was attenuated by increasing amounts of Foxp2. Co-immunoprecipitation and mammalian two-hybrid assays confirmed a physical interaction between Nkx2.1 and Foxp2 mediated through the Nkx2.1 homeodomain. Formation of an Nkx2.1 complex with an SP-C oligonucleotide was inhibited dose-dependently by recombinant Foxp2. These findings demonstrate that direct interaction between Foxp2 and Nkx2.1 inhibits Nkx2.1 DNA-binding and transcriptional activity and suggest a mechanism for down-regulation of SP-C (and probably other AT2 cell genes) during transition of AT2 cells to an AT1 cell phenotype.
PMCID: PMC2396252  PMID: 18239190
alveolar epithelium; transcriptional regulation; forkhead box; Nkx2.1; differentiation
5.  Crystallization and preliminary X-ray analysis of the Pax6 paired domain bound to the Pax6 gene enhancer 
The mammalian Pax6 paired domain has been cocrystallizaed with a 25 bp DNA fragment of the Pax6 gene enhancer.
Pax6 is a member of the Pax family of transcription factors and is essential for eye development. Pax6 has two DNA-binding domains: the paired domain and the homeodomain. The Pax6 paired domain is involved in Pax6 gene autoregulation by binding to its enhancer. In this study, crystallization and preliminary X-ray diffraction analysis of the mammalian Pax6 paired domain in complex with the Pax6 gene enhancer was attempted. The Pax6 paired domain complexed with an optimized 25 bp DNA fragment was crystallized by the hanging-drop vapour-diffusion method. The crystal diffracted synchrotron radiation to 3.0/3.7 Å resolution and belongs to the monoclinic space group P21, with unit-cell parameters a = 62.21, b = 70.69, c = 176.03 Å, β = 90.54°. Diffraction data were collected to 3.7 Å resolution.
PMCID: PMC1978128  PMID: 16511221
Pax6 paired domain; Pax6 gene enhancer; Pax-family proteins
6.  Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5 
PLoS ONE  2011;6(9):e24812.
Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggested that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a “shifting” site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity.
PMCID: PMC3171482  PMID: 21931855
7.  Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease 
Journal of Clinical Investigation  2000;106(2):299-308.
CSX/NKX2.5 is an evolutionarily conserved homeodomain-containing (HD-containing) transcription factor that is essential for early cardiac development. Recently, ten different heterozygous CSX/NKX2.5 mutations were found in patients with congenital heart defects that are transmitted in an autosomal dominant fashion. To determine the consequence of these mutations, we analyzed nuclear localization, DNA binding, transcriptional activation, and dimerization of mutant CSX/NKX2.5 proteins. All mutant proteins were translated and located to the nucleus, except one splice-donor site mutant whose protein did not accumulate in the cell. All mutants that had truncation or missense mutations in the HD had severely reduced DNA binding activity and little or no transcriptional activation function. In contrast, mutants with intact HDs exhibit normal DNA binding to the monomeric binding site but had three- to ninefold reduction in DNA binding to the dimeric binding sites. HD missense mutations that preserved homodimerization ability inhibited the activation of atrial natriuretic factor by wild-type CSX/NKX2.5. Although our studies do not characterize the genotype-phenotype relationship of the ten human mutations, they identify specific abnormalities of CSX/NKX2.5 function essential for transactivation of target genes.
PMCID: PMC314312  PMID: 10903346
8.  Physical and Functional Interactions between Homeodomain NKX2.1 and Winged Helix/Forkhead FOXA1 in Lung Epithelial Cells▿  
Molecular and Cellular Biology  2007;27(6):2155-2165.
