CBFA2/RUNX1 Regulates Human Platelet 12-Lipoxygenase: Studies in Runx1 Haplodeficiency.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3648-3648
Author(s):  
Gurpreet Kaur ◽  
Gauthami Jalagadugula ◽  
A. Koneti Rao
Keyword(s):  
Arachidonic Acid ◽  
Protein Binding ◽  
Binding Sites ◽  
Normal Subjects ◽  
Specific Protein ◽  
The Other ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Hel Cells ◽  
12 Lipoxygenase

Abstract Core binding factor 2 (CBFA2), also known as AML1 and RUNX1, is a transcription factor that regulates the expression of genes involved in hematopoiesis, through highly conserved DNA binding region, called RUNT homology domain (RHD). We have previously reported a patient with a mutation (haplodeficiency) in the conserved region of RUNX1/CBFA2 associated with mild thrombocytopenia and impaired platelet function. Expression profiling of patient platelets revealed ∼5 fold decreased mRNA expression of 12-lipoxygenase (12-LO, gene ALOX12) (Sun L. et al. J Thromb Haemost, 5:146–154, 2006). 12-LO catalyzes 12-hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid (AA) upon platelet activation. We have performed studies to determine whether ALOX-12 is regulated by CBFA2. We studied 12-HETE production in patient platelets and the regulation of ALOX-12 by CBFA2 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic (MK) transformation. 12-HETE production was decreased in patient platelets in response to 10 U/ml of thrombin (19.5 ng/10 8 platelets, normal subjects: range 29–306, n=9) and 100 μM of arachidonic acid (5.4, normal subjects: 67– 442, n=10). Three CBFA2 consensus binding sites were identified (−1498/−1493, −708/−70, −526/−521 from ATG) by computer analysis within 2 kb of ALOX-12 5′ upstream region. The binding site at −1489 is a 13nt palindromic sequence with two CBFA2 motifs. The other two CBFA2 binding sites at −708 and −526 bp overlap AP2 binding sites. Luciferase reporter studies in HEL cells using a construct carrying ∼ 1600 bp of 5′ upstream region indicate a greater than 10 fold increase in activity in PMA treated HEL cells relative to untreated cells. Truncation at − 873 bp in PMA-treated HEL cells resulted in a ∼10 fold decrease in activity with only minimal decreases with truncations at −705 or −438 to delete the other CBFA2 binding sites. Mutation of each of the putative CBFA2 binding sites individually resulted in 5–10 fold decrease in activity. Gel shift studies using a 30-mer probe (−1507/−1478) and PMA-treated HEL extracts revealed specific protein binding that was eliminated by CBFA2 antibody and by mutating CBFA2 site from TGGGGT to TGCATT. Specific protein binding was observed with probes containing putative CBFA2 sites at −708 and −526, but it was not altered by the antibody. Chromatin immunoprecipitation (ChIP) analysis using HEL cells demonstrated (PCR amplification) in vivo binding of CBFA2 to ALOX-12 promoter in the region −1507/−1478 but not the other two sites. Conclusions: CBFA2 haplodeficiency is associated with decreased platelet 12-HETE production and 12-LO activity. ALOX-12 is regulated by CBFA2 in megakaryocytes/platelets. These findings are important because of the role of 12-lipoxygenase in platelet arachidonate metabolism and function.

PMA Responsive Sequence (s) in Gαq Promoter during Megakaryocytic Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4246-4246
Author(s):  
Gauthami S. Jalagadugula ◽  
Danny Dhanasekharan ◽  
A.Koneti Rao
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Reporter Gene ◽  
Transcriptional Control ◽  
Nuclear Protein ◽  
Genomic Region ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Megakaryocytic Differentiation ◽  
Hel Cells

