Limits...
Therapeutic implications of activation of the host gene (Dleu2) promoter for miR-15a/16-1 in chronic lymphocytic leukemia.

Kasar S, Underbayev C, Yuan Y, Hanlon M, Aly S, Khan H, Chang V, Batish M, Gavrilova T, Badiane F, Degheidy H, Marti G, Raveche E - Oncogene (2013)

Bottom Line: Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1.CLL patient B-cell expression levels of BSAP were increased compared with control sources of B cells.With the use of small interfering RNA-mediated repression, the levels of BSAP were decreased in vitro in the NZB-derived malignant B-1 cell line, LNC, and in ex vivo CLL patient peripheral blood mononuclear cells (PBMCs).

View Article: PubMed Central - PubMed

Affiliation: 1] New Jersey Medical School, Rutgers, Newark, NJ, USA [2].

ABSTRACT
Genetic lesions and other regulatory events lead to silencing of the 13q14 locus in a majority of chronic lymphocytic leukemia (CLL) patients. This locus encodes a pair of critical proapoptotic microRNAs, miR-15a/16-1. Decreased levels of miR-15a/16-1 are critical for the increased survival exhibited by CLL cells. Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1. In this paper, we have identified additional molecular mechanisms regulating miR-15a/16-1 levels and have shown that the transcription factor BSAP (B-cell-specific activator protein) directly interacts with Dleu2, the host gene containing the miR-15a/16-1 loci, and by negative regulation of the Dleu2 promoter, results in repression of miR-15a/16-1 expression. CLL patient B-cell expression levels of BSAP were increased compared with control sources of B cells. With the use of small interfering RNA-mediated repression, the levels of BSAP were decreased in vitro in the NZB-derived malignant B-1 cell line, LNC, and in ex vivo CLL patient peripheral blood mononuclear cells (PBMCs). BSAP knockdown led to an increase in the expression of miR-15a/16-1 and an increase in apoptosis, and a cell cycle arrest in both the cell line and patient PBMCs. Moreover, using Dleu2 promoter analysis by chromatin immunoprecipitation assay, we have shown that BSAP directly interacts with the Dleu2 promoter. Derepression of the Dleu2 promoter via inhibition of histone deacetylation combined with BSAP knockdown increased miR-15a/16-1 expression, and also increased malignant B-cell death. In summary, therapy targeting enhanced host gene Dleu2 transcription may augment CLL therapy.

Show MeSH

Related in: MedlinePlus

Dleu2 Promoter Occupancy by BSAP in Human and MouseA) Representative data of promoter occupancy in human B cell line (Daudi) following immunoprecipitation with IgG (grey) or BSAP (black) antibody. B) Delta % Input = (% Input BSAP IP − % Input IgG), indicates promoter enrichment over pull down with non-specific IgG antibody in Daudi cells. C) Cumulative data of promoter enrichment ChIP experiments in CLL PBMC, n=4, D) Promoter analysis in mouse, schematic of the Dleu2 promoter in mouse, the transcription start site (Dleu2 TSS), the flanking upstream (U) and downstream (D) regions are indicated and the total region is approximately 1kb. The BSAP binding site is shown as a star. Chart is enrichment of CD19, GAPDH, Dleu2 D and Dleu2 U fragments in LNC, the NZB mouse CLL cell line (mean values of 3 independent experiments). *p<0.05, Student’s t-test. Error bars indicate ± SEM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4508006&req=5

Figure 4: Dleu2 Promoter Occupancy by BSAP in Human and MouseA) Representative data of promoter occupancy in human B cell line (Daudi) following immunoprecipitation with IgG (grey) or BSAP (black) antibody. B) Delta % Input = (% Input BSAP IP − % Input IgG), indicates promoter enrichment over pull down with non-specific IgG antibody in Daudi cells. C) Cumulative data of promoter enrichment ChIP experiments in CLL PBMC, n=4, D) Promoter analysis in mouse, schematic of the Dleu2 promoter in mouse, the transcription start site (Dleu2 TSS), the flanking upstream (U) and downstream (D) regions are indicated and the total region is approximately 1kb. The BSAP binding site is shown as a star. Chart is enrichment of CD19, GAPDH, Dleu2 D and Dleu2 U fragments in LNC, the NZB mouse CLL cell line (mean values of 3 independent experiments). *p<0.05, Student’s t-test. Error bars indicate ± SEM.

