The activity of eukaryotic genomes is regulated by dynamic changes in chromatin structure. A multitude of nucleosome remodeling enzymes, histone modifying activities and chromatin binding proteins cooperate to establish, maintain and reprogram chromatin structures that determine genome activity.
heat shock (HS) genes provide a textbook example of how dramatic changes in the organismal and cellular environment affect chromatin structure in a manner that promotes transcriptional activation of genes coding for molecular chaperones required during the HS response. Upon temperature shift, the HS loci of polytene chromosomes form transcriptionally active “puffs”. This rapid chromatin decondensation correlates with a strong decrease in nucleosome density 
. Puff formation can be uncoupled from transcription and much of the nucleosome loss at the hsp70
gene occurs prior to the first round of transcription 
. Recently, heat shock factor (HSF), GAGA factor and poly-[ADP-ribose] polymerase (PARP) have been shown to be required for the rapid removal of nucleosomes upon activation of the hsp70
. In addition, HS puffs accumulate PARylated proteins and puff formation depends on PARP activity 
. The mechanisms underlying PARP action during HS gene activation are not clear. It has been suggested that PARylation may be removing proteins, including histones - which are themselves a good PARP substrate - thereby promoting chromatin opening 
. The accumulation of PARylated proteins at HS loci has recently been proposed to build up a “transcription compartment” which hinders the diffusion of proteins into and out of the compartment, thus favouring factor recycling 
. In addition to histone displacement and transcription compartment formation at HS genes, recent evidence suggests that PARylation could also act as a signaling scaffold for the recruitment of PAR-sensing factors during DNA damage. In mammals PARylation at DNA damage sites can mediate the recruitment of several ATP-dependent nucleosome remodeling enzymes 
. Here we sought to address whether and how nucleosome remodelers may be recruited to PARP activation sites upon environmental stresses other than DNA damage. We have investigated a paradigm of environmental stress, the activation of HS loci in Drosophila
and have analyzed the mechanism through which the nucleosome remodeler dMi-2 is recruited to HS genes.
Mi-2 (CHD3/CHD4) is a conserved ATP-dependent nucleosome remodeler. In both vertebrates and invertebrates, it is a subunit of Nucleosome Remodeling and Deacetylation
(NuRD) complexes. NuRD complexes repress cell type specific genes during differentiation 
. dMi-2 is also a subunit of the Drosophila-specific Mep-1 complex
(dMec) which represses neuron-specific genes during differentiation of the peripheral nervous system 
Mi-2 containing complexes lack subunits with sequence-specific DNA binding activity. Two main mechanisms for their recruitment to chromatin have been suggested. First, NuRD complexes contain subunits with methylated DNA binding domains
(MBD) which direct NuRD to methylated DNA 
. This is unlikely to be a major recruitment mechanism for Drosophila
Mi-2 complexes, however, given the low and transient levels of DNA methylation in this organism 
. A second mode of Mi-2 recruitment involves interactions with DNA bound transcription factors 
. In addition, SUMOylation of transcription factors can increase their affinity for Mi-2 complexes 
Despite its well established role in repression, dMi-2 localises to actively transcribed chromosome regions suggesting an unexpected potential function of dMi-2 in transcription 
. Here we sought to establish how dMi-2 is recruited to actively transcribed chromatin and to clarify its role in transcriptional activation using genetic, biochemical and pharmacological assays. We show that dMi-2 rapidly associates with activated HS loci, covering the entire transcribed region of the hsp70
gene. dMi-2 recruitment is not affected when transcriptional elongation is blocked but is abrogated when PARP is inhibited. Indeed, we find that dMi-2 specifically binds PARP's oligomeric product PAR in vitro.
Significantly, a dMi-2 mutant unable to bind PAR is not recruited to active HS loci in vivo
. We have identified several regions of dMi-2 that bind PAR in vitro
. These include the chromodomains and a series of K/R-rich motifs near the N-terminus. Further, dMi-2 depletion or expression of an inactive enzyme greatly decreases transcript levels, suggesting that dMi-2 actively supports efficient HS gene expression. Indeed, dMi-2 associates with nascent hsp70
transcripts in vivo
and ablation of dMi-2 function results in inefficient RNA processing. RNA and PAR compete for dMi-2 binding suggesting a two step process of dMi-2 association with HS genes: intial recruitment of dMi-2 is effected by its binding to PAR which is produced prior to the onset of transcription, dMi-2 then switches to interacting with the emerging nascent transcripts. Taken together, our results uncover PAR binding as a novel mechanism for the recruitment of the nucleosome remodeler dMi-2 to targeted sites of PARP activitation upon environmental stress and demonstrate that dMi-2 acts as a co-activator for the full transcriptional activation of HS genes. This study provides the first evidence for an in vivo
function of PARylation in promoting the recruitment of a nucleosome remodeler to support the transcription of stress induced genes.