NKX2.1 is a homeodomain transcription factor that controls development of the brain, lung, and thyroid. In the lung, Nkx2.1 is expressed in a proximo-distal gradient and activates specific genes in phenotypically distinct epithelial cells located along this axis. The mechanisms by which NKX2.1 controls its target genes may involve interactions with other transcription factors. We examined whether NKX2.1 interacts with members of the winged-helix/forkhead family of FOXA transcription factors to regulate two spatially and cell type-specific genes, SpC and Ccsp. The results show that NKX2.1 interacts physically and functionally with FOXA1. The nature of the interaction is inhibitory and occurs through the NKX2.1 homeodomain in a DNA-independent manner. On SpC, which lacks a FOXA1 binding site, FOXA1 attenuates NKX2.1-dependent transcription. Inhibition of FOXA1 by small interfering RNA increased SpC mRNA, demonstrating the in vivo relevance of this finding. In contrast, FOXA1 and NKX2.1 additively activate transcription from Ccsp, which includes both NKX2.1 and FOXA1 binding sites. In electrophoretic mobility shift assays, the GST-FOXA1 fusion protein interferes with the formation of NKX2.1 transcriptional complexes by potentially masking the latter's homeodomain DNA binding function. These findings suggest a novel mode of selective gene regulation by proximo-distal gradient distribution of and functional interactions between forkhead and homeodomain transcription factors.
PMCID: PMC1820505  PMID: 17220277
9.  Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription. 
Molecular and Cellular Biology  1996;16(11):6372-6384.
We recently showed that the cardiogenic homeodomain factor Nkx-2.5 served as a positive acting accessory factor for serum response factor (SRF) and that together they provided strong transcriptional activation of the cardiac alpha-actin promoter, depending upon intact serum response elements (SREs) (C. Y. Chen, J. Croissant, M. Majesky, S. Topouz, T. McQuinn, M. J. Frankovsky, and R. J. Schwartz, Dev. Genet. 19:119-130, 1996). As shown here, Nkx-2.5 and SRF collaborated to activate the endogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts by a mechanism in which SRF recruited Nkx-2.5 to the alpha-actin promoter. Activation of a truncated promoter consisting of the proximal alpha-actin SRE1 occurred even when Nkx-2.5 DNA-binding activity was blocked by a point mutation in the third helix of its homeodomain. Investigation of protein-protein interactions showed that Nkx-2.5 was bound to SRF in the absence of DNA in soluble protein complexes retrieved from cardiac myocyte nuclei but could also be detected in coassociated binding complexes on the proximal SRE1. Recruitment of Nkx-2.5 to an SRE depended upon SRF DNA-binding activity and was blocked by the dominant negative SRFpm1 mutant, which allowed for dimerization of SRF monomers but prevented DNA binding. Interactive regions shared by Nkx-2.5 and SRF were mapped to N-terminal/helix I and helix II/helix III regions of the Nkx-2.5 homeodomain and to the N-terminal extension of the MADS box. Our study suggests that physical association between Nkx-2.5 and SRF is one way that cardiac specified genes are activated in cardiac cell lineages.
PMCID: PMC231639  PMID: 8887666
10.  Crystallization and diffraction of an Lhx4–Isl2 complex 
A complex comprising the LIM domains of the LIM-homeodomain protein Lhx4 tethered to a peptide region of Isl2 has been engineered, purified and crystallized. Crystals of this intramolecular complex diffracted to 2.16 Å resolution.
A stable intramolecular complex comprising the LIM domains of the LIM-homeodomain protein Lhx4 tethered to a peptide region of Isl2 has been engineered, purified and crystallized. The monoclinic crystals belonged to space group P21, with unit-cell parameters a = 46.8, b = 88.7, c = 49.9 Å, β = 111.9°, and diffracted to 2.16 Å resolution.
PMCID: PMC2635870  PMID: 19194008
Lhx4; Isl2; Lim domains; Lim-homeodomain transcription factors
11.  Crystallization and diffraction of an Isl1–Ldb1 complex 
A complex comprising the LIM domains of the LIM-homeodomain protein Isl1 tethered to a peptide region of Ldb1 has been engineered, purified and crystallized. Crystals of this intramolecular complex diffracted to 3.10 Å resolution.