Abstract Human erthroleukemia cells (HEL) differentiate towards megakaryocytic (MK) phenotype when stimulated with phorbol 12-myristate-13-acetate (PMA). We observed that the expression of Gq, a protein that plays a major role in platelet signal transduction, is increased in PMA-treated HEL cells. Western blotting revealed that Gq is upregulated in PMA-treated cells relative to untreated cells. Gq gene induction by PMA treatment was investigated with respect to transcriptional control. Serial 5′-truncations of the upstream region (upto 2727 bp from the ATG) of Gq gene were fused to a luciferase (Luc) reporter gene vector, PGL-3 Basic, and were transiently transfected into HEL cells in the absence and presence of PMA (10 nM). After 24 h, reporter gene activities were measured using Dual Luciferase Reporter Assay System (Promega). A reporter plasmid −1042 bp-Luc with a genomic region −1042/−1 showed a 12 fold activity in PMA treated cells and 4 fold activity in untreated cells. Its truncated plasmid with the genomic region −1036/−1 showed a decrease in luciferase activity by 50% in treated cells; and the activity became identical to that in untreated cells. Further truncation between −1036 and −1011 caused a complete loss of activity in both the cells. Thus, a PMA responsive element was localized to a region between −1042 and −1037 bp. Transcription factor data base search (TFSEARCH) predicted two consensus sites for early growth response factor EGR-1 at -1042/−1031 and −1026/−1015. Gel shift studies were performed with two oligos, −1042/−1012 and −1036/−1012, and nuclear extracts from PMA- treated and untreated cells. The studies with −1042/−1012 probe and extracts from treated cells showed that there was nuclear protein binding, which was abolished by competition with the consensus EGR-1 sequence. In extracts from untreated cells, the protein binding was observed but was not competed with consensus EGR-1 sequence. This suggests EGR-1 binding to the region −1042/−1012 in PMA-treated cells and role for this transcription factor in inducing Gq promoter activity. Moreover, studies on the region −1036/−1012 showed nuclear protein binding that was identical between extracts of untreated and treated cells, and it was not competed with consensus EGR-1 sequence. These findings suggest that, EGR-1 binding is localized to −1042/−1037, but not to −1036/−1012. Conclusion: A PMA responsive sequence (−1042/−1037) was identified in the Gq promoter. Our studies suggest that EGR-1 binding to this sequence confers the PMA responsive activity. These studies provide further evidence that EGR-1 plays an important role in the upregulation of Gq expression during PMA induced megakaryocytic differentiation.

Regulation of Platelet PLC-β2 Expression by NF-κB: Studies in Human Platelet PLC-β2 Deficiency.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 699-699 ◽  
Author(s):  
Guangfen Mao ◽  
Satya P. Kunapuli ◽  
A. Koneti Rao
Keyword(s):  
Protein Binding ◽  
Consensus Sequence ◽  
Pcr Amplification ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Mrna Levels ◽  
Luciferase Gene ◽  
Wild Type ◽  
Control Subjects ◽  
Hel Cells

Abstract We have previously described a patient with platelet phospholipase C (PLC)-β2 deficiency characterized by impaired platelet responses to activation with multiple G-protein coupled receptor agonists. The PLC-β2 coding sequence was normal and platelet PLC-β2 mRNA levels were decreased in the patient (Blood, 2002, 99:905). Very little is currently known regarding the transcriptional regulation of PLC-β2. PCR-amplification of patient leukocyte DNA and sequencing of the PLC-β2 5′-upstream region revealed a heterozygous 13-bp deletion (−1645 to −1633 bp from ATG) that encompasses a consensus binding site (GGGAATTCCC) for nuclear factor-κB, NF-κB. This deletion was present in the propositus and her affected son, but not in control subjects. PCR amplification of region −1791 to −1606 bp of genomic DNA revealed one band in 5 control subjects (size ~186 bp) on agarose gel electrophoresis but 2 bands in the patient and her son, consistent with a heterozygous defect. Luciferase reporter gene studies were performed in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA, 30 nM) to induce megakaryocytic transformation. Genomic fragment (−1648/−23 nt) of PLC-β2 5′-upstream sequence and its truncated form without the 13 nt region (−1633/−23 nt) were inserted upstream of luciferase gene in a promoterless expression vector PGL3-basic (Promega) and transiently transfected into HEL cells. Truncation of the wild-type −1648/−23 fragment at 1631 bp resulted in a consistent decrease in promoter activity by ~ 25% (6 experiments, p<0.05). Protein binding assay (EMSA) was performed using PMA-treated HEL cell nuclear extracts and oligonucleotide probes (−1652/−1628 bp) with wild-type and mutated NF-κB consensus sites. Specific protein binding to the wild-type oligonucleotide was abolished when the NF-κB consensus sequence was deleted or mutated. Protein binding to wild-type probe was not competed by the unlabeled mutant oligonucleotide lacking NF-κB consensus sequence. In supershift assay, antibody targeted against the p65 subunit of NF-κB abolished protein binding, indicating a role for NF-κB. In summary, our studies demonstrate in the 5′-upstream region of PLC-β2 gene of the patient a 13-bp deletion that has a consensus site for NF-κB. Luciferase gene promoter assays demonstrate loss of activity when the 13-bp site is truncated. These studies provide evidence that impaired regulation of PLC-β2 gene by NF-κB may be the basis for the PLC-β2 deficiency in our patient. They show for the first time that PLC-β2, the most abundant β-PLC in platelets, is regulated by NF-κB. These findings are highly relevant because of the important role of PLC-β2 in platelet function, and of NF-κB in megakaryocytic differentiation and atherosclerosis.