Mentions: It is currently unknown whether BSAP regulates Dleu2 promoter directly or indirectly. Promoter analysis using CONSITE failed to show any canonical BSAP binding sites in the human Dleu2 promoter. However, we cannot rule out the possibility that BSAP might be present in a complex with some of its binding partners at the Dleu2 promoter. Binding sites for some of the proteins that BSAP has been known to interact with were found in the human Dleu2 promoter (c-Myb, AML1, E2F). We performed a ChIP assay on Daudi cells to assess BSAP-Dleu2 promoter interactions. Daudi is a B lineage cell line (Burkitt’s Lymphoma) and has BSAP expression [28]. Similar to the positive control CD19 promoter, the Dleu2 promoter was enriched in the BSAP pulldown. No enrichment of the negative control Kras promoter was observed (Fig. 4A, B). The ChIP assay was performed employing 4 CLL PBMC samples in 4 independent pulldown experiments (Fig 4C). The Dleu2 promoter region was also enriched in the CLL samples and was similar to a known promoter, which encodes a BSAP binding site, the CD19 promoter. In contrast, the Kras promoter, which does not encode a BSAP binding site, was not pulldowned in CLL samples (Fig 4C). This data suggests that a BSAP binding site is present at the Dleu2 promoter in human B cells. However, a canonical BSAP binding site was found at +676 position in the mouse Dleu2 gene. A ChIP assay was performed on murine B cells, the LNC cells, to assess BSAP-Dleu2 promoter interactions. Since, we knew the position of the putative BSAP binding site in mouse Dleu2; we were able to design a ChIP assay having an internal control in addition to the GAPDH control as shown in Fig. 4D. Indeed, the Dleu2 promoter fragments were enriched in the BSAP pulldown and the amount of enrichment was inversely proportional to the distance from the BSAP binding site (Fig. 4E). The upstream sequences (U) were not as enriched by the BSAP pulldown as were the downstream sequences (D) which contained the BSAP binding site. The presence in the BSAP pulldown of the Dleu2 U region may be due to DNA fragments which contain both the D and U region. The human data indicating BSAP interaction with the Dleu2 promoter were validated in the mouse system which has a canonical BSAP binding site in its promoter.


Therapeutic implications of activation of the host gene (Dleu2) promoter for miR-15a/16-1 in chronic lymphocytic leukemia.

Kasar S, Underbayev C, Yuan Y, Hanlon M, Aly S, Khan H, Chang V, Batish M, Gavrilova T, Badiane F, Degheidy H, Marti G, Raveche E - Oncogene (2013)

Dleu2 Promoter Occupancy by BSAP in Human and MouseA) Representative data of promoter occupancy in human B cell line (Daudi) following immunoprecipitation with IgG (grey) or BSAP (black) antibody. B) Delta % Input = (% Input BSAP IP − % Input IgG), indicates promoter enrichment over pull down with non-specific IgG antibody in Daudi cells. C) Cumulative data of promoter enrichment ChIP experiments in CLL PBMC, n=4, D) Promoter analysis in mouse, schematic of the Dleu2 promoter in mouse, the transcription start site (Dleu2 TSS), the flanking upstream (U) and downstream (D) regions are indicated and the total region is approximately 1kb. The BSAP binding site is shown as a star. Chart is enrichment of CD19, GAPDH, Dleu2 D and Dleu2 U fragments in LNC, the NZB mouse CLL cell line (mean values of 3 independent experiments). *p<0.05, Student’s t-test. Error bars indicate ± SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4508006&req=5

