5. Discussion
5.2. Discussion objective 2
5.2.3 The role of CHD4 in transcriptional activation upon TNFα stimulation
stable Mi-2-containing complex has been discovered so far. Additionally, only vague hints exist that there could be a functional role for CHD4 outside the NuRD complex.305 Recently, it was shown that dMi-2 is also part of the newly identified complex dMec.
This complex constitutes the major Mi-2 containing complex in Drosophila.
Furthermore, it was shown not to rely on histone deacetylation to effect transcriptional repression. 306
In this study, due to time limitation I could not test whether CHD4 or the entire NuRD complex is required for gene activation. To evaluate the hypothesis that the entire NuRD complex is needed, it would be reasonable to perform sequential knockdown experiments for all NuRD subunits.
In both known Mi-2-containing complexes, NuRD and dMec, CHD4 was strongly linked to transcriptional repression.307 Therefore, a requirement for gene activation, as shown here, is quite surprising and suggests a role in active transcription. Only few studies have hitherto implicated CHD4 in transcriptional activation. For example, during T-cell development, Mi-2 associates with the CD4 enhancer resulting in
304 Ramirez-Carrozzi VR et al, A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell (2009).
305 As example, see Williams CJ et al, The chromatin remodeler Mi-2beta is required for CD4 expression and T cell development. Immunity. (2004).
306 Kunert N et al, dMec: a novel Mi-2 chromatin remodelling complex involved in transcriptional repression. EMBO J. (2009).
307 Murawska M and Brehm A, CHD chromatin remodelers and the transcription cycle. Transcription (2011).
recruitment of the E-box binding protein HEB and the histone acetyltransferase p300 to the CD4 enhancer. This causes histone H3-hyperacetylation of the regulatory region and enables CD4 transcription. With both factors, CHD4 was shown to interact in a HDAC independent manner suggesting this activity to be independent from NuRD.308 Furthermore, CHD4 was implicated to be involved in rRNA activation in the nucleolus.309 Recently, an activating role of dMi-2 in Drosophila heat shock gene induction was demonstrated. dMi-2 was recruited to Drosophila heat shock genes in a PAR–dependent manner. There, it associated with nascent heat shock gene transcripts and its catalytic activity was required for efficient transcription and co-transcriptional RNA processing.310 Whether a further role of Mi-2 exists in splicing is far from clear but it might recruit splicing machinery to RNA via binding to nascent transcripts.311
In summary, at least in some circumstances CHD4 appeared to be involved in transcriptional activation.
Several mechanisms are conceivable as to how CHD4 could act in transcriptional activation of TNFα-induced genes.
One possible mechanism could be a recruitment of CHD4 to the target genes followed by binding to nascent RNA and involvement in co-transcriptional RNA processing similar to the observed scenario upon heat shock gene activation in Drosophila.
Interestingly, PARP-1 was demonstrated as interacting with NF-κB upon LPS stimulation in mammalian cells312 and TNFα-induced transcriptional activation of a subset of NF-κB target genes was shown to be defective in PARP-1-deficient mice.313 Thus, PARP might also be a candidate to modulate CHD4 action in the context of TNFα pathway.
Moreover, as described for the CD4 locus, CHD4 could associate with a histone acetyltransferase, thereby establishing activating histone marks. One might argue that
308 Williams CJ et al, The chromatin remodeler Mi-2beta is required for CD4 expression and T cell development. Immunity. (2004).
309 Shimono K et al, Microspherule protein 1, Mi-2beta, and RET finger protein associate in the nucleolus and up-regulate ribosomal gene transcription. J Biol Chem. (2005).
310 Murawska M et al, Stress-induced PARP activation mediates recruitment of Drosophila Mi-2 to promote heat shock gene expression. PLoS Genet. (2011).
311 Murawska M and Brehm A, CHD chromatin remodelers and the transcription cycle. Transcription (2011).
312 Ullrich O et al, Regulation of microglial expression of integrins by poly(ADP-ribose) polymerase-1.
Nat Cell Biol. (2001).
313 Oliver FJ et al, Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP-ribose) polymerase-1 deficient mice. EMBO J. (1999).
the observed high basal levels of active histone modifications like acetylation at primary response genes dispense with the requirement for additional histone modifications because the chromatin is already in an open conformation.314 However, the histone acetyltransferase p300 was shown to be required for early target gene induction upon TNFα stimulation,315 thus an additional requirement of activating histone modifications might be feasible. An approach to validate or exclude this hypothesis would be a co-immunoprecipitation of CHD4 and the most abundant acetyltransferases like p300.
In order to explore any direct mechanism in this study, a recruitment of CHD4 to the CXCL2 gene upon TNFα stimulation was tested by ChIP experiments. Such recruitment could not be demonstrated in these experiments. That does not exclude any recruitment because I studied only one model gene. For a reliable result more target genes would have to be studied. Furthermore, the used antibody was not established in ChIP experiments in our laboratory beforehand. In addition, experiments were performed after 20 (data not shown), 40 and 60 minutes. If CHD4 had been recruited in the very initial phase of TNFα-pathway activation and released soon thereafter, detection could have been missed. Furthermore, if our ChIP results indeed represent a very low but steady binding of CHD4 to CXCL2, it might undergo activation by a post-translational modification upon stimulation.
As a second approach, an interaction of CHD4/BRG1 and the NF-κB subunit RelA upon TNFα stimulation was evaluated revealing no robust interaction between the chromatin remodelers and RelA. These results do not support the view that the remodelers are recruited to the target genes via interaction with DNA-bound transcription factors and are acting directly at the affected genes. But so far, a technical cause cannot be excluded as a reason for my results. Because the key to all possible direct mechanisms is almost exclusively the demonstration of a positive recruitment to the tested target genes, for further studies this question should be re-addressed with considerable effort.
314 Ramirez-Carrozzi VR et al, A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell (2009).
315 Covic M et al, Arginine methyltransferase CARM1 is a promoter-specific regulator of NF-kappaB-dependent gene expression. EMBO J. (2005).
In principle, any siRNA-mediated off-target effect can cause irregularities in gene expression. Considering the unaffected four-hour-response and housekeeping gene expression, these explanations become less likely. In addition, identical off-target effects for two siRNAs targeting different regions of the same transcript, as used in this study, seem implausible. Were there to be any remaining doubt, rescue experiments could be performed to ultimately exclude this kind of confounder. 316