Platelet/Megakaryocyte PKC-Θ Is a Transcriptional Target of RUNX1/ CBFA2: Studies in Human RUNX1 Haplodeficiency.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1846-1846
Author(s):  
Gauthami S Jalagadugula ◽  
Gurpreet Kaur ◽  
Guangfen Mao ◽  
Danny Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Platelet Function ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Mrna Levels ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
Hel Cells ◽  
Transcriptional Target

Abstract Protein kinase C Θ (PKC-Θ) is an important signaling molecule and regulates platelet responses to activation including aggregation and secretion. In a patient with lifelong thrombocytopenia, impaired platelet aggregation and secretion, we have shown (Gabbeta et al 1996, Blood 87:1368–1376) that phosphorylation of pleckstrin (a PKC substrate) and myosin light chain (MLC) is impaired along with diminished GPIIb-IIIa activation. Platelet protein and mRNA levels of PKC-Θ were decreased with normal levels of other PKC isozymes. These findings were associated with a heterozygous nonsense mutation in transcription factor RUNX1 (also known as CBFA2 or AML1) (Sun et al 2004, Blood 103:948–54). RUNX1 is transcription factor that plays a major role in megakaryopoiesis, megakaryocytic maturation, and platelet production. Haplodeficiency of RUNX1 has been associated with familial thrombocytopenia, impaired megakaryopoiesis, impaired platelet function and predisposition to acute myeloid leukemia. Because of the important role of PKC-Θ in platelet activation and of RUNX1 in hematopoiesis, we addressed the hypothesis that PKC-Θ is a direct transcriptional target of RUNX1. Studies were performed using human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. Chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody revealed RUNX1 binding to chromatin in the PKC-Θ 5’ upstream region −1225/−1056 bp from ATG codon. This region includes a RUNX1 consensus binding site ACCGCA at −1081/−1076 bp identified by TFSEARCH. We performed electrophoretic mobility shift assay (EMSA) using 20-mer probe −1088/−1069 containing the RUNX1 site and nuclear extracts from PMA-treated HEL cells. Protein binding to the probe was observed, which was competed by excess unlabelled probe, and anti-RUNX1 antibody inhibited this binding, indicating that RUNX1 was involved in the DNA binding. Moreover, protein binding to the wild type probe was not competed by an oligo with 4 nucleotides deleted from the RUNX1 consensus site. To determine the functional relevance of RUNX1 binding to PKC-Θ, transient transfections were performed in HEL cells with luciferase reporter constructs. The full length construct −1085/−206 showed ~14-fold activity compared to empty vector. A mutant construct with deletion of the RUNX1 site resulted in a ~50% decrease in activity indicating that the site was functional. siRNA-mediated knockdown of RUNX1 in HEL cells was associated with a decrease in both RUNX1 and PKC-Θ protein. Conclusion: These results and our findings in the patient provide the first evidence that PKC-Θ gene transcription in the megakaryocyte/platelet is regulated by RUNX1. They provide a cogent mechanism for the platelet PKC-Θ downregulation associated with RUNX1 haplodeficiency in our patient. RUNX1 dysregulation of PKC-Θ in megakaryocytic cells is an important aspect of the abnormal platelet function and production associated with human RUNX1 mutations.

EVALUATION OF TISSUE STEROID BINDING IN VITRO

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S223-S246 ◽  
Author(s):  
C. R. Wira ◽  
H. Rochefort ◽  
E. E. Baulieu
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Experimental System ◽  
Specific Protein ◽  
Coupling Mechanism ◽  
Target Tissue ◽  
Protein Binding Sites ◽  
Definition Of ◽  
Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

scholarly journals Maximal Activity of an Erythroid-specific Enhancer Requires the Presence of Specific Protein Binding Sites in Linked Promoters

1998 ◽  
Vol 273 (22) ◽  
pp. 13593-13598 ◽  
Author(s):  
Persis J. Amrolia ◽  
Wesley Gabbard ◽  
John M. Cunningham ◽  
Stephen M. Jane
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Maximal Activity ◽  
Specific Protein ◽  
Protein Binding Sites

Abstract 4023: Aspirin “Resistance”: Role of Pre-existent Platelet Reactivity and Correlation Between Tests

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Andrew L Frelinger ◽  
Youfu Li ◽  
Matthew D Linden ◽  
Inge Tarnow ◽  
Marc R Barnard ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Normal Subjects ◽  
Aspirin Resistance ◽  
The Other ◽  
List Type ◽  
Platelet Function Test ◽  
Function Test

Background: Aspirin “resistance” (i.e. hyporesponsiveness to aspirin in a platelet function test) has been widely reported, but the underlying mechanism is unclear. We examined the role of pre-existent platelet hyperreactivity in aspirin “resistance”. We also determined the correlation between aspirin resistance defined by serum thromboxane (TX) B 2 (the most specific test of aspirin’s effect) and other assays of platelet function. Methods: Platelet function measured before and after aspirin 81 mg daily for 7 days was analyzed by Spearman’s rank correlation. Normal subjects (n=165) were studied because virtually all clinically relevant patients are already taking aspirin. An additional advantage of the use of normal subjects is that the platelet response to stimuli is not influenced (with resultant increased scatter of the data) by an underlying disease, e.g. coronary artery disease, which causes platelet hyperreactivity. Results: The proportion of the post-aspirin platelet function predicted by the pre-aspirin platelet function was 28.3 ± 7.5% (mean ± asymptotic standard error) for serum TXB 2 , 39.3 ± 6.8% for urinary 11-dehydro TXB 2 , 4.4 ± 7.7% for arachidonic acid-induced platelet aggregation, 40.4 ± 7.1% for ADP-induced platelet aggregation, 26.3 ± 9.2% for the VerifyNow Aspirin Assay®, and 45.0 ± 10.9% for the TEG® PlateletMapping ™ System with arachidonic acid. Spearman rank order correlations were highly significant for comparisons between assays when both pre-aspirin and post-aspirin results were included in the analysis. However, residual serum TXB 2 levels post-aspirin treatment were not significantly associated with post-treatment results of any of the other assays. Platelet count correlated with pre-aspirin serum TXB 2 and VerifyNow Aspirin Assay, but not with any post-aspirin platelet function test. Conclusions: Aspirin “resistance” (i.e. hyporesponsiveness to aspirin in a laboratory test) is in part unrelated to aspirin but is the result of underlying platelet hyperreactivity prior to the institution of aspirin therapy. Individuals identified as aspirin “resistant” defined by serum TXB 2 are not the same individuals identified by the other tests.

scholarly journals Identification of regulatory sequences and protein-binding sites in the liver-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

1991 ◽  
Vol 11 (2) ◽  
pp. 1099-1106 ◽  
Author(s):  
F P Lemaigre ◽  
S M Durviaux ◽  
G G Rousseau
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Specific Protein ◽  
Regulatory Sequences ◽  
Alternative Promoters ◽  
Gene Encoding ◽  
Protein Binding Sites ◽  
Cis Acting ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.

scholarly journals Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Gαq promoter during megakaryocytic differentiation

2008 ◽  
Vol 100 (05) ◽  
pp. 821-828 ◽  
Author(s):  
Gauthami Jalagadugula ◽  
Danny N. Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Promoter Sequence ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Luciferase Reporter Gene ◽  
Nuclear Extracts ◽  
Upstream Promoter ◽  
Hel Cells ◽  
The Difference ◽  
Responsive Element ◽  
Increased Activity

SummaryGαq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Gαq is upregulated during phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic transformation of human erythroleukemia (HEL) cells and regulated by EGR-1, an early growth transcription factor. These studies focused on the initial 238 bp of the 5’ upstream region of the Gαq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5’ upstream of the Gαq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Gαq 5’ upstream promoter sequence -1042/-1 showed an about four-fold increased activity in PMA-treated compared to untreated cells. Deletion of 6-nt-1042/-1037 eliminated the difference. Gel-shift studies on Gαq probe (-1042/-1012 bp) revealed binding of EGR-1 with PMA-treated but not untreated nuclear extracts, and this was dependent on the sequence –1042/-1037.Silencing of endogenous EGR-1 inhibited Gαq induction by PMA. MEK/ERK inhibitor U0126 blocked PMA effect on promoter activity of the -1042/-1 construct. In conclusion, EGR-1 binding to sequence –1042/-1037 bp in Gαq promoter mediates the induction of Gαq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Gαq promoter, and new insights into regulation of Gαq gene by EGR-1.