A stable intramolecular complex comprising the LIM domains of the LIM-homeodomain protein Isl1 tethered to a peptide region of Ldb1 has been engineered, purified and crystallized. The orthorhombic crystals belonged to space group P2221, with unit-cell parameters a = 57.2, b = 56.7, c = 179.8 Å, and diffracted to 3.10 Å resolution.
PMCID: PMC3515390  PMID: 23143258
Isl1–Ldb1 complex; LIM domains
12.  Identification of known and novel pancreas genes expressed downstream of Nkx2.2 during development 
The homeodomain containing transcription factor Nkx2.2 is essential for the differentiation of pancreatic endocrine cells. Deletion of Nkx2.2 in mice leads to misspecification of islet cell types; insulin-expressing β cells and glucagon-expressing α cells are replaced by ghrelin-expressing cells. Additional studies have suggested that Nkx2.2 functions both as a transcriptional repressor and activator to regulate islet cell formation and function. To identify genes that are potentially regulated by Nkx2.2 during the major wave of endocrine and exocrine cell differentiation, we assessed gene expression changes that occur in the absence of Nkx2.2 at the onset of the secondary transition in the developing pancreas.
Microarray analysis identified 80 genes that were differentially expressed in e12.5 and/or e13.5 Nkx2.2-/- embryos. Some of these genes encode transcription factors that have been previously identified in the pancreas, clarifying the position of Nkx2.2 within the islet transcriptional regulatory pathway. We also identified signaling factors and transmembrane proteins that function downstream of Nkx2.2, including several that have not previously been described in the pancreas. Interestingly, a number of known exocrine genes are also misexpressed in the Nkx2.2-/- pancreas.
Expression profiling of Nkx2.2-/- mice during embryogenesis has allowed us to identify known and novel pancreatic genes that function downstream of Nkx2.2 to regulate pancreas development. Several of the newly identified signaling factors and transmembrane proteins may function to influence islet cell fate decisions. These studies have also revealed a novel function for Nkx2.2 in maintaining appropriate exocrine gene expression. Most importantly, Nkx2.2 appears to function within a complex regulatory loop with Ngn3 at a key endocrine differentiation step.
PMCID: PMC2799404  PMID: 20003319
13.  DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1 
Nucleic Acids Research  2000;28(12):2389-2395.
NKX3.1 is a member of the NK class of homeodomain proteins and is most closely related to Drosophila NK-3. NKX3.1 has predominantly prostate-specific expression in the adult human. Previous studies suggested that NKX3.1 exerts a growth-suppressive effect on prostatic epithelial cells and controls differentiated glandular functions. Using a binding site selection assay with recombinant NKX3.1 protein we identified a TAAGTA consensus binding sequence that has not been reported for any other NK class homeoprotein. By electromobility shift assay we demonstrated that NKX3.1 preferentially binds the TAAGTA sequence rather than the binding site for Nkx2.1 (CAAGTG) or Msx1 (TAATTG). Using mutated binding sites in competitive gel shift assays, we analyzed the nucleotides in the TAAGTA consensus sequence that are important for NKX3.1 binding. The consensus binding site of a naturally occurring polymorphic NKX3.1 protein with arginine replaced by cysteine at position 52 was identical to the wild-type binding sequence. The binding affinities of wild-type and polymorphic NKX3.1 for the TAAGTA consensus site were very similar, with values of 20 and 22 nM, respectively. Wild-type and polymorphic NKX3.1 specifically repressed transcription of luciferase from a reporter vector with three copies of the NKX3.1-binding site upstream from a thymidine kinase promoter. The data show that among NK family proteins NKX3.1 binds a novel DNA sequence and can behave as an in vitro transcriptional repressor.
PMCID: PMC102730  PMID: 10871372
14.  Functional Characterization of a Novel Mutation in NKX2-5 Associated with Congenital Heart Disease and Adult-Onset Cardiomyopathy 
Circulation. Cardiovascular genetics  2013;6(3):10.1161/CIRCGENETICS.113.000057.
The transcription factor NKX2-5 is crucial for heart development and mutations in this gene have been implicated in diverse congenital heart diseases (CHD) and conduction defects (CD) in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown.
Methods and Results
Mutation screening was performed in 220 probands with adult-onset dilated cardiomypathy (DCM). Six NKX2-5 coding sequence variants were identified, including 3 non-synonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain (HD), was identified in one family. A subset of family members had CHD, but there was an unexpectedly high prevalence of DCM. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes, due to reduced degradation by the ubiquitin-proteasome system (UPS). In functional assays, DNA binding activity of I184M was reduced, resulting in impaired activation of target genes, despite increased expression levels of mutant protein.
Certain NKX2-5 HD mutations show abnormal protein degradation via the UPS and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function, and contribute to NKX2-5-related cardiomyopathies with graded severity.
PMCID: PMC3816146  PMID: 23661673
dilated cardiomyopathy; transcription factors; gene mutations; ubiquitin-proteasome system; NKX2-5
15.  Functional Activation of ATM by the Prostate Cancer Suppressor NKX3.1 
Cell reports  2013;4(3):10.1016/j.celrep.2013.06.039.
The prostate tumor suppressor NKX3.1 augments response to DNA damage and enhances survival after DNA damage. Within minutes of DNA damage, NKX3.1 undergoes phosphorylation at tyrosine 222, which is required for a functional interaction with ataxia telangiectasia mutated (ATM) kinase. NKX3.1 binds to the N-terminal region of ATM, accelerates ATM activation, and hastens the formation of γhistone2AX. NKX3.1 enhances DNA-dependent ATM kinase activation by both the MRN complex and H2O2 in a DNA-damage-independent manner. ATM, bound to the NKX3.1 homeodomain, phosphorylates NKX3.1, leading to ubiquitination and degradation. Thus, NKX3.1 and ATM have a functional interaction leading to ATM activation and then NKX3.1 degradation in a tightly regulated DNA damage response specific to prostate epithelial cells. These findings demonstrate a mechanism for the tumor-suppressor properties of NKX3.1, demonstrate how NKX3.1 may enhance DNA integrity in prostate stem cells and may help to explain how cells differ in their sensitivity to DNA damage.
PMCID: PMC3838670  PMID: 23890999
16.  Crystallization and preliminary X-ray crystallographic analysis of dihydrouridine synthase from Thermus thermophilus and its complex with tRNA 
Crystals of dihydrouridine synthase from Thermus thermophilus and its complex with tRNA were obtained and X-ray diffraction data were collected to 1.70 and 3.51 Å resolution, respectively.
Dihydrouridine synthase (Dus) is responsible for catalyzing dihydrouridine formation in RNA by the reduction of uridine. To elucidate its RNA-recognition mechanism, Dus from Thermus thermophilus (TthDus) and its complex with tRNA were crystallized. Diffraction data sets were collected from crystals of native and selenomethionine-substituted TthDus to resolutions of 1.70 and 2.30 Å, respectively. These crystals belonged to space group P1. Preliminary X-­ray crystallographic analysis showed that two molecules of TthDus were contained in an asymmetric unit. In addition, diffraction data were collected to 3.51 Å resolution from a crystal of selenomethionine-substituted TthDus in complex with tRNA, which belonged to space group P41212. Preliminary structural analysis showed that the asymmetric unit contained two TthDus–tRNA complexes.
PMCID: PMC3107143  PMID: 21636912
dihydrouridine synthase; tRNA; flavin mononucleotide; Thermus thermophilus
17.  The atrial natriuretic factor promoter is a downstream target for Nkx-2.5 in the myocardium. 
Molecular and Cellular Biology  1996;16(9):4648-4655.
