ATP-dependent chromatin remodeling by the CHD family of proteins plays an important role in the regulation of gene transcription. Here we report that full-length CHD8 interacts directly with ␤-catenin and that CHD8 is also recruited specifically to the promoter regions of several ␤-catenin-responsive genes. Our results indicate that CHD8 negatively regulates ␤-catenin-targeted gene expression, since short hairpin RNA against CHD8 results in the activation of several ␤-catenin target genes. This regulation is also conserved through evolution; RNA interference against kismet, the apparent Drosophila ortholog of CHD8, results in a similar activation of ␤-catenin target genes. We also report the first demonstration of chromatin remodeling activity for a member of the CHD6-9 family of proteins, suggesting that CHD8 functions in transcription through the ATP-dependent modulation of chromatin structure.The alteration of chromatin structure provides a key regulatory step for all processes that act upon DNA (41). The factors that regulate this structure, commonly referred to as chromatin remodeling enzymes, can be grouped into two broad categories: complexes that alter chromatin structure via the covalent modification of histones (25,42,83) and complexes that use the energy of ATP hydrolysis to alter the structure or position of the nucleosome (6,47,57,67).ATP-dependent chromatin remodeling enzymes modulate the contacts between histones and DNA. In vitro, these enzymes catalyze structural changes that allow factors to access nucleosomal DNA, reposition nucleosomes on a template, transfer histone octamers to donor DNA, and replace histones with histone variants (27,43,80). In vivo, these activities are crucial for transcription, replication, repair, and recombination of the eukaryotic genome (2,21,59,65). These remodeling enzymes can be divided into numerous families based on domain architecture. One such family is the CHD (chromodomain, helicase, DNA binding) group of proteins, which are critical regulators of chromatin structure (23,26,29,49). These enzymes are characterized by tandem chromodomains N-terminal to their catalytic Snf2 helicase domain.The CHD family can be further subdivided into three subfamilies: CHD1-2, CHD3-5, and CHD6-9. While the first two subfamilies have been extensively studied, very little is known about the CHD6-9 family (29, 49). Previous studies have indicated that CHD8 may regulate the Wnt signaling pathway, since an N-terminal fragment of CHD8 was previously identified as a protein in Rattus norvegicus that binds ␤-catenin both in vivo and in vitro (61). This N-terminal fragment, termed Duplin, contains only the chromodomains and lacks the Snf2 helicase domain and C-terminal sequences. Overexpression of this N-terminal fragment results in inhibition of Tcf4-dependent transcription, and studies of Xenopus embryos demonstrated that this fragment inhibited axis formation and ␤-catenin-mediated axis duplication (61).The "canonical" Wnt signaling pathway functions by controlling the soluble pool ...

A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, we performed genomewide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.

Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing.

Handgrip strength is decreased in the RASopathies. The etiology of the reduced muscle force is unknown, but likely multifactorial.

a b s t r a c tChromodomain, helicase, DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin remodeling enzyme that has been demonstrated to exist within a large protein complex which includes WDR5, Ash2L, and RbBP5, members of the Mixed Lineage Leukemia (MLL) histone modifying complexes. Here we show that CHD8 relocalizes to the promoter of the MLL regulated gene HOXA2 upon gene activation. Depletion of CHD8 enhances HOXA2 expression under activating conditions. Furthermore, depletion of CHD8 results in a loss of the WDR5/Ash2L/RbBP5 subcomplex, and consequently H3K4 trimethylation, at the HOXA2 promoter. These studies suggest that CHD8 alters HOXA2 gene expression and regulates the recruitment of chromatin modifying enzymes. Structured summary:MINT-7542810: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with RbBP5 (uniprotkb:Q15291) by anti tag coimmunoprecipitation (MI:0007) MINT-7542794: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007) MINT-7542820: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with ASH2L (uniprotkb:Q9UBL3) by anti tag coimmunoprecipitation (MI:0007) MINT-7542769: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0914) with RbBP5 (uniprotkb:Q15291), ASH2L (uniprotkb:Q9UBL3) and WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)