Limits...
Recruitment of the ATP-dependent chromatin remodeler dMi-2 to the transcribed region of active heat shock genes.

Mathieu EL, Finkernagel F, Murawska M, Scharfe M, Jarek M, Brehm A - Nucleic Acids Res. (2012)

Bottom Line: Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs.We find that dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A.Rather, dMi-2 binding is restricted to transcribed regions.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Tumor Research, Philipps-University, Emil-Mannkopff-Strasse 2, 35037 Marburg, Germany.

ABSTRACT
The ATP-dependent chromatin remodeler dMi-2 can play both positive and negative roles in gene transcription. Recently, we have shown that dMi-2 is recruited to the hsp70 gene in a heat shock-dependent manner, and is required to achieve high transcript levels. Here, we use chromatin immunoprecipitation sequencing (ChIP-Seq) to identify other chromatin regions displaying increased dMi-2 binding upon heat shock and to characterize the distribution of dMi-2 over heat shock genes. We show that dMi-2 is recruited to the body of at least seven heat shock genes. Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs. We find that dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A. Rather, dMi-2 binding is restricted to transcribed regions. Our results suggest that dMi-2 distribution over active heat shock genes are determined by transcriptional activity.

Show MeSH

Related in: MedlinePlus

dMi-2 does not associate with other stress-induced genes. (A) Upper panel: RT-qPCR analysis of MtnA expression in control cells (0 µM) and cells treated with CdCl2 (100 µM). Values are expressed relative to the value in control cells. (Lower panel) ChIP analyses of dMi-2 binding to the MtnA gene in control cells (0 µM) and cells treated with CdCl2 (100 µM). IgG was used as a negative control. Error bars denote standard deviations from triplicate samples. (B) (Upper panel) schematic representation of Dam fusion cDNA under control of hsp70 promoter. Regions analysed by RT-qPCR and ChIP-qPCR are indicated. (Left) HS-induced transcriptional activation of endogenous hsp70 genes and hsp70-Dam fusion reporter gene were determined by RT-qPCR and are displayed as fold expression (expression level under NHS conditions was set to 1). (Right) dMi-2 and IgG ChIP-qPCR on reporter gene under HS and NHS conditions. (C) Model. dMi-2 distribution over active heat shock genes is closely correlated with transcription.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3367206&req=5

gks178-F5: dMi-2 does not associate with other stress-induced genes. (A) Upper panel: RT-qPCR analysis of MtnA expression in control cells (0 µM) and cells treated with CdCl2 (100 µM). Values are expressed relative to the value in control cells. (Lower panel) ChIP analyses of dMi-2 binding to the MtnA gene in control cells (0 µM) and cells treated with CdCl2 (100 µM). IgG was used as a negative control. Error bars denote standard deviations from triplicate samples. (B) (Upper panel) schematic representation of Dam fusion cDNA under control of hsp70 promoter. Regions analysed by RT-qPCR and ChIP-qPCR are indicated. (Left) HS-induced transcriptional activation of endogenous hsp70 genes and hsp70-Dam fusion reporter gene were determined by RT-qPCR and are displayed as fold expression (expression level under NHS conditions was set to 1). (Right) dMi-2 and IgG ChIP-qPCR on reporter gene under HS and NHS conditions. (C) Model. dMi-2 distribution over active heat shock genes is closely correlated with transcription.

Mentions: Like HS genes, the metallothionein genes are strongly induced under stress conditions. Addition of Cd+ resulted in a 25-fold activation of MtnA transcription (Figure 5A, upper panel). We used ChIP followed by qPCR to assess dMi-2 binding to the transcribed part of the MtnA gene under non-induced and induced conditions. dMi-2 binding to the MtnA gene was not significantly altered following its activation (Figure 5B, lower panel). Taken together, these results indicate that neither strong constitutive transcription nor the strong induction of an inactive gene is sufficient to recruit dMi-2. Recruitment of dMi-2 to HS genes requires, therefore, additional signals.Figure 5.


Recruitment of the ATP-dependent chromatin remodeler dMi-2 to the transcribed region of active heat shock genes.

Mathieu EL, Finkernagel F, Murawska M, Scharfe M, Jarek M, Brehm A - Nucleic Acids Res. (2012)

dMi-2 does not associate with other stress-induced genes. (A) Upper panel: RT-qPCR analysis of MtnA expression in control cells (0 µM) and cells treated with CdCl2 (100 µM). Values are expressed relative to the value in control cells. (Lower panel) ChIP analyses of dMi-2 binding to the MtnA gene in control cells (0 µM) and cells treated with CdCl2 (100 µM). IgG was used as a negative control. Error bars denote standard deviations from triplicate samples. (B) (Upper panel) schematic representation of Dam fusion cDNA under control of hsp70 promoter. Regions analysed by RT-qPCR and ChIP-qPCR are indicated. (Left) HS-induced transcriptional activation of endogenous hsp70 genes and hsp70-Dam fusion reporter gene were determined by RT-qPCR and are displayed as fold expression (expression level under NHS conditions was set to 1). (Right) dMi-2 and IgG ChIP-qPCR on reporter gene under HS and NHS conditions. (C) Model. dMi-2 distribution over active heat shock genes is closely correlated with transcription.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367206&req=5

gks178-F5: dMi-2 does not associate with other stress-induced genes. (A) Upper panel: RT-qPCR analysis of MtnA expression in control cells (0 µM) and cells treated with CdCl2 (100 µM). Values are expressed relative to the value in control cells. (Lower panel) ChIP analyses of dMi-2 binding to the MtnA gene in control cells (0 µM) and cells treated with CdCl2 (100 µM). IgG was used as a negative control. Error bars denote standard deviations from triplicate samples. (B) (Upper panel) schematic representation of Dam fusion cDNA under control of hsp70 promoter. Regions analysed by RT-qPCR and ChIP-qPCR are indicated. (Left) HS-induced transcriptional activation of endogenous hsp70 genes and hsp70-Dam fusion reporter gene were determined by RT-qPCR and are displayed as fold expression (expression level under NHS conditions was set to 1). (Right) dMi-2 and IgG ChIP-qPCR on reporter gene under HS and NHS conditions. (C) Model. dMi-2 distribution over active heat shock genes is closely correlated with transcription.
Mentions: Like HS genes, the metallothionein genes are strongly induced under stress conditions. Addition of Cd+ resulted in a 25-fold activation of MtnA transcription (Figure 5A, upper panel). We used ChIP followed by qPCR to assess dMi-2 binding to the transcribed part of the MtnA gene under non-induced and induced conditions. dMi-2 binding to the MtnA gene was not significantly altered following its activation (Figure 5B, lower panel). Taken together, these results indicate that neither strong constitutive transcription nor the strong induction of an inactive gene is sufficient to recruit dMi-2. Recruitment of dMi-2 to HS genes requires, therefore, additional signals.Figure 5.

Bottom Line: Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs.We find that dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A.Rather, dMi-2 binding is restricted to transcribed regions.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Tumor Research, Philipps-University, Emil-Mannkopff-Strasse 2, 35037 Marburg, Germany.

ABSTRACT
The ATP-dependent chromatin remodeler dMi-2 can play both positive and negative roles in gene transcription. Recently, we have shown that dMi-2 is recruited to the hsp70 gene in a heat shock-dependent manner, and is required to achieve high transcript levels. Here, we use chromatin immunoprecipitation sequencing (ChIP-Seq) to identify other chromatin regions displaying increased dMi-2 binding upon heat shock and to characterize the distribution of dMi-2 over heat shock genes. We show that dMi-2 is recruited to the body of at least seven heat shock genes. Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs. We find that dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A. Rather, dMi-2 binding is restricted to transcribed regions. Our results suggest that dMi-2 distribution over active heat shock genes are determined by transcriptional activity.

Show MeSH
Related in: MedlinePlus