The recently described NK2 family of homeodomain proteins are key developmental regulators. In Drosophila melanogaster, two members of this family, bagpipe and tinman, are required for visceral and cardiac mesoderm formation, respectively. In vertebrates, tinman appears to represent a family of closely related NK2 genes, including Nkx-2.5, that are expressed at an early stage in precardiac cells. Consistent with a role for Nkx-2.5 in heart development, inactivation of the Nkx-2.5 gene in mice causes severe cardiac malformations and embryonic lethality. However, little is known about the molecular action of Nkx-2.5 and its targets in cardiac muscle. In this paper, we report the identification and characterization of a functional and highly conserved Nkx-2.5 response element, termed the NKE, in the proximal region of the cardiac atrial natriuretic factor (ANF) promoter. The NKE is composed of two near-consensus NK2 binding sites that are each able to bind purified Nkx-2.5. The NKE is sufficient to confer cardiac cell-specific activity to a minimal TATA-containing promoter and is required for Nkx-2.5 activation of the ANF promoter in heterologous cells. Interestingly, in primary cardiocyte cultures, the NKE contributes to ANF promoter activity in a chamber- and developmental stage-specific manner, suggesting that Nkx-2.5 and/or other related cardiac proteins may play a role in chamber specification. This work provides the identification of a direct target for NK2 homeoproteins in the heart and lays the foundation for further molecular analyses of the role of Nkx-2.5 and other NK2 proteins in cardiac development.
PMCID: PMC231464  PMID: 8756621
18.  Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5 
Molecular and Cellular Biology  1999;19(1):526-536.
Csx/Nkx2.5, a member of the homeodomain-containing transcription factors, serves critical developmental functions in heart formation in vertebrates and nonvertebrates. In this study the putative nuclear localization signal (NLS) of Csx/Nkx2.5 was identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 was mutated a significant amount of the cytoplasmically localized Csx/Nkx2.5 was unphosphorylated, in contrast to the nuclearly localized Csx/Nkx2.5, which is serine- and threonine-phosphorylated, suggesting that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicated that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, we detected a Csx/Nkx2.5 kinase whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increased Csx/Nkx2.5 DNA binding. Serine-to-alanine mutation at the CKII phosphorylation site reduced transcriptional activity when the carboxyl-terminal repressor domain was deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, as this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes.
PMCID: PMC83910  PMID: 9858576
19.  Crystallization and initial X-ray diffraction studies of the flavoenzyme NAD(P)H:(acceptor) oxidoreductase (FerB) from the soil bacterium Paracoccus denitrificans  
The flavin-dependent enzyme FerB from P. denitrificans has been purified and both native and SeMet-substituted FerB have been crystallized. The two variants crystallized in two different crystallographic forms belonging to the monoclinic space group P21 and the orthorhombic space group P21212, respectively. X-ray diffraction data were collected to 1.75 Å resolution for both forms.
The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8 Å resolution. The crystals of native FerB belonged to space group P21, with unit-cell parameters a = 61.6, b = 110.1, c = 65.2 Å, β = 118.2° and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P21212, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5 Å and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress.
PMCID: PMC2852337  PMID: 20383015
flavoenzymes; quinone reductases; Paracoccus denitrificans
20.  Biochemical Analyses of Csx/Nkx2.5 Mutants and Their Structure–Function Relationship 
A homeodomain-containing transcription factor Csx/Nkx2.5 is an important regulator of cardiogensis in mammals. There has been considerable interest in understanding determinants of the diverse cardiac phenotypes associated with Csx/Nkx2.5 mutations situated within or around the homeodomain in patients. To make clear of the functional properties of the regions out of homeodomain, we found that mutants locate outside of the homeodomain retained intact nuclear localization and have nearly normal or increased transcriptional activity but impaired DNA binding capability, the C-terminus region exhibits an inhibitory function on transcriptional activity of wild type Csx/Nkx2.5, and the NK2-Specific Domain is likely to facilitate both DNA binding and protein-protein interaction. In the current study, deletion mutant in homeodomain displayed extremely different biological appearance from the mutants with deletion outside of the homeodomain, these may explain the clinical observation that patients with missense situated outside the homeodomain were not associated with atrioventricular conduction disturbance.