Figure 4: Dleu2 Promoter Occupancy by BSAP in Human and MouseA) Representative data of promoter occupancy in human B cell line (Daudi) following immunoprecipitation with IgG (grey) or BSAP (black) antibody. B) Delta % Input = (% Input BSAP IP − % Input IgG), indicates promoter enrichment over pull down with non-specific IgG antibody in Daudi cells. C) Cumulative data of promoter enrichment ChIP experiments in CLL PBMC, n=4, D) Promoter analysis in mouse, schematic of the Dleu2 promoter in mouse, the transcription start site (Dleu2 TSS), the flanking upstream (U) and downstream (D) regions are indicated and the total region is approximately 1kb. The BSAP binding site is shown as a star. Chart is enrichment of CD19, GAPDH, Dleu2 D and Dleu2 U fragments in LNC, the NZB mouse CLL cell line (mean values of 3 independent experiments). *p<0.05, Student’s t-test. Error bars indicate ± SEM.
Mentions: It is currently unknown whether BSAP regulates Dleu2 promoter directly or indirectly. Promoter analysis using CONSITE failed to show any canonical BSAP binding sites in the human Dleu2 promoter. However, we cannot rule out the possibility that BSAP might be present in a complex with some of its binding partners at the Dleu2 promoter. Binding sites for some of the proteins that BSAP has been known to interact with were found in the human Dleu2 promoter (c-Myb, AML1, E2F). We performed a ChIP assay on Daudi cells to assess BSAP-Dleu2 promoter interactions. Daudi is a B lineage cell line (Burkitt’s Lymphoma) and has BSAP expression [28]. Similar to the positive control CD19 promoter, the Dleu2 promoter was enriched in the BSAP pulldown. No enrichment of the negative control Kras promoter was observed (Fig. 4A, B). The ChIP assay was performed employing 4 CLL PBMC samples in 4 independent pulldown experiments (Fig 4C). The Dleu2 promoter region was also enriched in the CLL samples and was similar to a known promoter, which encodes a BSAP binding site, the CD19 promoter. In contrast, the Kras promoter, which does not encode a BSAP binding site, was not pulldowned in CLL samples (Fig 4C). This data suggests that a BSAP binding site is present at the Dleu2 promoter in human B cells. However, a canonical BSAP binding site was found at +676 position in the mouse Dleu2 gene. A ChIP assay was performed on murine B cells, the LNC cells, to assess BSAP-Dleu2 promoter interactions. Since, we knew the position of the putative BSAP binding site in mouse Dleu2; we were able to design a ChIP assay having an internal control in addition to the GAPDH control as shown in Fig. 4D. Indeed, the Dleu2 promoter fragments were enriched in the BSAP pulldown and the amount of enrichment was inversely proportional to the distance from the BSAP binding site (Fig. 4E). The upstream sequences (U) were not as enriched by the BSAP pulldown as were the downstream sequences (D) which contained the BSAP binding site. The presence in the BSAP pulldown of the Dleu2 U region may be due to DNA fragments which contain both the D and U region. The human data indicating BSAP interaction with the Dleu2 promoter were validated in the mouse system which has a canonical BSAP binding site in its promoter.

Bottom Line: Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1.CLL patient B-cell expression levels of BSAP were increased compared with control sources of B cells.With the use of small interfering RNA-mediated repression, the levels of BSAP were decreased in vitro in the NZB-derived malignant B-1 cell line, LNC, and in ex vivo CLL patient peripheral blood mononuclear cells (PBMCs).

View Article: PubMed Central - PubMed

Affiliation: 1] New Jersey Medical School, Rutgers, Newark, NJ, USA [2].

ABSTRACT
Genetic lesions and other regulatory events lead to silencing of the 13q14 locus in a majority of chronic lymphocytic leukemia (CLL) patients. This locus encodes a pair of critical proapoptotic microRNAs, miR-15a/16-1. Decreased levels of miR-15a/16-1 are critical for the increased survival exhibited by CLL cells. Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1. In this paper, we have identified additional molecular mechanisms regulating miR-15a/16-1 levels and have shown that the transcription factor BSAP (B-cell-specific activator protein) directly interacts with Dleu2, the host gene containing the miR-15a/16-1 loci, and by negative regulation of the Dleu2 promoter, results in repression of miR-15a/16-1 expression. CLL patient B-cell expression levels of BSAP were increased compared with control sources of B cells. With the use of small interfering RNA-mediated repression, the levels of BSAP were decreased in vitro in the NZB-derived malignant B-1 cell line, LNC, and in ex vivo CLL patient peripheral blood mononuclear cells (PBMCs). BSAP knockdown led to an increase in the expression of miR-15a/16-1 and an increase in apoptosis, and a cell cycle arrest in both the cell line and patient PBMCs. Moreover, using Dleu2 promoter analysis by chromatin immunoprecipitation assay, we have shown that BSAP directly interacts with the Dleu2 promoter. Derepression of the Dleu2 promoter via inhibition of histone deacetylation combined with BSAP knockdown increased miR-15a/16-1 expression, and also increased malignant B-cell death. In summary, therapy targeting enhanced host gene Dleu2 transcription may augment CLL therapy.

Show MeSH
Related in: MedlinePlus