Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein

2009 ◽  
Vol 39 (3) ◽  
pp. 195-201 ◽  
Author(s):  
Hee Keun Lee ◽  
Mee Hyun Song ◽  
Myengmo Kang ◽  
Jung Tae Lee ◽  
Kyoung-Ah Kong ◽  
...  
Keyword(s):  
Dna Binding ◽  
Inner Ear ◽  
Molecular Mechanisms ◽  
Tertiary Structure ◽  
In Vitro Assays ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dna Binding Domains ◽  
Binding Domains

X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.

scholarly journals Fused protein domains inhibit DNA binding by LexA.

1992 ◽  
Vol 12 (7) ◽  
pp. 3006-3014 ◽  
Author(s):  
E A Golemis ◽  
R Brent
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
In Vitro Assays ◽  
Dimerization Domain ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Operator Binding

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

Ikaros 6 Immortalizes Murine Hematopoietic Precursors In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4354-4354
Author(s):  
Anna Ruiz ◽  
Hugh J.M. Brady
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Zinc Finger Protein ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Delayed Cell Death

Abstract The Ikaros transcription factor has been shown to play an important role in the differentiation of both the myeloid and lymphoid lineages. The ikaros gene encodes for a zinc finger protein containing seven exons that can be alternatively spliced generating several isoforms with differing functional properties. Isoforms with less than three DNA binding domains act as dominant negative (DN) by forming complexes with longer isoforms and interfering with their DNA binding and transcriptional activation ability. Mice heterozygous for a DN ikaros isoform develop T cell leukemia and lymphoma with 100% penetrance. Overexpression of DN Ikaros isoforms has been found in some forms of leukemias. We have previously reported overexpression of the DN Ikaros6 (Ik6) isoform in a subset of leukemia patients harboring t(4;11) translocations. In addition, we inducibly expressed Ik6 in BaF3 cells and found that Ik6 overexpression delayed cell death after IL-3 withdrawal. To further investigate the leukemogenic properties of Ik6 overexpression, we have transduced murine hematopoietic precursors with a retroviral Ik6 expression vector and have analysed the effects on proliferation and differentiation of these precursors by in vitro colony formation assays. We have found that Ik6 can immortalize murine hematopopietic precursors in these in vitro assays. We are currently analysing the leukemogenic potential of Ik6 in vivo by transplanting Ik6 expressing cell lines into NOD/SCID mice.

Fused protein domains inhibit DNA binding by LexA

1992 ◽  
Vol 12 (7) ◽  
pp. 3006-3014
Author(s):  
E A Golemis ◽  
R Brent
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
In Vitro Assays ◽  
Dimerization Domain ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Operator Binding

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

scholarly journals The Chromokinesin Kid Is Required for Maintenance of Proper Metaphase Spindle Size

2005 ◽  
Vol 16 (11) ◽  
pp. 5455-5463 ◽  
Author(s):  
Noriko Tokai-Nishizumi ◽  
Miho Ohsugi ◽  
Emiko Suzuki ◽  
Tadashi Yamamoto
Keyword(s):  
Dna Binding ◽  
Coiled Coil ◽  
Metaphase Plate ◽  
Wild Type ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Chromosome Alignment ◽  
Spindle Microtubules ◽  
Similar Domain

The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes—imprecise chromosome alignment at metaphase and shortened spindles— exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65

1993 ◽  
Vol 13 (2) ◽  
pp. 852-860
Author(s):  
M B Toledano ◽  
D Ghosh ◽  
F Trinh ◽  
W J Leonard
Keyword(s):  
Dna Binding ◽  
Point Mutations ◽  
Terminal Region ◽  
Sequence Motif ◽  
Structural Determinants ◽  
Nf Kappa B ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Binding Specificity

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.

scholarly journals RNA polymerase II transcription factors H4TF-1 and H4TF-2 require metal to bind specific DNA sequences.

1987 ◽  
Vol 7 (12) ◽  
pp. 4582-4584 ◽  
Author(s):  
L Dailey ◽  
S B Roberts ◽  
N Heintz
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Chelating Agents ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Rna Polymerase Ii Transcription

Specific DNA-binding and in vitro transcription activities of H4TF-1 and H4TF-2 are inactivated by chelating agents. Binding activity is restored by addition of Zn2+, and H4TF-2 is also reactivated by Fe2+. In contrast, preformed factor-DNA complexes are resistant to chelators. Therefore, metal ions are a required component of the H4TF-1 and H4TF-2 DNA-binding domains.