PMCID: PMC3685383
Csx/Nkx2.5; sporadic human congenital heart diseases (CHD); regions out of homeodomain; biochemical characteristics
21.  Purification, crystallization and preliminary X-ray diffraction experiments on the breakage-reunion domain of the DNA gyrase from Mycobacterium tuberculosis  
The breakage-reunion domain of M. tuberculosis DNA gyrase was crystallized using the hanging-drop vapour-diffusion method. One of the four crystal forms obtained belonged to space group C2 and diffraction data were collected to a resolution of 2.7 Å.
Mycobacterium tuberculosis DNA gyrase, a nanomachine that is involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target for fluoroquinolone action. The breakage-reunion domain of the A subunit plays an essential role in DNA binding during the catalytic cycle. Two constructs of 53 and 57 kDa (termed GA53BK and GA57BK) corresponding to this domain have been overproduced, purified and crystallized. Diffraction data were collected from four crystal forms. The resolution limits ranged from 4.6 to 2.7 Å depending on the crystal form. The best diffracting crystals belonged to space group C2, with a biological dimer in the asymmetric unit. This is the first report of the crystallization and preliminary X-ray diffraction analysis of the breakage-reunion domain of DNA gyrase from a species containing one unique type II topoisomerase.
PMCID: PMC2777054  PMID: 19923746
tuberculosis; Mycobacterium tuberculosis; type II topoisomerase; DNA gyrase; antibiotic resistance
22.  Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-Dmo-I in complex with its target DNA 
I-Dmo-I is a well characterized homing endonuclease from the archaeon Desulfurococcus mobilis. The enzyme was cloned and overexpressed in Escherichia coli. Crystallization experiments of I-Dmo-I in complex with its DNA target in the presence of Ca2+ and Mg2+ yielded crystals that were suitable for X-ray diffraction analysis.
Homing endonucleases are highly specific DNA-cleaving enzymes that recognize long stretches of base pairs. The availability of these enzymes has opened novel perspectives for genome engineering in a wide range of fields, including gene therapy, by taking advantage of the homologous gene-targeting enhancement induced by a double-strand break. I-Dmo-I is a well characterized homing endonuclease from the archaeon Desulfurococcus mobilis. The enzyme was cloned and overexpressed in Escherichia coli. Crystallization experiments of I-Dmo-I in complex with its DNA target in the presence of Ca2+ and Mg2+ yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 106.75, b = 70.18, c = 106.85 Å, α = γ = 90, β = 119.93°. The self-rotation function and the Matthews coefficient suggested the presence of three protein–DNA complexes per asymmetric unit. The crystals diffracted to a resolution limit of 2.6 Å using synchrotron radiation at the Swiss Light Source (SLS) and the European Synchrotron Radiation Facility (ESRF).
PMCID: PMC2344106  PMID: 18084082
homing endonucleases; I-Dmo-I
23.  The Ambiguous Role of NKX2-5 Mutations in Thyroid Dysgenesis 
PLoS ONE  2012;7(12):e52685.
NKX2-5 is a homeodomain-containing transcription factor implied in both heart and thyroid development. Numerous mutations in NKX2-5 have been reported in individuals with congenital heart disease (CHD), but recently a select few have been associated with thyroid dysgenesis, among which the p.A119S variation. We sequenced NKX2-5 in 303 sporadic CHD patients and 38 families with at least two individuals with CHD. The p.A119S variation was identified in two unrelated patients: one was found in the proband of a family with four affected individuals with CHD and the other in a sporadic CHD patient. Clinical evaluation of heart and thyroid showed that the mutation did not segregate with CHD in the familial case, nor did any of the seven mutation carriers have thyroid abnormalities. We tested the functional consequences of the p.A119S variation in a cellular context by performing transactivation assays with promoters relevant for both heart and thyroid development in rat heart derived H10 cells and HELA cells. There was no difference between wildtype NKX2-5 and p.A119S NKX2-5 in activation of the investigated promoters in both cell lines. Additionally, we reviewed the current literature on the topic, showing that there is no clear evidence for a major pathogenic role of NKX2-5 mutations in thyroid dysgenesis. In conclusion, our study demonstrates that p.A119S does not cause CHD or TD and that it is a rare variation that behaves equal to wildtype NKX2-5. Furthermore, given the wealth of published evidence, we suggest that NKX2-5 mutations do not play a major pathogenic role in thyroid dysgenesis, and that genetic testing of NKX2-5 in TD is not warranted.