RUNX1/CBFA2 Regulates Myosin Light Chain 9 (MYL9) in Megakaryocytic Cells: Decreased MYL9 Expression in Human RUNX1 Haplodeficiency.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1831-1831
Author(s):  
Gauthami S Jalagadugula ◽  
Gurpreet Kaur ◽  
Guangfen Mao ◽  
Danny Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Platelet Function ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Construct ◽  
Hel Cells

Abstract RUNX1 (also known as CBFA2 or AML1) is a transcription factor that plays a major role in hematopoiesis. Haplodeficiency of RUNX1 has been associated with familial thrombocytopenia, impaired megakaryopoiesis, impaired platelet function and predisposition to acute myeloid leukemia. We have reported a patient with inherited thrombocytopenia and abnormal platelet function (Gabbeta et al, Blood87:1368–76, 1996). The patient platelets showed impaired phosphorylation of pleckstrin and myosin light chain, diminished GPIIb-IIIa activation and decreased platelet protein kinase C-𝛉. This was associated with a heterozygous nonsense mutation in transcription factor RUNX1 (Sun et al, Blood103: 948–54, 2004). Platelet transcript profiling showed a striking downregulation of myosin light chain 9 (MYL9) by ~77-fold relative to normal platelets (Sun et al, J. Thromb Haemost.5: 146–54, 2007). Myosin light chains (MLCs) play an important role in platelet responses to activation, in platelet biogenesis, and are involved in cellular processes such as cytokinesis, cell adhesion, cell contraction, cell migration. We have addressed the hypothesis that MYL9 is a direct transcriptional target of RUNX1. Studies were performed in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. To determine endogenous interaction of RUNX1 with MYL9 promoter, we performed chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody. These studies revealed RUNX1 binding to MYL9 chromatin at −742/−529 bp upstream of the ATG codon. TFSEARCH revealed four RUNX1 sites within this region. We performed electrophoretic mobility shift assay (EMSA) using probes containing each of the RUNX1 motifs and PMA-treated nuclear extracts from HEL cells. With each probe, protein binding was observed that was competed by excess unlabelled probe and inhibited by anti-RUNX1 antibody indicating RUNX1 as the protein involved. This protein binding was not competed by oligos containing mutations in the specific RUNX1 sites. No binding was noted directly to the mutant probes. To further corroborate our findings, we performed transient-ChIP analysis where wild type luciferase reporter construct −691/+4 and constructs with each of the RUNX1 sites individually mutated were transiently transfected into HEL cells. ChIP was performed using these cells and anti-RUNX1 antibody, and the expression analyzed by PCR amplification with a forward primer from MYL9 promoter sequence and reverse primer from luciferase vector sequence. Amplification was observed with immunoprecipitated wild type construct but not with any of the mutant constructs. Thus, RUNX1 interacts in vivo with MYL9 promoter, and the multiple RUNX1 sites interact with each other, as also shown for other genes. To test the functional relevance, the wild type construct −691/+4 containing all 4 RUNX1 sites or mutant constructs with each site individually deleted were cloned into firefly luciferase reporter gene vector and transfected into HEL cells. Deletion of RUNX1 site 1, 2, 3 or 4 caused ~60–90% reduction in the activity indicating that each site was functional. Lastly, siRNA mediated knock down of RUNX1 in HEL cells was associated with a decrease in both RUNX1 and MYL9 protein. Conclusions: Our results provide the first evidence that MYL9 gene is transcriptionally regulated by RUNX1. They provide evidence for the presence of multiple RUNX1 sites in MYL9 promoter, as also observed in other genes. Moreover, these studies provide a cogent mechanism for the MYL9 transcript downregulation and the impaired MLC-phosphorylation we have previously described in association with RUNX1 haplodeficiency.

Sign in / Sign up

Export Citation Format

Share Document