Targeting DNA Repair Gene, RAD52, Induces Exhaustion of the Proliferating CML-CP Leukemia Stem Cells Carrying Oxidative DNA Damage

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 447-447
Author(s):  
Kimberly Cramer ◽  
Elisabeth Bolton ◽  
Margaret Nieborowska-Skorska ◽  
Sylwia Flis ◽  
Tomasz Skorski
Keyword(s):  
Stem Cells ◽  
Dna Binding ◽  
Leukemia Stem Cells ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Dsb Repair ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Fold Reduction ◽  
Clonogenic Potential

Abstract Abstract 447 BCR-ABL1 transforms hematopoietic stem cells (HSCs) into leukemia stem cells (LSCs) to induce chronic myeloid leukemia in chronic phase (CML-CP). We detected that the most primitive LSCs display elevated levels of reactive oxygen species (ROS) and accumulate excessive numbers of potentially lethal DNA double-strand breaks (DSBs). We also reported that BCR-ABL1-transformed cells exhibit enhanced RAD51-mediated homologous recombination repair (HRR) activity occurring in S and G2/M cell cycle phases. In normal cells initiation of RAD51-mediated HRR is directed either by BRCA1– or RAD52–dependent mechanisms. Since BCR-ABL1 kinase downregulated BRCA1, LSCs containing high number of DSBs should depend more on RAD52 to promote HRR to repair lethal DSBs. We found that in vivo leukemogenic potential of BCR-ABL1 –positive RAD52−/− hematopoietic cells is abrogated in comparison to their BCR-ABL1 -positive RAD52+/+ counterparts. The absence of RAD52 in BCR-ABL1 –positive cells reduced the percentage of Lin−Kit+Sca1+ cells by >2-fold and inhibited their clonogenic potential and proliferation by >10-fold. In addition RAD52 knockout caused approximately 2-fold reduction of Lin−Kit+Sca1+CD34−Flt3− long-term LSCs (LT-LSCs) and Lin−Kit+Sca1+CD34+Flt3− short-term LSCs (ST-LSCs). Conversely, 4-fold accumulation of BCR-ABL1 –positive RAD52−/− Lin−Kit+Sca1+eFluor670max quiescent cells was detected in comparison to BCR-ABL1 –positive RAD52+/+ counterparts. These effects were accompanied by 2-fold reduction of the percentage of BCR-ABL1 –positive RAD52−/− cells in S and G2/M and 7-fold increase of these cells in sub-G1 when compared to BCR-ABL1 –positive RAD52+/+ counterparts. BCR-ABL1-positive RAD52−/− Lin−Kit+Sca1+ cells accumulated more DSBs than BCR-ABL1 –positive RAD52+/+ cells. These differences were not observed between non-transformed RAD52−/− and RAD52+/+ cells. Expression of the wild-type RAD52 reduced the accumulation of lethal DSBs and rescued the clonogenic potential and proliferation of BCR-ABL1-positive RAD52−/− Lin−Kit+Sca1+ cells. Downregulation of ROS with antioxidants vitamin E (VE) and N-acetyl-cysteine (NAC) exerted similar effect as restored expression of RAD52. Thus it appears that RAD52 is necessary to repair the extensive ROS-induced DSBs in LSC-enriched Lin−Kit+Sca1+ cells. BCR-ABL1 kinase does not affect the expression of RAD52 protein, but phosphorylates RAD52 on Y104. However, expression of RAD52(Y104F) phosphorylation-less mutant reduced the number of DSBs and rescued the clonogenic potential of BCR-ABL1-positive RAD52−/− Lin−Kit+Sca1+ cells in a similar way to the wild-type RAD52. Accordingly, RAD52-mediated DSB repair activity in CML-CP cells should not be affected by imatinib treatment. RAD52 mediates the annealing of complementarry DNA strands during DSB repair. To exert this function RAD52 has two DNA binding domains. Expression of RAD52(F79A) and RAD52(K102A) DNA binding-deficient mutants (each amino acid substitution inactivated different DNA binding domain) failed to prevent the accumulation of DSBs and did not rescue the clonogenic and proliferative potential of BCR-ABL1-positive RAD52−/− cells. In addition, RAD52(F79A), but not RAD52(Y104F) inhibited DSB repair by HRR. Therefore DNA binding capability of RAD52 appears essential for BCR-ABL1 –mediated leukemogenesis, but it is dispensable in normal hematopoietic cells. The “addiction” of BCR-ABL1 leukemia cells to RAD52 was confirmed by demonstration that RAD52(F79A) mutant inhibited clonogenic potential of CD34+ CML-CP cells, but not normal counterparts. Furthermore, to determine if RAD52 DNA binding domains could be targeted to selectively inhibit CML-CP, peptide aptamers containing RAD52 DNA binding domain amino acids sequence surrounding F79 were employed as potential decoys for RAD52 DNA binding. Aptamer containing F79, but not the A79 substitution, diminished the number of RAD52 foci and reduced the clonogenic potential and proliferation of CD34+ cells from CML-CP, but not from normal donors. In conclusion, we postulate that RAD52 is essential for BCR-ABL1 –mediated leukemogenesis and that DNA binding domains of RAD52 may be targeted for selective elimination of the proliferating CML-CP LSCs. Disclosures: No relevant conflicts of interest to declare.

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