PMCID: PMC3532205  PMID: 23285148
24.  The Role of Nkx2.5 in Keratinocyte Differentiation 
Annals of Dermatology  2009;21(4):376-381.
Nkx2.5 is a homeodomain-containing nuclear transcription protein that has been associated with acute T-lymphoblastic leukemia. In addition, Nkx2.5 has an essential role in cardiomyogenesis. However, the expression of Nkx2.5 in the skin has not been investigated.
In an attempt to screen the differentially regulated genes involved in keratinocyte differentiation, using a cDNA microarray, we identified Nkx2.5 as one of the transcription factors controlling the expression of proteins associated with keratinocyte differentiation.
To investigate the expression of Nkx2.5 during keratinocyte differentiation, we used a calcium-induced keratinocyte differentiation model.
RT-PCR and Western blot analysis revealed that the expression of Nkx2.5, in cultured human epidermal keratinocytes, increased with calcium treatment in a time-dependent manner. In normal skin tissue, the expression of Nkx2.5 was detected in the nuclei of the keratinocytes in all layers of the epidermis except the basal layer by immunohistochemistry. In addition, the expression of Nkx2.5 was significantly increased in psoriasis and squamous cell carcinoma, but was barely detected in atopic dermatitis and basal cell carcinoma.
These results suggest that Nkx2.5 may play a role in the change from proliferation to differentiation of keratinocytes and in the pathogenesis of skin disease with aberrant keratinocyte differentiation.
PMCID: PMC2861273  PMID: 20523827
Keratinocyte differentiation; Nkx2.5; Transcription factor
25.  Transcriptional Defect of an Inherited NKX2-5 Haplotype Comprising a SNP, a Nonsynonymous and a Synonymous Mutation, Associated with Human Congenital Heart Disease 
PLoS ONE  2013;8(12):e83295.
Germline mutations in cardiac-specific transcription factor genes have been associated with congenital heart disease (CHD) and the homeodomain transcription factor NKX2-5 is an important member of this group. Indeed, more than 40 heterozygous NKX2-5 germline mutations have been observed in individuals with CHD, and these are spread along the coding region, with many shown to impact protein function. In pursuit of understanding causes of CHD, we analyzed n = 49 cardiac biopsies from 28 patients and identified by direct sequencing two nonsynonymous NKX2-5 alterations affecting alanine 119, namely c.356C>A (p.A119E) and c.355G>T, (p.A119S), in patients with AVSD and HLHS, respectively. In functional assays, a significant reduction in transcriptional activities could be determined for the NKX2-5 variants. Importantly, in one family the mother, besides p.A119E, carried a synonymous mutant allele in the homeodomain (c.543G>A, p.Q181), and a synonymous dbSNP (c.63A>G, p.E21) in the transactivation domain of the protein, that were transmitted to the CHD daughter. The presence of these variants in-cis with the p.A119E mutation led to a further reduction in transcriptional activities. Such difference in activity may be in part related to reduced protein expression for the double variant c.356C>A and c.543G>A. We propose changes in mRNA stability and folding, due to a silent mutation and a dbSNP in the NKX2-5 coding region to contribute to the functional defect. Although the clinical significance of the NKX2-5 haplotype identified in the CHD patients remains to be ascertained, we provide evidence of an interaction of a dbSNP, with synonymous and nonsynonymous mutations to negatively impact NKX2-5 transcriptional activity.
PMCID: PMC3869772  PMID: 24376681

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