Figure 3
Figure 3. Recruitment of polycomb and RNAi machinery components on NFI-A gene. ChIP assays performed in HL60 cells treated (RA) or not (CTR) with RA 1μM, and in K562 cells treated (Ara-C) or not (CTR) with Ara-C 0.5μM for the indicated times. (A) Diagram of the YY1 binding sites (black stars), CpG islands (gray boxes), and CpG clusters (represented by a single ○) in the 5′end regions 1, 2, and 3 of the NFI-A gene promoter region. Numbers indicate nucleotides relative to transcription start sites (+1). ChIP with antibodies anti-YY1, -Suz12, -Dicer1, -Ago1, and -Ago2, and PCR primers as in Figure 2. (B) PCR of NFI-A gene regions 1, 2, and 3. (C) Quantitative RT-PCR of NFI-A region 1 on separate experiments, including the 12-hour time point, and of Ara-C–treated K562 cells. Ratio of the values obtained in RA- or Ara-C–treated versus untreated cell samples. Error bars represent SD of 3 independent evaluations *P < .05. **P < .01. (D) Coimmunoprecipitation/immunoblotting experiments: Ip indicates indicates the antibody used for the coimmunoprecipitation. Rabbit control IgG was used as nonspecific antibody. Coimmunoprecipitates were analyzed by immunoblotting anti-Dicer1 and YY1. Bottom panel: Densitometric analysis by ImageQuant Version 5.2 software of coimmunoprecipitated YY1 in relation to immunoprecipitated Dicer1 after normalization with their respective inputs. (E) ChIP assays carried out on immature human CD34+ HSCs/HPCs and mature CD34− hematopoietic cells isolated from healthy donors, immunoprecipitated with antibodies anti-YY1, anti-Dicer1, and anti-H3K27me3. Recovered DNA was analyzed by PCR using primers described in Figure 2. (F) Quantitative RT-PCRs performed to amplify NFI-A region 1 in immature CD34+ HSCs/HPCs, in CD34− myeloid populations, and in AML patients 7, 8, and 14 treated or not with RA for 72 hours. Error bars represent SD of 3 independent evaluations. In primary human HPCs and AML patient blasts (patients 7 and 8), statistical significance was calculated with respect to immature CD34+ HSCs/HPCs, and for APL patient 14 with respect to untreated APL blasts. *P < .05. **P < .01.

Recruitment of polycomb and RNAi machinery components on NFI-A gene. ChIP assays performed in HL60 cells treated (RA) or not (CTR) with RA 1μM, and in K562 cells treated (Ara-C) or not (CTR) with Ara-C 0.5μM for the indicated times. (A) Diagram of the YY1 binding sites (black stars), CpG islands (gray boxes), and CpG clusters (represented by a single ○) in the 5′end regions 1, 2, and 3 of the NFI-A gene promoter region. Numbers indicate nucleotides relative to transcription start sites (+1). ChIP with antibodies anti-YY1, -Suz12, -Dicer1, -Ago1, and -Ago2, and PCR primers as in Figure 2. (B) PCR of NFI-A gene regions 1, 2, and 3. (C) Quantitative RT-PCR of NFI-A region 1 on separate experiments, including the 12-hour time point, and of Ara-C–treated K562 cells. Ratio of the values obtained in RA- or Ara-C–treated versus untreated cell samples. Error bars represent SD of 3 independent evaluations *P < .05. **P < .01. (D) Coimmunoprecipitation/immunoblotting experiments: Ip indicates indicates the antibody used for the coimmunoprecipitation. Rabbit control IgG was used as nonspecific antibody. Coimmunoprecipitates were analyzed by immunoblotting anti-Dicer1 and YY1. Bottom panel: Densitometric analysis by ImageQuant Version 5.2 software of coimmunoprecipitated YY1 in relation to immunoprecipitated Dicer1 after normalization with their respective inputs. (E) ChIP assays carried out on immature human CD34+ HSCs/HPCs and mature CD34− hematopoietic cells isolated from healthy donors, immunoprecipitated with antibodies anti-YY1, anti-Dicer1, and anti-H3K27me3. Recovered DNA was analyzed by PCR using primers described in Figure 2. (F) Quantitative RT-PCRs performed to amplify NFI-A region 1 in immature CD34+ HSCs/HPCs, in CD34− myeloid populations, and in AML patients 7, 8, and 14 treated or not with RA for 72 hours. Error bars represent SD of 3 independent evaluations. In primary human HPCs and AML patient blasts (patients 7 and 8), statistical significance was calculated with respect to immature CD34+ HSCs/HPCs, and for APL patient 14 with respect to untreated APL blasts. *P < .05. **P < .01.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal